A Novel Population of Phytophthora, Similar to P. infestans, Attacks Wild Solanum Species in Ecuador.

ABSTRACT Twenty-six isolates of a Phytophthora population from two wild solanaceous species, Solanum tetrapetalum (n 11) and S. brevifolium (n = 15), were characterized morphologically, with genetic and phenotypic markers, and for pathogenicity on potato and tomato. Based on morphology, ribosomal internal transcribed spacer region 2 (ITS2) sequence, and pathogenicity, all isolates closely resembled P. infestans and were tentatively placed in that species. Nonetheless, this population of Phytophthora is novel. Its primary host is neither potato nor tomato, and all isolates had three restriction fragment length polymorphism (RFLP) bands (probe RG57) and a mitochondrial DNA haplotype that have not been reported for P. infestans. All the isolates were the A2 mating type when tested with a P. infestans A1 isolate. The A2 mating type has not been found among isolates of P. infestans from potato or tomato in Ecuador. Geographical substructing of the Ecuadorian A2 population was detected. The three isolates from the village of Nono, identical to the others in all other aspects, differed by three RFLP bands; those from Nono lacked bands 10 and 16, but possessed band 19. Most of the Ecuadorian A2 isolates were nonpathogenic on potato and tomato, but a few caused very small lesions with sparse sporulation on necrotic tissue. Cluster analysis of multilocus genotypes (RFLP, mating type, and two allozymes) dissociated this A2 population from genotypes representing clonally propagated populations of P. infestans worldwide. The current hypotheses for the historical global movements of P. infestans do not satisfactorily explain the origin or possible time of introduction into Ecuador of this A2 population. Assuming the population is P. infestans, its presence in Ecuador suggests either a hitherto unreported migration of the pathogen or an indigenous population that had not previously been detected.

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium.

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with ß-1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.

Cytology of induced systemic resistance of tomato to Phytophthora infestans.

The infection of tomato leaves by Phytophthora infestans was followed using cytological methods. Fungal ingress and plant reactions in untreated and induced resistant plants were studied. Systemic disease resistance was induced by a local pre-infection with the same fungus. Induction retarded fungal progress at the leaf surface, epidermis and in the mesophyll. The reduced numbers of germinated cysts indicate the presence of fungitoxic substances on the leaf surface of induced plants. Frequency of fungal penetration through the outer epidermal cell wall was reduced, but only in plants exhibiting a high level of induced resistance. Autofluor-escent material, indicating the presence of lignin-like substances, accumulated rapidly beneath some of the appressoria, but this plant response was similar in induced and non-induced plants. Staining with aniline blue indicated that callose deposition was not involved in induced resistance. Thus, none of the cytologically investigated plant reactions correlated with the reduced penetration frequency observed. In the mesophyll, however, the cytological picture corresponding to a hypersensitive reaction occurred more often in induced plants. It is concluded that reduction of disease severity by induction is the result of the combined action of several successive defence reactions.

Actin and tubulin cytoskeletons in germlings of the oomycete fungus Phytophthora infestans.

Actin and tubulins of Phytophthora infestans germlings were detected with monoclonal antibodies on Western blots of crude extracts separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Mr of actin was approximately 43,000, whereas alpha- and beta-tubulin, which migrated as a single band, had an Mr of 53,000. Rhodamine-phalloin revealed peripheral patches of actin in ungerminated cysts. In young germlings, actin fibers were visible in the conversion zone between cyst and germ tube and as connections between actin patches and the incipient germ tube. Actin patches also occurred throughout the peripheral cytoplasm of longer germ tubes, except for the hyphal apex, which commonly contained actin fibers, but actin patches only exceptionally. Associations between patches and fibers were frequent. A monoclonal antibody specific for actin also stained fibers, but in addition it revealed diffuse staining of the apex and fine granular structures, indicative of the presence of G-actin or of single actin filaments. Cysts incubated with a monoclonal antibody against tubulin contained an array of cytoplasmic microtubules (MTs) that arise from a nucleus-associated center. Some of these MTs circumflexed the nucleus, whereas others extended to the cyst periphery. In germ tubes, axially oriented MT bundles extended from the nucleus-associated center into the proximal and distal cytoplasm. Their density was highest near the nucleus, and their number decreased towards the tip, with only a few remaining at the extreme apex. Bundles of MTs were continuous from the nucleus to the subapical region, reaching lengths of up to 20 microns. Ultrastructurally the bundles consisted of as many as 10 MTs. The architecture of the actin and tubulin cytoskeletons in germ tubes of P. infestans bolsters the hypothesis that they maintain the spatial organization of the hyphal protoplast and support or accomplish intrahyphal movements.

[Frequency of Escherichia coli resistance in Switzerland]. / Resistenzhäufigkeit von Escherichia coli in der Schweiz.

