Differences among Endophytic Fungal Communities Isolated from the Roots of Cephalanthera longibracteata Collected from Different Sites in Korea

Orchidaceous plants have symbiotic relationships with endophytic fungi, including mycorrhizal fungi, which play important roles in the seed germination and growth of the host plants. In this study, endophytic fungal communities isolated from the roots of Cephalanthera longibracteata collected from three different sites in Korea were analyzed, and it was determined whether fungal communities were preferentially correlated with the sites. The fungal isolates were identified by sequence analysis of the internal transcribed spacer regions of rDNA. In total, 30 species of endophytic fungi, including two species of mycorrhizal fungi belonging to the genus Tulasnella, were identified. Leptodontidium orchidicola showed the highest frequency and was isolated from all root samples. Species diversity and richness were not significantly different among sites. However, the community structure of the endophytic fungi significantly differed among sites, suggesting that the site characteristics affected the community composition of the endophytic fungi colonizing the roots of C. longibracteata. Our findings will aid in developing methods involving the use of symbiotic fungi for orchid conservation and restoration in native habitats.

Isolation and identification of fungal endophytes from Ophiopogon japonicus and their antimicrobial activities / 中草药

Objective: To explore the resource diversity and specificity of fungal endophytes from the Ophiopogon japonicas and their antimicrobial activities. Methods: Fungal endophytes were isolated from roots and leaves of wild and cultivated O. japonicas by tissue block method. Those fungal endophytes were identified by morphological methods and phylogenetic analysis of ITS sequences. And the antimicrobial activities of those fungal endophytes were estimated by agar disk containing mycelium method. Results: Twenty-seven fungal endophytes strains were isolated from O. japonicas. There was more fungal endophytes diversity in wild plants (22 strains) than cultivated plants (5 strains). Likewise, there was more fungal endophytes diversity in roots of O. japonicas (17 strains) than the leaves (10 strains). And Fusarium and Aspergillus were the dominant genus in O. japonicas. The F. spp. fungal endophytes only isolated from roots while A. spp. from both roots and leaves. In addition, 40% fungal endophytes displayed potent inhibition of Moniliaalbican. Only one or two fungal endophytes strains showed obvious inhibition of Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Conclusion: O. japonicas is rich in fungal endophytes and those fungi distributed with relative specificity characteristics on environment and space. Those fungal endophytes display the antimicrobial activities, especially stain H5, 031p, and H8 with potent inhibition activity are worthy of further research, development, and application.

Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential

Bauhinia forficata is native to South America and used with relative success in the folk medicine in Brazil. The diversity, antibacterial activity, and extracellular hydrolytic enzymes of endophytic fungi associated with this plant were studied. Plant samples, which included leaves, sepals, stems, and seeds, were used. Ninety-five endophytic fungal were isolated (18 from leaves, 22 from sepals, 46 from stems, and nine from seeds), comprising 28 species. The most frequently isolated species were Acremonium curvulum (9.5%), Aspergillus ochraceus (7.37%), Gibberella fujikuroi (10.53%), Myrothecium verrucaria (10.53%) and Trichoderma piluliferum (7.37%). Diversity and species richness were higher in stem tissues, and Sorensen’s index of similarity between the tissues was low. Eleven fungi showed antibacterial activity. Aspergillus ochraceus, Gibberella baccata, Penicillium commune, and P. glabrum were those with the greatest antibacterial activity against Staphylococcus aureus and/or Streptococcus pyogenes. Thirteen species showed proteolytic activity, particularly Phoma putaminum. Fourteen species were cellulase positive, particularly the Penicillium species and Myrmecridium schulzeri. All isolates tested were xylanase positive and 10 showed lipolytic activity, especially Penicillium glabrum. It is clear that the endophytic fungi from B. forficata have potential for the production of bioactive compounds and may be a source of new therapeutic agents for the effective treatment of diseases in humans, other animals, and plants. To our knowledge, this is the first study of endophytic fungi from different tissues of B. forficata and their biotechnological potential.

Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes

In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications.

