Molecular characterization of some lignicolous species from fungal culture collection

Culture collections of microorganisms, including fungi, are strain deposits recognised as Biological Resource Centers (BRCs) with a great importance in science, industry and education. Their objective is to preserve the purity, viability and genomic integrity of every single strain as a member of such collection. Since improvement of molecular methods nowadays brought many novel approaches in manipulation with strains of microorganisms, they can also be useful for characterization of existing stored strains. ITS1 region in nuclear DNA is preferred barcoding marker for taxon identification, which can be explained by its great inter-species variability. This paper presents results from analysing ITS1 region sequences (17) obtained from fungal DNA of culture collection of autochthonous, lignicolous genera Piptoporus, Pleurotus, Ganoderma and Schizophyllum cultured on malt agar plates for 14 days at 25°C. BLAST (Basic Local Alignment Search Tool) was used for comparison with online databases, while alignment of sequences was made with MEGA 5.10 software. Morphological determination of species or genus was confirmed for 13 cultures, while the others were disproved. The resulting alignment indicated small intra-species variability of ITS1 region and pointed to it as an ideal marker for verification of fungal culture collections' authenticity. [Projekat Ministarstva nauke Republike Srbije, br. III43002 and by the Provincial Secretariat for Science and Technological Development, Vojvodina, Serbia APV 114-4513592/2013-03: Molecular and phenotypic diversity of taxa of economical and epidemiological importance, and endangered and endemic species in Europe

Antifungal activity of surfactant ionic liquids on mycotoxigenic molds

Traditionally, mold control usually involves usage of highly toxic compounds, and this practice must be replaced with more environmentally friendly solutions (green chemistry). In this paper, antifungal activity of five newly synthesized ionic liquids (IL) was tested, in vitro. All ionic liquids are based on N-decyl- N, N, N-trimethylammonium chloride which differs only by a single substituent on a quaternary N- atom. The antifungal activity of IL was tested on Fusarium, Aspergillus, Alternaria, Trichoderma and Penicillium strains using the microdilution method by determining the minimal inhibitory and minimal fungicidal concentrations. Selected genera are major problem in food industry since they are the primary pathogens of agronomically important plants, as well as mycotoxin producers. All five IL used in this study showed antifungal effect in the range of 0.002 mol/dm3 to 0.036 mol/dm3. The greatest antifungal activity was observed when strains were treated with (C2OH)C10DMACI and (C2OOEt)C10DMACI IL. Results obtained in this study showed that all examined ILs have the potential to be used as effective antifungal agents

Ectomycorrhizal fungi modulate biochemical response against powdery mildew disease in Quercus robur L.

In light of climate change, pedunculate oak (Q. robur L.) was marked as the most threatened European tree species. Pedunculate oak is particularly jeopardized by powdery mildew disease caused by Erysiphe alphitoides. We hypothesized that priming of this tree species with ectomycorrhizal fungi could mitigate biotic stress and produce bioprotective properties against the disease. In this study, we have compared oaks’ foliar physiological and biochemical responses upon infection with E. alphitoides in the presence and absence of ectomycorrhizal fungi (ECM). The main aim of this study was to inspect how ECM modulate an oak’s biochemical response to infection with E. alphitoides, particularly at the level of the accumulation of the main polyamines (putrescine, spermidine, and spermine), soluble osmolytes (proline and glycine betaine), and phenolics (total phenolic content, flavonoids, and condensed tannins). A polyamine quantification was performed after derivatization by using high-performance liquid chromatography (HLPC) coupled with fluorescent detection. Oak seedlings inoculated with ECM fungi exhibited significantly higher levels of putrescine, spermine, and proline compared to non-inoculated seedlings, indicating the priming properties of the ECM. E. alphitoides caused an increase in individual and total polyamine content and lipid peroxidation in oak leaves regardless of the effect of ECM, while causing a decrease in physiological and antioxidative parameters and water use efficiency (WUE). Common biochemical parameters may contribute to understanding the underpinning plant defense mechanisms in three-way interactions among plants and pathogenic and ectomycorrhizal fungi and can be used as reliable adaptability descriptors in the context of climate change

