Utilization of oligosaccharides by three pathogenic isolates of colletotrichum gloeosporioides

Summary Utilization of six oligosaccharides (viz., sucrose, maltose, cellobiose, lactose, raffinose and melezitose) by the isolates ofColletotrichum gloeosporioides Penz. causing leaf-spot diseases ofCarissa carandas L.,Eucalyptus robusta Sm. andBougainvillaea glabra Choizy has been studied. Daily chromatographic analysis of the culture medium revealed that the utilization of sucrose, maltose, cellobiose and raffinose was through a hydrolytic pathway. In case of lactose and melezitose no hydrolytic product could be detected. During the utilization of sucrose two oligosaccharides (Rf 0.27 and 0.2) made their appearance. TheBougainvillaea-isolate synthesized two oligosaccharides (Rf 0.18 and 0.1) in a maltose medium although no glucose could be traced, while in case of the other two isolates, in addition to these synthetic oligosaccharides, glucose also appeared. When theCarissa-isolate ofColletotrichum gloeosporioides was grown on a cellobiose medium, in addition to glucose, two synthetic oligosaccharides (Rf 0.15 and 0.11) were formed. Other two isolates showed the formation of one oligosaccharide (Rf 0.15) and no glucose. The dry weight of mycelium recorded an increase on oligosaccharides with slow rate of consumption, while on oligosaccharides with rapid rate of utilization it tended to become constant or showed a decline towards the end of the incubation.     

Studies on the nutrition of the genus pestalotiopsis

Four isolates ofPestalotiopsis, viz.P. gracilis, P. paeoniae isolates I and II, andP. royenae were isolated from the leaf spots ofAchras sapota, Anona squamosa, Syzygium cumini andCarissa congesta respectively. Studies on the nutrition of the isolates were carried out in liquid basal meduim in stationary cultures. Preliminary experiments had shown that a pH of 5.5 and a temperature of 25° C± 1° C were the most suitable for the growth of the present organisms. The growth response of the various species, as judged by their mycelial dry weight on 26 different carbon sources was studied.Among the pentoses, D-xylose supported poor growth of all the four isolates whereas L-arabinose and L-rhamnose were as good as glucose in efficiency, or occasionally better. Among the hexoses, D-fructose and D-mannose were superior to glucose (except forP. royenae). Galactose and L-sorbose were poor sources, the latter being poorest among all the carbohydrates used in this study. The disaccharides were generally superior to glucose except lactose which was poor. AlsoP. royenae had slighthly inferior growth on trehalose than on glucose. Maximum growth, among disaccharides, was attained on cellobiose. The nutritional value of trisaccharide, raffinose, was maximum for all butP. royenae which too grew better on it than on glucose. The three polysaccharides, viz. soluble starch, dextrin and inulin were much superior than glucose except forP. royenae. Generally sugar alcohols were equal to or better than glucose in efficiency. Glycerol, however, was a very poor source. The acids supported very poor growth of all the isolates.     

Utilization of oligosaccharides by three imperfect fungi

Summary The utilization of oligosaccharides (maltose, sucrose, lactose and raffinose) by three imperfect fungi viz.Alternaria tenuis Auct.,Colletotrichum capsici (Syd.)Butler &Bisby andColletotrichum gloeosporioides Penz. isolated from diseased leaves of tomato,Scindapsus pictus andPolyscias balfuriana respectively, was studied chromatographically. Except for lactose, all the sugars were utilizzed through a hydrolytic pathway and almost all proved to be a suitable source of carbon for the growth of fungi. Along with the utilization of sucrose, a simultaneous synthesis of an oligosaccharide was also observed in case of two species ofColletotrichum. C. gloeosporioides, however, also synthesized an oligosaccharide, maltotriose if it is grown in the medium containing maltose. During the utilization of raffinose, melibiose and fructose were detectted in the culture medium ofA. tenuis. Melibiose was further broken down into glucose and galactose by the two species ofColletotrichum.All the sugars were consumed from the media within 15 days except for lactose which persisted in the media even after 15 days.     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.     

