Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.     

The Endophytic Mycoflora of Bark,Leaf, and Stem Tissues of <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss (Neem) from Varanasi (India)

A systematic study was made of the endophytes of Azadirachta indica A. Juss (the neem tree) growing in several of its natural habitats in India. A total of 233 isolates of endophytic fungi representing 18 fungal taxa were obtained from segments of bark, stem, and leaves of this tree. Hyphomycetes (62.2%) were the most prevalent followed by the Coelomycetes (27.4%) and Mycelia Sterilia (7.7%). As mathematically determined, the maximum species richness and frequency of colonization of endophytes appeared in leaf segments rather than stem and bark tissues from each location. Endophytic colonization frequency was also greater in leaves (45.5%) than bark (31.5%). The leaf samples from all locations were nearly constant in their endophytic composition, whereas bark samples showed maximum diversity at different locations. Inter-site comparisons for endophytic diversity, however, were not significantly different with Loc1 and Loc2 having a maximum of 66.67% J _c. The smallest similarity was between Loc2 and Loc3 of 54.17% J _c. The dominant endophytic fungi isolated were Phomopsis oblonga, Cladosporium cladosporioides, Pestalotiopsis sp., Trichoderma sp, and Aspergillus sp. Genera such as Periconia, Stenella, and Drechslera are reported here for the first time as endophytes from this host plant. This report illustrates the value of sampling different tissues of a given plant in several locations to obtain the greatest species diversity of endophytes. The rich and sizeable collection of endophytic fungi from this specific plant may represent a unique source of one or more of the interesting and useful bioactive compounds normally associated with A. indica such as the azadirachtins and related tetranortriterpenoids.     

Endophytic fungi from the medicinal plant <Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham. and their antimicrobial activity

Endophytic fungi were isolated from healthy tissues of Lippia sidoides Cham. (Verbenaceae), a medicinal plant used as an antiseptic in the northeast of Brazil. From 480 fragments of leaves (240) and stems (240), a total of 203 endophytic fungi were isolated, representing 14 species belonging to the groups Ascomycota, Coelomycetes and Hyphomycetes. Endophytic colonization was greater in leaves (50.4%) than in stems (35.4%). Colletotrichum gloeosporiodes had the maximum colonization frequency (12.3%), followed by Alternaria alternata (7.08%), Guignardia bidwelli (6.87%) and Phomopsis archeri (5.41%). Some species showed specificity for the host tissue: Curvularia pallescens, Dreschlera dematioidea, G. bidwellii, Microascus desmosporum, Peacilomyces variotti, Periconia byssoides and Ulocladium oudemansii were only isolated from leaves while Fusarium lateritium and Phoma tracheiphila were only isolated from stems. Through a preliminary screening and fermentation assay, 16 isolates where found to produce antimicrobial metabolites against bacterial and fungi. The diversity and role of endophytes in medicinal plants is briefly discussed. In conclusion, endophytic fungi from L. sidoides have pharmaceutical potential and can be seen as an attractive source of biologically active compounds.     

Antimicrobial potentials of endophytic fungi residing in <Emphasis Type="Italic">Quercus variabilis</Emphasis> and brefeldin A obtained from <Emphasis Type="Italic">Cladosporium</Emphasis> sp.

Among 67 endophytic fungi isolated from Quercus variabilis, 53.7% of endophytic fungal fermentation broths displayed growth inhibition on at least one test microorganism, such as pathogenic fungi (Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis) and bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens). Moreover, 19.4% of strains showed a broader antimicrobial spectrum, such as Aspergillus sp., Penicillium sp., Alternaria sp., 20.9% of strains showed strong inhibition (+++) to pathogenic bacteria, while only 7.5% displayed that to test fungi. The most active antifungal strain I(R)9-2, Cladosporium sp. was selected and fermented. From the broth, a secondary metabolite, brefeldin A was obtained. This is the first report on the antimicrobial potentials of endophytic fungi residing in Q. variabilis and isolation of brefeldin A produced by Cladosporium sp.     

Isolation and characterization of endophytic taxol-producing fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

This study investigated the endophytic fungi diversity of Taxus chinensis and screened the taxol-producing fungi in the host. A total of 115 endophytic fungi isolates obtained from bark segments of T. chinensis were grouped into 23 genera based on the morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in T. chinensis are diverse and abundant. Diaporthe, Phomopsis (anamorph of Diaporthe), Acremonium, and Pezicula were the dominant genera, whereas the remaining genera were infrequent groups. The 13 representative species of the distinct genera were capable of producing taxol verified by reverse-phase high performance liquid chromatography (HPLC). Among the taxol-producing fungi, the yield of taxol produced by the Metarhizium anisopliae, H-27 was 846.1 μg l⁻¹ in reformative potato dextrose liquid medium, and the fungal taxol was further validated by mass spectrometry (MS). The taxol-producing fungi (92.3%) were infrequent communities, suggesting that infrequent fungi associated with T. chinensis might be a fascinating reservoir of taxol-generating fungi.     