rDNA based analysis of autochtonous fungal species from Serbia

Determination of fungal species by traditional morphological approach can often be problematic. In the phylum Basidiomycota, sporocarps of different species can share very similar morphoanatomical characteristics. Using molecular markers and phylogenetic species concept this problem can be reduced. In this study identification of six autochtonous fungal species, collected from several locations in Serbia (Tara, Kopaonik, Stara planina) was done by comparison between morphological and molecular data of fungal species, as well as information obtained from phylogenetic tree. ITS sequences amplified from 11 specimens of two genera of ph. Basidiomycota: Marasmius and Ganoderma, were compared with ITS sequences from database using basic local alignment search tool (BLAST). Phylogenetic tree was constructed using Neighbor joining method based on differences between analyzed ITS sequences. Our results showed that within genera Marasmius and Ganoderma morphological and molecular determinations are usually in accordance, but for proper species delimitation both approaches should be used. [Projekat Ministarstva nauke Republike Srbije, br. III43002 and by the Provincial Secretariat for Science and Technological Development, Vojvodina, Serbia (APV 114-4513592/2013-03: Molecular and phenotypic diversity of taxa of economical and epidemiological importance, and endangered and endemic species in Europe

Do <i>Ganoderma</i> Species Represent Novel Sources of Phenolic Based Antimicrobial Agents?

Ganoderma species have been recognized as potential antimicrobial (AM) agents and have been used in traditional Chinese medicine (TCM) for a long time. The aim of this study is to examine the AM potential of autochthonous Ganoderma species (G. applanatum, G. lucidum, G. pfeifferi and G. resinaceum) from Serbia. The extraction of fungal material was prepared in different solvents (ethanol—EtOH, water—H2O, chloroform—CHCl3). Antibacterial activity (ABA) was determined using disk-diffusion, agar-well diffusion, and micro-dilution method, while for antifungal properties disk-diffusion and pour plate method were applied. Antiviral activity was tested on model DNA virus LK3 and determined by plaque assay. Statistical PCA analysis was applied for detection of correlation effects of phenolics and AM activities, while LC-MS/MS was performed for phenolics quantification. G. resinaceum CHCl3 extract expressed the most potent ABA against P. aeruginosa (MIC = 6.25 mg/mL), probably due to presence of flavonoids and 2,5-dihydroxybenzoic acid. Among H2O extracts, the highest ABA was determined for G. pfeifferi against both E. coli and S. aureus (21 and 19 mm, respectively). EtOH extracts of G. pfeifferi and G. resinaceum were the most effective against A. niger (23.8 and 20.15 mm, respectively), with special impact of phenolic acids and flavonoid isorhamnetin, while C. albicans showed the lowest susceptibility. The most potent antiviral inhibitor was G. lucidum (70.73% growth inhibition) due to the high amount of phenolic acids. To the best of our knowledge, this is the first report of a methodical AM profile of G. pfeifferi and G. resinaceum from the Balkan region including PCA analysis

A Fine-Tuning of the Plant Hormones, Polyamines and Osmolytes by Ectomycorrhizal Fungi Enhances Drought Tolerance in Pedunculate Oak