Hexose phosphate metabolism and exopolysaccharide formation inPseudomonas cepacia

When grown on solid medium containing excess glucose, glucose dehydrogenase-deficient (Gcd^–) mutants ofPseudomonas cepacia 249 formed large amounts of an exopolysaccharide comprised of galactose, glucose, mannose, glucuronic acid, and rhamnose. The Gcd⁺ parent strain failed to accumulate comparable amounts of exopolymer from glucose because of its rapid conversion of glucose to gluconic and 2-ketogluconic acids and its lower content of enzymes related to glucose-1-phosphate synthesis. Both Gcd⁺ and Gcd^– strains ofP. cepacia accumulated exopolymer when substrates such as mannitol and glycerol were substituted for glucose. A survey of clinical isolates from patients with cystic fibrosis indicated that there was no correlation between ability ofP. cepacia to colonize the respiratory tracts of such individuals and increased capacity to form exopolymer related to glucose dehydrogenase deficiency.     

In-feed 0.6% ivermectin formulation for treatment of wild boar in the Moslavina hunting ground in Croatia

An in-feed 0.6% ivermectin formulation was administered for 7 days to wild boar piglets at three sites of the Moslavina hunting ground in Croatia. Examination of internal organs and skin of five piglets that died immediately before the start of administration of the ivermectin formulation revealed the presence of Metastrongylus apri and Metastrongylus pudendotectus in the lungs, and of Ascarops strongylina, Physocephalus sexalatus and Globocephalus urosubulatusin in the gastrointestinal tract. Coccidial oocysts were found in the feces of all animals. Sarcoptes scabiei var. suis was identified in the skin of four piglets. The efficacy of treatment was assessed by examining fecal samples before start of therapy (day 0) and on days 7 and 14. Before treatment strongylid-type eggs were detected in 70–100% of fecal samples (210–505 EpG) The eggs of Strongyloides ransomi, Trichuris suis, Ascaris suum, Ascarops strongylina and Physocephalus sexalatus were identified in 10–50% of fecal samples at an intensity of 5–45 EpG. On day 14 after the start of the treatment, strongylid-type eggs were detected in 10% of fecal samples from one of the three sites only. Eggs of other helminth species were not detected at any of the three sites. This confirmed the successful therapeutic efficacy of the in-feed 0,6% ivermectin formulation.     

Interactions between freshwater bacteria andAnkistrodesmus braunii in batch and continuous culture

Batch and continuous cultures ofAnkistrodesmus braunii were established in an inorganic medium with growth rate limited by P. In batch culture, inoculation of lake water bacterial isolates ofPseudomonas sp. andFlavobacterium sp. showed that thePseudomonas isolate was capable of more rapid growth on algal exudates of lytic products than was theFlavobacterium isolate. When inoculated singly into a continuous culture (D=0.267 day^–1; P level, 2M), theFlavobacterium isolate initially caused a decrease in the population density of the alga, but then steady states for both organisms were obtained. ThePseudomonas isolate under the same conditions caused a rapid washout of the algal culture, and steady-state conditions were never obtained. When thePseudomonas isolate was added to the two-member, steady-state system ofA. braunii andFlavobacterium, the algal population again washed out of the vessel, followed by theFlavobacterium and then thePseudomonas isolate. A transient increase in the P concentration to 200M in the culture vessel caused the low algal population level to increase, followed by increases in the bacterial isolates when the algal population was high enough to supply the required organic carbon source. The system demonstrated that competition for P between the alga and the bacteria can occur, and the results were dependent on the algal and bacterial relative growth rates. The bacterial growth rates were limited initially by organic substrates produced by the alga, and the different bacterial isolates competed for these substrates.     

Effects of some carbon and nitrogen sources on spore germination,production of biomass and antifungal metabolites by species of Trichoderma and Gliocladium virens antagonistic to Sclerotium cepivorum

Conidia of Trichoderma pseudokoningii (IMI 322662) and T. viride (IMI 322659) were incubated in 1% bacteriological peptone at 25° C for 20 h and more than 95% of the spores germinated. In the same medium, only 35% of the conidia of Gliocladium virens (G20) and T. viride (IMI 322663) germinated but when 1% glucose was added, germination was increased to 70%. In the presence of glucose as a carbon source, maximal biomass production of G. virens (G20) after seven days at 25°C was obtained with either potassium nitrate or L‐alanine as the nitrogen source, whereas the Trichoderma isolates needed an organic nitrogen source. With L‐alanine as a nitrogen source, glucose, galactose and sucrose were readily utilized for biomass production by all fungal isolates. Maltose utilization by G. virens (G20) and T. pseudokoningii was incomplete after 21 days incubation, whereas glucose utilization was complete by this time. G. virens, T. pseudokoningii and T. viride (IMI 322663) produced antifungal metabolites which were effective at reducing radial growth of Rhizoctonia solani, Botrytis cinerea as well as S. cepivorum. The metabolites produced by G. virens were very active against all three pathogens but the metabolites produced by T. pseudokoningii and T. viride (IMI 322663) were less active. T. viride (IMI 322659) was a very poor antifungal metabolite producer.     

