Isolation and Identification of Fungal Endophytes Associated with Leaves of Rhizophora mucronata Lamk.

Microorganisms associated with medicinal plants are of great interest as they are the producers of important bioactive compounds effective against common and drug-resistant pathogens. The characterization and biodiversity of fungal endophytes of the Petiveria alliacea plant and their antimicrobial production potential are of great interest as they are known for their antimicrobial and anticancer properties. In this study, we investigated the endophytic fungal microbiome associated with P. alliacea, and the endophytic fungal isolates were classified into 30 morphotypes based on their cultural and morphological characteristics. Ethyl acetate extract of fungal endophytes was obtained by liquid–liquid partitioning of culture broth followed by evaporation. The crude extract dissolved in dimethyl sulfoxide was screened for antimicrobial activity against three bacterial strains (Escherichia coli ATTC 25902, Staphylococcus aureus ATTC 14775, Bacillus subtilis NRRL 5109) and two fungal strains (Candida albicans ATTC 10231 and Aspergillus fumigatus NRRL 5109). Among the crude extracts from endophytes isolated from leaves, 65% of them showed antimicrobial activity against the bacteria tested. Similarly, 71 and 88% of the fungal crude extracts from endophytes isolated from root and stem, respectively, showed inhibitory activities against at least one of the bacterial strains tested. Crude extracts (at a concentration of 10 mg/mL) from ten of the fungal isolates have shown a zone of inhibition of more than 12 mm against both Gram-positive and negative bacteria tested. Sequenced data from isolates showing strong inhibitory activity revealed that Fusarium solani, F. proliferatum, and Fusarium oxysporium are the major endophytes responsible for bioactive potential. These results indicate that Petiveria alliacea harbors fungal endophytes capable of producing antimicrobial metabolites. Future studies need to focus on testing against drug-resistant bacteria (ESKAPE group) and other pathogenic bacteria and fungi.

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus. Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.

Biodiversity and biogeography of leaf-inhabiting endophytic fungi have not been resolved yet. This is because host specificity, life cycles and species concepts, in this heterogeneous ecological guild of plant-associated microfungi, are far from being understood. Even though it is known that culture-based collection techniques are often biased, this has been the method of choice for studying fungal endophytes. Isolation of fungal endophytes only through culture-based methods could potentially mask slow growing species as well as species with low prevalence, preventing the capture of the communities’ real diversity and composition. This bias can be partially resolved by the use of cultivation-independent approaches such as direct sequencing of plant tissue by next generation techniques. Irrespective of the chosen sampling method, an efficient analysis of community ecology is urgently needed in order to evaluate the driving forces acting on fungal endophytic communities. In the present study, endophytic ascomyceteous fungi from three different plant genera (Vasconcellea microcarpa, Tillandsia spp., and Hevea brasiliensis) distributed in Peru, were isolated through culture-based sampling techniques and sequenced for their ITS rDNA region. These data sets were used to assess host preferences and biogeographic patterns of endophytic assemblages. This study showed that the effect of the host’s genetic background (identity) has a significant effect on the composition of the fungal endophytic community. In other words, the composition of the fungal endophytic community was significantly related to their host’s taxonomic identity. However, this was not true for all endophytic groups, since we found some endophytic groups (e.g. Xylariales and Pleosporales) occurring in more than one host genus. Findings from this study promote the formulation of hypotheses related to the effect of altitudinal changes on the endophytic communities along the Eastern Andean slopes. These hypotheses and perspectives for fungal biodiversity research and conservation in Peru are addressed and discussed.

Endophytic fungi residing in the host plants are well known for the production of industrially significant enzymes. This study deals with the isolation of fungal endophytes and the screening of extracellular enzymes from nine different plants of the family Lamiaceae with potent medicinal properties. Overall, 40 endophytic fungi (LCJ401 - LCJ440) were isolated from selected medicinal plants. The isolated endophytic fungi were evaluated for their capability to synthesize industrially important enzymes like amylase, protease, cellulase, laccase and L-Asparaginase in specific solid media. The results indicated that 45%, 37.5%, 35%, 32.5% and 52.5% of isolates could produce amylase, protease, cellulase, laccase and L-Asparaginase enzymes respectively. Based on the primary screening, the L-Asparaginase enzyme was more dominant and therefore the promising L-Asparaginase producing isolates were estimated by the nesselarization method. Of these, LCJ413, LCJ428, LCJ432 and LCJ438 were known to produce a high amount of L-Asparaginase. These isolates were subject to molecular analysis by 18S rRNA sequencing and MEGA software was used to construct a phylogenetic tree. The endophytic fungi identified were LCJ413 (Curvularia sp.), LCJ428 (Arxotrichum sp.), LCJ432 and LCJ438 (Fusarium sp.). Findings here revealed that fungal endophytes of Lamiaceae taxa have high L-Asparaginase-producing potency and can be exploited as a substitute reservoir for anticancer enzyme production.

