Phylogenetic Implications of Phasmid Absence in Males of Three Genera in Heteroderinae

Absence of the phasmid was demonstrated with the transmission electron microscope in immature third-stage (M3) and fourth-stage (M4) males and mature fifth-stage males (M5) of Heterodera schachtii, M3 and M4 of Verutus volvingentis, and M5 of Cactodera eremica. This absence was supported by the lack of phasmid staining with Coomassie blue and cobalt sulfide. All phasmid structures, except the canal and ampulla, were absent in the postpenetration second-stage juvenile (J2) of H. schachtii. The prepenetration V. volvingentis J2 differs from H. schachtii by having only a canal remnant and no ampulla. This and parsimonious evidence suggest that these two types of phasmids probably evolved in parallel, although ampulla and receptor cavity shape are similar. Absence of the male phasmid throughout development might be associated with an amphimictic mode of reproduction. Phasmid function is discussed, and female pheromone reception ruled out. Variations in ampulla shape are evaluated as phylogenetic character states within the Heteroderinae and putative phylogenetic outgroup Hoplolaimidae.     

Fine structure of body wall cuticle of females of Meloidodera charis,Atalodera lonicerae,and Sarisodera hydrophila (Heteroderidae)

The body wall cuticle of adult females of Meloidodera charis, Atolodera lonicerae, and Sarisodera hydrophila is examined by transmission electron and light microscopy for comparison with Heterodera schachtii and previous observations of additional species of Heterodera, Globodera, and Punctodera. The cuticle of M. charis is least complex, consisting of layers A, B, C (with A outermost), and varies in overall thickness from 3 to 8 μm. As in other species, the cuticle is thickest in mature specimens. The cuticle of A. lonicerae is 6-9 μm thick; unlike M. charis it has an innermost layer, D, in addition to A, B, and C. The cuticle of S. hydrophila varies from 14 to 30 μm thick and includes a D layer similar to A. lonicerae; layer C is subdivided into additional zones relative to other heteroderids, and the external portion of the cuticle is infused with an electron-dense material. The presence of a D layer in A. lonicerae and S. hydrophila is a character state which is shared with Globodera spp. and Punctodera sp. The electron-dense material in the outer layers of S. hydrophila also occurs in Globodera spp. and Punctodera sp. On the other hand, H. schachtii resembles other Heterodera spp. as well as M. charis by the absence of a D layer and lack of electron-dense material in the outer layers. The pattern of occurrence of shared character states, including those of the cuticle, may be useful for phylogenetic analysis of Heteroderidae.     

Fine Structure of Sperm of Ekphymatodera thomasoni (Heteroderinae,Nemata)

Fine structure of developing sperm of the monospecific genus, Ekphymatodera, was compared with other Heteroderinae as part of a study to recognize diversity and phylogenetically informative characters within the subfamily. Sperm of Ekphymatodera originate from germ cells connected to a central rachis, a character which is shared with Globodera, but not with other Heteoderinae. In Ekphymatodera, and cyst-forming genera, a layer of cortical microtubules lies just beneath the surface of the plasma membrane. Sperm of Ekphymatodera are unique among Heteroderinae examined by the presence of spiral surface elevations on the filopodia, a character that may prove to be a synapomorphy for Sarisoderini. Fibrous bodies are abundant in spermatids; however, they do not persist in sperm of Ekphymatodera as they do in Meloidodera and Verutus. The male gonad of Ekphymatodera is lined by epithelial cells, which are greatly enlarged near the ejaculatory canal. These enlarged cells contain vesicles with concentric lamellar inclusions, not observed in other genera of the subfamily. Sperm of Heteroderinae are rich in diversity, and examination of additional representative species may indicate new phylogenetically informative characters.     

Histopathology of Okra and Ridgeseed Spurge Infected with Meloidodera charis

Histological observations of okra Abelomoschus esculentus ''Clemson Spineless'' and ridgeseed spurge Euphorbia glyptosperma (a common weed) infected with Meloidodera charis Hopper, indicated that the juvenile nematode penetrated the roots intercellularly. Within 5 days after plant emergence the nematode positioned its body in the cortical tissue parallel to the vascular system. By 10 days after plant emergence the juvenile had extended its head into the vascular system and initiated giant cell formation, generally in protophloem tissue. Giant cells were one celled and usually multi-nucleate. Eggs were observed in the female body 30 days after plants emerged and juveniles were found within the female body by 40 days. Nematode development progressed equally in the root system of either host plant. Generally, throughout the nematode''s life cycle its entire body remained inside the cortical tissue of okra. In ridgeseed spurge, however, the posterior portion of the female erupted through the host epidermis as early as 15 days after plant emergence; only the head and neck remained embedded in the host. The nematode caused extensive tissue disruption in the cortical and vascular system of both plant species. Corn, Zea mays, was another host of the nematode.     

