Endophytic Bacteria isolated from the leaf of Langusei (Ficus minahassae Tesym. & De Vr.) and their antibacterial activities

Endophytic bacteria colonizing the internal tissues of plants are being studied for its potential beneficial properties including antibacterial activity. The aims of this study were to identify the endophytic bacteria residing in the leaf of Ficus minahassae, a native plant of North Sulawesi, as well as their antibacterial activities against laboratory-adapted reference bacterial strains, Staphylococcus aureus, and Escherichia coli. The isolate identification was conducted using the 16S rRNA gene marker and the antibacterial activity test was done using a well diffusion method. A yellow-pigmented (designated as YL1) and a beige-pigmented (designated as YL2) bacteria were isolated from the internal tissue of langusei. Using the BLAST search, the isolate YL1 had a 100% identity with Brachybacterium muris, while YL2 has 99% identity with Pseudacidovorax intermedius. Ez-Taxon database confirmed that YL1 was B. muris with 99.81% similarity, while YL2 was P. intermedius with 99.80% similarity. Isolate YL2 was able to inhibit the growth of S. aureus but not E. coli. Isolate YL1 did not inhibit both reference bacteria. This study represents the first investigation of endophytic bacteria isolated from langusei leaf and their antibacterial activity.


Introduction
In recent years, the mining of microbial resources contained in plant tissue has begun to receive much attention. These endophytic bacteria colonize the intercellular spaces and vascular systems of the plants. These microbes have been studied for various purposes. Compounds produced by endophytes have many benefits, such as antibacterials. The active compound of plant endophytic microbes often has a stronger activity than that produced by their host plants. The abilities of the active compounds of plant endophytic bacteria still need to be explored to discover new bacterial strains that have the potential to be further explored [1].
Several studies have reported that endophytic bacteria can be found in ficus plants, for example in Ficus carica [2] and F. racemosa [3]. One of the ficus plants native to North Sulawesi is F. minahassae. The local people names this plant as langusei. This plant is found in the primary forest, especially along the river, to a height of about 135 meters above the sea. Dichloromethane extract of the leaves contained 2-hydroxyethyl benzoate, phytyl fatty acid ester, squalene, and β-sitosterol [4]. The increasing emergence of bacterial resistance creates demands in focusing on new and more potent antimicrobial sources. Therefore, this study was aimed to identify endophytic bacteria in F. minahassae and to assay their antibacterial activity.

Sample Preparation
The leaf of langusei was obtained from the Batukota river bank in Manado. The leaf was surface sterilized by incubation in 5% NaOCl for 10 min, followed by in 70% alcohol for 2 min, and rinsed three times in sterile dH2O. To check the sterilisation procedure, 100 ul aliquot of dH20 taken from the rinsing of the sterilized leaf was spread over the nutrient agar (NA) media, incubated at 37°C for 2 days, and examined for the presence or absence of the growth of microorganisms.

Isolation of the Endophytes
The plant leaf was acceptically ground in a sterile mortar and pestle until producing juice. One hundred microlitters of the juice were spread over the NA media. The plates were incubated at 37°C for 2 to 4 days. The growing bacterial isolates were grouped based on their morphology and characteristics of their colonies such as size, shape, and colour. Each separated colony was picked using Oose and streaked on NA media, incubated at 37°C for 2 days. Each separately grown colony was considered as a pure isolate to be used for further examination.

Identification of the Endophytes
The pure selected isolates were identified using Gram staining followed by identification based on the 16S rRNA gene. The DNA genome was isolated using the Plant Genomic DNA mini kit (Geheaid). MyTaq HS Red Mix (Bioline) was used for PCR amplification, with 10 μM of each BKXF and BKXR primer. The PCR was performed using the following condition: initial denaturation at 95 o C for 3 min followed by 35 cycles of denaturation of 95 o C for 30 sec, annealing at 50 o C for 30 sec, elongation at 72 o C for 90 sec, and final elongation at 72 o C for 1 min. The PCR products were sent to 1st BASE Malaysia for sequencing. The aligned contiguous consensus sequences were used for the homology search by BLAST software (http://blast.ncbi.nlm.nih.gov) algorithm at the National Center for Biotechnology Information (NCBI). The identification was further confirmed using the Ez-Taxon database (https://www.ezbiocloud.net/) [5].

Phylogenetic Analysis
The phylogenetic analysis was performed using the stepwise procedure described previously [6]. The chromatograms obtained from 1st BASE Malaysia were processed using Geneious 10.1.3. The primer sequences were removed by trimming approximately 50 nucleotides at the beginning of DNA sequences. Errors of the reading of the nucleotides were corrected accordingly. By using the pairwise alignment of forward and reverse sequences, the consensus sequences were generated. All similar sequences obtained from Genebank were aligned using multiple sequence alignment with hierarchical clustering [7], which is available online at (http://multalin.toulouse.inra.fr/multalin/), and trimmed accordingly to obtain the core sequences of 16S rRNA. By using the Neighbor Joining method integrated into MEGA v10.0.4 [8], the phylogenetic tree was reconstructed.

