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.     

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.     

Factors Affecting the Biology and Pathogenicity of Heterodera schachtii on Sugarbeet

A direct relationship exists between soil temperature and Heterodera schachtii development. The average developmental period of two nematode populations from Lewiston, Utah, and Rupert, Idaho, from J2 to J3, J4, adult, and the next generation J2 at soil temperatures of 18-28 C were 100, 140,225, and 399 degree-days (base 8 C), respectively. There was a positive relationship (P < 0.05) between nematode Pi, nematode generations, and sugarbeet yields. The greatest sugarbeet growth inhibition (87%) occurred when sugarbeets were exposed to a Pi of 12 eggs/cm³ soil for five generations (1,995 degree-days), compared with a 47% inhibition when plants were exposed to the same Pi for two generations. There was a negative correlation (P < 0.05) between the Pi, Pf, and sugarbeet yield for each population threshold. The smaller the Pi, the greater the sugarbeet yields and the greater the Pf. Root yields were 80 and 29 t /ha and Pf were 8.4 and 3.6 eggs/cm³ soil when sugarbeet seeds were planted at Pi of 0.4 and 7.9 eggs/cm³. respectively, at a soil temperature of 8 C. The number of years rotation with a nonhost crop required to reduce the nematode population density below a damage threshold level of 2 eggs/cm³ depends on the Pi. A Pi of 33.8 eggs/cm³ soil required a 5-year crop rotation, whereas a Pi of 8.4 eggs/cm³ soil required a 2-year crop rotation.     

Identification of Meloidogyne Species on the Basis of Head Shape and,Stylet Morphology of the Male

Head shape and stylet morphology of males of 90 populations of M. arenaria, M. hapla, M. incognita, and M. javanica from geographic regions of the world were compared by light microscopy (LM). In addition, stylets of one population each of M. arenaria, M. incognita, and M. javanica and three different chromosomal forms of M. hapla race A and two of race B were excised and examined with a scanning electron microscope (SEM). Differences among species occurred in both head and stylet morphology. Head morphology differed in size and shape of the head cap, annulation of the head region, and width of the head region relative to the first body annule. Differences in stylets occurred in size and shape of the cone, shaft, and knobs. All populations of M. hapla, except one, had similar head morphology, but stylet morphology was different between cytological races A and B. Populations of M. javanica varied with respect to the presence of head annulations. Head shape and stylet morphology of males are recommended as additional characters useful in the identification of root-knot nematodes.     

Fungi Associated with Females and Cysts of Heterodera glycines in a Florida Soybean Field

Fungal colonization was determined for females and cysts of Heterodera glycines on soybean roots or in rhizosphere soil from a Florida soybean field. A total of 1,620 females and cysts were examined in 1991, and 1,303 were examined in 1992. More than 35 species of fungi were isolated from females and cysts. The frequency of fungi colonizing white and yellow females was low, but a high frequency of fungi was encountered in brown cysts, which increased with time of exposure of the cysts to the soil. No single fungal species predominated in the nematode females or cysts in this field. Rarely was a female or cyst colonized by more than one fungus. The common fungi isolated from the females and cysts were Neocosmospora vasinfecta, Fusarium solani, Fusarium oxysporum, Dictyochaeta coffeae, Dictyochaeta heteroderae, Pyrenochaeta terrestris, Exophiala pisciphila, Gliocladium catenulatum, Stagonospora heteroderae, and a black yeast-like fungus. The communities of common fungal species isolated from cysts in several regions in the southeastern United States appear to be similar.     