The Genera of the Subfamily Heteroderinae (Nematoda: Tylenchoidea) with a Description of Two New Genera

The family Heteroderidae, its two subfamilies Heteroderinae and Meloidogyninae and the nominal genera of Heteroderinae (Heterodera Schmidt, 1871; Meloidodera Chitwood, Hannon &Esser, 1956; and Cryphodera Colbran, 1966) are rediagnosed. Meloidoderita Pogosyan, 1966 is considered a genus inquirenda. Two new genera from southern California are described in the subfamily Heteroderinae. A key to the genera, illustrations and a phylogeny of the Heteroderinae are proposed.     

Cuticle Ultrastructure of Criconemella curvata and Criconemella sphaerocephala (Nemata: Criconematidae)

Cuticle ultrastructure of Criconemella curvata and C. sphaerocephala females is presented; males were available only in the second species. Ultrathin sections revealed three major zones: cortical, median, and basal. The cortical zone in the females consists of an external and internal layer. In C. curvata the external layer is trilaminate and at each annule it is covered by a multilayered cap. In C. sphaerocephala the trilaminate layer is lacking and the external cortical layer includes an osmophilic coating. In both species the internal layer consists of alternate striated and unstriated sublayers. The median zone is fibrous with a central lacuna and the zone is interrupted between the annules. The basal zone of the cuticle is striated and narrower between each annule. The cuticle of the C. sphaerocephala male is typical of Tylenchida, except under both lateral fields; the striated layer becomes forked at the first incisure and the innermost two prongs of the fork overlap each other, resulting in a continuous striated band.     

Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida)

Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status.     

Morphology of Second-stage Juveniles and Males of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

Morphological comparisons with light microscopy and scanning electron microscopy were made among second-stage juveniles (J2) and males of several isolates of the three subspecies of the tobacco cyst nematode complex, Globodera tabacum sspp. tabacum, virginiae, and solanacearum. Observations focused on the anterior region, (including head shape, lip pattern, and stylet morphology) and the tail region (including tail shape in J2 and spicules in males). The three subspecies could not be separated on the basis of any of these characters.     

Characterization of Biocontrol Traits in Heterorhabditis floridensis: A Species with Broad Temperature Tolerance

Biological characteristics of two strains of the entomopathogenic nematode, Heterorhabditis floridensis (332 isolated in Florida and K22 isolated in Georgia) were described. The identity of the nematode’s symbiotic bacteria was elucidated and found to be Photorhabdus luminescens subsp. luminescens. Beneficial traits pertinent to biocontrol (environmental tolerance and virulence) were characterized. The range of temperature tolerance in the H. floridensis strains was broad and showed a high level of heat tolerance. The H. floridensis strains caused higher mortality or infection in G. mellonella at 30°C and 35°C compared with S. riobrave (355), a strain widely known to be heat tolerant, and the H. floridensis strains were also capable of infecting at 17°C whereas S. riobrave (355) was not. However, at higher temperatures (37°C and 39°C), though H. floridensis readily infected G. mellonella, S. riobrave strains caused higher levels of mortality. Desiccation tolerance in H. floridensis was similar to Heterorhabditis indica (Hom1) and S. riobrave (355) and superior to S. feltiae (SN). H. bacteriophora (Oswego) and S. carpocapsae (All) exhibited higher desiccation tolerance than the H. floridensis strains. The virulence of H. floridensis to four insect pests (Aethina tumida, Conotrachelus nenuphar, Diaprepes abbreviatus, and Tenebrio molitor) was determined relative to seven other nematodes: H. bacteriophora (Oswego), H. indica (Hom1), S. carpocapsae (All), S. feltiae (SN), S. glaseri (4-8 and Vs strains), and S. riobrave (355). Virulence to A. tumida was similar among the H. floridensis strains and other nematodes except S. glaseri (Vs), S. feltiae, and S. riobrave failed to cause higher mortality than the control. Only H. bacteriophora, H. indica, S. feltiae, S. riobrave, and S. glaseri (4-8) caused higher mortality than the control in C. nenuphar. All nematodes were pathogenic to D. abbreviatus though S. glaseri (4-8) and S. riobrave (355) were the most virulent. S. carpocapsae was the most virulent to T. molitor. In summary, the H. floridensis strains possess a wide niche breadth in temperature tolerance and have virulence and desiccation levels that are similar to a number of other entomopathogenic nematodes. The strains may be useful for biocontrol purposes in environments where temperature extremes occur within short durations.     

