Antagonistic Activity of Endophytic Fungi Isolated from Globba patens Miq. Rhizome against Human Pathogenic Bacteria

The emergence of bacterial infections caused by resistant strains poses a threat to the development of new antibiotics. The majority of antibiotics being produced has been accelerated through the finding of newly reported natural products, especially those originated and produced by biological sources. Endophytic fungi residing in medicinal plants may be regarded as potential sources and encourage the exploration of more plant species for their antimicrobial activity. Our current study reports on the assemblage of endophytic fungi that colonize the rhizomes, using Globba patens a representative of Zingiberaceous species from North Sumatra. Twenty-six fungal morphotypes were obtained and differentiated by their morphological features. Each isolate was tested against human pathogenic bacteria namely Staphylococcus aureus ATCC® 29213TM, Methicillin-resistant S. aureus (MRsA) AtCC® 43300TM, Escherichia coli AtCC® 25922TM, and Enteropathogenic E. coli (EPEC) K11 in a dual culture assay. The results revealed that the majority of fungal isolates were strong antagonists against S. aureus and E. coli but not against MRSA and EPEC. Isolate Gp07 was the most potential fungus with a wide range of antibacterial activities and was subjected to further species-level identification based on its morphological characteristics and DNA sequence in the ITS-rDNA region. The isolate Gp07 was identified as Colletotrichum siamense, yet the presence of C. siamense in the rhizome of G. patens is not fully understood while possibly being characterized as the antibiotics-producing agent in the future.


INTRODUCTION
The emergence and rapidly increasing incidence of bacterial resistance limited the discovery and development of antibiotics for treating infections. During the past two decades, there have been very numerous reports of untreatable infections caused by multidrugresistant bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) and its resistant strains as also of serious infections by diarrheagenic Escherichia coli strains 1,2 . Ninety-one years after the discovery of Penicillin in 1929, scientists from each region of the world continue to explore and investigate the many sources of new natural products. Natural products are significant sources that deserve investigation as alternative sources of antibiotics and are commonly derived or synthesized by plants, bacteria, and fungi. One study of bioactive compounds discovered between 1981 and 2010 reported that half of the molecules discovered originated from natural sources 3 .
In most cases, fungi are known as notable producers of natural products. Furthermore, half of the novel natural products derived from fungal sources was from endophytic origins, with the most studies on their antimicrobials in specific to antibacterial activities 4 . Endophytic fungi are new sources for the discovery of bioactive compounds with potential development in biotechnology, although their true potential may not be fully investigated due respect of their unknown and unlimited host species 5,6 . Interaction of endophytic fungi with medicinal plants as their hosts has revealed some beneficial features such as promotion of plant growth, increase of stress resistances, and increased bioactive accumulation of molecules by the plants 7 . The medicinal properties of directly consumed or consumption of processed medicinal plants may be studied thoroughly, particularly their endophytic fungal colonization. This may broaden the scale of the discovery of natural products, especially in the tropical regions that are rich with many plant species.
Zingiberaceae is one of the most studied families of pharmacological plants growing in the tropical region including Indonesia for its bioactive compounds and some of its endophytic associates. Globba spp. are examples and the third-largest genus of Zingiberaceae distributed in Southeast Asia. It comprises about 100 species in that region 8 . Members of the genus Globba have been used in many ethnobotanical practices by the local communities of India, Malaysia, and Indonesia 9 . Although most ginger species are known to exhibit antimicrobial activities, the reports in this respect on the species of Globba are still limited 10 . Furthermore, information regarding the microbial associates, especially endophytic fungi colonizing the rhizomes of Globba species is even more limited and, therefore, merit investigation.
In North Sumatra, four species of Globba are found, namely, Globba aurantiaca, G. paniculata, G. patens, and G. pendula 11 . Our preliminary investigation on endophytic fungi began with G. pendula rhizome and some of the fungal morphotypes found in it exhibited potential antibacterial activities against S. aureus ATCC 29213 12 . Our study was undertaken to isolate endophytic fungi from the rhizomes of Globba patens and use the dual culture assay to test them for their antagonistic activities against human pathogenic bacteria. Screening of endophytic fungi from G. patens for antibacterial activities will provide the basis for the discovery and development of new antibiotic agents.

Plant materials
Globba patens Miq. was collected during an exploration in Sibolangit Forest located in Deli Serdang District, North Sumatra, Indonesia. The asymptomatic G. patens plants were uprooted from the intact soil and stored in sterile plastic bags, then transported to the laboratory at Universitas Sumatera Utara (Fig. 1). The plant samples were kept in cold temperature until further analysis. Duplicate samples were submitted to Herbarium Medanese for species authentication. Fungal isolation was performed within 48 h of sample collection.

Isolation of endophytic fungi
The procedure of isolating endophytic fungi was based on a modified surface sterilization method 12 . Rhizomes were gently cleaned under running tap water and cut into segments 10 cm long. The segments were surface sterilized in 75% EtOH for 2 min, 5.3% NaOCl for 5 min, and 75% EtOH for 1 min followed by three washings in sterile distilled water, each for about 1 min.
The sterilized rhizome segments were cut into 1-2-cm fragments and placed on Petri dishes (90 mm) containing Potato Dextrose Agar (PDA) supplemented with Chloramphenicol (0.01%, w/v). Plates were incubated at 25°C ± 3°C and monitored daily for any fungal colonies emerging from the rhizome fragments. Individual fungal colonies were picked from the edge of growth and transferred to new PDA plates. Stock cultures were made by sub-culturing of fungal colonies for maintenance and further analysis. Each isolate was differentiated based on the features of their colonies such as form, elevation, margin, upperlower color, texture, concentric rings (+/-), and radial line (+/-). Each isolate was assigned a code and maintained for further analysis.

