Non-sporulating Mycelia Research Articles

Keratinolytic and Opportunistic Pathogenic Fungi from Carpet Dust in Mosques and Residentialhouses in Duhok, Kurdistan Region, Iraq

One hundred samples of carpet dusts (50 samples from residential houses and 50 samples from mosques) were collected from different sites in Duhok province during the year 2014 to 2015 for the objective of the study of the occurrence of keratinolytic and other potentially pathogenic fungi using hair baiting method. A total of 24 fungal species (17 species from house dust) and (12 species from mosque dust) in addition to non-sporulating mycelia and yeasts were isolated and identified. The keratinolytic species Arthroderma cuniculi, Chrysosporium tropicum and Gymnoascus ressii were detected. Potentially pathogenic fungi in the genera Aspergillus, Arthrographis,Geomyces, Microascus,Scopulariopsis and Neoscytalidium were also able to colonize and grow on baited hairs.

Structural characterization of a putative chitin synthase gene in Phytophthora spp. and analysis of its transcriptional activity during pathogenesis on potato and soybean plants.

Although chitin is a major component of the fungal cell wall, in oomycetes (fungal-like organisms), this compound has only been found in very little amounts, mostly in the cell wall of members of the genera Achlya and Saprolegnia. In the oomycetes Phytophthora infestans and P. sojae the presence of chitin has not been demonstrated; however, the gene putatively encoding chitin synthase (CHS), the enzyme that synthesizes chitin, is present in their genomes. The evolutionary significance of the CHS gene in P. infestans and P. sojae genomes is not fully understood and, therefore, further studies are warranted. We have cloned and characterized the putative CHS genes from two Phytophthora spp. and multiple isolates of P. infestans and P. sojae and analyzed their phylogenetic relationships. We also conducted CHS inhibition assays and measured CHS transcriptional activity in Phytophthora spp. during infection of susceptible plants. Results of our investigations suggest that CHS contains all the motifs that are typical in CHS genes of fungal origin and is expressed, at least at the mRNA level, during in vitro and in planta growth. In infected tissues, the highest levels of expression occurred in the first 12h post inoculation. In addition, results from our inhibition experiments appear to suggest that CHS activity is important for P. infestans normal vegetative growth. Because of the considerable variation in expression during infection when compared to basal expression observed in in vitro cultures of non-sporulating mycelium, we hypothesize that CHS may have a meaningful role in Phytophthora pathogenicity.

Fungi associated with grapevine (Vitis vinifera L) decline in middle of Iraq

Fungal species associated with three (Vitis vinifera L.)cultivars exhibited decline grown in the main grapevine production area in Salahaldin province, middle Iraq were surveyed during 2012 – 2013. Based on microscopical and cultural characteristics, a total of 24 species in addition to non-sporulating mycelia were identified. The most frequently isolated fungi from shoots were Aspergillus niger, Cladosporium cladosporoides, Cadophora spp., Clonostachys rosea, Penicillium spp. Phaeoacremonium sp. I, Neocytalidium dimidiatum and Stachybotrys atra, whereas, Fusarium spp., Acremonium sp., Cylindrocladiella viticola, Cylindrocarpon spp., and Phaeoacremonium sp. 2, were the most frequently isolated fungi from roots. C. viticola is recorded for the first time from Iraq.

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.

Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

The 18mer peptaibols from Trichoderma virens elicit plant defence responses

SUMMARY Peptaibols, the products of non-ribosomal peptide synthetases (NRPS), are linear peptide antibiotics produced by Trichoderma and other fungal genera. Trichoderma virens strain Gv29-8, a well-known biocontrol agent and inducer of plant defence responses, produces three lengths of peptaibols, 11, 14 and 18 residues long, with several isoforms of each. Disruption of the NRPS gene, tex1, encoded by a 62.8-kb uninterrupted open reading frame, results in the loss of production of all forms of 18-residue peptaibols. Tex1 is expressed during all Trichoderma developmental stages (germinating conidia, sporulating and non-sporulating mycelia) examined on solid media. Expression analysis by reverse transcriptase PCR shows that in Gv29-8 wild-type the abundance of tex1 transcript is greater during co-cultivation with cucumber seedling roots than when grown alone. Cucumber plants co-cultivated with T. virens strains disrupted in tex1 show a significantly reduced systemic resistance response against the leaf pathogen Pseudomonas syringae pv. lachrymans, and reduced ability to produce phenolic compounds with inhibitory activity to the bacteria as compared with plants grown in the presence of wild-type. Two synthetic 18-amino-acid peptaibol isoforms (TvBI and TvBII) from Gv29-8 when applied to cucumber seedlings through the transpiration stream can alone induce systemic protection to the leaf pathogenic bacteria, induce antimicrobial compounds in cucumber cotyledons and up-regulate hydroxyperoxide lyase (hpl), phenylalanine ammonia lyase (pal1) and peroxidase (prx) gene expression. These data strongly suggest that the 18mer peptaibols are critical in the chemical communication between Trichoderma and plants as triggers of non-cultivar-specific defence responses.