Morphometrics of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

A morphometric evaluation of second-stage juveniles (J2), males, females, cysts, and eggs of several isolates of the tobacco cyst nematode (TCN) complex, Globodera tabacum tabacum (GTT), G. t. virginiae (GTV), and G. t. solanacearum (GTS) is presented. Morphometrics of eggs, J2, and males are considerably less variable than of females and cysts. No measurements of eggs and J2 are useful for identification of the three subspecies. Distance from the median bulb and excretory pore to the head end in J2 and males is quite stable. Stylet knob width of males is useful for identifying GTV isolates and tail length in separating males of GTT isolates from GTV and GTS. Body length/width (L/W) ratio of females and cysts discriminates GTT from GTV and GTS; stylet knob width is an auxiliary character for identifying GTV. This subspecies complex has a continuum of values for the other characters. Data suggest a close relationship between GTV and GTS, which also occur in close proximity in Virginia.     

Ultrastructural Variation of Cuticular Layers in Cephalobinae (Nemata: Rhabditida)

The ultrastructure of the body wall cuticle in Acrobeles complexus, Cervidellus alutus, and Zeldia punctata was studied as a step toward understanding biological diversity within Cephalobinae, and to discover new characters for phylogeny-based classification of the suborder. In each species the cuticle consists of cortical, median, and basal layers. The cortical layer includes an external trilaminate and internal granular zone; the basal layer is striated. In Z. punctata the median layer is electron-lucent, vacuolar, and penetrates the cortical layer; it also includes periodically dense columns that apparently correspond to punctuations visible with light microscopy. In contrast, the median layer of the body wall cuticle in A. complexus and C. alutus is bisected by a zone that undulates parallel to the nematode surface and with periodicity corresponding to annuli. Phylogenetic analysis, using derived cuticle patterns of Cephalobinae, requires an understanding of ecological pressures that could result in convergent evolution of cuticle characters.     

Morphology of Females and Cysts of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

Detailed morphological comparisons with light and scanning electron microscopy were made of white females and cysts of several isolates of Globodera tabacum sspp. tabacum (GTT), virginiae (GTV), and solanacearum (GTS). Observations focused on body shape, anterior region including head shape, lip pattern, stylet morphology, and the terminal area in females; and body shape and terminal area of cysts. The most useful characters to separate the three subspecies were forms of the female body, cyst, stylet knobs, tail region, perineal tubercles, anal-fenestral ridge patterns, and the distinctiveness of the anus. GTT is characterized by having round females and cysts, sharply back sloped stylet knobs, clumped perineal tubercles in the vulval region, tight parallel ridges in the cyst anal-fenestral region, and a uniformly conoid tail region. GTV is characterized by its ovoid to ellipsoid female and cyst shape, the "Dutch shoe" shape of the dorsal stylet knob, the more dispersed perineal tubercles, a maze-like pattern of ridges in the anal-fenestral region, and an indistinct anus. GTS is characterized by its ovoid to ellipsoid female and cyst shape, moderately backward sloped stylet knobs, more widely separated ridges, a distinct anus, and a usually crescent shaped tail region. Much variability in shape and patterns is visible among all the isolates of the different subspecies. Tubercles in the neck, as well as bullae, are reported, and their taxonomic value is discussed.     

Fine Structure of Body Wall Cuticle of Females of Eight Genera of Heteroderidae

Body wall cuticle of adult females of eight genera within the Heteroderidae was examined by transmission electron microscopy for comparison with previously studied species within the family. Cuticle structure was used to test some current hypotheses of phylogeny of Heteroderidae and to evaluate intrageneric variability in cuticle layering. Verutus, Rhizonema, and Meloidodera possess striated cuticle surfaces and have the simplest layering, suggesting that striations have not necessarily arisen repeatedly in Heteroderidae through convergent or parallel evolution. Atalodera and Thecavermiculatus possess similar cuticles with derived characteristics, strengthening the hypothesis that the two genera are sister groups. Similarly, the cuticle of Cactodera resembles the specialized cuticle of Globodera and Punctodera in having a basal layer (D) and a surface layer infused with electron-dense substance. Heterodera betulae has a unique cuticle in which the thickest layer (C) is infiltrated with an electron-dense matrix. Little intrageneric difference was found between cuticles of two species of Meloidodera or between two species of Atalodera. However, Atalodera ucri has a basal layer (E) not found in other Heteroderidae. The most striking intrageneric variation in cuticle structure was observed between the thin three-layered cuticle of Sarisodera africana and the much thicker four-layered cuticle of Sarisodera hydrophila; results do not support monophyly of Sarisodera.     