Diversity and Plant Growth Promoting Capacity of Endophytic Fungi Associated with Halophytic Plants from the West Coast of Korea

Five halophytic plant species, Suaeda maritima, Limonium tetragonum, Suaeda australis, Phragmites australis, and Suaeda glauca Bunge, which are native to the Muan salt marsh of South Korea, were examined for fungal endophytes by sequencing the internal transcribed spacer (ITS) region containing ITS1, 5.8S rRNA, and ITS2. In total, 160 endophytic fungal strains were isolated and identified from the roots of the 5 plant species. Taxonomically, all 160 strains belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The most dominant genus was Fusarium, followed by the genera Penicillium and Alternaria. Subsequently, using 5 statistical methods, the diversity indices of the endophytes were determined at genus level. Among these halophytic plants, P. australis was found to host the greatest diversity of endophytic fungi. Culture filtrates of endophytic fungi were treated to Waito-C rice seedlings for plant growth-promoting effects. The fungal strain Su-3-4-3 isolated from S. glauca Bunge provide the maximum plant length (20.1 cm) in comparison with wild-type Gibberella fujikuroi (19.6 cm). Consequently, chromatographic analysis of the culture filtrate of Su-3-4-3 showed the presence of physiologically active gibberellins, GA(1) (0.465 ng/mL), GA(3) (1.808 ng/mL) along with other physiologically inactive GA(9) (0.054 ng/mL) and GA(24) (0.044 ng/mL). The fungal isolate Su-3-4-3 was identified as Talaromyces pinophilus.

Antimicrobial and Cytotoxic Activities of Endophytic Fungi Isolated from Artemisia annua L.

This study was aimed to investigate antimicrobial and cytotoxic effects of endophytic fungi isolated from A. annua L. The ethyl acetate extracts were obtained by liquid-liquid partition of fermentation broth of endophytes followed by evaporation. Antimicrobial assay was determined by disc diffusion method. Cytotoxicty assay was conducted against T47D, WiDr and Vero using MTT assay. Data generated were used to determine IC50. While DP2 extract did not show any inhibition against all tested bacteria, DP6 extract at 200 μg inhibited the growth of B. subtilis, P. aeruginosa and S. thypi with the highest inhibition against B. subtilis. Similarly, E2 extract inhibited the growth of B. subtilis, E. coli and S. thypi, with the highest inhibition against B. subtilis. The three extracts (DP2, DP6 and E2) were found to be cytotoxic against T47D and WiDr with E2 extract to be the most cytotoxic. These compounds were more selective against T47D cell line with IC50 of 91.7, 74.8 and 12.0 μg/mL, respectively. The IC50 against Vero cell line are 444.4, 140.3 and 87.7 μg/mL, respectively.

Antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz & Pav

Objective:To investigate antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz&Pav (P. crocatum). Methods:Ethyl acetate extracts were obtained by liquid-liquid partition of fermentation broth of endophytes followed by evaporation. The antimicrobial activities were determined by diffusion techniques followed by thin layer chromatography-bioautography. Cytotoxicity studies were conducted using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Data generated were used to plot a dose-response curve, of which the concentration of extract required to kill 50%of cell population (IC50) was determined. Results: Two endophytes were isolated from leaves and stem of P. crocatum, designated as DS1 and BS1. Ethyl acetate extracts of BS1 was found to inhibit the growth of Bacillus subtilis, Escherichia coli and Staphylococcus aureus at minimum dose of 31.25, 125 and 250 μg, respectively. thin layer chromatography-bioautography of this extract resulted in at least two inhibition zones. BS1 extract also inhibited the growth of WiDr and T47D cell lines with IC50 of 120.38 μg/mL and 37.43 μg/mL, respectively. Conclusions: BS1 extract produced by endophytic fungi of P. crocatum is potential to be developed as source of novel bioactive compounds.

Culturable Fungal Endophytes Isolated from the Roots of Coastal Plants Inhabiting Korean East Coast

Twelve plant species were collected from the east coast of Korea to identify culturable endophytes present in their roots. The fungal internal transcribe spacer (ITS) region (ITS1-5.8SrRNA-ITS2) was used as a DNA barcode for identification of fungi. A total of 194 fungal strains were identified and categorized into 31 genera. The genus Penicillium accounted for the largest number of strains, followed by the genus Aspergillus. Furthermore, using 5 statistical methods, the diversity indices of the fungi were calculated at the genus level. After comprehensive evaluation, the endophytic fungal group from Phragmites australis ranked highest in diversity analyses. Several strains responsible for plant growth and survival (Penicillium citrinum, P. funiculosum, P. janthinellum, P. restrictum, and P. simplicissimum), were also identified. This study provides basic data on the sheds light on the symbiotic relationship between coastal plants and fungi.

Antimicrobial Activity of Crude Extracts of Endophytic Fungi Isolated from Medicinal Plant Trichilia elegans A. Juss.

