Isolation Purification and Characterization of Antimicrobial Peptides from <Emphasis Type="Italic">Cuminum cyminum</Emphasis> L. Seeds

In this work, antimicrobial peptides from Cuminum cyminum L. seeds were isolated and purified for the first time by 50% ethanol extraction, C18 reverse phase column chromatography and ion exchange chromatography for separation different peptides fraction. Then isolated fractions were characterized by Gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography and the peptides components and molecular weights were determined by liquid chromatography and mass spectrometry. The extracts were tested against some strains of bacteria (E. coli and Staphylococcus aureus) and one strain of fungi (Candida albicans) using well diffusion and broth dilution assays. The extracts from C. cyminum L. seeds demonstrated a high degree of activity (some antibacterial effect) against the bacteria strains and аntifungal activity against the Candida albicans. However, the study indicates that the crude peptide extracts from C. cyminum L. seeds have promising antimicrobial and antioxidant activities that can be harnessed as leads for potential bioactive compounds.     

In silico design of polycationic antimicrobial peptides active against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Antimicrobial peptides (AMPs) have the potential to become valuable antimicrobial drugs in the coming years, since they offer wide spectrum of action, rapid bactericidal activity, and low probability for resistance development in comparison with traditional antibiotics. The search and improvement of methodologies for discovering new AMPs to treat resistant bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are needed for further development of antimicrobial products. In this work, the software Peptide ID 1.0^® was used to find new antimicrobial peptide candidates encrypted in proteins, considering the physicochemical parameters characteristics of AMPs such as positive net charge, hydrophobicity, and sequence length, among others. From the selected protein fragments, new AMPs were designed after conservative and semi-conservative modifications and amidation of the C-terminal region. In vitro studies of the antimicrobial activity of the newly designed peptides showed that two peptides, P3-B and P3-C, were active against P. aeruginosa Escherichia coli and A. baumannii with low minimum inhibitory concentrations. Peptide P3-C was also active against K. pneumoniae and S. aureus. Furthermore, bactericidal activity and information on the possible mechanisms of action are described according to the scanning electron microscopy studies.     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.     

Identification and Characterization of Novel Antimicrobial Peptide from <Emphasis Type="Italic">Hippocampus comes</Emphasis> by In Silico and Experimental Studies

Antimicrobial peptides (AMPs) have attracted attentions as a novel antimicrobial agent because of their unique activity against microbes. In the present study, we described a new, previously unreported AMP, moronecidin-like peptide, from Hippocampus comes and compared its antimicrobial activity with moronecidin from hybrid striped bass. Antibacterial assay indicated that gram-positive bacteria were more sensitive to moronecidin and moronecidin-like compared with gram-negative bacteria. Furthermore, both AMPs were found to exhibit effective antifungal activity. Comparative analysis of the antimicrobial activity revealed that moronecidin-like peptide has higher activity against Acinetobacter baumannii and Staphylococcus epidermidis relative to moronecidin. Both moronecidin-like and moronecidin peptides retained their antibacterial activity in physiological pH and salt concentration. The time-killing assay showed that the AMPs completely killed A. baumannii and S. epidermidis isolates after 1 and 5 h at five- and tenfold above their corresponding MICs, respectively. Anti-biofilm assay demonstrated that peptides were able to inhibit 50% of biofilm formation at sub-MIC of 1/8 MIC. Furthermore, moronecidin-like significantly inhibited biofilm formation more than moronecidin at 1/16 MIC. Collectively, our results revealed that antimicrobial and anti-biofilm activities of moronecidin-like are comparable to moronecidin. In addition, the hemolytic and cytotoxic activities of moronecidin-like were lower than those of moronecidin, suggesting it as a potential novel therapeutic agent, and a template to design new therapeutic AMPs.     

Antimicrobial properties and death-inducing mechanisms of saccharomycin,a biocide secreted by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.     