Gametogenesis and Reproduction of Meloidogyne graminis and M. ottersoni (Nematoda: Heteroderidae)

Oogenesis and spermatogenesis of seven populations of Meloidogyne graminis and one population of M. ottersoni (formerly Hypsoperine spp.) were of the meiotic type. When males were abundant, reproduction was by amphirnixis. In most greenhouse cultures, however, males were rare and reproduction was by meiotic parthenogenesis. M. graminis and M. ottersoni are closely related to each other and to M. graminicola and M. naasi, but differ in some respect from other Meloidogyne species. It is suggested that these four species be treated together as a group of species, either in the genus Meloidogyne or in the genus Hypsoperine.     

Labellar anatomy and secretion in Bulbophyllum Thouars (Orchidaceae: Bulbophyllinae) sect. Racemosae Benth. & Hook. f.

Background and Aims

Floral secretions are common in Bulbophyllum Thouars, and the labella of a number of Asian species are said to produce secretions rich in lipids that act as food rewards for insect pollinators. Although some of these reports are based on simple histochemical tests, a much greater number are anecdotal and, hitherto, neither the ultrastructure of the labellum nor the secretory process has been investigated in detail. Furthermore, sophisticated histochemical approaches have generally not been applied. Here, both the labellar structure and the secretory process are investigated for four species of Asian Bulbophyllum sect. Racemosae Benth. & Hook. f., namely Bulbophyllum careyanum (Hook.) Spreng., B. morphologorum Kraenzl., B. orientale Seidenf. and B. wangkaense Seidenf., and compared with those of unequivocal lipid-secreting orchids.

Methods

Labellar, secretory tissue was investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry.

Key Results

The adaxial median longitudinal groove of the labellum contained secretory tissue comprising palisade-like epidermal cells, similar to those of certain lipid-secreting Oncidiinae Benth. However, these cells and their secretions gave positive results mainly for protein and mucilage, and their organelle complement was consistent with that of cells involved in protein and mucilage synthesis. Sub-cuticular accumulation of secretion resulted in cuticular distension and blistering. The sub-epidermal layer of isodiametric parenchyma contained starch and, like the epidermal cells, ultrastructure consistent with mucilage synthesis. Lipids were mainly confined to the cuticle, and hardly any intracellular lipid droplets were observed.

Conclusions

It is proposed that mucilage is produced by dictyosomes present in the palisade-like epidermal cells. Mucilage precursors may also be produced by these same organelles in sub-epidermal cells and are thought to pass along the symplast via plasmodesmata into the adjoining palisade-like secretory cells, which contain abundant arrays of rough endoplasmic reticulum. Here, they become chemically modified and form a protein-rich, mucilaginous secretion that, following vesicle-mediated transport across the cytoplasm, traverses the cell wall and accumulates in blisters formed from the distended cuticle. Rupture of these blisters releases the secretion onto the labellar surface. However, in certain species, there is some evidence that the secretion may traverse the cuticle via cuticular pores, and micro-channels may permit the passage of fragrance. Hydrolysis of sub-epidermal starch probably generates the carbohydrate and, together with mitochondria, much of the energy required for the secretory process. This anatomical organization resembles that found in certain lipid-secreting, Neotropical species of Bulbophyllum and Oncidiinae, but since the chemical composition of their secretions is different, and these taxa occur on a separate continent and have different insect pollinators, parallelism of floral anatomy is likely.     

Descriptions of Meloidogyne camelliae n.sp. and M. querciana n.sp (Nematoda:Meloidogynidae), with SEM and Host-Range Observations

Meloidogyne camelliae n.sp. on camellia (Camellia japonica) from Japan and M. querciana n.sp. on pin oak (Quercus palustris) from Virginia, USA, are described and illustrated. M. camelliae n.sp. is distingnishable from other species of the genus especially by its striking perineal pattern having heavy ropelike striae forming a squarish to rectangular outline with shoulders or projections, appearing sometimes ahnost starlike. M. querciana differs from other species by its characteristic perineal pattern round to oval in outline, sometimes with a low arch, and sunken vulva surrounded by a prominent obovate area devoid of striae. M. querciana shows some relationship to M. ovalis, but differs further fxom the latter by longer larvae, absence of annules on head of larvae, and rarity of males. Examination of specimens of M. camelliae n.sp. and M. querciana n.sp. with the scanning electron microscope confirmed observations made by optical microscopy and revealed diagnostic and other structures in greater detail. In greenhouse host tests, M. camelliae infected camellia heavily, showed moderate infection on oxalis, only a trace infection on tomato, and no infection on five other plants tested; and M. querciana attacked pin oak, red oak, and American chestnut heavily, but did not infect nine other test plants. In another test, pin oak seedlings did not become infected when heavily inoculated with and grown in the presence of two populations of M. incognita incognita and one of M. incognita acrita. The common names "camellia root-knot nematode" and "oak root-knot nematode" are respectively proposed for M. camelliae and M. querciana.     

