Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes

During the MICROMAT project, the fungal diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics and anticancers. In all, 160 filamentous fungi belonging to fifteen different genera and 171 yeasts were isolated from 11 lakes, classified and cultivated in different media and at different temperatures. Filamentous fungi were then screened to discover novel antimicrobial and cytotoxic compounds. A total of 1422 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 47 (29%) filamentous fungi showed antimicrobial activity; most of them inhibited the growth of gram-positive Staphyloccus aureus (14%), gram-negative E. coli (10%), and of yeasts Candida albicans (11%) and Cryptococcus neoformans (8%). Less activity was detected against representatives of enterobacteria and filamentous fungi. The most productive in terms of bioactivities were cold-tolerant cosmopolitan hyphomycetes such as Penicillium, Aspergillus, Beauveria and Cladosporium. Two bioactive bis-anthraquinones (rugulosin and skyrin) were identified by LC–MS as the main products in a strain of Penicillium chrysogenum isolated from a saline lake in the Vestfold Hills. LC–MS fractionation of extracts from two diverse species of Aspergillus, that exhibited relatively potent antimicrobial activities, evidenced a chemical novelty that was further investigated. To our knowledge, this is the first report of new antibiotics produced by fungi from benthic microbial mats from Antarctic lakes. It can be concluded that these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as drugs.     

Lignicolous fungi from northern Serbia as natural sources of antioxidants

As a result of an interest in natural derived metabolites, lignicolous fungi have taken on great importance in biochemical investigations. In the present study, antioxidative screening analyses have included in vitro testing of different extracts (aqueous, methanol, chloroform) of four fungal species using three different assays: Fe²⁺/ascorbate-induced lipid peroxidation by TBA assay, the neutralisation of OH· radicals and the radical scavenging capacity with the DPPHk]assay. TLC analysis confirmed the existance of phenolics in the extracts, but also indicates the presence of some other compounds. The obtained results indicate that MeOH extracts manifested a degree of activity higher than that of CHCl₃ extracts. With respect to antioxidative activity, the extracts can be ranged in the following declining order: G. lucidum, G. applanatum, M. giganteus and F. velutipes. These results suggest that analyzed fungi are of potential interest as sources of strong natural antioxidants that could be used in the food industries and nutrition.     

Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material

Molecular phylogenetic studies were carried out based on ITS-5.8S rDNA, the D1–D2 region of the large subunit rRNA gene, RPB2, and combined data of D1–D2 and RPB2 as well as these three genes on 36 species among 7 genera for Lachnum and allied genera in the family Hyaloscyphaceae. In the combined data of all three regions, seven strongly supported clades were obtained. The same clades were also recognized in most of the trees based on each gene, and the combined data of D1–D2 and RPB2, although some of them were not strongly supported. Four clades represented Albotricha, Brunnipila, Incrucipulum, and Lachnellula, respectively, whereas Lachnum was distributed to the remaining three clades. The molecular phylogenies strongly supported a group of species with granulate hairs, and we suggest the concept of Lachnaceae should be restricted to these species. Based on the molecular phylogenetic analysis, three new combinations—Incrucipulum longispineum, I. radiatum, and Lachnellula pulverulentum from Lachnum—are proposed.     

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions

Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1‐aminocyclopropane‐1‐carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l^?1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l^?1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l^?1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase‐producing strain Pseudomonas putida UW4 (AcdS⁺), its mutant unable to produce the enzyme (AcdS^?), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l^?1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro‐organism on the photosynthetic efficiency were evaluated. The AcdS⁺ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase‐producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS⁺ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.     

Changes in biochemical constituents of paddy straw during degradation by white rot fungi and its impact on in vitro digestibility

Aims: To improve the digestibility of paddy straw to be used as animal feed by means of selective delignification using white rot fungi. Methods and Results: Solid state fermentation of paddy straw was carried out with some white rot fungi for 60 days. Different biochemical analyses, e.g. total organic matter (TOM) loss, hemicellulose loss, cellulose loss, lignin loss and in vitro digestibility, were carried out along with laccase, xylanase and carboxymethyl cellulase activity. The results were compared with that of a widely studied fungus Phanerochaete chrysosporium, which degraded 464 g kg^?1 TOM and enhanced the in vitro digestibility from 185 to 254 g kg^?1 after 60 days of incubation. Straw inoculated with Phlebia brevispora possessed maximum crude protein. Conclusions: All the tested white rot fungi efficiently degraded the lignin and enhanced the in vitro digestibility of paddy straw. Phlebia brevispora, Phlebia radiata and P. chrysosporium enhanced the in vitro digestibility almost to similar levels, while the loss in TOM was much lesser in P. brevispora and P. radiata when compared to P. chrysosporium. Significance and Impact of the Study: The study reflects the potential of P. brevispora and P. radiata as suitable choices for practical use in terms of availability of organic matter with higher protein value, selective ligninolysis and better digestibility.     

Development and optimization of an 18S rRNA‐based oligonucleotide microarray for the fungal order Eurotiales

Aims: For identification of members of the fungal order Eurotiales, an 18S rRNA gene‐based oligonucleotide microarray was developed and optimized. Methods and Results: Eurotiales‐specific probes covering most members of the Eurotiales as well as species‐specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. Conclusions: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species‐specific probes were also capable of species‐level detection. Significance and Impact of the Study: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.     

Dynamics in the microbiology of maize silage during whole‐season storage

Aims: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole‐year storage. Methods and Results: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time‐points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time‐points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. Conclusions: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5–7 months after ensiling and lowest after 11 months. Significance and Impact of the Study: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole‐season storage is applied.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants

Endophytic filamentous fungi from coffee plants (Coffea arabica and C. robusta) deposited in the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI) were characterized taxonomically by using molecular tools and investigated concerning their antimicrobial activity against different human pathogenic bacteria. Thirty-seven out of 39 CBMAI strains investigated were identified to at least at genus level by ITS and rDNA D1/D2 sequencing and phylogenetic analyses. Bioactivity screening of fungal extracts against Salmonella choleraesuis (CBMAI 484), Staphylococcus aureus (CBMAI 485), Pseudomonas aeruginosa (CBMAI 489) and against four different Escherichia coli serotypes showed that 17 fungi inhibited at least one of the bacteria studied. The endophytic fungi Trichoderma harzianum (CBMAI 43), Guignardia sp. (CBMAI 69) and Phomopsis sp. (CBMAI 164) inhibited from four to five bacterial species, while five fungi were active against all pathogenic bacteria tested and were identified as Aspergillus versicolor (CBMAI 46), Fusarium oxysporum (CBMAI 53), Glomerella sp. (CBMAI 63) and Cladosporium spp. (CBMAI 64 and CBMAI 66). The Minimal Inhibitory Concentration (MIC) for the fungus extracts varied from 0.025 to 1.0 mg ml⁻¹, demonstrating antimicrobial potential of some of these fungi.