Production and Germination of Conidia of Trichoderma stromaticum, a Mycoparasite of Crinipellis perniciosa on Cacao.

ABSTRACT Growth characteristics of the fungus Trichoderma stromaticum, a mycoparasite on the mycelium and fruiting bodies of Crinipellis perniciosa, the causal agent of witches'-broom disease of cacao, were evaluated under controlled environmental conditions. The ability of T. stromaticum to produce conidia and germinate on dry brooms was evaluated at three constant temperatures (20, 25, and 30 degrees C) and two constant relative humidities (75 and 100%). T. stromaticum produced abundant conidia on brooms at 100% relative humidity and incubation temperatures of 20 and 25 degrees C, but none at 30 degrees C. Sporulation of T. stromaticum was not observed at 75% relative humidity at any temperature. At 100% relative humidity and either at 20 or 25 degrees C, treatment of brooms with T. stromaticum suppressed C. perniciosa within 7 days. In contrast, at 30 degrees C, treatment with T. stromaticum had no effect on the pathogen in brooms maintained at either 75 or 100% relative humidity. Mycelium of C. perniciosa grew from brooms at all temperatures at 100% relative humidity. Conidial germination on broom tissue approximated 80% at temperatures from 20 to 30 degrees C. Results suggest that applying T. stromaticum under high-moisture conditions when the air temperature is below 30 degrees C may enhance the establishment of this mycoparasite in cacao plantations.

Peptide synthetase gene in Trichoderma virens.

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.

Seed Treatment Using Pre-infiltration and Biocontrol Agents to Reduce Damping-off of Corn Caused by Species of Pythium and Fusarium.

Bioassays were conducted in a greenhouse at 18°C to determine the effectiveness of a seed treatment used in combination with biocontrol agents for the reduction of corn damping-off caused by species of Pythium and Fusarium. Corn seeds were infiltrated with tap water, drained, air-dried, and then coated with biomass of an antagonistic fungus, Gliocladium virens isolate Gl-3, or an antagonistic bacterium, Burkholderia cepacia isolates Bc-B or Bc-1, or a combination of Gl-3 with each of the bacterial isolates. A nonsterile field soil was infested with a combination of pathogens: Pythium ultimum, P. arrhenomanes, and Fusarium graminearum at 2 inoculum rates (1× and 4×). Pre-infiltration enhanced (P ≤ 0.05) disease control with most treatments at both inoculum rates. Treatments with biocontrol agents alone or in combination, as well as the fungicide captan, effectively reduced the disease at a pathogen inoculum rate of 1×, resulting in greater (P ≤ 0.05) seedling stands, plant height, and fresh weight, and lower (P ≤ 0.05) root rot severity compared with untreated seeds in infested soil. At a pathogen inoculum rate of 4×, stands were lower (P ≤ 0.01) and root-rot severity was higher (P ≤ 0.01) compared to those at 1× for all treatments. Nevertheless, coating seeds with all biocontrol agents (alone or in combination), except with Bc-1 alone, reduced disease (P ≤ 0.05) compared to untreated seeds in infested soil. At both inoculum rates of 1× and 4×, coating seeds with Gl-3 + Bc-B was more effective (P ≤ 0.05) in disease control than any other treatment, resulting in stands, growth rate (plant height and fresh weight), and root rot severity similar to plants from untreated seeds in noninfested soil. In addition, when the exudate from a 2-h infiltration of corn seed was added to the seeds during seed coating, seedling stand was often lower and root rot severity was often higher than those from infiltrated seeds (P ≤ 0.05). These results indicated that the infiltration process removed certain exudates, including nutrients and/or stimulants (not detected in this study) that might be utilized by pathogens to initiate seed infection. A thin-layer chromatography (TLC) profile of the exudates showed the presence of eight amino acids and three major carbohydrates.

Seed Treatment with a Fungal or a Bacterial Antagonist for Reducing Corn Damping-off Caused by Species of Pythium and Fusarium.

