Renato N. Inserra

Schistonchus caprifici parasitizing caprifig (Ficus carica sylvestris) florets and the relationship with its fig wasp (Blastophaga psenes) vector

Studies were conducted in Italy on the biology of Schistonchus caprifici (Nemata: Aphelenchoididae). This nematode parasitizes and reproduces on caprifig (Ficus carica sylvestris) inflorescences and also in the haemocoel of the fig pollinator wasp Blastophaga psenes. Staminate and pistillate florets of caprifig infected by S. caprifici exhibited necrosis and cavities in the cortical parenchyma of floret peduncles and stamen filaments. Schistonchus caprifici attacked pistillate florets of commercial fig (F. carica); however, S. caprifici population densities were less (P= 0.05) in commercial fig than in caprifig (550 nematodes vs. 12,000 nematodes/g fresh floret tissues, respectively). Schistonchus caprifici was spread from infected to healthy caprifig inflorescences through oviposition behaviour of the wasp, Blastophaga psenes. Wasp development occurred in inflorescences concomitantly infected with nematodes, which colonized and started reproducing in the haemocoel of wasp larvae. Schistonchus caprifici persisted in the pupae and winged females that developed in the pistillate florets. Wingless male wasps were not infected by the nematode. Commercial fig inflorescences were not receptive to wasp development, although these inflorescences were visited by ovipositing wasps which vectored nematodes in their florets. Nematode infection may result in premature inflorescence decay. There was no evidence of any adverse effect on B. psenes due to S. caprifici infection. The association between S. caprifici and B. psenes on caprifig can be defined as a parasitic relationship because of nematode reproduction in the wasps haemocoel. Nematode populations from wasps and caprifig florets did not differ in their morphology.

Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae)

Phylogenetic analysis of five gene fragments: ITS-rRNA, D2 and D3 of 28S rRNA, 18S rRNA, Hsp90 and actin, of Heterodera species and two representative Afenestrata species, A. koreana and A. orientalis , form a clade with H. cynodontis, H. bifenestra and an unidentified Heterodera sp. infecting grasses. Based on these results and the consideration that the key diagnostic characters of Afenestrata are convergent and do not define a clade, synonymisation of Afenestrata with Heterodera is proposed. The following new combinations are made: H. africana comb. n., H. axonopi comb. n., H. koreana comb. n., and H. orientalis comb. n. Furthermore, H. (= Afenestrata) sacchari is renamed as H. saccharophila nom. nov. to avoid homonymy. All these species, together with H. bamboosi , are regarded as members of a paraphyletic ‘Afenestrata group’ within Heterodera . Whilst recognised as artificial, the Afenestrata group is nevertheless an aid to discussion about these similar species. Morphological and molecular characterisation of populations of H. koreana comb. n. from Florida and H. orientalis comb. n. from Florida and Guatemala verify the identification of these populations as valid representatives for molecular studies of the species. Light and SEM observations also provide new detail and a broader understanding of the morphological range of both species. These include a longer stylet for females of H. koreana comb. n. and H. orientalis comb. n. than reported in the original descriptions. In addition, previously unreported tuberculate ridges are noted on the surface of vulval lips of H. orientalis comb. n. The lip region of second-stage juveniles of H. koreana comb. n. and H. orientalis comb. n. both include fused adjacent submedian lips that also fuse with the labial disc and the second lip annulus. The ITS-rRNA gene sequences of H. orientalis comb. n. populations from Florida and Guatemala were similar to those from the Russian type locality. Diagnostic PCR-RFLP of ITS-rRNA profiles with six enzymes for H. orientalis comb. n. and H. koreana comb. n. are given. A key for the morphological identification of species of the Afenestrata group is provided.

Pratylenchus Pseudocoffeae From Florida and Its Relationship With P. Gutierrezi and P. Coffeae

Morphology and morphometrics of Pratylenchus sp. from aster in Florida, mum and artemisia in Japan (P pseudocoffeae), coffee in Costa Rica and Guatemala (putative populations of P gutierrezi), citrus in Florida (P coffeae), and coffee in Indonesia (P coffeae) were studied to: i) compare the morphologies of the lesion nematodes from aster and those of P. gutierrezi, P. pseudocoffeae, and P coffeae, ii) describe the morphological variability of putative P gutierrezi populations originating from coffee in Costa Rica and Guatemala, and iii) characterize the en face lip morphology of P. coffeae from the type host and near the type locale in Java, Indonesia. Pratylencl2us coffeae from citrus in Florida differed from all other populations, including P coffeae from coffee in Indonesia, in having medial and lateral lips that were fused rather than separate, when viewed en face with scanning electron microscopy. Pharyngeal overlap length and distance from the base of the oesophageal gland lobe to the head end were the only morphometric characters of diagnostic value to separate female P. p.seudocoffeae from P gutierrezi and P coffeae from Indonesia. Pratylenchus pseudocoffeae females also had subhemispherical or hemispherical tails with smooth termini vs subcylindrical tails with bluntly rounded and sometimes coarsely annulated termini for P. gutierrezi., The Pratylenchus population from aster in Florida most closely resembled P pseudocoffeae based on tail and en,face lip morphology, long pharyngeal overlap (60.5-114.5 pm), long distance between the base of the oesophageal gland lobe to the head end (140.5-209.5 pm), and multivariate analyses of morphometric variables. The head pattern of P. coffeae from coffee in Indonesia differed from patterns previously attributed to this species and provides sufficient evidence to reconsider the taxonomic status of P coffeae, P gutierrezi, P. pseudocoffeae, and closely related species.