Some biochemical characteristics of fungi isolated from salmonid eggs

Fungal isolates from salmonid eggs displayed apparently unique patterns of biochemical characteristics at both the generic and specific levels. of the five genera examinedAchlya andPythium were able to assimilate 13–16 out of 19 carbohydrates.Aphanomyces was able to assimilate only glucose and starch, which was assimilated by all isolates. Members ofSaprolegnia displayed identical patterns of carbohydrate assimilation, except forS. hypogyna, which was also able to assimilate melibiose, in common withAchlya, Pythium, andLeptolegnia. Pythium was the only genus capable of assimilating salicin. OnlyAchlya andP. monospermum were able to assimilate rhamnose. In terms of amino acid assimilation isolates ofSaprolegnia ferax andS. diclina displayed an identical patterns, as did isolates ofS. parasitica andS. hypogyna. OnlyAphanomyces frigidophilus isolate was capable of assimilating cysteine. All genera exceptPythium assimilated glutamine, a fundamental amino acid. All isolates exhibited lipase and fatty acid esterase activities but no cellulase acitivity. The biochemical characteristics discovered in this study offer possibilities for identification and classification of these fungi, which are discussed herein.     

Observations on gymnoascaceae. IX. A new species of pseudoarachniotus from soil-burial beds

Summary A new species ofPseudoarachniotus P. marginosporus, is described and illustrated. The developmental morphology of this new ascomycete is discussed. On a standard medium such as YpSs,P. marginosporus can be distinguished from other species of the genus by colonial coloration which varies from ochre-rust to very pale yellow at different stages of maturation. The colony ofP. roseus is red, red-orange or orange-yellow;P. citrinus is lemon yellow to brownish yellow in age;P. reticulatus is red orange andP. hyalinosporus is yellowish and usually with a great deal of green pigmentation in the mycelium and in the medium. Furthermore ascospores ofP. marginosporus are oblate, small and thin-walled, measuring 2.2–3.0×4.0–4.4 µ, with a minute elevated rim around the long axis, while ascospores ofP. reticulatus are globose and reticulate, and those ofP. roseus andP. citrinus are large and thick-walled, measuring about 4.4×7.7 µ. Ascospores ofP. hyalinosporus are similar in size and shape to those ofP. marginosporus, but lack a rim or other markings.     

Double-stranded RNA in isolates ofDiscula destructiva from the Eastern United States

Phenol-chloroform extraction and CF-11 chromatography were used to examine 83 isolates ofDiscula sp. for the presence of dsRNA. Agarose-gel electrophoresis revealed three major size classes: large (ca. 8–12 kbp), medium (ca. 3–4 kpb), and small (ca. 1.5–2.5 kbp). Additional segments were present in two isolates. Double-stranded RNA was demonstrated in 80/80 isolates ofDiscula destructiva, but not in three isolates of an unidentifiedDiscula sp. commonly referred to asDiscula, Type 2. Twenty-two isolates contained one or more large segments. Isolates with large segments were more common in collection sites on the eastern slope of the Appalachian Mountains. The most common banding profile consisted of two medium and two small bands.     