Bioassays were conducted under greenhouse conditions to test the efficacy of antagonists applied to corn (Zea mays) seed for protection against seed rot and seedling damping-off at 18 and 25°C in a field soil artificially infested with a combination of Pythium ultimum, P. arrhenomanes, and Fusarium graminearum. Biomass of Gliocladium virens isolates Gl-3 or Gl-21, Trichoderma viride isolate Tv-1, or peat-based slurry of Burkholderia cepacia isolates Bc-B, Bc-T, or Bc-1 was coated individually onto corn seeds in one test, and Gl-3 or Bc-B at four inoculum levels was used in another test. Seed treatments with most of the biocontrol agents, as well as with the fungicide captan, significantly (P ≤ 0.05) increased seedling stand, plant height and fresh weight, and decreased root rot severity compared with untreated seeds in pathogen-infested soil. Coating seeds with the biocontrol fungus G. virens isolate Gl-3 was the most effective treatment, resulting in greater (P ≤ 0.05) seedling stand, plant height, and fresh weight, and lower (P ≤ 0.05) severity of root rot than those parameters from seeds treated with captan or other antagonists at both temperatures. The results from the seeds treated with Gl-3 were similar to those of untreated seeds in noninfested soil. In treatments with Bc-1, Bc-T, Bc-B, or Tv-1, incubation temperature affected plant emergence, root rot severity, plant height, and fresh weight (P ≤ 0.01). Conversely, in seeds coated with Gl-3 or Gl-21, these parameters were similar at both temperatures. The minimum number of propagules needed per corn seed to obtain plant emergence comparable to that from captan-treated seeds was between 104 and 105 CFU for Gl-3 and >108 for Bc-B. When propagules of Gl-3 were applied at a rate >106 CFU per seed, seedling emergence was greater (P ≤ 0.05) than that from captan-treated seeds.

Reappraisal of the mucosal epithelial space associated with the surface of Hymenolepis diminuta and its effect on transport parameters.

Using nonpermeating, radiolabeled solutes to estimate the magnitude of the "unstirred water layer" (="mucosal epithelial space") of the surface of Hymenolepis diminuta, a value approximating 1% of the worm's fluid volume (0.011-0.022 ml/g wet tissue) was obtained. This value was compared with those previously reported by other workers which were greater by an order of magnitude. The difference between these results appears to be related to the use in previous studies of a permeating marker (mannitol), and a failure to divest the surface of nonspecifically adherent bathing fluid in excess of the actual "unstirred layer". These parameters must be considered in future studies on this useful model for the study of transport.

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta.

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.

Phloretin inhibition of glucose transport by the tapeworm Hymenolepis diminuta: a kinetic analysis.

Phloretin non-competitively inhibits glucose and methionine absorption by Hymenolepis diminuta (Ki = 0.24 and 1.48 mM, respectively). Inhibition of glucose transport by phloretin is reversible. Phloretin and phlorizin binding sites on the surface of H. diminuta are distinct. Phloretin does not inhibit Na+ transport independent of glucose transport in H. diminuta. The data suggest that phloretin inhibits absorptive functions across the surface of H. diminuta via non-specific binding sites associated with the lipid portion of the worm outer membrane.

Histochemical, cytochemical and autoradiographic studies on the rostellum of Hymenolepis diminuta.

Histochemical studies on the rostellum of Hymenolepis diminuta revealed diastase-stable, protein/neutral carbohydrate-rich material localized in the rostellum tegument. The remainder of the rostellum, primarily composed of the glycogen-rich myocytons of the rostellum musculature, is protein-poor, but rich in diastase-labile, neutral and acidic carbohydrates. Ultrastructural cytochemical studies, using the periodic acid-thiocarbohydrazide osmium (PATCO) technique, indicated that the granules of the rostellar tegumental cytons and distal cytoplasm are carbohydrate-rich. Lipids are present in the rostellar myocytons but not in the tegumental cytons. Autoradiography using a pulse-labeling with [3H]leucine revealed an apical translocation of tegumental granules, but at a slower rat than had been reported for the strobilar tegument of the same organism. Neither [3H]galactose nor [3H]glucose were incorporated into the rostellar tegumental granules. The function of the secretory glycoprotein(s) produced in the rostellar tegument and its (their) possible role in the regulation of maturation and/or strobilization remain enigmatic.

The microanatomy and fine structure of the rostellum of Hymenolepis diminuta.