Influence of Heterodera glycines on Interspecific and Intraspecific Competition Associated with Glycine max and Chenopodium album

The influence of Heterodera glycines (soybean cyst nematode) on the interspecific and intraspecific competition associated with Glycine max (soybean) and Chenopodium album (common lambsquarters) was studied in 1988 and 1989 in three de Wit replacement series experiments in growth chambers and microplots. Glycine max was grown alone (1 plant/experimental unit), in intraspecific competition (2 plants/experimental unit), in interspecific competition with C. album, and in presence or absence of H. glycines. No significant effects of H. glycines and C. album on G. max growth were observed 14 days after planting. By 42 days after planting, both H. glycines and C. album had a negative (P = 0.05) influence on the growth of G. max. Relative crowding coefficients for G. max were lower and deviated (P = 0.05 and P = 0.001) from 1.0 in the presence of H. glycines, compared to that of C. album and early emerged C. album in the absence of the nematode, respectively. Glycine max, therefore, became less competitive than C. album. There was a trend that the presence of H. glycines decreased the competitiveness of G. max on measures of the aggressivity and relative mixture response. Heterodera glycines decreased the aggressivity of G. max (ca. 150-350%) and increased the relative effects of intraspecific interference on G. max (ca. 10-50%) and interspecific interference (ca. 60-350%) after 42 days of plant growth, compared with plants grown in the absence of H. glycines. No H. glycines x C. album interactions were detected. Observations showed that H. glycines and early emerged C. album inhibited the growth of G. max 5-13%, as measured by plant dry weight.     

Description and SEM Observations of a New Species of Cyst Nematode Heterodera goldeni (Nematoda: Heteroderidae) Attacking Panicum coloratum in Egypt

A cyst nematode, Heterodera goldeni n. sp., is photographed and described from Qasabagrass roots (Panicum coloratum L.) in Alexandria, Egypt. It is characterized in having second-stage juveniles with body length of 546 µm (450-612), stylet length of 22.6 µm (22-23.5) with anchor-shaped knobs, lateral field with 3 lines, tail 60-75 µm, hyaline tail terminus 38.4 µm (33-43); cysts are lemon-shaped, dark to light brown with an extensive sub-crystalline layer covering the entire cyst, cuticular midbody pattern zig-zag, cysts ambifenestrate, well-developed underbridge with finger-like projections, bullae present, vulva slit measuring 44-48 µm long. Males are absent, and females have heavy punctations on the cuticle. Its relationship to H. graminophila described from Florida and Louisiana and H. leuceilyma described from Florida are discussed. The present known distribution is restricted to Alexandria, Egypt. Its economic importance in rangeland grasses and cultivated crops such as rice is not known.     

Description of Bakernema dauniense n. sp. (Nematoda: Criconematidae) from Italy

Bakernema dauniense n. sp., a bisexual species from the rhizosphere of Pinus halepensis Mill. in Italy, is described and illustrated. Primary differentiating characteristics of the female are body annules bearing short, membranous projections 1.4-2.2 μm long, an anterior vulval lip slightly overlapping the posterior, and a linearly arcuate vagina. The juvenile differs markedly from adults by having annules with a beaded margin, which lack membranous projections. Generic and specific relationships of B. dauniense n. sp. are discussed. This occurrence is the first for a species of Bakernema in Europe.     

Emendation of the Genus Paurodontella Husain &Khan, 1968 and Description of P. auriculata n. sp. from Western Canada (Nematoda: Paurodontidae)

Males and females of Paurodontella auriculata n. sp. are described and illustrated and the genus emended. The species is characterized by a short, robust body of 355-525 μm, a stylet length of 7-8 μm, lateral field with 5-6 incisures, and a conoid, attenuated tail 1.5 times the vulva-anus distance. Primary characters used in emending the genus are asymmetrical stylet knobs, absence of lateral lips, a deep slit-like amphid dividing the submedian lips to base of head, where open lateral sides are covered by a large auriform cuticular flap, and variable length of the basal esophageal bulb stem and male bursa.     

Ultrastructure of the Z-organ and Parts of the Female Genital Tract in Xiphinema coxi coxi

Ultrastructure of the Z-organ and associated apophyses in Xiphinema coxi coxi was studied by transmission electron microscopy to determine their structural origin and relationship with other parts of the genital tract. The Z-organ of X. coxi coxi is oval-shaped, ca. 30 µm long and 16 µm wide. It is clearly distinguished from the other parts of the female genital tract by its thick muscular outer wall, epithelium-lined lumen, and 4-5 centrally located apophyses. Each apophysis is continuous with the epithelial lining of the Z-organ, suggesting that it originated from epithelium. The apophyses appear as thickened and densely folded masses forming numerous interlaced pores and (or) chambers containing mucous-like materials and electron-dense crystals. These apophyses are characteristic of a typical Z-organ; no globular structures characteristics of the pseudo-Z-organ were observed. The thickness of the muscular layer of the oviduct and uterus varied with position. The overall Z-organ ultrastructure of this study, including body wall and internal apophyses, was comparable to the typical Z-organ of X. ifacolum. This suggests that X. coxi coxi should be classified as a Xiphinema species that contains the typical Z-organ.     

