Fine Structure of the Cone of Heterodera schachtii with Emphasis on Musculature and Fenestration

Fine structure of the posterior cone of monoxenically cultured Heterodera schachtii is examined. The cone is not evident at the end of the fourth molt, but as the female matures the cone elongates, vulval lips enlarge, and cuticular patterns on the lips are modified. Body wall cuticle (BW) of the cone includes layers A and B, but C is modified or replaced by a network of fibers which correspond to the semifenestrae. Vaginal lining is continuous with the BW and terminates at the cuticular underbridge near the uterus. Vaginal musculature includes 48 dilatores vaginae (DV) as well as a sphincter vaginae (SV). The DV include a contractile and noncontractile region with abundant actin and glycogen. A distinct anal depressor muscle is present. In the cyst, only bullae, the underbridge, vagina lining, and traces of the SV muscle persist. Detailed morphology of the cone of H. schachtii provides insight into characters which, when compared with other heteroderines, will be useful in phylogenetic analysis of Heteroderinae.     

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.     

In vitro Cultivation of the Entomogenous Nematode Filipjevimermis leipsandra

The mermithid nematode, Filipjevimermis leipsandra, was successfully cultivated to the preadult stage in Schneider''s Drosophila medium supplemented with 20% fetal bovine serum. Upon transfer to a solid substrate the preadults continued to develop into ovipositing adult females. Four molts were observed. The first molt occurred in the egg. The second occurred after 6-8 days in culture during which the very thin cuticle was shed completely. The third molt occurred after 18-20 days in culture; the cuticle was retained by the third-stage nematode. This stage was considered comparable to the preadult stage that emerges from host larva, Diabrotica spp. The fourth molt occurred within 12 days after the preadult was transferred from the liquid medium to a solid substrate. Adult females began ovipositing viable eggs 1-3 days after the final molt.     

In Vitro Embryo Explant Cultures of Peanut to Evaluate Resistance to Ditylenchus destructor

Population densities of D. destructor on embryo explants of 22 peanut genotypes grown in vitro were compared with those in roots and seeds of the same genotypes grown in the greenhouse. During the first 8 weeks after inoculation, the optimum incubation period was 6 weeks for maximum reproduction of Ditylenchus destructor on embryo explants of peanut (Arachis hypogaea L. cv. Sellie) inoculated with 250 nematodes at 25 C. Nematode numbers increased 17-fold. Deletion of MnSO₄ H₂O and a higher KH₂PO₄ concentration in the medium resulted in higher nematode reproduction. Resistance or susceptibility to D. destructor was observed in seeds of several genotypes but was not matched by differences in host suitability in roots. The results indicate that the factor for resistance or susceptibility to D. destructor is synthesized in the seeds of peanut but is not translocated to the roots. Use of embryo explant cultures of peanut as a rapid method to evaluate resistance to D. destructor did not work under the conditions described.     

Population Dynamics of Dactylella oviparasitica and Heterodera schachtii: Toward a Decision Model for Sugar Beet Planting

A series of experiments were performed to examine the population dynamics of the sugarbeet cyst nematode, Heterodera schachtii, and the nematophagus fungus Dactylella oviparasitica. After two nematode generations, the population densities of H. schachtii were measured in relation to various initial infestation densities of both D. oviparasitica and H. schachtii. In general, higher initial population densities of D. oviparasitica were associated with lower final population densities of H. schachtii. Regression models showed that the initial densities of D. oviparasitica were only significant when predicting the final densities of H. schachtii J2 and eggs as well as fungal egg parasitism, while the initial densities of J2 were significant for all final H. schachtii population density measurements. We also showed that the densities of H. schachtii-associated D. oviparasitica fluctuate greatly, with rRNA gene numbers going from zero in most field-soil-collected cysts to an average of 4.24 x 10⁸ in mature females isolated directly from root surfaces. Finally, phylogenetic analysis of rRNA genes suggested that D. oviparasitica belongs to a clade of nematophagous fungi that includes Arkansas Fungus strain L (ARF-L) and that these fungi are widely distributed. We anticipate that these findings will provide foundational data facilitating the development of more effective decision models for sugar beet planting.     

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.     