Effect of salt stress on the antimicrobial activity of <Emphasis Type="Italic">Ruta chalepensis</Emphasis> essential oils

In the current investigation, the biological activities of essential oils obtained from organs of Ruta chalepensis plants grown under salt stress (0, 50 and 100 mM NaCl) were analyzed. Their chemical composition was often investigated by GC/FID and GC–MS and the antimicrobial activities towards eight bacteria (Salmonella All, Salmonella K, Escherichia coli 45AG, Escherichia coli 45AI, Staphylococcus aureus 9402, Staphylococcus aureus 02B145, Listeria 477 and Pseudomonas aeruginosa ATCC 10145) and five fungi strains (Aspergillus, Saccharomycee crvisiale, Streptomyces griseus, Fusarium solani and Penicillium thomii) were studied. Results revealed that salt increased essential oil production in leaves at 50 and 100 mM NaCl. A total of 20 compounds were identified in leaves, undecan-2-one, nonan-2-one and geijerene being the dominant ones. In stems, 21 compounds were found; they were dominated by decan-2-one, geijerene, nonan-2-one and undecan-2-one. In contrast, roots exhibited a large variation with 25 volatile compounds and octyl acetate, methyl decanoate, phytyl acetate were the major ones. Salt stress induced significant antibacterial activity changes, mainly in leaves and stems. In leaves, the minimum inhibitory and bactericidal concentration decreased at 100 mM NaCl against Listeria 477, the two strains of E. coli (45AG and 45AI) and P. aeruginosa but it increased versus other bacteria. In stems, salt increased oil antibacterial activity against all strains except P. aeruginosa ATCC 10145. Root oil showed the least antibacterial activity under saline conditions versus Listeria 477 and P. aeruginosa ATCC 10145. As regards antifungal activity, NaCl reduced the antifungal activity of essential oils against the majority of fungi strains.     

Endophytism and bioactivity of endophytic fungi isolated from <Emphasis Type="Italic">Combretum lanceolatum</Emphasis> Pohl ex Eichler

We report the isolation and identification of endophytic fungi from Combretum lanceolatum Pohl ex Eichler. Further, we evaluated the relationships of fungi with the host plant and tested bioactivities of isolates. The fungi were isolated from disinfected root fragments and plated onto potato dextrose agar. Root pieces were also used to quantify fungal structures associated with the roots. Identification of fungi was carried out by characterization of morphological features and sequencing of the ITS region. Endophytism was confirmed by inoculation of endophyte-free seedlings followed by microscopic examination. The extract was obtained by maceration of the mycelium in ethyl acetate for antioxidant and antimicrobial evaluations. A total of 112 strains belonging to nine different species were isolated, the major classes were Dothideomycetes and Sordariomycetes. C. lanceolatum is colonized by dark septate endophytes (DSE), evidenced by the presence of microsclerotia and melanized hyphae. There is also co-colonization with mycorrhizal fungi in the same root fragments. Seedling inoculation experiments revealed that C. perangustum-95C and M. phaseolina-46C showed association with the seedlings of C. lanceolatum and differentiated microsclerotia and dark septate hyphae, indicating that these species are DSE. In the antimicrobial test, the D. phaseolorum-92C extract had the highest zones of inhibition against S. aureus and E. coli, respectively. The results showed that 100 % of the extracts have antioxidant activity ranging from low to moderate. All endophyte species had antioxidant and antimicrobial activities that were directly proportional to the dose-responses. Future research will involve chemical characterization and structural elucidation of bioactive compounds.     