Insecticidal and nematicidal properties of microbial metabolites

Summary Metabolites from 942 microbial isolates were screened for insecticidal and nematicidal properties. The isolates included 302 streptomycetes, 502 novel actinomycetes including representatives of 18 genera, 28 unidentified aerobic actinomycetes, 70 fungi and 40 bacteria other than actinomycetes. When diluted 10-fold, the metabolites from 55 isolates caused nearly 100% mortality in mosquito larvae (Aedes aegypti) within 24 h. These isolates included 27 isolates ofStreptomyces, four ofActinoplanes, three isolates each ofActinomadura andStreptoverticillium, two isolates each ofMicromonospora, Bacillus andPaecilomyces, and one isolate each ofMicropolyspora, Nocardiopsis, Streptosporangium, Oerskovia, Thermomonospora, Chainia, Pseudomonas, Fusarium, Monilia andSyncephalestrum. Two fungal isolates could not be identified to the generie level. Extracts from the culture broth of 18 isolates caused 100% mortality in mosquito larvae within 15 min to 24 h at a concentration of 1000 ppm. The LC₅₀ of partially purified products from two isolates was 1–2.5 ppm and that of the semipurified preparations from four other isolates was 50 ppm. Valinomycin was identified as an active component in the culture broth from one isolate. The culture broth from 15 isolates of aerobic actinomycetes and 4 of fungi were toxic toPanagrellus redivivus (Nematoda); these included 12 isolates with selective nematicidal properties.Michigan Agriculture Experiment Station, Journal Article No. 12321.     

Morphological and physiological comparisons ofHelicobasidium mompa andH. purpureum

Morphological and physiological comparisons were made of sevenHelicobasidium mompa isolates and fourH. purpureum isolates. Colonies of theH. mompa isolates were thin, dense, or hard and dense, and most were pale brown to brown or dark brown, while that of isolate 344c was pinkish. Colonies ofH. purpureum isolates were hard and dense, and their colonies were dark brown. Diameters of hyphae were similar forH. mompa andH. purpureum. Dimensions of conidia and morphology of conidiophores ofH. mompa isolate 344c were close to those ofH. purpureum reported previously.H. mompa isolates grew well at 23°C, 25°C or 27°C, while all isolates ofH. purpureum grew well at 23°C. Growth rates ofH. purpureum isolates was almost the same as those ofH. mompa isolates with slow growth. Polygaracturonase activity at pH 3 was variable among the isolates for bothH. mompa andH. purpureum. Itaconic acid was produced abundantly by three isolates ofH. mompa but not produced by isolate AH130, whereas all isoaltes ofH. purpureum produced a small amount of itaconic acid.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

Chemical and cytological changes during the autolysis of yeasts

Summary Cell suspensions ofSacharomyces cerevisiae, Kloeckera apiculata andCandida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25–35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3–7%; organic acids 3–6%; protein 12–13%; free amino acids 8–12%; nucleic acid products 3–5%; and lipids 1–12%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85–90% of cellular RNA and 25–40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neutral lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization withinS. cerevisiae andC. stellata. There were differences in the autolytic behavior ofK. apiculata compared withS. cerevisiae andC. stellata.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Development ofPuccinia striiformis West. on nutrient agar

Uredospores of a barley isolate ofPuccinia striiformis West. were germinated on glucose peptone agar and the development of colonies was observed with a microscope. A small number (0.1–1%) of germ tubes did not abort during the first 20 h of incubation and either differentiated an infection structure or continued slowly to elongate for several days. In 4 out of 9 experiments some of these sporelings produced colonies that reached maximum size (0.15–0.20 mm) after 3 weeks at 15° C.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Fungal succession in the early decomposition process of pine cones on the floor ofPinus densiflora forests

The fungal succession on pine cones on the floor ofPinus densiflora forest was investigated in the early decomposition process (within ca. 30% decrease in dry weight). The fungal flora was examined by both washing and surface-sterilization methods on artificially placed cones and naturally fallen cones. The decomposition rates of artificially placed cones were 0.081–0.082 yr^–1. On withered cones still attached to the tree,Pestalotiopsis spp. were dominant. These fungi also occurred with higher frequencies after cones had lain on the floor and on cones in the L and FH horizons.Xylaria sp. andPhomopsis sp., which seem to colonize the interior of the tissue, occurred with higher frequencies on the cones on the tree, but their occurrence frequencies decreased after cones had lain on the forest floor. Conversely,Mortierella spp. andTrichoderma spp. newly occurred or their occurrence frequencies increased on lying cones. Of these,Trichoderma koningii increased rapidly and showed high occurrence frequencies.Thysanophora penicillioides, which prefers coniferous substrates, showed higher occurrence frequencies in the early stages of lying on the forest floor. On cones lying on the floor, the fungal flora did not significantly change during the investigation period.