Anticancer effects of 6-<Emphasis Type="Italic">o</Emphasis>-palmitoyl-ascorbate combined with a capacitive-resistive electric transfer hyperthermic apparatus as compared with ascorbate in relation to ascorbyl radical generation

The aim of the present study is to determine the anti-proliferative activity of 6-o-palmitoyl-l-ascorbic acid (Asc6Palm) that is a lipophilic derivative of l-ascorbic acid (Asc), on human tongue squamous carcinoma HSC-4 cells by combined use of hyperthermia in comparison to Asc. Asc6Palm or Asc were administered to HSC-4 cells for 1 h, to which hyperthermia at 42 °C was applied for initial 15 min. After further 1–72 h incubation at 37 °C, cell proliferation was determined with Crystal Violet staining. Ascorbyl radical (AscR) in HSC-4 cell suspension was measured by electron spin resonance (ESR), and cell morphology was observed with scanning electron microscopy (SEM). At 37 °C, 4 mM Asc or 0.35 mM Asc6Palm were enough to suppress proliferation of HSC-4 cells. By combined use of hyperthermia at 42 °C, cell proliferation was decreased when compared to 37 °C. After Asc of 4 mM was incubated with HSC-4 cell suspensions at 37 °C or 42 °C for 0–180 min, the signal intensity of ascorbyl radical (AscR) by ESR was not different regardless of the presence or absence of cells at 37 °C, whereas AscR signal was enlarged in the presence of HSC-4 cells at 42 °C. It was suggested that oxidation of Asc occurred rapidly in HSC-4 cells by hyperthermia, and thereby enhanced the anti-proliferative activity. By SEM observation, the surface of HSC-4 cells treated with Asc6Palm revealed distinct morphological changes. Thus, the combined regimen of Asc6Palm and hyperthermia is expected to exert a marked antitumor activity.     

Redescription of Robustodorus megadorus with Molecular Characterization and Analysis of Its Phylogenetic Position within the Family Aphelenchoididae

Based on a new record of the rare species Robustodorus megadorus from Utah, the generic diagnosis was amended to include the following characters: a labial disc surrounded by six pore-like sensilla; the absence of a cephalic disc; a lobed cephalic region devoid of annulation; a hexagonal inner cuticular structure of the pouch surrounding the stylet cone; large stylet knobs, rounded in outline and somewhat flattened on their lateral margins; a large spermatheca with an occluded lumen and lacking sperm; the excretory pore located between the median bulb and nerve ring. The stylet orifice consists of an open, ventral, elongate slit or groove. These characters distinguish the genus from the closely related genus Aphelenchoides. A lectotype and paralectotypes were designated. Results of phylogenetic analyses of the 18S and D2-D3 of 28S rRNA gene sequences revealed that R. megadorus occupies a basal position within one of the two main clades of the subfamily Aphelenchoidinae and shares close relationships with a species group of the genus Aphelenchoides that includes A. blastophthorus, A. fragariae, A. saprophilus, A. xylocopae, and A. subtenuis. Several specimens in our collection of R. megadorus were infected with Pasteuria sp. as were some of the paralectotypes.     

Tylenchulus palustris Parasitizing Peach Trees in the of nited States

Most morphological characteristics of three populations of a Tylenchulus sp. from peach roots in Alabama, Arkansas, and Georgia did not differ from those of T. palustris paratypes. However, some mature females differed slightly from those of T. palustris paratypes from Florida. These mature females were more swollen in the posterior portion of their bodies, and they possessed digitate postvulval body sections with round rather than conoid termini. These morphological variants had a wide postvulval section core (PVSC), as do T. palustris paratypes; they did not differ from the paratypes in other characteristics. Second-stage juveniles and males were less morphologically variable and were not different from the paratypes. No males were found in populations from Alabama and Georgia. The Tylenchulus sp. from three peach sites was determined to be T. palustris. This is the first report of T. palustris on an economically important crop.     