It is a known fact that the bacterial and fungal endophytes inhabit the plant tissues besides aiding in the better growth and health of the plants. The bark and leaves of Taxus wallichiana have drawn a lot of interest in recent years since they are the richest source of taxol, an anticancer drug. As it is a slow-growing tree that can only be regenerated via vegetative propagation, it has been classified as a critical rare species due to its extensive collection for medicinal and other purposes. Nonetheless, the use of endophytes as plant growth promoters is gaining much importance among environmentalists and agronomists because of their imperative role in crop production. Even then, there is hardly any information available regarding the growth-promoting endophytes isolated from bark and leaves associated with T. wallichiana commonly known as Himalayan Yew. Therefore, the present study was undertaken to isolate fungal and bacterial endophytes from T. wallichiana and to classify the growth-promoting properties of these endophytes. In total, seven fungal and ten bacterial endophytes were obtained from different parts of T. wallichiana. All of the isolated fungal and bacterial endophytes produced indole acetic acid while most of them also produced ammonia. Besides, the fungal and bacterial endophytes were also screened for antimicrobial and various enzymatic activities. Based on the above results, the two fungal endophytes were selected for their possible ability to promote seed growth. The results showed that the fungal endophytes isolated from T. wallichiana played an active role in increasing growth in other plant species and therefore, can be used as potential plant growth promoters.

ABSTRACTAim: The aim of this study was to evaluate the heavy metal resistance potentiality of endophytic fungi isolated from the leaves of Boswellia ovalifoliolata,an endemic medicinal plant of Tirumala Hills.Methods: Initially, isolation of fungal endophytes was carried out. Isolated fungi were screened for the heavy metal resistance against Co, Cd, Cuand Zn using growth and evaluated their maximum tolerant capacity. Molecular identification of endophytic fungi was carried out by 18S rRNA geneamplification and Sanger’s nucleotide sequencing. Phylogenetic tree was constructed using NCBI Clustal W.Results: Ten different endophytic fungi were isolated from the leaves of B. ovalifoliolata. Among the isolated endophytic fungi, five showed resistanceto Co, Cd, Cu, and Zn. The most resistant fungus was identified as Lasiodiplodia theobromae based on 18S rRNA gene sequencing.Conclusions: L. theobromae was isolated from B. ovalifoliolata and identified as one of the useful fungi involved in mycoremediation against heavymetal toxicity.Keywords: Heavy metals, Endophytic fungi, Endemic plant, Bioremediation.

The impact of pH on the microbial colonisation of endophytes on two plants (Buchanania and Celastrus sp.) was studied. The leaf and stem of two plant species were subjected to two PDA (Potato Dextrose Agar) medium at pH levels (4.5 and 9) for 5 weeks for isolation of fungal endophytes in aseptic condition. Over the testing period, pH 4.5 produced the highest fungal colony. At different pH values, the fungal populations were noticeably different, with consistently higher counts at pH 4.5 and 9, respectively. At pH 4.5 isolated fungi were Aspergillus niger, Alternaria, Curvularia, Fusarium, Rhizopus, Mucor, and Penicillium, and at pH 9.0 the isolated fungi were Aspergillus fumigatus, Penicillium, Alternaria, Curvularia, Fusarium, and Rhizopus respectively. It demonstrates that varied pH levels led to changes in the fungal populations in plant species, leaves and stems (resistance/ susceptibility to environmental influences).