Description of Meloidoderita polygoni n. sp. (Nematoda: Meloidoderitidae) from USA and Observations on M. kirjanovae from Israel and USSR

Meloidoderita polygoni n. sp. is described and illustrated from roots of smartweed (Polygonum hydropiperoides) from Beltsville, Maryland. This new species is similar to M. kirjanovae but differs especially in having larger spines on the cystoid bodies, females with the anus much closer to the vulva, and more posterior excretory pore. M. polygoni differs from M. safrica particularly in having females with a shorter stylet, a DGO much closer to base of stylet, greater distance between vulva and anus, and larger cystoid bodies. LM and SEM observations showed only three incisures in lateral fields of juveniles and males and no bursa in males. Morphometric data and illustrations are given for M. kirjanovae from mint (Mentha longifolia) in Israel and some details on a limited number of specimens from Armenian SSR. LM examination of juveniles from both these areas indicated only three incisures in lateral fields. Males from Israel had no detectable bursa and appeared to have only three incisures in lateral fields. (Males from Armenian SSR not observed.)     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

Ultrastructure of Gonionchus australis (Xyalidae,Nematoda)

Observations are reported on the ultrastructure of the buccal cavity, body cuticle, spermatids, spermatozoa, male genitalia, and caudal glands of Gonionchus australis. The buccal cuticle is a continuation of the pharyngeal cuticle. Anteriorly it is secreted by arcade tissue and overlaps the mouth rim; laterally it forms longitudinal tooth ridges. The non-annulated cephalic cuticle differs sharply from the remainder of the body wall cuticle. The cortical and basal zones become much thinner, while a largely structureless, lucent median zone expands to fill the bulk of the lips and lip flaps. Spermatids possess fibrous bodies, multimembrane organelles, mitochondria, and compact chromatin. The spermatozoa of G. australis resemble those of most other nematodes by the absence of the nuclear envelope and presence of fibrous bodies, mitochondria, and compact chromafin. The ejaculatory duct possesses microvilli. Two ejaculatory glands lie beside the duct. Two neurons are located within each spicule and each part of the paired gubernaculum. Caudal gland nuclei are large, with dispersed chromatin. The ducts of all three caudal glands are filled with secretory vesicles.     

Relationship of Resistance to Meloidogyne chitwoodi (race 2) and M. hapla in Alfalfa

In the Pacific Northwest, alfalfa (Medicago sativa) is host to two species of root-knot nematodes, including race 2 of the Columbia root-knot nematode (Meloidogyne chitwoodi) and the northern root-knot nematode (Meloidogyne hapla). In addition to the damage caused to alfalfa itself by M. hapla, alfalfa’s host status to both species leaves large numbers of nematodes available to damage rotation crops, of which potato is the most important. A nematode-resistant alfalfa germplasm release, W12SR2W1, was challenged with both nematode species, to determine the correlation, if any, of resistance to nematode reproduction. Thirty genotypes were screened in replicated tests with M. chitwoodi race 2 or M. hapla, and the reproductive factor (RF) was calculated. The distribution of natural log-transformed RF values was skewed for both nematode species, but more particularly for M. chitwoodi race 2, where more than half the genotypes screened were non-hosts. Approximately 30 percent of genotypes were non-hosts or very poor hosts of M. hapla, but RF values for M. hapla on susceptible genotypes were generally much higher than RF values for genotypes susceptible to M. chitwoodi race 2. The Spearman rank correlation was positive (0.52) and significant (p-value = 0.003), indicating there is some relationship between resistance to these two species of root-knot nematode in alfalfa. However the relationship is not strong enough to suggest genetic loci for resistance are identical, or closely linked. Breeding for resistance or immunity will require screening with each species separately, or with different DNA markers if marker-assisted breeding is pursued. A number of genotypes were identified which are non-hosts to both species. These plants will be intercrossed to develop a non-host germplasm.