Methods for in vitro evaluating antimicrobial activity: A review

Summary
•Dark septate endophytes (DSE) frequently colonize roots in the natural environment, but the effects of these fungi on plants are obscure, with previous studies indicating negative, neutral or positive effects on plant performance
•In order to reach a consensus for how DSE influence plant performance, meta-analyses were performed on data from 18 research articles, in which plants had been inoculated with DSE in sterile substrates
•Negative effects of DSE on plant performance were not recorded Positive effects were identified on total, shoot and root biomass, and on shoot nitrogen (N) and phosphorus contents, with increases of 26–103% in these parameters for plants inoculated with DSE, relative to uninoculated controls Inoculation increased total, shoot and root biomass by 52–138% when plants had not been supplied with additional inorganic N, or when all, or the majority, of N was supplied in organic form Inoculation with the DSE Phialocephala fortinii was found to increase shoot and root biomass, shoot P concentration and shoot N content by 44–116%, relative to uninoculated controls
•The analyses here suggest that DSE enhance plant performance under controlled conditions, particularly when all, or the majority, of N is available in organic form

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1469-8137.2010.03611.x

A meta-analysis of plant responses to dark septate root endophytes

Rapid colorimetric assay for cell growth and survival in application of immunotoxicology

 Premise of the study: The soil - inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass ( Panicum virgatum) and haricot beans ( Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia.  Methods: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed.  Key results: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1 – 2 d). Plants inoculated with M. robertsii Δ MAD2 (plant adhesin gene) took signifi cantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant.  Conclusions: These results suggest that M. robertsii is not only rhizosphere competent but also displays a benefi cial endophytic association with plant roots that results in the proliferation of root hairs.

The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development.

The National Sciences and Engineering Research Council of Canada and Saint Mary’s University, Halifax, NS, provided financial support for this project

The effects of fungal root endophytes on plant growth: a meta-analysis.

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

Antimicrobial Activity and Biodiversity of Endophytic Fungi in Dendrobium devonianum and Dendrobium thyrsiflorum from Vietman

The seed germination of orchids under natural conditions requires association with mycorrhizal fungi. Dendrobium nobile and Dendrobium chrysanthum are threatened orchid species in China where they are considered medicinal plants. For conservation and application of Dendrobium using symbiosis technology, we isolated culturable endophytic and mycorrhizal fungi colonized in the protocorms and adult roots of two species plants and identified them by morphological and molecular analyses (5.8S and nrLSU). Of the 127 endophytic fungi isolated, 11 Rhizoctonia-like strains were identified as Tulasnellales (three strains from protocorms of D. nobile), Sebacinales (three strains from roots of D. nobile and two strains from protocorms of D. chrysanthum) and Cantharellales (three strains from roots of D. nobile), respectively. In addition, species of Xylaria, Fusarium, Trichoderma, Colletotrichum, Pestalotiopsis, and Phomopsis were the predominant non-mycorrhizal fungi isolated, and their probable ecological roles in the Dendrobium plants are discussed. These fungal resources will be of great importance for the large-scale cultivation of Dendrobium plants using symbiotic germination technology and for the screening of bioactive metabolites from them in the future.

Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of Dendrobium (Orchidaceae).

Drug resistance in bacteria has become a global concern and the search for new antibacterial agents is urgent and ongoing. Endophytes provide an abundant reservoir of bioactive metabolites for medicinal exploitation, and an increasing number of novel compounds are being isolated from endophytic fungi. Ophiopogon japonicus, containing compounds with antibacterial activity, is a traditional Chinese medicinal plant used for eliminating phlegm, relieving coughs, latent heat in the lungs, and alleviating diabetes mellitus. We investigated the antimicrobial activities of 30 strains of O. japonicus. Fungal endophytes were isolated from roots and stems of O. japonicus collected from Chongqing City, southwestern China. Mycelial extracts (MC) and fermentation broth (FB) were tested for antimicrobial activity using peptide deformylase (PDF) inhibition fluorescence assays and MTT cell proliferation assays. A total of 30 endophytic strains were isolated from O. japonicus; 22 from roots and eight from stems. 53.33% of the mycelial extracts (MC) and 33.33% of the fermentation broths (FB) displayed potent inhibition of PDF. 80% of MC and 33.33% of FB significantly inhibited Staphylococcus aureus. 70% of MC and 36.67% of FB showed strong activities against Cryptococcus neoformans. None showed influence on Escherichia coli. The secondary metabolites of endophytic fungi from O. japonicus are potential antimicrobial agents.

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Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae)

Dendrobium spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several Dendrobium species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of Dendrobium (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven Dendrobium species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each Dendrobium species were greatly different, though common xylariaceous fungi were found in several Dendrobium species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery.

/pdf/diversity-and-taxonomy-of-endophytic-xylariaceous-fungi-from-39r58d19sy.pdf

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

A dark-septate endophytic (DSE) fungus EF-37 was isolated from the roots of Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant. The molecular identification of the fungus was based on internal transcribed spacer regions and the result showed that EF-37 was congeneric to Mycocentrospora. This study was conducted to clarify the influence of the root endophyte EF-37 on the host plant S. involucrata using material grown in a sterile culture bottle. After cultivation for 40 days, fungal hyphae were found to be branching repeatedly and forming “hyphae nets” in the epidermal layers. Significant differences were detected between the study groups in plant dry weight, plant height, root dry weight, shoot dry weight, and the number of hair root tips. There was a positive effect of endophyte EF-37 on plant root development, with results showing that cortical cells dissolved and formed aerate structures. There was a positive effect of endophyte EF-37 on plant growth, but chlorophyll fluorescence analysis showed that there were no significant differences between the study groups. In addition, analysis of the chemical composition of seedlings showed that the level of rutin was higher in plants cultivated with the EF-37 fungus compared to the controls. This study helps to establish a basis for germplasm conservation and for further investigation of the interaction between dark-septate fungi and this alpine plant.

Shun-Xing Guo

Papers

Antimicrobial Activity and Biodiversity of Endophytic Fungi in Dendrobium devonianum and Dendrobium thyrsiflorum from Vietman

Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of Dendrobium (Orchidaceae).

Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae)

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

The promoting role of an isolate of dark-septate fungus on its host plant Saussurea involucrata Kar. et Kir.