Introduction/Objective Background: Synthesizing metal nanoparticles (NPs) by chemical or physical methods is difficult due to energy and time consumption, equipment costs, and chemical toxicity. Chemosynthesis-derived NPs are unstable, limiting their biological applications. Thus, identifying alternative green sources to address these drawbacks is difficult scientifically. The hardest part of green metal nanoparticle synthesis is finding a nontoxic natural product and an eco-friendly solvent. Objectives: Isolation of fungal endophytes from Eucalyptus sideroxylon leaves to synthesize ZnO and Au nanoparticles. Methods/Case Report Materials and methods: We used healthy Eucalyptus sideroxylon plant samples from the desert research center to separate endophytes. All fungi were grown on malt extract broth on a rotary shaker at 28 °C (120 rpm) for 96 hours after isolation. Biomass components were removed by filtration and washing in distilled water. Nanoparticles came from biomass cell filtrate. 0.1 M, 1 mL of Zn SO4, and HAuCl4 were added to 10 mL of fungal filtrate and agitated for 24 hours in the dark at room temperature to make Zn and Au nanoparticles separately. Characterization of nanoparticles shows a green synthesis of ZnONPs and AuNPs. Results (if a Case Study enter NA) Results: Fungal endophytes' filtrates were mixed with 1mM Zn SO4 and HAuCl4 solutions to test their extracellular biosynthesis of ZnO and Au NPs. The fungal filtrate became colorless and violet after mixing with Zn SO4 and HAuCl4. Only FI-03 is from 13 (FI-01 to FI-013). The most potent fungal strain for Zn O NP and Au NP nanoparticle synthesis was Fusarium Chlamydosporum. Pure, mostly spherical ZnO and Au NPs averaged 19.3 and 22.1 nm. UV-Vis absorption: Excitonic absorption peaks of ZnO NPs were between 300 and 400 nm. ZnO NPs' maximum absorption peak was 305 nm, while Au NPs' peak was 520 nm. One absorption peak indicates metallic gold nanoparticles. Au and ZnO NPs aggregate the most due to their hydrophilicity, HD of 34.88 and 15.77 nm, and PdI of 0.77, 1.00, and 0.952. Gold face-centered cubic fcc structure dominates Au NPs' XRD pattern. EDX shows 64.61 and 35.39 percent Zn and O, respectively, in both nanoparticles. The optical absorption band of gold nanocrystallites peaked at 2.2 keV in energy-dispersive X-rays. Conclusion Endophytic fungus biosynthesizes ZnO and Au NPs, which are bioactive compounds for medicine and food without toxic chemicals and are environmentally friendly.

In the present investigation, we have carried out the isolation of fungal endophytes from Centella asiatica Linn leaves followed by fermentation and extraction of fungal endophytes with non-polar solvents such as chloroform, ethyl acetate and n-butanol. Preliminary phytochemical investigation of endophytic crude fractions of leaves were also determined to detect the presence of primary and secondary metabolites followed by invitro free radical scavenging activity by reducing power, DPPH and hydroxyl radical assay. The chloroform fungal endophytic fractions were subjected to column chromatography by gradient elution technique for isolation of possible secondary metabolite. Reducing power of endophytic extracts of C. asiatica Leaf (CAL-1) (50-450μg/ml) increased with increase in concentration. Reaction with DPPH radicals of CAL-1 showed good scavenging activity. The IC50 values for Ascorbic acid, chloroform extract, ethyl acetate extract and n- butanol extract were found to be 30.33 μg /ml, 66.58 μg/ml, 79.33 μg /ml and 96.39 μg/ml respectively. In hydroxyl radical assay, The IC50 values for mannitol, chloroform extract, ethyl acetate extract and n- butanol extract were found to be 121.06 μg / ml, 141.21 μg/ml, 181.80 μg/ml and 189.90 μg/ml respectively. The endophytic crude fractions of ethyl acetate exhibited potent antioxidant activity as compared to other fractions. Hence, ethyl acetate fungal endophytic fractions of Centella asiatica Linn leaves can be employed as a potential antioxidant in the prevention of oxidative stress caused by the free radicals.

Endophytic fungi were isolated from black spruce (Picea mariana (Mill.) BSP) dormant buds and needles of four age-classes. Only one isolate was obtained from 400 buds. A total of 914 isolates were obtained from the needles. With increasing needle age the rate of colonization increased between current-year and 3-year-old needles from 4 to 90%, respectively. The needle segment attached to the twig was colonized more often (p < 0.05) than other segments overall and for 6 of the 11 taxa isolated. The first endophyte from current-year needles was isolated on July 14, 1988, but endophytes were obtained from needles of the other age-classes (1, 2, and 3 years) on all sampling dates between June 10 and September 16, 1988. Key words: endophyte, Picea mariana, dormand buds, needles.

