Structural investigations and biological activity of inositol sphingophospholipids from Phytophthora capsici.

Inositol sphingophospholipids that protect pepper (Capsicum annuum c.v. Yolo Wonder) against pathogen have been isolated by chromatographic methods from the mycelium of Phytophthora capsici. The structure of the major compound was determined by chemical methods and mass spectrometry. Phosphodiester bond cleavage of the phospholipid by mild alkaline hydrolysis liberated a ceramide which contained a C16-sphingosine. This long-chain base was identified by gas chromatography and mass spectrometry of its trimethylsilyl derivative. One of the amide-linked fatty acids was found to be 4-hydroxy-2 docosenoic acid. Fast-atom-bombardment mass spectrometry and fast-atom-bombardment collison-induced tandem mass spectrometry were used to characterize the ceramide as N(4-hydroxy-2-docosenoyl)C16-sphingosine. These sphingolipids have a protective effect on cotyledons of young peppers against necrotic lesions induced by the pathogen P. capsici.

Comparative studies on the DNA of Pythium species and some possibly related taxa.

DNA from 14 Pythium species, Verrucalvus flavofaciens and Zoophagus insidians was characterized by CsCl-bisbenzimide density gradients in order to investigate its taxonomic potential. A few incomplete analyses were made for other species. All clearly assignable Pythium species produced three DNA bands in the gradient. Pythium undulatum along with Verrucalvus and Zoophagus produced only two bands. Another possible exception, which needs further investigation, is P. vexans. The DNA had a relatively constant banding pattern in CsCl gradients. The small number (eight) of DNA criteria that were available were subjected to cluster analysis to assess the relationships between replicates and species. This restricted database, similar in size to the number of criteria used in morphological taxonomy, provided an independent assessment of the values that have been attached to generic and subgeneric classifications. This approach enabled assessments to be made of relationships between species that have incomplete life-histories and which therefore lack features essential for traditional taxonomic decisions.

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis.

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen.

Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell wall formation in zoospores of Allomyces arbuscula. III. Carbohydrate composition of cell walls during development from meiospores to hyphae.

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.

Evidence for the presence of messenger ribonucleic acid in Allomyces macrogynus mitospores.

Sucrose density gradient analysis was used to show that polysomes were present in the mitospores of Allomyces macrogynus. Fifty percent of the spore monosomes were shown to be resistant to dissociation by 0.8 M KCl, indicating that messenger ribonucleic acid (mRNA) was bound to them. These polysomes and all the spore ribosomes were contained in the nuclear cap. Only 4S RNA could be demonstrated in the extra-cap fraction. Hybridization studies using 3H-labeled polydeoxythymidylic acid indicated that polyadenylate was present to the extent of 0.08% of the total spore RNA. Sixty-eight percent of the polyadenylic acid is found in the nuclear cap, and 32% is found in the extra-cap fraction. It was demonstrated that [3H]uridine was taken up by the spores and converted to uridine triphosphate. Lack of incorporation of 3H into RNA indicated that the spores do not synthesize RNA. Thus, the mRNA found in spores is synthesized prior to spore formation.

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity.

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.