Characterization of Antimicrobial Peptides Isolated from the Processing by-Products of African Catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis>

Antimicrobial peptides (AMPs) as components of innate immunity system have been isolated from fish and other species. In this study, the crude proteins extracted with gradient ammonium sulfate precipitation technique from the processing by-products of African catfish Clarias gariepinus (C. gariepinus) were purified by size-exclusion chromatography and all the four obtained fractions, Clarias antimicrobial peptides I(CAP-I), CAP-II, CAP-III and CAP-IV, showed antimicrobial activity. Among of these fractions, CAP-IV showed the highest antimicrobial activity against Staphylococcus aureus, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli by agar diffusion plate test and the diameter of inhibition zone was 8.34, 9.27, 6.76, 6.13 mm, respectively. The molecular weight of main peptides of CAP-IV was around 4.1 KD by SDS-PAGE analysis. CAP-IV showed antimicrobial activity against both gram-negative and gram-positive bacterial pathogens at minimum inhibitory concentrations (MICs) ranging from 105 to 420 μg/mL. The antimicrobial activity of CAP-IV was stable at wide pH range, 3–11 and was also heat-stable when temperature was below 80 °C. Freeze-thawing treatment also only had slight effects on the antimicrobial activity of CAP-IV. Besides, CAP-IV was not sensitive to the hydrolysis by pepsin and trypsin, except for protease K. These results suggest that CAP-IV isolated from C. gariepinus is potential to be developed as a new antimicrobial peptide and may partially explain the high disease resistance of African catfish C. gariepinus.     

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.     

Molecular Cloning of Cathelicidin-like cDNA from <Emphasis Type="Italic">Andrias davidianus</Emphasis>

Antimicrobial peptides (AMPs) as part of host defense systems has been widely recognized in most organisms. Cathelicidin is an important family of AMPs acting as multifunctional effector molecules in innate immunity and exists in organisms with cathelicidin-like precursor. Andrias davidianus (A. davidianus) is a unique species in China and the biggest amphibians in the world. With the rapid growth of A. davidianus aquaculture, pathogens of bacteria, virus and fungus were reported, however little is known about antimicrobial peptides derived from A. davidianus. To investigate antimicrobial peptides of cathelicidin-like in A. davidianus, cathelicidin-like precursor gene cloning and bioinformatic analysis was carried out. The results showed that 1106 bp full-length cDNA of cathelicidin-like precursor was obtained, which was including a 35 bp 5' terminal UTR, a 546 bp open reading frame (ORF) and a 525 bp 5' terminal UTR. The cathelicidin-like precursor amino acid (AA) sequence of A. davidianus comprised N-terminal signal peptide (21 AA), highly conserved cathelin domain and C-terminal mature peptide. The cathelicidin-like precursor gene nucleotide sequence showed low identify with other cathelicidin-like sequences, while AA sequence displayed relatively higher similarity with cathelicidin-like isolated from other species. Phylogenetic tree indicated cathelicidinlike precursor of A. davidianus was firstly clade with Tylototrition verrucosus, which also belonged to Caudata, Amphibian. The precursor gene expression was detected by RT-qPCR. The result displayed this gene was abundant expression in A. davidianus skin. According the specificity proteases cleavage and characteristic of cathelicidin, five putative mature cathelicidin were predicted. This study confirms the presence of cathelicidin in A. davidianus. Their results not only reveal innate immune system of A. davidianus but also enlarge the AMP knowledge of urodele amphibians.     

Phylogeny,antimicrobial, antioxidant and enzyme-producing potential of fungal endophytes found in <Emphasis Type="Italic">Viola odorata</Emphasis>

Viola odorata, a medicinal plant, is traditionally used to treat common cold, congestion and cough. Given its medicinal properties and occurrence in the northwestern Himalayas, we isolated and characterized endophytic fungi from this plant morphologically, microscopically and by internal transcribed spacer-based rDNA sequencing. In total, we isolated 27 morphotypes of endophytes belonging to phyla Ascomycota and Basidiomycota. The roots showed the highest diversity of endophyte as well as fungal dominance, followed by leaves and leaf nodes. The fungal extract of VOR16 (Fusarium oxysporum) displayed potent antimicrobial activity against Salmonella typhimurium, Klebsiella pneumoniae and Escherichia coli, with a minimum inhibitory concentration of 0.78, 0.78 and 1.56 μg/mL, respectively, while fungal extract VOLF4 (Aspergillus sp.) exhibited promising antioxidant activity (IC₅₀ of 17.4 μg/mL). To identify the components responsible for various bioactivities, we analyzed the content of penicillin G in the extract of bioactive endophytes. The results suggested that the expression of penicillin G under the fermentation conditions applied was too low to display antimicrobial effects. Thus, the activity may be contributed by a different, novel secondary metabolite. The antioxidant activity of VOLF4 may be attributed to its high content of flavonoids. Of the endophytic fungi assessed, 27% were found to be enzyme producers. The highest zone of clearance was observed in VOLN5 (Colletotrichum siamense) for protease production. Only VOR5 (Fusarium nematophilum) was found to be a producer of cellulase, glutenase, amylase and protease. In summary, this is the first report of the isolation of endophytes, namely Fusarium nematophilum, Colletotrichum trifolii, C. destructivum, C. siamense and Peniophora sp., from V. odorata and their bioactive and enzyme-producing potential.     