Carbon Dioxide and Temperature Gradielits in Baermann Funnel Extraction of Rotylenchulus reniformis

Vermiform Rotylenchulus reniformis were anesthetized in water by 10-40% CO₂ but were fully motile for 24 hours in water below 5% CO₂. When air containing 2.5% CO₂ was blown onto agar, nematodes accumulated at the point of highest CO₂ concentration. Nematodes also accumulated when chilling (0.2-1 C) of agar by the gas flow at the accumulation point was offset with heat from a fiber optic. In Baermann funnels containing R. reniformis in silt loam and sandy clay loam soils, CO₂ in funnel water increased during 24 hours from 0 to ca. 1%; more CO₂ accumulated below the soil layer than above. Bubbling air with 2.5% CO₂ into water below soil in covered funnels increased the CO₂ gradient and increased nematode extraction, whereas bubbling air without CO₂ below soil purged CO₂ from the water and decreased nematode extraction. Manipulation of CO₂ within funnels usually increased extraction by only 30% and never by more than 3-fold. Controlling temperature gradients consistently increased extraction by 2-30-fold.     

Pathological Interaction of a Combination of Heterodera schachtii and Meloidogyne hapla on Tomato

Increased culturing of a tomato population of Heterodera schachtii (UT1C) on tomato for 480 days (eight inoculation periods of 60 days each) significantly increased virulence to ''Stone Improved'' tomato. A synergistic relationship existed between Meloidogyne hapla and H. schaehtii on tomato. A combination of H. schachtii (UTIC) and M. hapla significantly reduced tomato root weights by 65, 64, and 61% below root weights of untreated controls, and single inoculations of M. hapla and H. schachtii, respectively. This corresponded to root reductions of 42, 44, and 46% from a combination of H. schachtii (UT1B) and M. hapla. Antagonism existed between H. schachtii and M. hapla with regard to infection courts and feeding sites. The root-knot galling index dropped from 6.0 with a single inoculation of M. hapla to 4.3 and 3.3 with combined inoculations of M. hapla plus UT1B and M. hapla plus UTIC cyst nematode populations. The pathological virulence of H. schachtii to sugarbeet was not lost by extended culturing on tomato; there were no differences in penetration, maturation, and reproduction between sugarbeet populations continually cultured on sugarbeet and the population continually cultured on tomato.     

Comparative Morphometrics of Eggs and Second-Stage Juveniles of Heterodera schachtii and a Race of H. trifolii Parasitic on Sugarbeet in The Netherlands

Measurements of second-stage juveniles of Heterodera schachtii from California and The Netherlands and a race of H. trifolii from The Netherlands were obtained and compared to determine if these populations can be differentiated by morphometrics. Juvenile lengths of 10 specimens from each of 10 cysts of each population were measured. Dimensions of tail regions of 20 juveniles from individual cysts of H. schachtii (California) and a like number of juveniles of H. trifolii (The Netherlands) were also obtained. The mean lengths of juveniles of H. schachtii from California and The Netherlands were not significantly different, but similar measurements of H. schachtii and H. trifolii were different (P = 0.05). Mean dimensions of tail lengths, tail widths, tail hyaline lengths, and tail length/tail width were significantly greater for H. trifolii than for H. schachtii. Also, dimensions of eggs of H. trifolii were significantly greater than dimensions of H. schachtii eggs. The investigations established that H. schachtii can be readily differentiated from H. trifolii by morphometrics of eggs and juveniles, Minimum sample sizes required for specified confidence intervals for each criterion measured are provided.     

Interrelationship of Heterodera schachtii and Meloidogyne hapla on Tomato

Invasion of tomato (Lycopersicon esculentum L.) roots by combined and sequential inoculations of Meloidogyne hapla and a tomato population of Heterodera schachtii was affected more by soil temperature than by nematode competition. Maximum invasion of tomato roots, by M. hapla and H. schachtii occurred at 30 and 26 C, respectively. Female development and nematode reproduction (eggs per plant) of M. hapla was adversely affected by H. schachtii in combined inoculations of the two nematode species. Inhibition of M. hapla development and reproduction on tomato roots from combined nematode inoculations was more pronounced as soil temperature was increased over a range of 18-30 C and with prior inoculation of tomato with H. schachtii. M. hapla minimally affected H. schachtii female development, but there was significant reduction in the buildup of H. schachtii when M. hapla inoculation preceded that of H. schachtii by 20 days.     

