Ultrastructure of Nematode-Trapping Fungi

Capture cells differ ultrastructurally from vegetative cells in the nematode-trapping fungi, Dactylella drechslerii, Monacrosporium rutgeriensis and Arthrobotrys dactyloides, which capture prey by means of adhesive knobs, adhesive hyphal networks, and constricting rings, respectively. Adhesive knobs and adhesive networks contain dense inclusions not found in hyphal tips or subapical regions of the vegetative hyphae, and rough- and smooth-surfaced membranes are abundant in these trap cells. The fine structure of constricting rings differs from that of adhesive traps, and it is altered by closure. In the open configuration, there are membrane-bound inclusions, labyrinthine networks, and electron-lucent regions between the protoplasts and cell wall, all localized on the luminal side of the ring cells. After closure, these features no longer are evident and the cytoplasm of trap cells stains less densely.     

Nematode-Trapping Fungi in Biological Control of Dictyocaulus viviparus

Larvae of the cattle lungworm Dictyocaulus viviparus were cultured in experimental units of 200 g cattle faeces placed in semi-transparent trays in the laboratory. In each of 4 experimental series using this experimental unit, chlamydospores (chl) of the nematode-trapping fungus Duddingtonia flagrans were admixed to half of the faecal cultures in a concentration of 50.000 chl/g. In all 4 series there was a significant reduction in the development and subsequent release of infective lungworm larvae from faecal cultures containing chlamydospores. The average reduction in larval release, caused by fungal spores, was 86%.     

Influence of Cadmium, Zinc, and Lead on Growth, Trap Formation, and Collagenase Activity of Nematode-Trapping Fungi

Growth and morphogenesis of seven species of nematode-trapping fungi and the activity of a collagenase produced by Arthrobotrys amerospora were measured in the presence of various concentrations of divalent cadmium, zinc, or lead. In general, growth varied with species and was dependent on the metal present and the concentration at which it was tested. Cadmium was found to exhibit the greatest toxicity followed by zinc and lead, respectively. In most cases, inhibition of growth was directly correlated with a decreased capacity to form traps. However, in a few cases, trap formation was inhibited either more or less than was growth. The activity of the collagenase was less sensitive than was growth or trap formation to heavy-metal inhibition.     

Arachidonic Acid Production by Fungi

After preliminary screening, Mortierella alpina and Mortierella elongata were compared with respect to arachidonic acid content. M. alpina ATCC 16266 produced 2.1 g of arachidonic acid per liter in media containing 10% glucose while the highest percentage of arachidonic acid in lipid (43.3%) was observed at a glucose concentration of 2%. Arachidonic acid content in lipids increased to 66% during storage.     

l-Lysine Production by Uestilaginales Fungi

From a screening of a few Ustilaginales fungi for free lysine accumulation, Sphacelotheca sorghi ITCC No. 591 was found to accumulate 50μg/ml of free lysine in the culture medium. However, it was later seen that the free lysine content of the culture broths could not be increased much unless cell lysis took place.

Experiments were also carried out to increase the free lysine content of the culture broths of U. maydis PRL No. 1092. It was found that U. maydis maintained on sterilised corn, yields up to 400 μg of free-l-lysine/ml in its culture medium in five days.     

Peptone Induction and Rifampin-Insensitive Collagenase Production by Vibrio alginolyticus

Vibrio alginolyticus produces an extracellular collagenase which requires specific induction by collagen or its high-molecular-weight fragments. Peptone also induces collagenase during the late exponential and early stationary growth phases. The peptone inducers have been shown to have a broad molecular weight range between 1,000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability, whereas digestion with trypsin or alpha-chymotrypsin did not. This indicated that induction by the inducers required the presence of collagenase-sensitive bonds. Prolonged digestion of the inducers with collagenase did not completely eliminate the inducing ability of the inducers. The peptone inducers acted as inhibitors of collagenase. A minimal medium induction system has been developed which involves resuspending cells at high density in a medium containing succinate, (NH(4))(2)SO(4), KH(2)PO(4), and the peptone inducer. Cells grown in minimal medium induce earlier than cells grown on peptone, Casamino Acids, or tryptone. Collagenase production was shown to occur for 30 to 60 min in the presence of rifampin at levels which completely inhibit the incorporation of [(3)H]uracil into trichloroacetic acid-precipitable material. Chloramphenicol completely and immediately abolished collagenase production, which together with labeling studies has confirmed that collagenase production involves de novo synthesis of the enzyme. Both glucose and Casamino Acids repressed collagenase production, although synthesis of the enzyme continued for 30 to 60 min after their addition. The repression of collagenase production by glucose and Casamino Acids was more severe than the inhibition of enzyme formation due to addition of rifampin.     