Mycelial responses of the soil fungus, Mortierella isabellina, to grazing by Onychiurus armatus (collembola)

Hyphal morphology and extracellular enzyme production of the fungus Mortierella isabellina were studied during grazing by a soil Collembola, Onychiurus armatus. Grazing induced switching from a “normal” hyphal mode, with appressed growth and sporulating hyphae, to fan shaped sectors of fast growing and nonsporulating mycelium which developed extensive aerial mycelium. Specific protease and α-amylase activities were several times higher in grazed cultures where switching occurred compared with plates without switching. Increased protease activity was localized to switched sectors within the mycelium. On the basis of inhibitor studies, the protease of “normal” mycelium was classified as a metalloprotease. Switching induced activity of an additional serine protease. Anion-exchange chromatography showed that the two proteases had similar charges and, according to SDS-gclatine-PAGE. the molecular weight of the serine protease was 80.000. Switching to a fast growing hyphal mode could be one explanation for compensatory growth of grazed fungi.

Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants

Methods were developed for the reproducible production of high yields of stable protoplasts from monokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. This is a prerequisite for mutation, cell fusion, and transformation of this mycorrhizal fungus. Protoplasts from Hebeloma cylindrosporum, Hebeloma edurum, Hebeloma sinapizans, and Suillus bellini were released by using cellulase Onozuka R 10 and driselase as lytic enzymes. Production was strongly influenced by the nature and molarity of the osmotic stabilizer and by the mycelial age. In spite of the high percentage of anucleate protoplasts (42%) produced, the regeneration rate averaged 15%. Fungicide-resistant mutants from monokaryotic mycelia of H. cylindrosporum were isolated following ultraviolet irradiation of protoplasts. This indicates that protoplasts will be useful to select physiological mutants from nonsporulating mycelia and to create new strains by mating complementary mycelia or by interspecific cell fusion.

Changes in the lipid composition during the photo-induced conidiation of Trichoderma viride.

The sporulating and nonsporulating mycelium of Trichoderma viride exhibited significant differences in the levels of triacylglycerols, sterol esters. fatty acids, gangliosides, phosphatidylethanolamine and sphingomyelin. Quantitative changes in the contents of polar and nonpolar lipids were observed also during the analyses of one-, two-, three-day-old sporulation zones in colonies subjected to repeated photo-induction. These findings are similar to the changes observed during the differentiation of other species of fungi.

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.

Changes in activities of the embden-meyerhof-parnas and pentose phosphate pathways during the growth cycle of Aspergillus niger

The specific activities of certain enzymes of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways varied in cultures of Aspergillus niger in relation to the stage of the growth cycle and the nature of the growth medium. At 24 h from spore inoculation the levels of PP pathway enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transketolase were higher in extracts from non-sporulating than from sporulating mycelium. Correspondingly, the levels of most EMP pathway enzymes were higher in extracts from sporulating than from non-sporulating mycelium. These fluctuations in enzyme levels occurred well in advance of any morphological evidence of sporulation. At later stages of growth (48–96 h) the levels of the enzymes were similar in extracts from both types of mycelium. Radiorespirometric patterns for the oxidation of specifically labelled [14C] glucose implied that the EMP and PP pathways were active in both types of mycelium. The contribution of the PP pathway was highest in young non-sporulating mycelium whereas the contribution of the EMP pathway was highest in sporulating mycelium of corresponding age.