Suppression of Heterodera schachtii Populations by Dactylella oviparasitica in Four Soils

The effects of Dactylella oviparasitica strain 50 applications on sugarbeet cyst nematode (Heterodera schachtii) population densities and plant weights were assessed in four agricultural soils. The fungus was added to methyl iodide-fumigated and nonfumigated portions of each soil. The soils were seeded with Swiss chard. Four weeks later, soils were infested with H. schachtii second-stage juveniles (J2). Approximately 1,487 degree-days after infestation, H. schachtii cyst, egg and J2 numbers and plant weights were assessed. In all four fumigated soils, D. oviparasitica reduced all H. schachtii population densities and increased most of the plant weights compared to the nonamended control soils. In two of the nonfumigated soils (10 and SC), D. oviparasitica reduced H. schachtii population densities and increased most plant weight values compared to the nonamended control soils. For the other two nonfumigated soils (44 and 48), which exhibited pre-existing levels of H. schachtii suppressiveness, fungal applications had relatively little impact on H. schachtii population densities and plant weights. The results from this study combined with those from previous investigations suggest that D. oviparasitica strain 50 could be an effective biological control agent.     

Ultrastructure of the Head Region of Molting Second-Stage Juveniles of Heterodera glycines with Emphasis on Stylet Formation

The morphology and alterations of infective juvenile (J2) body components with emphasis on the body wall, stomatal wall, stylet, and sensilla of Heterodera glycines were observed. During the molt of J2 to J3, the J2 hypodermis separates from the J2 cuticle and forms an extracellular space, continuous with an invagination of the anterior, center of the J3. The space between the J2 cuticle and the enlarged J3 hypodermal cells is filled with electron-dense material resembling a fluid observed in insects during molt. Regeneration of the J2 during molt was traced in a series of ultrathin sections. The site of stylet regeneration is in the hypodermal and myoepithelial tissues of the invaginated anterior, center of the J3. Four arcade-like cells are related to specific components of the stomatal wall, the stylet cone, and the stylet shaft of the J3. The first and second arcade-like cells are primarily related to stomatal wall development, whereas the third and fourth arcade-like cells are related to stylet cone and shaft development. Spherical, electron-translucent vacuoles that occur in myoepithelial cells just posterior to the arcade-like cells appear to be progenitors of the stylet knobs. Early stages of protractor muscle attachment to the vacuolar membrane were observed.     

Resistance of Trisomic and Diploid Hybrids of Beta vulgaris and B. procumbens to the Sugarbeet Nematode,Heterodera schachtiis Arnold

Trisomic and diploid hybrids of sugarbeet (Beta vulgaris L.) X wild beet (B. procumbens Chr. Sin.) inherited the gene for resistance to Heterodera schachtii Schm. from B. procumbens. The hybrids showed partial resistance to H. schachtii, manifested in failure of larvae to reach maturity. Although significantly greater numbers of female nematodes developed on plants inoculated with populations from the Netherlands or Italy than on plants inoculated with a population from the Salinas Valley, California. the totals for all populations on resistant plants were small. Greater numbers of males than females developed on root-slice cultures of resistant hyhrids when compared to a susceptible cultivar.     

Comparisons of Mitochondrial DNA from the Sibling Species Heterodera glycines and H. schachtii

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.     

A Comparison of the Hatching of Juveniles from Cysts of Heterodera schachtii and H. trifolii

The effects of root diffusates of selected plants within the families Chenopodiaceae and Cruciferae and the hatching agent zinc chloride were tested for their effects on hatching and emergence of juveniles from cysts of Heterodera schachtii and a race of H. trifolii parasitic on Chenopodaceae and Cruciferae in The Netherlands. Although all diffusates strongly stimulated hatching of juveniles of H. schachtii, their effects on H. trifolii were less evident.     