Characterisation of populations of Longidorus orientalis Loof, 1982 (Nematoda: Dorylaimida) from date palm (Phoenix dactylifera L.) in the USA and other countries and incongruence of phylogenies inferred from ITS1 rRNA and coxI genes

Needle nematode populations of Longidorus orientalis associated with date palm, Phoenix dactylifera, and detected during nematode surveys conducted in Arizona, California and Florida, USA, were characterised morphologically and molecularly. The nematode species most likely arrived in California a century ago with propagative date palms from the Middle East and eventually spread to Florida on ornamental date palms that were shipped from Arizona and California. This is the first validated continental record of this needle nematode species in the USA and the Americas. The USA populations of L. orientalis contained a small number of males that were not reported in the original description and are herein described. Longidorus orientalis was able to survive for at least 4 years at very low numbers in the warm and humid environment of Florida on date palms imported from California and Arizona. Association of L. orientalis with L. africanus was observed in all of the surveyed sites, indicating that date palm is a host of both nematodes. Phylogenetic relationships of L. orientalis with closely related Longidorus species, in addition to relationships between populations of L. orientalis from the USA, Greece, Iran and Spain, were inferred from the analyses of D2-D3 of 28S rRNA, ITS1 rRNA and partial coxI gene sequences. The PCR-D2-D3 expansion segments of 28S rDNA-RFLP diagnostic profile is provided. Longidorus orientalis populations display a high level of intraspecific variation (up to 15.5%) in coxI mtDNA sequences. Analysis of phylogenetic relationships of nematode populations revealed incongruence of the ITS1 rRNA and coxI mtDNA gene trees, which might be the result of selective introgression of mtDNA through gene flow between previously isolated populations introduced simultaneously into new geographical regions.

Pratylenchus speijeri n. sp. (Nematoda: Pratylenchidae), a new root-lesion nematode pest of plantain in West Africa.

A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, is described and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has been assessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new species with 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-D3 expansion 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 species complex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musa spp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae with non-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijeri n. 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 of P. 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.

Diversity and phylogenetic relationships within the spiral nematodes of Helicotylenchus Steiner, 1945 (Tylenchida: Hoplolaimidae) as inferred from analysis of the D2-D3 expansion segments of 28S rRNA gene sequences

The spiral nematodes of the genus Helicotylenchus are globally distributed and associated with the root system of diverse groups of plants in cultivated and uncultivated areas. Several species are considered serious parasites of crops. The identification of many Helicotylenchus species is not always reliable, in part because many species share very similar diagnostic characters and high intraspecific variation. To verify species identification of geographically distant populations of Helicotylenchus , we tested monophyly of some classical morphospecies and studied their phylogenetic relationships; specifically, we conducted sequence and phylogenetic analysis of 89 sequences of the D2-D3 expansion segments of 28S rRNA gene sequences from 54 Helicotylenchus isolates, including species identified as H. brevis , H. digonicus , H. dihystera , H. labiodiscinus , H. leiocephalus , H. martini , H. multicinctus , H. platyurus , H. pseudorobustus and H. vulgaris , together with three outgroup taxa. Phylogenetic analysis distinguished nine highly or moderately supported major clades within Helicotylenchus. Using the molecular approach we were able to confirm congruence with morphologicalbased identification of samples of H. dihystera and H. multicinctus . However, sequence and phylogenetic analysis using Bayesian inference and maximum parsimony analysis showed that isolates collected in different countries and morphologically identified as H. pseudorobustus , H. digonicus or H. vulgaris were each representative of several different and, sometimes, unrelated lineages. Further detailed comparative morphometrics and morphological studies will help to elucidate if there is some misidentification or if putative species actually comprise a complex of cryptic species. Molecular analysis also revealed that 14 samples were classified as representatives of 11 unidentified species. Molecular characterisation of known Helicotylenchus species especially, using samples collected from type localities, is needed for future reliable identification of species of this genus.