Isolation of fungal endophytes from root samples of trees blown over at the Royal Botanic Gardens, Kew, during the 1987 hurricane

Ceriops tagal is a mangrove plant species with potential as a source of endophytic fungal isolates. Known for its resilience in extreme environments, such as high salinity, Ceriops tagal provides an ideal habitat for endophytic fungi. The endophytic fungi isolated from Ceriops tagal are capable of producing bioactive compounds with promising potential. This study aims to isolate and characterize endophytic fungi in Ceriops tagal tissues. Conducted in August 2024, the research follows an exploratory descriptive method. The study stages include surface sterilization of samples, isolation of endophytic fungi, purification of fungal isolates, and morphological identification of endophytic fungi macroscopically and microscopically. The results reveal that isolation and characterization of endophytic fungi from the roots, branches, and leaves of Ceriops tagal yielded five isolates, four of which belong to the genus Aspergillus and one to the genus Curvularia.

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.

Endophytes were first reported by the Freeman (1904) from dunel grass. 3,00,000 plant species known which exist on earth, all are host to one or more endophytes [1]. Endophytes are found in roots, leaves, stems, fruits, tubers, ovules etc. (Hallmann et al., 1997; Sturzet al., 1997: Hurek et al., 2002, Benhizia et al., 2004; Iniguez et al., 2004). Many different types of alkaloids, terpenoids, flavonoids and steroids are produced by endophytic fungi and these metabolites of endophytic fungi have antimicrobial, antiviral, anticancer, and antioxidant potential. The root of the Hibiscus rosa is used for cough treatment. The leaves of the H. rosa are used as a laxative and flowers has anti – diarrhea properties and are considered as demulcent, emollient, refrigerant, aphrodisiac and emmenagogue. The high level of antioxidants in H. rosa sinensis can slow down the aging process by eliminating the free radicals and neutralizing their harmful effects on the cells of human body. Hibiscus flowers are used as a remedy for relieving respiratory problem and to improve the health of respiratory system.
 This work reveals that the endophytic bacteria and fungi were isolated from healthy leaves and stem of medicinal plant, H. rosa-sinensis which were first sterilized in 70% ethanol. The bacterial endophytes were isolated on Nutrient Agar Medium and fungal endophytes were isolated on Potato Dextrose Agar Medium. Colonies showing similar morphology on one plate were considered as single colony and so the frequency of each colony is calculated and isolated in pure form. By pure culture isolation technique, 4 different isolates of bacterial endophytes and 2 different isolates of fungal endophytes from stem of H. rosa-sinensis and 3 different isolates of fungal endophytes from leaves of H. rosa-sinensis were obtained. Bacterial endophytes were identified on the basis of Gram Stain and biochemical characterization whereas the identification of fungal endophytes was carried out at Plant Pathology Department of Forest Research Institute (FRI), Dehradun. The secondary metabolites were isolated from isolated bacterial and fungal endophytes by using two different solvents (Ethyl acetate, chloroform) in the ratio 1:1 left for 15 minutes, the organic phase were collected and kept for drying at 37C. The antimicrobial activities were done against human pathogen (Stayphylococcus aureus and Klebsiella pneumonia) by using Disc diffusion method of isolated secondary metabolites.

Plant diseases and pests reduce crop yields, accounting for global crop losses of 30% to 50%. In conventional agricultural production systems, these losses are typically controlled by applying chemical pesticides. However, public pressure is mounting to curtail agrochemical use. In this context, employing beneficial endophytic microorganisms is an increasingly attractive alternative to the use of conventional chemical pesticides in agriculture. A multitude of fungal endophytes are naturally present in plants, producing enzymes, small peptides, and secondary metabolites due to their bioactivity, which can protect hosts from pathogens, pests, and abiotic stresses. The use of beneficial endophytic microorganisms in agriculture is an increasingly attractive alternative to conventional pesticides. The aim of this study was to characterize fungal endophytes isolated from apparently healthy, feral wine grapes in eastern Canada that have grown without agrochemical inputs for decades. Host plants ranged from unknown seedlings to long-lost cultivars not widely propagated since the 1800s. HPLC-MS was used to identify unique endophyte-derived chemical compounds in the host plants, while dual-culture competition assays showed a range in endophytes’ ability to suppress the mycelial growth of Botrytis, which is typically controlled in viticulture with pesticides. Twelve of the most promising fungal endophytes isolated were identified using multilocus sequencing and morphology, while DNA barcoding was employed to identify some of their host vines. These fungal endophyte isolates, which consisted of both known and putative novel strains, belonged to seven genera in six families and five orders of Ascomycota. Exploring the fungal endophytes in these specimens may yield clues to the vines’ survival and lead to the discovery of novel biocontrol agents.