Antibacterial Test of the Isolates
The antimicrobial activity of the isolates was determined using the agar well-diffusion method. Nutrient agar media was poured into plates equipped with stainless-steel cylinders. After the media were solidified, the cylinders were removed leaving a 5 mm diameter of wells. Two hundred microliters of an overnight culture of indicator strains (Escherichia coli (Migula) Castellani and Chalmers (ATCC ® 25922™) and Staphylococcus aureus subsp. aureus Rosenbach (ATCC ® 25923™)) containing approximately 10 6 cells/ml were used for this purpose. Bacterial isolates were grown overnight, heatkilled by incubating at 80 o C for 1 hour, and the supernatant was collected by centrifuging at 6000 rpm for 5 min. One hundred microlitters of each isolate was added into each well. Inhibitory zone of the isolates was checked after 24 hours incubation at 37 o C. Fifty µg/ml 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid was used as positive control and sterile dH2O as the negative control.

Endophytic Bacterial Isolation
Of several endophytic bacteria that were successfully isolated and purified from the surface-disinfected leaf of langusei, two isolates (YL1 and YL2) were further investigated. Gram staining indicated that YL1 was a positive-Gram bacterium. The isolate was yellow-pigmented on nutrient agar (Fig. 1a). YL2 was a negative-Gram bacterium and beige-pigmented on nutrient agar (Fig. 1b). a b Figure 1. a) Colonies of isolate YL1 and b) colonies of isolate YL2 on nutrient agar media
To the best of our knowledge, there has been no report on the isolation of endophytes from langusei, therefor this finding is the first report of the isolation of P.   [25], and agrobacteria which were detected in many healthy benjamin fig [31].

Phylogenetic Analysis
The construction of a phylogenetic tree through the Neighbor joining method (Fig. 2) demonstrates the differences between taxa of the endophytes found in the leaf of F. minahassae. Brachybacterium muris belong to Actinobacteria and P. intermedius belongs to β-Proteobacteria. Endophytic bacteria of fig tree reported previously were from α-Proteobacteria, ɣ-Proteobacteria, and Firmicutes. Most of the endophytic bacteria of rice were α-Proteobacteria (46%) and Actinomycetales (16%) [11]. Bacterial endophytes are polyphyletic, belonging to a broad range of taxa, including Actinobacteria, Firmicutes, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria [32]. Of these, Pseudomonas sp. has frequently found as dominant bacterial endophytes [33].

Antimicrobial Test
The antibacterial activities of B. muris YL1 and P. intermedius YL2 were examined using the well diffusion method. B. muris did not inhibit the growth of S. aureus nor E. coli. P. intermedius displayed anti-bacterial activity against S. aureus (Fig 3a) but not against E. coli (Fig 3b). The diameter of the inhibition zone was 18.75 ± 0.096. The similar finding showed that Brachybacterium sp. isolated from D. cochinchinensis did not inhibit bacteria including S. aureus, E. coli, B. subtilis, K. pneumoniae, and fungi including A. niger and A. fumigatus, but inhibited plant fungus Gibberella fujikuroi [13]. The current study suggests that endophytic bacteria of langusei are a source of potential novel antibacterial. Endophytic bacteria from F. variegata produced antibacterial compounds [30]. Streptomyces sp. isolated from leaves of F. racemosa showed an antibacterial activity [3]. Pseudacidovorax intermedius was confirmed as nitrogen-fixing bacterium [21,22,24]. Nitrogen-fixing nitrogenase (nifH) homolog gene was also found in Pseudacidovorax sp. isolated from potato [27]. Nitrogenase is an enzyme used by some organisms to fix atmospheric nitrogen gas (N2).
Identification using NMR, dichloromethane extract of the leaf of F. minahassae contains 2hydroxyethyl benzoate, phytyl fatty acid ester, squalene, and β-sitosterol [4]. These compounds are probably induced by endophytic bacteria living in the intracelullar tissues of the leaves. Polyethylene terephthalate (PET) hydrolase is produced by bacteria hydrolase PET to release among others is 2hydroxyethyl benzoate (HEB) [34]. Phytyl esters, an intracellular wax esters, are also found in bacteria and generally considered to be energy storage components [35]. This compound has an antimicrobial property [36]. Squalene hopene cyclases have been found in bacteria [37]. Squalene-hopene cyclase (SHC) catalyzes the conversion of acyclic squalene into the pentacyclic hopene and hopanol in prokaryotes. Squalene has antimicrobial properties [38,39]. β-sitosterol, one of the severol phytosterols, also has antibacterial activity [40]. This compound has been isolated from endophytic fungi of Amazon plants [41] and the Chinese medicinal plant Arisaema erubescens [42]. It may also be produced by endophytic bacteria.

Conclusion
Two endophytic bacteria isolated from F. minahassae were confirmed as B. muris (YL1) and P. intermedius (YL2) based on molecular identification using 16S rRNA gene marker. Among the two, only P. intermedius showed activity against the growth of S. aureus. Further studies are required to reveal the potential of these endophytes as antibacterial agents.

Conflict of Interest
There is no conflict of interest.