Cuticular Collagenous Proteins of Second-stage Juveniles and Adult Females of Meloidogyne incognita: Isolation and Partial Characterization

Cuticles isolated from second-stage juveniles and adult females of Meloidogyne incognita were purified by treatment with 1% sodium dodecyl sulfate (SDS). The juvenile cuticle was composed of three zones differing in their solubility in β-mercaptoethanol (BME). Proteins in the cortical and median zones were partially soluble in BME, whereas the basal zone was the least soluble. The BME-soluble proteins from the juvenile cuticle were separated into 12 bands by SDS-polyacrylamide gel electrophoresis and characterized as collagenous proteins based on their sensitivity to collagenase and amino acid composition. The adult cuticle consisted of two zones which were dissolved extensively by BME. The basal zone was completely solubilized, leaving behind a network of fibers corresponding to the cortical zone. The BME-soluble proteins from the adult cuticle were separated by electrophoresis into nine bands one of which constituted > 55% of the total BME-soluble proteins. All bands were characterized as collagenous proteins. Collagenous proteins from juvenile cuticles also contained glycoproteins which were absent from the adult cuticles.     

Cuticle Ultrastructure of Hemicycliophora arenaria,Aphelenchus avenae,Hirschmanniella gracilis and Hirschmanniella belli

Ultrathin sections of all parasitic stages of Hemicycliophora arenaria revealed two major divisions in the body covering. The outermost was a seven-layered sheath and the innermost a five-layered cuticle comprising three zones; an outer trilaminate cortex, a fibrillar matrix and a striated basal layer. The body covering of the nonparasitic males also exhibited two major divisions: the outer, a relatively thin four-layered sheath and the inner, a six-layered cuticle consisting of three zones; an outer trilaminate cortex, a two-layered matrix and a striated basal layer. The cuticles of all stages of Aphelenchus avenae were similar, consisting of five layers divisible into three zones; an outer trilaminate cortex, a fibrillar matrix and a striated basal layer. Hirschmanniella gracilis and H. belli cuticles were also similar in all stages examined, consisting of six layers divisible into three zones; an outer trilaminate cortex, a two-layered matrix and a striated basal layer.     

Host Suitability of Rapeseed for Heterodera schachtii

Because rapeseed, especially canola, has the potential to be grown in rotation with sugarbeet in the north-central region of the United States, this study was initiated to assess its susceptibility to infection by Heterodera schachtii and to develop a screening method for Brassica germplasm. Existing methodology was adapted for growing Brassica juncea, B. napus, B. rapa, Brassica hybrids, and sugarbeet, Beta vulgaris, in H. schachtii-infested soil to count the females that developed on the roots. Cysts on sugarbeet contained a mean of 130 eggs compared with 240 for B. napus, lowest for the Brassica. Viability of eggs produced was assessed in soil planted with Brassica and sugarbeet and infested with with 0, 100, 1,000, 3,000, and 5,000 eggs to count resulting females and cysts. Number of females (y) was related linearly to infestation rate (x) by the regression equations y = 2.82 + 0.07(x) for the Brassica lines (R² = 0.79; P < 0.001) and y = 0.43 + 0.04(x) for sugarbeet (R² = 0.69; P < 0.007). These data indicated the potential for H. schachtii population increase if the two crops are used in rotation. All of the 111 germplasm lines tested were susceptible. The methodology developed during this research would benefit attempts to develop rapeseed cultivars resistant to H. schachtii.     

Cuticle Formation in Hemicycliophora arenaria,Aphelenchus avenae and HirschmannIella gracilis

The onset of molting in all stages of Hemicycliophora arenaria was preceded by the appearance of numerous, discrete globular structures which were termed "molting bodies" because they were present in the hypodermis only during the production of the new cuticle. In all parasitic stages the molt commenced with the separation of the cuticle from the hypodermis from which the new sheath and cuticle were differentiated. Following completion of the new sheath and cuticle most of the old outer covering was apparently absorbed before ecdysis. Electronmicrographs of body wall cross sections in molting L4 male specimens revealed the final molt to be a double molt in which an additional sixth cuticle was produced. Since both a new sheath and cuticle were produced during the molt of each stage, the sheath must be considered as an integral part of the cuticle and not as a residual cuticle or the result of an incomplete additional molt. Molting in Aphelenchus avenae and Hirschmanniella gracilis was less complex and "molting bodies" were not observed. After cuticle separation the hypodermis gave rise to a new trilaminate zone, the future cortex, and (later) the matrix and striated basal layers.