Antagonistic activity of endophytic fungi against human pathogenic bacteria
Each isolate was tested against four representative human pathogenic bacteria, namely, Staphylococcus aureus ATCC® 29213™, Methicilinresistant S. aureus ATCC® 43300™, Escherichia coli ATCC® 25922™, and Enteropathogenic E. coli K11 using a modified dual culture method 12,14 . Three 6-mm mycelial plugs were cut from active-growing fungal cultures and placed on top of PDA + 1% yeast extract (w/v) that were previously seeded with 1-2×10 8 CFU/mL of bacterial suspensions. Plates were incubated at 37°C for 2 days. Clear zones surrounding mycelial plugs indicated the antagonistic activities and measured in millimeters (mm) using digital calipers.

Molecular identification of the antagonistic fungus
The potential antagonistic fungal isolate, Gp07 was identified to the genus level based on the sequence of the internal transcribed spacer (ITS) rDNA. For extracting DNA, the manufacturer's prescribed Protocol provided with Wizard® Genomic DNA Purification Kit (United States) was followed. Polymerase chain reaction (PCR) was performed using universal primers for fungi i.e ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3) 15

RESULTS
Twenty-six fungal isolates were successfully recovered from the rhizomes of G. patens and differentiated based on the characteristics of their colonies ( Table 1). The number of isolates found is higher than the 16 (sixteen) fungal isolates derived in the previous study of G. pendula 12 . Also, the number of isolates was more than in the cases of other genera that Also, plants of a species at different stages of life-cycle may harbor distinct endophytic fungal communities over time. Younger parts of plants tend to accumulate a higher concentration of bioactive and anti-herbivory compounds which may reduce the opportunity for colonization by endophytic fungi. On the contrary, an endophytic study of Dioscorea zingiberensis showed that the number of endophytic fungal isolates may be accompanied by the higher concentration of saponins accumulated in the rhizome 23 . Another study on Alpinia officinarum using a metagenomic approach revealed that there was a connection between the colonization by endophytic fungal communities and two types of accumulated chemicals, namely, volatile oils and galangin which constructed the whole plant-microbe interaction in the rhizome 24 . However, no such link has so far been discussed for other Zingiberaceous species, including Globba spp., which may open the possibility of further investigation in the future due to the metabolite-rich rhizomes and their connection to endophytic fungi colonization.
The endophytic fungal isolates were subjected to antagonistic test against human pathogenic bacteria using dual culture assay. The majority of isolates were antagonists of S. aureus Gp07 stood out as the most potential isolate that exhibits a wide range of activities antagonistic to all the tested pathogenic bacteria based on the diameter of the clear zone (mm). Also, the isolate Gp07 was the only isolate which exhibited inhibitory activities towards the growth of MRSA, which was the only drug-resistant strain used in this study. The antibacterial activity demonstrated by each fungal isolate was attributable to certain metabolites diffusible in agar plugs and not the mycoparasitism of the isolates. These results may lead to the identification of the anti-MRSA compound and the significance of Gp07 for its production through fermentation. Meanwhile, the discovery of antibacterial compounds, particularly those with special potential as anti-MRSA, has been reported for endophytic fungi from other sources. Dothideomycete sp. isolated from a Thai medicinal plant species, Tiliacora triandra showed potential antibacterial activities against both S. aureus ATCC 25923 and MRSA ATCC 33591 by producing Dothideomycetide A with minimum inhibitory concentration (MIC) from 128 to 256 µg/mL 25 . Pestalotia sp. isolated from a mangrove medicinal plant species in Bangladesh, Heritiera fomes, was reported to produce oxysporone and xylitol that have the anti-MRSA properties with MIC from 32 to 128 µg/mL 26 . The major portion of antibacterial components produced by Gp07 may be identified in the future for the possibility of finding new compounds to combat bacterial infections.
The identification of isolate Gp07 was based on its macroscopic and microscopic morphological features confirmed with a   molecular identification in the region of ITS-rDNA (Fig. 2, 3). Based on the preliminary identification, the isolate Gp07 was preliminarily assigned as the member of Colletotrichum, for having the unique morphological characteristics, i.e, colony color, absence of setae, and cylindrical conidia 27 . However, some discrepancies in morphological features may exist due to the variation of the species and variations across the range of the host species. A phylogenetic tree was then constructed to reveal the species-level identification which assigned the Gp07 position as C. siamense, a fungal species described firstly from Thailand. However, no information is available so far about this species, especially the one extracted from Zingiberaceous species. Also, this is the first report from Indonesia on finding C. siamense as an endophytic fungus from G. patens. The ITS sequence of C. siamense Gp07 was then submitted to GenBank and allotted the accession number of MT974212.1. The majority of recent reports on this species are regarding its being found in new host species, mainly as a newly reported phytopathogenic fungus causing anthracnose on various horticultural plants grown in the Southeast Asia region 28,29 . Antibacterial or bioactive compound-producing Colletotrichum strains derived from a range of hosts and origins as endophytes have been documented. Colletotric acid produced by C. gloeosporioides from Artemisia mongolica in China was reported to inhibit a wide range of human pathogenic bacteria with MIC from 25 to 50 μg/mL 30 . Two novel compounds, apigenin-8-Cβ-D-glucopyranoside, and 2-(hydroxymethylthio) ethanol were identified as produced by an endophytic Colletotrichum sp. from Chinese Ginkgo biloba through fermentation 31 . However, the presence of C. siamense in the rhizome of G. patens is not fully understood though it is, possibly, an antibiotics-producing agent of the future.