Monitoring the drug resistance of bacteria is necessary at regional, national and international levels. Over a two-week period in October 1978, 2561 strains of Escherichia coli and coliforms were isolated from stool specimens obtained from 19 civilian institutions (1338 strains) and from 9 army training camps (1223 strains) located all over Switzerland. Only a few of the donors had undergone recent antimicrobial therapy. The bacterial strains were rapidly distributed among 7 Swiss army microbiological field laboratories and tested for resistance against ampicillin, gentamycin, tetracycline and cotrimoxazole by standard disc diffusion techniques. Criteria for the interpretation of inhibition zones were adapted from WHO and NCCLS guidelines. E. coli ATCC 25922 served as a control strain. The overall resistance rates of E. coli and coliforms were 18% for ampicillin, 0.3% for gentamycin, 33% for tetracycline and 4% for cotrimoxazole. Multiple drug resistance patterns involving these four drugs plus sulfisoxazole, streptomycin, kanamycin, chloramphenicol, cefalothin and colistin were analyzed. Many combinations of drug resistances occurred in far higher numbers than was expected from individual drug resistance frequencies, a fact which suggested linkage of resistance genes. The resistance frequencies observed were shown by several tests of reproducibility to be subject to a modest overall error of approximately 20%.

Freeze-fracture examination of the plasma membrane of the cellular slime mould Polysphondylium pallidum during microcyst formation and germination.

Freeze-fracture observations of the plasma membrane of the amoebae of the cellular slime mould Polysphondylium pallidum during encystment showed a rapid increase in the density of intramembrane particles, especially in the EF face. This density remained high during the formation of the microcyst wall and then fell abruptly to the much lower values typical of the resting microcyst. During germination there was a corresponding increase in intramembrane particle density which continued to a level typical of vegetative amoebae. These results are discussed in the light of recent biochemical studies of microcyst formation, cellulose biosynthesis and microcyst germination.

Plasma membrane alterations as a result of heat activation in Dictyostelium spores.

At the end of heat activation the distribution of spore plasma membrane particles between the two fracture faces (PF and EF) is drastically changed. While in dormant spores the particle number ratio of PF/EF was about 1;1, it increased up to 9:1 in heat activated sproes, indicating a subtle change in plasma membrane properties. The permeability of spores increased within 30 min following heat activation as determined by efflux measurements of radioactively labelled spores. At the onset of swelling this efflux was accelerated. During germination the osmotically active material within the spores increased, part of which could be recovered from the supernatant. The combined experiments point to the plasma membrane as possible target site of heat activation in this system.

Hyphal tip growth in Phytophthora. Gradient distribution and ultrahistochemistry of enzymes.

Germinating cysts and isolated walls from germinating cysts incorporated 14C-UDPG into wall material of which 22.5 and 15% respectively were insoluble in boiling 1 N HCl, indicating that part of the synthetase activity is located in the wall itself. A combination of Urografin and Ficoll density gradients was used to separate various intracellular fractions. A consistent separation of beta-glucanase and UDPG-transferase enriched fractions was achieved. The beta-glucanase fraction contained dictyosome vesicles and fragments along with some plasma membranes. The UDPG-transferase fraction was relatively rich in membranes resembling rough and smooth ER. The results suggest the two enzymes are transported to the wall by different intracellular routes, and two types of vesicle may be involved. Alkaline phosphatase, beta-glucosidase and acid phosphatase were found extracellularly and their distribution in density gradients determined. The results of histochemical staining for acid phosphatase, alkaline phosphatase and polysaccharide are described and compared with the biochemical data. beta-1,3-glucanase, found intra- and extracellularly, induced distorted growth of germ tubes and also removed most of the apical wall when added to the incubation medium. None of these responses were observed with cellulase. Determinations of the osmotic pressure of germinating cysts and incubation medium revealed that the turgor of germinating cysts amounts to about 1.8 at under the conditions used.

Ultrastructural changes and a second mode of flagellar degeneration during ageing of Phytophthora palmivora sporangia.

Ageing of sporangia in Phytophthora palmivora is accompanied by a gradual breakdown of flagella, a transformation of the 'fingerprint' vacuoles, extensive vacuolization of the cytoplasm, accumulation of bundles of microtubules (mastigonemes)contained within cisternae of the rough endoplasmic reticulum, formation of a germination wall lining the inside of the sporangial wall and production of secondary sporangia. The mechanism of flagellar degeneration differs from that observed in directly germinating sporangia. In ageing sporangia and in sporangia induced to germinate directly with germ tubes, there is a close correlation between loss of competence for zoosporogenesis and breakdown of the flagella.

Histochemical and biochemical evidence for the presence of microbodies in phytophthora palmivora.

Using the diaminobenzidine (DAB) reaction catalase activity could be demonstrated histochemically in cytoplasmic structures of Phytophthora palmivora bearing general ultrastructural features of microbodies. These socalled U-bodies sediment together with the catalase activity in Ficoll-Sorbitol-Sucrose gradients following prior purification by differential centrifugation.

Ultrastructural changes during germination of Dictyostelium discoideum spores.

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.