The drought sensitivity of the pedunculate oak (Quercus robur L.) poses a threat to its survival in light of climate change. Mycorrhizal fungi, which orchestrate biogeochemical cycles and particularly have an impact on the plant’s defense mechanisms and metabolism of carbon, nitrogen, and phosphorus, are among the microbes that play a significant role in the mitigation of the effects of climate change on trees. The study’s main objectives were to determine whether ectomycorrhizal (ECM) fungi alleviate the effects of drought stress in pedunculate oak and to investigate their priming properties. The effects of two levels of drought (mild and severe, corresponding to 60% and 30% of field capacity, respectively) on the biochemical response of pedunculate oak were examined in the presence and absence of ectomycorrhizal fungi. To examine whether the ectomycorrhizal fungi modulate the drought tolerance of pedunculate oak, levels of plant hormones and polyamines were quantified using UPLC-TQS and HPLC-FD techniques in addition to gas exchange measurements and the main osmolyte amounts (glycine betaine-GB and proline-PRO) which were determined spectrophotometrically. Droughts increased the accumulation of osmolytes, such as proline and glycine betaine, as well as higher polyamines (spermidine and spermine) levels and decreased putrescine levels in both, mycorrhized and non-mycorrhized oak seedlings. In addition to amplifying the response of oak to severe drought in terms of inducible proline and abscisic acid (ABA) levels, inoculation with ECM fungi significantly increased the constitutive levels of glycine betaine, spermine, and spermidine regardless of drought stress. This study found that compared to non-mycorrhized oak seedlings, unstressed ECM-inoculated oak seedlings had higher levels of salicylic (SA) and abscisic acid (ABA) but not jasmonic acid (JA), indicating a priming mechanism of ECM is conveyed via these plant hormones. According to a PCA analysis, the effect of drought was linked to the variability of parameters along the PC1 axe, such as osmolytes PRO, GB, polyamines, and plant hormones such as JA, JA-Ile, SAG, and SGE, whereas mycorrhization was more closely associated with the parameters gathered around the PC2 axe (SA, ODPA, ABA, and E). These findings highlight the beneficial function of the ectomycorrhizal fungi, in particular Scleroderma citrinum, in reducing the effects of drought stress in pedunculate oak

Ectomycorrhizal Fungi Modulate Pedunculate Oak’s Heat Stress Responses through the Alternation of Polyamines, Phenolics, and Osmotica Content

The physiological and biochemical responses of pedunculate oaks (Quercus robur L.) to heat stress (HS) and mycorrhization (individually as well in combination) were estimated. One-year-old Q. robur seedlings were grown under controlled conditions in a pot experiment, inoculated with a commercial inoculum of ectomycorrhizal (ECM) fungi, and subjected to 72 h of heat stress (40 °C/30 °C day/night temperature, relative humidity 80%, photoperiod 16/8 h) in a climate chamber, and they were compared with seedlings that were grown at room temperature (RT). An in-depth analysis of certain well-known stress-related metrics such as proline, total phenolics, FRAP, ABTS, non-protein thiols, and lipid peroxidation revealed that mycorrhized oak seedlings were more resistant to heat stress (HS) than non-mycorrhized oaks. Additionally, levels of specific polyamines, total phenolics, flavonoids, and condensed tannins as well as osmotica (proline and glycine betaine) content were measured and compared between four treatments: plants inoculated with ectomycorrhizal fungi exposed to heat stress (ECM-HS) and those grown only at RT (ECM-RT) versus non-mycorrhized controls exposed to heat stress (NM-HS) and those grown only at room temperature (NM-RT). In ectomycorrhiza inoculated oak seedlings, heat stress led to not only a rise in proline, total phenols, FRAP, ABTS, non-protein thiols, and lipid peroxidation but a notable decrease in glycine betaine and flavonoids. Amounts of three main polyamines (putrescine, spermine, and spermidine) were quantified by using high-performance liquid chromatography coupled with fluorescent detection (HPLC/FLD) after derivatization with dansyl-chloride. Heat stress significantly increased putrescine levels in non-mycorrhized oak seedlings but had no effect on spermidine or spermine levels, whereas heat stress significantly increased all inspected polyamine levels in oak seedlings inoculated with ectomycorrhizal inoculum. Spermidine (SPD) and spermine (SPM) contents were significantly higher in ECM-inoculated plants during heat stress (approximately 940 and 630 nmol g−1 DW, respectively), whereas these compounds were present in smaller amounts in non-mycorrhized oak seedlings (between 510 and 550 nmol g−1 DW for Spd and between 350 and 450 nmol g−1 DW for Spm). These findings supported the priming and biofertilizer roles of ectomycorrhizal fungi in the mitigation of heat stress in pedunculate oaks by modification of polyamines, phenolics, and osmotica content