Endophytes are microorganisms which inhabit inside plants. These microorganisms are identified as being fungi or bacteria and can be useful for prospectionof bioactive compounds that may have medical and pharmaceutical applications.Trichilia elegans (Meliaceae) is a native tree in Brazil. Preparations using leaves, seeds, bark and roots and some members of the Trichilia genus are used in Brazilian popular medicine.The aim of the present work was to investigate biotechnological potential of fungal endophytes (Cordyceps memorabilis, Phomopsis longicolla, Dothideomycete sp. and one non-indentified) isolated from T. elegans, that have been assayed against five pathogenic bacteria. The fungi were incubated in Potato Dextrose and the secondary metabolites was extracted from fermentation medium with ethyl acetate, also was used directly extraction with methanolfrom mycelium. The extraction by ethyl acetate from C. memorabilis inhibited growing of Enterococus hirae, Micrococcus luteus and Escherichia coli. Fungal Phomopsis sp. inhibited M. luteus, E. hirae and Salmonella typhi, Dothideomycetes sp. and G8-25 inhibited M. luteus and E. hirae. Noextract by ethyl acetate inhibited Staphylococcus aureus and also no extract obtained by methanol inhibited the growing of tested bacteria. The present study helped justified the traditional use of T. elegans against human pathogenic bacteria.

Molecular Identification of Endophytic Fungi Isolated from Needle Leaves of Conifers in Bohyeon Mountain, Korea

Fungal endophytes are microfungi that live in plants without causing apparent symptoms of infection. This study was conducted to identify endophytic fungi isolated from leaves of coniferous trees in Bohyeon Mountain of Korea. We collected leaves of two species of coniferous trees, Pinus densiflora and Pinus koraiensis, from 11 sites in the study area. A total 58 isolates were obtained and identified using molecular and morphological characteristics. Four species of endophytic fungi were isolated from P. densiflora: Lophodermium conigenum, Leotiomycetes sp., Septoria pini-thunbergii, and Polyporales sp., while two fungal species were isolated from P. koraiensis: Eurotiomycetes sp. and Rhytismataceae sp. The most frequently isolated species were L. conigenum and S. pini-thunbergii.

Arbuscular mycorrhizal infection in two morphological root types of Araucaria araucana (Molina) K. Koch / Infección por micorrizas arbusculares en dos tipos de raíces de Araucaria araucana (Molina) K. Koch

Araucaria araucana (Molina) K. Koch is a conifer distributed in the Andean-Patagonian forests in the south of Argentina and Chile. The main objective of this work was to relate the different root classes appearing in A. araucana to mycorrhizal behavior. Samples were collected in three different sites in the Lanín National Park (NW Patagonia, Argentina). Two different root classes were present in A. araucana: longitudinal fine roots (LFR) and globular short roots (GSR). Both had extensive mycorrhizal arbuscular symbiosis (AM) and presented abundant hyphae and coils in root cells, a characteristic of the anatomical Paris-type. Dark septate fungal endophytes were also observed. Values of total AM colonization were high, with similar partial AM% values for each root class. Seasonal differences were found for total and partial colonization, with higher values in spring compared to autumn. Regarding the percentage of fungal structures between root classes, values were similar for vesicles and arbuscules, but higher coil percentages were observed in GSR compared to LFR. The percentages of vesicles increased in autumn, whereas the arbuscule percentages increased in spring, coinciding with the plant growth peak. Results show that both root classes of A. araucana in Andean-Patagonian forests are associated with AM fungi, which may have ecological relevance in terms of the importance of this symbiosis, in response to soil nutrient-deficiencies, especially high P-retention.

La conífera Araucaria araucana (Molina) K. Koch se encuentra distribuida en los bosques Andino-Patagónicos de Argentina y Chile. En este trabajo se relacionaron las diferentes clases morfológicas de raíces presentes en la especie con el comportamiento micorrícico. Las muestras fueron tomadas en tres sitios del Parque Nacional Lanín (NO de Patagonia, Argentina). Se observaron dos clases diferentes de raíces: raíces finas longitudinales (RFL) y raíces cortas globulares (RCG). Ambas clases presentaron una importante simbiosis micorrícico-arbuscular (MA), con abundante desarrollo de hifas intracelulares y rulos, característico del tipo anatómico Paris. También se observaron hifas marrones septadas. Los valores totales de colonización MA fueron altos, con porcentajes parciales similares en cada clase radicular. Se encontraron diferencias estacionales en los porcentajes de infección (totales y parciales), con valores mayores en primavera comparados con los de otoño. Los porcentajes de estructuras fúngicas presentes en cada clase radicular fueron similares para vesículas y arbúsculos, pero se observaron mayores valores de rulos en las RCG que en las RFL. Los porcentajes de vesículas aumentaron en otoño, mientras que los de arbúsculos lo hicieron en primavera, en coincidencia con el pico de crecimiento vegetativo de los árboles. Los resultados muestran una fuerte asociación simbiótica MA con A. araucana y su relevancia en términos ecológicos en respuesta a la deficiencia de nutrientes en el suelo, en especial, a la alta retención de fósforo.