The microanatomic arrangement of the muscular, nervous, and excretory systems as well as the organization of the tegument are described for the rostellum of Hymenolepis diminuta. An inner circular and an outer longitudinal layer of muscle comprise the rostellar capsule, delimiting the rostellum from the scolex proper. A similar muscular arrangement surrounds an apical invagination of the rostellar tegument, the anterior canal. Elements of the excretory and nervous systems enter the rostellum basally through a discontinuous region of the capsule. Although excretory canals extend into the rostellum, flame cells and their associated collecting ducts are absent. The rostellar nervous system is comprised of a single bilateral pair of ganglia, which provide motor innervation of the anterior canal, and the circular muscles of the rostellar capsule; it also receives dendrites from apical uniciliate sensory receptors. The tegument lining the anterior canal and covering the apical rostellum is syncytial and continuous with the tegument of the scolex proper. Twelve to 15 cytons are radially arranged around the anterior canal. They stain selectively with paraldehyde-fuchsin between days 3 and 35 postinfection, but are clearly tegumental, not neurosecretory elements. The appearance of ovoid granules in the rostellar tegumentary cytons coincides with the onset of fuchsinophilia, but the granules persist despite the subsequent loss of this staining characteristic. Intact granules are secreted into the lumen of the anterior canal, although their function has not been ascertained.

A unicellular endocrine gland in cestodes.

Unicellular glands are reported from the scolex and anterior neck region of Hymenolepis diminuta and H. nana. Despite positive staining reactions with the presumptive neurosecretory stains, paraldehyde-fuchsin and chrome-alum-hematoxylin, ultrastructurally these glands exhibit many non-neural characteristics. Glandular cell processes are frequently found in close proximity to muscular tissue, particularly in the suckers, suggesting a regulatory role in muscle modulation as a possible function. Two types of putative, neurosecretory cells are reported from the cephalic ganglia and the lateral nerve cords. Neurosecretory regulation of the unicellular endocrine glands is postulated based on the lack of direct innervation of the glands and the frequent close proximity of axons containing putative, neurosecretory granules.

Schistosoma mansoni: topochemical features of intrasporocyst cercariae.

The carbohydrate rich filamentous coat investing the mature Schistosoma manosini cercaria affects important physiological and antigenic properties of the larval body surface. The origin of the filamentous coat and intrinsic topochemical properties of ccrcariae were investigated by fine structural and cytochemical examination of intrasporocyst larvae of various developmental stages. Staining results achieved with concanavalin A-peroxidase, bismuth subnitrate, silver protein, cationic colloidal iron, and polycationic ferritin indicate the presence of both neutral and acidic glycans at the external surface of the trilaminar tegumental plasmalemma, the latter saccharide moieties conferring upon this surface a superficial electronegative charge. The filamentous coat, apparent only on relatively well-developed larvae, is rich in neutral glycans, but fails to stain with cationic cytochemical reagents. Appearance of the surface coat occurs coincident with the differentiation of tegumentary cytons, the elaboration of carbohydrate-containing vesicles by Golgi complexes within these cell bodies, and the translocation of vesicles from sites of formation to the tegumental syncytium. It is likely that those saccharides, glycoproteins, and/or glycolipids present within the neutral filamentous coat, and those which constitute the acidic layer immediately superticial to the larval body surface, are intrinsic molecular constituents of the cercarial tegumental plasmalemma. Both the neutral filamentous coat and subjacent acidic layer may be regarded as distinct functional elements of the larval body surface glycocalyx. The molecular architecture of this membrane complex apparently reflects the specializations necessary for survival in fresh water followed by rapid adaptation to the serum environment of the mammalian host.

Cytochemical characterization of tegument membrane-associated carbohydrates in Taenia crassiceps larvae.

The tegument of larval Taenia crassiceps possesses a surface coat rich in both neutral and acidic carbohydrates. Neutral glycans were detected in Golgi vesicles of the tegument perikarya, vesicles of the distal tegument, and on the surface of the plasma membrane. Autoradiographs indicated the tegument perikarya as major sites of 3H-galactose incorporation into acid-insoluble macromolecules. The labeled material is subsequently translocated to more superficial regions of the tegument, then concentrated in the brush border. Loss of radioactivity is appreciable within 6 hr of the synthesis of this material, indicating continual replacement of this tegument surface component.