In vitro assessment of antimicrobial activity of aqueous and alcoholic extracts of moss <Emphasis Type="Italic">Atrichum undulatum</Emphasis> (Hedw.) P. Beauv.

Bryophytes, the shade loving plants, have tremendous medicinal properties. The aqueous and alcoholic extracts of Atrichum undulatum (Hedw.) P. Beauv. were analysed for antimicrobial properties against the fungi Aspergillus fumigatus and Fusarium oxysporum and the bacteria Escherichia coli, Bacillus mycoides, Proteus mirabilis, Staphylococcus aureus and Salmonella typhi. The study is an attempt to investigate the medicinal properties of Atrichum undulatum (Hedw.) P. Beauv. using disc-diffusion method. No inhibition was observed against A. fumigatus and P. mirabilis. For bacteria S. typhi and E. coli (20 and 15 mm), aqueous and alcoholic extracts of Atrichum showed significant inhibition. However, alcoholic extract was found remarkably effective against bacteria rather than aqueous extract.     

Genes encoding hevein-like antimicrobial peptides WAMPs in the species of the genus <Emphasis Type="Italic">Aegilops</Emphasis> L.

Earlier, in the wheat Triticum kiharae Dorof. et Migusch., a new family of genes coding for the hevein-like antimicrobial peptides WAMPs, involved in the protection of wheat plants against pathogens, was discovered. In the present study, we examined the wamp homologs in plants belonging to ten di-, tetra-, and hexaploid species of the genus Aegilops L., among which there are donors of polyploid wheat genomes, as well as of the resistance genes to the most important wheat pathogens. Using PCR amplification with genomic DNA as a template and primers specific to the sequences of the wheat wamp genes, for the first time, nucleotide sequences of the protein-coding regions of wamp homologs were determined in the species of the genus Aegilops L. The wamp homologs were found in all species studied. It was demonstrated that the WAMP peptide precursors encoded by them differed in single nucleotide substitutions, as well as deletions/insertions of amino acid sequences. The most conserved region of the precursor is the mature peptide region, where, in addition to the variable position 34, deletions of amino acid sequences were found in a number of peptides. To elucidate the role of deletions in the antimicrobial activity of WAMPs, a recombinant WAMP-3 peptide with a deletion in the N-terminal region was produced by expression in E. coli cells, and it was shown that antimicrobial activity of the peptide was preserved. It was demonstrated that all the discovered wamp genes were expressed in seedlings of the studied Aegilops species. The results shed new light on the structural diversity of genes encoding the hevein-like antimicrobial peptides WAMPs.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Antimicrobial Peptide from <Emphasis Type="Italic">Bacillus</Emphasis> Strain K1R Exhibits Ameliorative Potential Against Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> Group of Organisms