Do Organic Amendments Enhance the Nematode-Trapping Fungi Dactylellina haptotyla and Arthrobotrys oligospora?

Soil cages (polyvinyl chloride pipe with mesh-covered ends) were used to determine how the quantity of two organic amendments affected the nematode-trapping fungi Dactylellina haptotyla and Arthrobotrys oligospora, which were studied independently in two different vineyards. Each cage contained 80 cm³ of field soil (120 g dry weight equivalent), fungal inoculum (two alginate pellets, each weighing 1.9 mg and containing assimilative hyphae of one fungus), and dried grape or alfalfa leaves (0, 360, or 720 mg equivalent to 0, 4,500, or 9,000 kg/ha) with a C:N of 28:1 and 8:1, respectively. Cages were buried in the vineyards, recovered after 25 to 39 days, and returned to the laboratory where fungus population density and trapping were quantified. Dactylellina haptotyla population density and trapping were most enhanced by the smaller quantity of alfalfa amendment and were not enhanced by the larger quantity of alfalfa amendment. Arthrobotrys oligospora population density was most enhanced by the larger quantity of alfalfa amendment, but A. oligospora trapped few or no nematodes, regardless of amendment. Trapping and population density were correlated for D. haptotyla but not for A. oligospora.     

Production of Mannitol by Fungi from Cotton Dust

Cotton dust associated with high pulmonary function decrements contains relatively high levels of mannitol. In this study, cotton leaf and bract tissue and dust isolated from cotton leaf tissue were examined by optical microscopy, scanning electron microscopy, and capillary gas chromatography. Alternaria alternata, Cladosporium herbarum, Epicoccum purpurascens, and Fusarium pallidoroseum were isolated from cotton leaf dust. The fungal samples, cotton dust, and cotton leaf contained mannitol. This study demonstrates that fungi from a late-fall harvest of cotton leaf material produce mannitol and are a probable source of the mannitol found in cotton dust.     

Fermentative Production of Exocellular Glucans by Fleshy Fungi

Two specimens of higher fungi produced exocellular β-1, 3-glucans when their mycelial forms were cultivated under submerged aerobic conditions. Plectania occidentalis NRRL 3137 consumed up to 6% glucose or xylose with about 30% conversion to polymer in a medium composed of hydrolyzed soy protein, salts, and thiamine. A 5% inoculum was used in a 10-day shaken fermentation. After dilution of the culture liquors and partial disruption of mycelia with a blender, solids were removed by centrifugation, and the polymer was precipitated by the admixture of 2 volumes of ethyl alcohol. A second polymer was formed in 40 to 65% yield by fermentation with Helotium sp. NRRL 3129, which in the imperfect stage would be identified as Monilia sp. It consumed up to 4% glucose, fructose, mannose, or sucrose in 60 to 72 hr. A 2% inoculum in a medium composed of commercial defatted soy flakes, phosphate, and thiamine in tap water gave a satisfactory fermentation. This polymer was precipitated by the addition of 0.5 volume of ethyl alcohol. Both organisms have a broad pH optimum on the slightly acidic side and did best at about 25 C.     

Mycorrhizal Fungi: Siderophore Production

Abstract

Mycorrhizal fungi, which commonly occur in natural as well as agricultural soils, are known to enhance plant uptake of nutrients, including metal ions present as trace concentrations. As mycorrhizal infection is a widespread feature of plant communities, it seems appropriate to review the data on mycorrhizal fungi and their potential to produce siderophores.

Based on a bioassay with Aureobacteriumflavescens JG-9 it was shown that a number of ectomycorrhizal fungi (EM) produce hydroxamate siderophores. Also an arbuscular mycorrhizal (AM) grass species, which showed greater iron uptake than nonmycorrhizal controls, tested positively when bioassayed for hydroxamate siderophores. Encoid mycorrhizal fungi, too, have been demonstrated to be capable of producing hydroxamate-type siderophores. However, only in the case of the eridoid mycorrhizal fungi the main siderophores have been isolated and subsequently identified as ferricrocin and fusigen, respectively. The biotechnological and ecological significance of studies of the siderophore biosynthesis by mycorrhizal fungi is discussed.     