Characterisation of a population of Pratylenchus hippeastri from bromeliads and description of two related new species, P. floridensis n. sp. and P. parafloridensis n. sp., from grasses in Florida

Morphological and molecular analyses confirmed the presence of P. hippeastri in regulatory samples collected in commercial bromeliad operations from genera Guzmania, Neoregelia and Vriesea in central and south Florida, USA. These P. hippeastri from bromeliads contained males which were not detected in the type population from amaryllis. The rDNA sequences of these males matched those of P. hippeastri female type material. Pratylenchus hippeastri and related root-lesion nematodes from several hosts in Florida were characterized at the morphological and molecular level, whereas other samples from Russia and South Africa at the molecular level only. Phylogenetic and sequence analysis using the ITS rRNA gene of these root-lesion nematodes revealed the presence of eight putative new species (spH1-H8) closely related to P. hippeastri. However, detailed morphological and molecular analyses are still required to confirm their unique species status. Here we describe two Florida representatives of the amphimictic root-lesion nematodes from Bahia grass (N1) and maidencane (N2), previously characterized by Inserra et al. (1996) and Duncan et al. (1999), as two new species phylogentically related to P. hippeastri and named Pratylenchus floridensis n. sp. and P. parafloridensis n. sp., respectively. The small round or oval, rarely rectangular and occasionally oblong and enlarged spermatheca and the bluntly pointed or subacute tail with smooth and occasionally indented terminus separate P. floridensis n. sp. from P. parafloridensis n. sp., which has a quadrangular spermatheca and a sub hemispherical or bluntly pointed tail with generally smooth and rarely indented terminus. However, these characters may overlap in some specimens making the morphological separation problematic without the use of molecular analysis. The close phylogenetic relationships shared by the species characterized in this study indicate that they are representatives of a P. hippeastri species complex.

Pratylenchus hippeastri n. sp. (Nematoda: Pratylenchidae) from amaryllis in Florida with notes on P. scribneri and P. hexincisus.

A new root-lesion nematode Pratylenchus hippeastri n. sp. is described from amaryllis (Hippeastrum spp.) in Florida. The new species is characterised by a combination of the following morphological features of females: slender body, flat, plain and smooth face, head with two lip annuli, an incomplete third annulus, and with second lip annulus thicker than first, ellipsoidal stylet knobs, rectangular empty spermatheca with large round cavity, and conoid tail with bluntly pointed terminus usually showing a ventral constriction, or subhemispherical and smooth. The ranges of morphometric characters of P. hippeastri n. sp. overlap with those of P. scribneri and P. hexincisus. These three species also share some morphological and biological features such as two lip annuli, an empty spermatheca and similar lateral fields, but differ in the morphology of head patterns (smooth face in P. hippeastri n. sp. vs divided face in the others) and tail termini (hemispherical and subhemispherical in P. scribneri and subdigitate in P. hexincisus vs bluntly pointed in P. hippeastri n. sp.). A reference population of P. scribneri from Ohio and one of P. hexincisus from Tennessee were obtained during the course of unsuccessful attempts to recover P. scribneri at the type locale in Tennessee. The D3 rDNA sequences of the reference populations matched GenBank sequences for these two species. Small morphological differences between the P. scribneri population from Ohio and the P. scribneri lectotype occurred in the shape of stylet knobs (ellipsoidal vs round) and spermatheca (round with a central cavity vs oblong) and also in the length of the pharyngeal overlap (longer in the reference population). These morphological differences cast doubt about the identity of the lectotype, which may be P. hexincisus. New morphological details were obtained for P. hexincisus from Tennessee, which included a divided face, an almost rectangular empty spermatheca with a central cavity and subdigitate tail termini. Based on our phylogenetic inferences from DNA sequences P. hippeastri n. sp. is more closely related to P. zeae and a Florida population of P. loosi than to P. scribneri or P. hexincisus.

Hemicaloosia vagisclera n. sp. (Nematoda: Caloosiidae) from Bermuda grass in Florida and its phylogenetic relationships with other criconematids

Hemicaloosia vagisclera n. sp. is described from Bermuda grass (Cynodon dactylon) in Florida. This new species is characterised by females with body slightly ventrally arcuate in death, face with a prominent elliptical oral disc, large amphids, slender stylet with mean length 64 μm, lateral field consisting of a single longitudinal line marked by continuous and discontinuous transverse striae, oval and full spermatheca, sclerotised vagina vera and tail annuli width greater than that of remaining body annuli. Diagnostic characters for the males are a C-shaped body, head with 4-5 faint lip annuli and pronounced oval oral disc, lateral field with two longitudinal lines intersected by transverse striae, tail digitate in the distal portion posterior to bursa, distinctly annulated and with a round terminus. Molecular characterisation of H. vagisclera n. sp. using the D2-D3 domain of 28S rRNA, partial 18S rRNA and ITS rRNA gene sequences is also provided. The phylogenetic relationships of this species with other representatives of the suborder Criconematina are presented and indicate that H. vagisclera n. sp. has sister relationships with Caloosia longicaudata supporting the classification of Caloosia together with Hemicaloosia as separate genera in the family Caloosiidae. A diagnostic PCR-ITS-RFLP profile for H. vagisclera n. sp. is also given together with an identification key for seven known species of Hemicaloosia.

Characterisation of a topotype and other populations of Hemicriconemoides strictathecatus Esser, 1960 (Nematoda: Criconematidae) from Florida with description of H. phoenicis sp. n. from the USA

Pablo Castillo and Juan E. Palomares-Rius acknowledge support from grant AGR-136 from ‘Consejeria de Economia, Innvovacion y Ciencia’ from Junta de Andalucia, and the European Social Fund and Sergei A. Subbotin acknowledges support from the Russian Foundation of Basic Research, project number 14-04-00953.