Microorganisms secrete antimicrobial peptides (AMPs) which are part of the innate immune system and rapidly increase in concentration in the host upon challenge by pathogens, which they produce themselves. Kimchi, a traditional Korean food fermented by Bacillus organisms, is found to be ideal for AMP production. Our aim was to investigate the therapeutic potential of antimicrobial substances produced by Bacillus species. Peptide K1R was subjected to fermentation in a culture media containing carbon and nitrogen sources and metal ions. A protein band around 4.6 kDa was detected in tricine-SDS-PAGE and confirmed by in situ inhibitory activity of the gel. Peptide K1R was stable over a broad range of pH (6.5–9), thermo tolerant up to 60?°C and showed unaltered activity at low temperatures (0–4?°C). The complete amino acid sequence of peptide K1R was AVQGTLEDALNLSKGALNQVQKAIQNGDXLTVXGXLGTIXLAVSX. The antagonistic effect of peptide K1R against multiple drug resistant (MDR) pathogens such as Salmonella Typhimurium and Enterococcus sp. verified its potential application in treating MDR cases. The antioxidant activity of peptide K1R was also comparable to that of standard ascorbic acid.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Effect of foliar application of salicylic acid,hydrogen peroxide and a xyloglucan oligosaccharide on capsiate content and gene expression associated with capsinoids synthesis in <Emphasis Type="Italic">Capsicum annuum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>

Capsinoids are non-pungent analogues of capsaicinoids in pepper (Capsicum spp). The absence of pungency, in addition to their biological activities similar to that of capsaicinoids such as anti-inflammatory, antimicrobial, and antioxidant properties, makes capsinoids an excellent option for increasing use in human and animal nutrition, as well as health and pharmaceutical industries. There are only few sources of pepper producing capsinoids, and one of them (accession 509–45-1), Capsicum annuum L., is a potential source for increasing capsinoids content using strategies as controlled elicitation during plant production in the greenhouse. In this research we evaluated the effect of weekly and one-day-before-harvest foliar applications of hydrogen peroxide, salicylic acid and a xyloglucan oligosaccharide on the concentration of capsiate in fruits of this pepper accession, as well as the gene expression of phenylalanine ammonia-lyase (pal), putative aminotransferase (pamt), capsaicin synthase (at3) and β-keto acyl synthase (kas). Results showed that the two tested concentrations of H₂O₂ significantly increased capsiate content and gene expression associated with capsaicinoids (pamt, at3 and kas) and the phenylpropanoids (pal) pathways. Plant yield was not affected using this induction strategy. Our results indicated that the pre-harvest and weekly application of hydrogen peroxide and xyloglucan oligosaccharide improved production of capsiate in C. annuum L.     

Fungi from leaves of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

In spite of the self-cleaning property of its leaves called the lotus effect, leaves of lotus (Nelumbo nucifera) provide a habitat for an unknown fungal diversity. The aim of this study was to detect and identify fungi from leaves of N. nucifera, including ectophytic, parasitic and endophytic fungi, in Taiwan using different collection strategies, as well as morphological and diverse molecular markers established in the different systematic groups of fungi. Among ectophytic and parasitic fungi, a new species of Dissoconium and of Pseudocercospora are described, respectively. Phyllosticta nelumbonis Sawada is transferred to Diaporthe. Among plant parasitic fungi, Erysiphe takamatsui and Ps. nymphaeacea are recorded in Taiwan for the first time. Euryale is recorded as a new host genus for Ps. nymphaeacea. The basidiomycetous yeast Fereydounia khargensis is recorded for the first time from living plants and in East Asia. Endophytic fungi from lotus were studied for the first time. From 1002 plant segments, 476 endophytic isolates were produced in culture, comprising 33 typical terrestrial species mainly belonging to the genera Colletotrichum (mainly C. siamense), Diaporthe (D. tulliensis and D. ueckerae) and Fusarium (F. solani species 6, hitherto known from clinical samples), as well as to Xylariaceae, but no Ingoldian fungi. Most isolates were from leaf laminas (71%) compared to those from petioles (29%). From this observation, we conclude that the fungi of the aquatic lotus plant appear to have terrestrial origin and, after dispersal by wind and in spite of the lotus effect, may enter the plant from the lamina. Only three species isolated as endophytes were also found as ectophytic or parasitic fungi.