H. Stuart Pankratz

The unique root-nodule symbiosis between Rhizobium and the aquatic legume, Neptunia natans (L. f.) Druce

We examined the development of the aquatic N2-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N2 fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N2-fixing root-nodule symbiosis with Neptunia natans.

Ultrastructural effects of Bacillus thuringiensis var. san diego on midgut cells of the cottonwood leaf beetle

Abstract Sequential observations of the ultrastructural effects of Bacillus thuringiensis var. san diego were made on midgut epithelial cells of the cottonwood leaf beetle, Chrysomela scripta F. Larvae imbibed a droplet of B. thuringiensis var. san diego containing δ-endotoxin and live spores. Sections of midgut were prepared for transmission electron microscopy at 15-min intervals during the first hour and then hourly for a duration of 3 hr. No signs of cellular disruption were observed until 2 hr after treatment. At this time midgut epithelial cells from treated larvae appeared more elongate and swollen than cells observed from control larvae, with the apical region of the cell bulging into the gut lumen. The cells contained large cytoplasmic spaces, expansion of the basal labyrinth, disruption of rough endoplasmic reticulum, and apical displacement of nuclei. No adverse effects were observed on microvilli, membranes, or organelles. After 3 hr cellular damage had increased, with cells ruptured at the apical surface and leakage of cytoplasmic materials into the gut lumen. B. thuringiensis var. san diego proliferated in the midgut lumen, producing additional δ-endotoxin crystals during sporulation. Although cellular swelling caused by B. thuringiensis var. san diego δ-endotoxin is similar to that reported for B. thuringiensis var. kurstaki δ-endotoxin in lepidopterans, some differences were observed. These differences included lack of membrane lesions and microvillar damage and comparatively slow response.

Comparison of rod- versus filament-type methanogenic granules: microbial population and reactor performance

Two types of methanogenic granules capable of high chemical oxygen demand removal rates were developed in laboratory-scale upflow reactors at 35° C. One granule type (R-granules) had a rod-type Methanothrix-like species as the predominant species whereas the other (F-granules) had a filament-type M. soehngenii-like acetate-utilizer as the predominant species. These two types of granules were compared in terms of operational performance, physical-chemical characteristics and microbial population. The R-granules had a higher density [65–70 vs 39–43 g suspended solids (SS)/l], specific gravity (1.03 vs 1.01) and specific volumetric methane production rate (180 vs 120 l CH4/l granules per day) than the F-granules. Acetate, propionate and butyrate degraders in both types of granules had similar specific growth rates. The most probable number enumeration indicated that both types of granule had the same population levels (cells/g SS) in terms of methanogens (H2-CO2-, formate- and acetate-utilizing) and syntrophic acetogens. Hydrolytic-fermentative bacteria were present in greater number in the F-granules than in the R-granules. The R-granules had a higher cell density than the F-granules. The differences in operational performance were due mainly to their different microbial composition, especially the predominant acetate-utilizing methanogens in the granules. The long-filamentous M. soehngenii-like rods in the F-granules appeared to be responsible for their lower density and large-sized granules.

Nosema scripta N. Sp. (Microsporida: Nosematidae), a Microsporidian Parasite of the Cottonwood Leaf Beetle, Chrysomela scripta (Coleoptera: Chrysomelidae)'

ABSTRACT. Nosema scripta (Microsporida: Nosematidae), a new species of microsporidian parasite, is described from the cottonwood leaf beetle, Chrysomela scripta F. (Coleoptera: Chrysomelidae), in North America. Studies using light and electron microscopy reveal that this species completes its life cycle in direct contact with the cytoplasm of cells within the fat body, midgut, hindgut, muscles, central nerve cord, Malpighian tubules, tracheal end cells, and ovaries. the microsporidium is monomorphic, all life stages are diplokaryotic, and spores develop from disporous sporonts. The mature spores are broadly oval and measure 4.2 ± 0.10 times 3.4 ± 0.04 μm. Spores contain a tubular polar filament that is arranged peripherally in a single layer of 13‐15 coils. Both horizontal transmission and vertical transmission have been demonstrated for this microsporidium in C. scripta in the laboratory. The Colorado potato beetle, Leptinotarsa decemlineata (Say), was susceptible to infection with this pathogen in cross‐infectivity studies.

Recognition and Infection of Clover Root Hairs by Rhizobium Trifolii

The infection of clover roots by the nitrogen-fixing bacterium,Rhizobium trifolii, involves interaction of both symbionts and is host specific. The host specificity expressed during root hair infection occurs before formation of the infection thread (Li, Hubbell 1969) and constitutes a model for cellular recognition between procaryotes and eucaryotes. A white clover glycoprotein lectin called trifoliin A (ca. 53 kD) accumulates on the surface of root hairs at the growing tip and appears to function as a cell recognition molecule (Dazzo et al. 1978; Dazzo, Truchet 1983). Therefore, our studies of recognition in this symbiosis have focused on trifoliin A on the root and complementary saccharide receptors on the bacterial symbiont. In this paper, we review the current understanding of interactions between these cell surface components as affected by symbiotic genes on the nodulation plasmid ofR. trifolii.