Ascochitine Production by Fungi Responsible for Ascochyta Diseases of Pea

Ascochitine production by 24 isolates of three fungal species causing the Ascochyta disease complex in pea plants, was examined. All eight isolates of Ascochyta pisi Lib. secreted ascochitine to liquid nutrient medium in amounts of 0.1 to 1.2 mg/l. However, no ascochitine was found in culture filtrates of Mycosphaerella pinodes (Berk. and Blox.) Vestergr. or of Phoma medicaginis var. pinodella (Jon.) Boerema although the aggressiveness of these two species as pathogens was shown to be higher than that of A. pisi. Individual isolates of each of the three fungal species examined did not differ in their pathogenicity.     

Laccase Production and Humic Acids Decomposition by Microscopic Soil Fungi

Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic substances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not established, and conditions of the enzyme production have been poorly studied. In the present work soil micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and foreststeppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied. Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was higher than without HA. Significant and weak extracellular laccase production in liquid medium was observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respectively. The level of laccase production by A. murorum was the same without inducers and in the presence of HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation requires further investigation.     

Itaconic Acid Production by Filamentous Fungi in Starch-Rich Industrial Residues

Several fungi and starch-rich industrial residues were screened for itaconic acid (IA) production. Out of 15 strains, only three fungal strains were found to produce IA, which was confirmed by HPLC and GC–MS analysis. These strains were identified as Aspergillus terreus strains C1 and C2, and Ustilago maydis strain C3 by sequencing of 18S rRNA gene and internal transcribed spacer regions. Cis-aconitate decarboxylase (cad) gene, which encodes a key enzyme in IA production in A. terreus, was characterized from strains C1 and C2. C1 and C2 cad gene sequences showed about 96% similarity to the only available GenBank sequence of A. terreus cad gene. 3-D structure and cis-aconitic acid binding pocket of Cad enzyme were predicted by structural modeling. Rice, corn and potato starch wastes were screened for IA production. These materials were enzymatically hydrolyzed under experimentally optimized conditions resulting in the highest glucose production of 230 mg/mL from 20% potato waste. On comparing the production potential of selected strains with different wastes, the best IA production was achieved with strain C1 (255.7 mg/L) using potato waste. Elemental composition as well as batch-to-batch variation in waste substrates were analyzed. The difference in IA production from two different batches of potato waste was found to inversely correlate with their phosphorus content, which indicated that A. terreus produced IA under phosphate limiting condition. The potato waste hydrolysate was deionized to remove inhibitory ions like phosphate, resulting in improved IA production of 4.1 g/L by C1 strain, which is commercially competitive.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

Acid Protease Production by Fungi Used in Soybean Food Fermentation

Growth conditions for maximum protease production by Rhizopus oligosporus, Mucor dispersus, and Actinomucor elegans, used in Oriental food fermentations, were investigated. Enzyme yields by all three fungi were higher in solid substrate fermentations than in submerged culture. The level of moisture in solid substrate must be at about 50 to 60%. Very little growth of these fungi was noted when the moisture of substrate was below 35%, whereas many fungi including most storage fungi generally grow well on solid substrate with that level of moisture. Among the three substrates tested—wheat bran, wheat, and soybeans—wheat bran was the most satisfactory one for enzyme production. The optimal conditions for maximum enzyme production of the three fungi grown on wheat bran were: R. oligosporus, 50% moisture at 25 C for 3 to 4 days; M. dispersus, 50 to 63% moisture at 25 C for 3 to 4 days; A. elegans, 50 to 63% moisture at 20 C for 3 days. Because these fungi are fast growing and require high moisture for growth and for enzyme synthesis, the danger of contamination by toxin-producing fungi would be minimal.     

Production and Degradation of Oxalic Acid by Brown Rot Fungi

Our results show that all of the brown rot fungi tested produce oxalic acid in liquid as well as in semisolid cultures. Gloeophyllum trabeum, which accumulates the lowest amount of oxalic acid during decay of pine holocellulose, showed the highest polysaccharide-depolymerizing activity. Semisolid cultures inoculated with this fungus rapidly converted ¹⁴C-labeled oxalic acid to CO₂ during cellulose depolymerization. The other brown rot fungi also oxidized ¹⁴C-labeled oxalic acid, although less rapidly. In contrast, semisolid cultures inoculated with the white rot fungus Coriolus versicolor did not significantly catabolize the acid and did not depolymerize the holocellulose during decay. Semisolid cultures of G. trabeum amended with desferrioxamine, a specific iron-chelating agent, were unable to lower the degree of polymerization of cellulose or to oxidize ¹⁴C-labeled oxalic acid to the extent or at the rate that control cultures did. These results suggest that both iron and oxalic acid are involved in cellulose depolymerization by brown rot fungi.