Description of Meloidogyne pini n. sp., a Root-Knot Nematode Parasitic on Sand Pine (Pinus clausa), with Additional Notes on the Morphology of M. megatyla

Meloidogyne pini n. sp. is described from sand pine, Pinus clausa, in Georgia. The perineal pattern of the female has a large cuticular ridge surrounding a deeply recessed perivulval area. The lateral fields are marked by transverse striae. The female stylet is 14.6 μm long, and the knobs are small, rounded, and set off from the straight and narrow shaft. The excretory pore is near the level of the base of the stylet. The labial disc of the male is large, rounded, and fused with the crescent-shaped medial lips. The head region is smooth, the styler is 20.8 μm long, and the cone is more than twice as long as the shaft. The knobs are rounded and set off from the shaft. In the second-stage juvenile, the labial disc, medial lips, and lateral lips form one smooth, continuous, ovoid head cap. Mean juvenile length is 434 μm, stylet length is 12.8 μm, and tail length is 44.4 μm. M. pini n. sp. also parasitizes loblolly and slash pine. Additional morphological details of M. megatyla are presented.     

Chitooligosaccharides--preparation with the aid of pectinase isozyme from Aspergillus niger and their antibacterial activity

An isozyme of pectinase from Aspergillus niger with polygalacturonase activity caused chitosanolysis at pH 3.5, resulting in low-molecular weight chitosan (86%), chitooligosaccharides (COs, 4.8%) and monomers (2.2%). HPLC showed the presence of COs with DP ranging from 2 to 6. Charcoal-Celite chromatography and re-N-acetylation of the COs followed by CD, IR, MALDI-TOF-MS and FAB-MS analyses revealed an abundance of chitobiose, chitotriose and chitotetraose. The COs-monomeric mixture showed a bactericidal effect towards Bacillus cereus and Escherichia coli more efficiently than native chitosan. Among the chitooligomers, the hexamer showed maximum antibacterial effect followed by the penta-, tetra-, tri- and dimers. Of the two monomers, only GlcN showed slight bacterial growth inhibition. SEM revealed bactericidal action patterns of COs-monomeric mixture towards B. cereus and E. coli.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Morphological aspects of interactions between microparticles and mammalian cells: intestinal uptake and onward movement

Uptake of ingested microparticles into small intestinal tissues and on to secondary organs has moved from being an anecdotal phenomenon to a recognised and quantifiable process, which is relevant to risk assessment of accidental exposure, treatment of multi-organ dysfunction syndrome and therapeutic uses of encapsulated drug or vaccine delivery. This review puts in context with the literature the findings of a morphological study of microparticle uptake, using two approaches.The first is a rat in vivo in situ model, appropriate to a study rooted in the exposure of human populations to microparticles. Latex microspheres 2 μm in diameter are the principal particle type used, although others are also investigated. Most data are based on microscopy, but analysis of macerated bulk tissue is also useful. Uptake occurs at early time points after a single dose and is shown to take place almost entirely at villous rather than Peyer's patch sites: however, multiple feeding and therefore a longer time-span produces a higher proportion of particles associated with Peyer's patches, albeit for very small total uptake at those later time points. Uptake is less affected by species, fasting and immunological competence than by age and reproductive status.The second approach uses in vitro methods to confirm the role of intercellular junctions in particle uptake. Particle-associated tight junction opening, in a Caco-2 monolayer, is reflected in changes in transepithelial resistance and particle uptake across the epithelial monolayer: Tight junction opening and particle uptake are both increased further by external irradiation, ethanol and sub-epithelial macrophages, but reduced by exposure to ice. An M cell model has looser tight junctions than Caco-2 cells, but a similar level of particle uptake. These results, along with the changes seen in junctional proteins after particle addition, confirm the role of tight junctions in uptake but suggest that adhering junctions are also important.