Fungal endophytes associated with the mistletoe <Emphasis Type="Italic">Phoradendron perrottettii</Emphasis> and its host tree <Emphasis Type="Italic">Tapirira guianensis</Emphasis>

The endophytic mycobiota of leaves and stems of the mistletoe Phoradendron perrottettii and its host tree Tapirira guianensis, two physiologically connected plant species of the Brazilian savannah in southeastern Brazil, were investigated to evaluate host and organ recurrence among endophytes. Leaves and stems of P. perrottettii and leaves of T. guianensis were sampled in the dry and wet season. Stems of T. guianensis were also sampled in the wet season. Endophytes were isolated by an adapted trituration and particle filtration protocol. A total of 1,615 isolates representing 99 species and 20 sterile morphotypes were recovered; 64 morphospecies occurred as singletons. The number of isolates and species was higher in the wet season. Leaves of P. perrottettii were less densely colonized than other organs studied, but were the most species-rich. Conversely, stems of T. guianensis yielded more isolates but were less species-rich. Both plants were found to harbor similar but distinguishable endophytic assemblages. The Jaccard’s index of similarity between the fungal assemblages of both plants was 0.82, higher than found for other plants in similar habitats. The fungal species composition seemed to be influenced by the collection season and organ type, as demonstrated by multivariate correspondence analysis. Paraconiothyrium brasiliense, P. sporulosum and Verticillium leptobactrum were the dominant species in P. perrottettii. In leaves of T. guianensis, Pseudocercospora sp., Phomopsis sp. 1 and Lecanicillium psalliotae were the most frequent, while Stagonospora sp. 1 and Phomopsis sp. 1 were the dominant endophytes in its stems. The results indicated that some of the dominant endophytic taxa isolated in this study colonize different hosts and plant organs while others seem to exhibit a high degree of host or organ recurrence. This study represents the first evaluation of diversity of fungal endophytes in natural vegetation of the Brazilian savannah and contributes information about the distribution and possible specificity of endophytes in tropical dicotyledoneous plants.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Molecular analysis of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within β-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.     

Latent infections of <Emphasis Type="Italic">Fomes fomentarius</Emphasis> in the xylem of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>)

Fungal colonisation originating from endophytic thalli in wood of healthy European beech trees (Fagus sylvatica) was studied. Fungi were isolated from wood immediately after felling and after incubation for 8, 16 and 24 weeks under two different drying regimes. Two media were used to isolate fungi: malt extract agar with and without thiabendazol. Thiabendazol was added to inhibit non-basidiomycetes. The two drying regimes had no influence on the species composition of the recovered mycobiota and the frequency of isolation of these species. Mycelia of basidiomycetes except Coniophora puteana emerged only from wood samples inoculated onto malt extract agar containing thiabendazol. Only a few isolates were obtained from freshly cut wood, but a great number of isolates was recovered already after eight weeks of wood incubation. Four taxa accounted for 88 % of the total number of isolates: Hypoxylon fragiforme, Trichoderma spp., and the basidio-mycetes Coniophora puteana and Fomes fomentarius. The latter had not been considered an endophyte before. The isolates of F. fomentarius were made exclusively from the stem and some large diameter branches, which are the locations of its basidiocarps on dying trees. Every isolate of F. fomentarius was genetically different as revealed by tests for somatic compatibility. Advantages of the presumed endophytic strategy of F. fomentarius are discussed. Microscopic investigations showed a great number of hyphae within the cell lumina of vessels and a distinct wood degradation already after eight weeks of wood incubation.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.     

Diversity and pathogenicity of ophiostomatoid fungi associated with <Emphasis Type="Italic">Tetropium</Emphasis> species colonizing <Emphasis Type="Italic">Picea abies</Emphasis> in Poland

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.     

Plant growth promoting potential of bacterial endophytes in novel association with <Emphasis Type="Italic">Olea ferruginea</Emphasis> and <Emphasis Type="Italic">Withania coagulans</Emphasis>

Microbially unexplored medicinal plants can have a genetically diverse microbial population with multi-functional plant growth promoting traits. In this aspect, 75 endophytic bacterial isolates with plant growth promoting traits were isolated from Withania coagulans Dunal and Olea ferruginea Royal. Many of these bacteria were able to solubilize phosphate, produce indole-3-acetic acid, ammonia as well as hydrogen cyanide, synthesize extracellular enzymes and show antagonistic activities against plant pathogenic fungi under in vitro conditions. These isolates were also characterized by morphological and biochemical analysis. Furthermore, four representative isolates with pronounced plant growth promoting activities were identified as Enterobacter cloacae, Enterobacter dissolvens, Enterobacter hormaechei and Cronobacter sakazakii by 16S rDNA sequencing analysis. This work for the first time, reported the isolation of endophytic bacteria, the novel association form selected plants, Withania coagulans and Olea ferruginea. The explored endophytes might have great potential in the field of biocontrol and plant growth promoting for sustainable agricultural practices.     

Screening of taxol-producing endophytic fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis> var. <Emphasis Type="Italic">mairei</Emphasis>

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography, and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compound-producing endophytic fungi, which can improve prominently screening efficiency. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 490–494. The text was submitted by the authors in English.     

Biologically Active Endophytic Streptomycetes from <Emphasis Type="Italic">Nothofagus</Emphasis> spp. and Other Plants in Patagonia

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.