Bioactive compounds and antibacterial activity of endophytic fungi isolated from Black Mangrove ( Avicennia africana ) leaves

The antibacterial potential of fungal endophytes from black mangrove against pathogenic marine bacteria was evaluated and the bioactive compounds identified and quantified. Black mangrove (Avicennia africana) healthy leaves were obtained from a Mangrove Forest in Eagle Island, Port Harcourt, Nigeria. The fungal endophytes were o cultured on acidified Potato dextrose agar plates for 5 days at 28 C. The isolated fungal endophytes were identified based on microscopic and colonial morphologies. Different concentrations (0, 20, 40, 60, 80 and 100 mg/ml) of ethyl acetate extract of the fungal isolates were screened against pathogenic marine bacteria (Salmonella spp., Staphylococcus aureus and Shigella spp.) via agar well diffusion assay. The most active isolate was identified using molecular method. Gas chromatography – Mass spectrometry was employed in the identification and quantification of its bioactive secondary products. Fungi of the genera Aspergillus, Penicillium, Fusarium, Collectotrichum, Phomopsis, Epicoccum and Rhizopus were isolated. Two bioactive compounds were identified in ethyl acetate extract of Fusarium sp. which was molecularly identified as Fusarium phyllophilum KU350622.1. Dibutyl phthalate (C H O ) is the major 16 22 4 compound at 55.926% peak area. The results showed that fungal endophytes from black mangrove exhibited antibacterial action against pathogenic marine bacteria. The bioactive secondary products identified have vast potentials for use in agriculture and industries.


Introduction
The mangrove ecosystem, therefore, is a Mangroves are extremely useful discrete environment inhabiting various groups ecosystem with different essential economic and of microbes (Thatol et al., 2013).Among such ecological functions (Bandaranayake, 2002).
microbes of unique significance are marine fungi which inhabit diverse marine ecosystems and are Black Mangrove (Avicennia africana), locally capable of producing a number of new bioactive known in Nigeria as Ogbun or Ofun (Odugbemi compounds with extensive biological activities and Akinsulire, 2008), is a grey -barked small (Amira et al., 2009).tree or shrub.The leaves have glands where salt A major group of fungi found in the is excreted.Yellow centred white flowers appear marine habitat is mangrove -endophytic fungi during the dry season at the axillary stalk which are found in most species of plants (Hyde, (Steentoft, 1988).This ecological unit is known 2008;Rodriguez et al., 2009).They colonize for intermittent tidal flooding which causes inner tissues of plants with no obvious harmful ecological factors like nutrient availability and effects (Darshan and Shishupala, 2014).They salinity to be greatly inconsistent with definite flourish under severe environment that cause environmental uniqueness (Holguin et al., 2006).them to develop unique metabolic pathways and traces were removed by rinsing with sterile generate distinctive chemicals that make it distilled water.The leaf fragments were dried on possible for them to bear such tense sterile blotting paper.The cut surfaces of the leaf environmental setting.A number of these tissue were placed aseptically on acidified potato chemicals are long-established to be of immense dextrose agar plates.The plates were incubated potential as a source of new agents for diverse at 28°C for 7 days.Tips of fungi, growing out of applications in industries (Eldeen and Effendy, the leaf tissue, were selected and cultivated in 2013).There is need for regular exploration for pure culture on potato dextrose agar.They were new antimicrobial compounds as a result of identified on the basis of their cultural and constant antibiotic resistance by pathogenic microscopic characteristics (Barnett and Hunter, microbes (Pucci and Bush, 2013).Through 1998).The pure cultures of endophytic fungal isolation of endophytic fungi, novel species that strains were maintained in potato dextrose agar are regarded as an exceptional source of slants at 4°C. bioactive compounds are being revealed.
Antibacterial potential of indigenous red Bioactive compounds extraction: Mycelial plug (1 mangrove-leaf fungal endophytes and their cm diameter) of 7 day -old culture of each fungal bioactive compounds have been documented isolate was inoculated into 1 L Erlenmeyer flask (Ariole and Akinduyite, 2016).In the present containing 300 ml sterile potato dextrose broth.
o study, endophytic fungi from the leaf of The flasks were incubated at 28 C for 21 days indigenous Avicennia africana (Black mangrove) under static state.The liquid culture of each flask were evaluated for their antibacterial potential.
was filtered using sterile cheesecloth.Then, The bioactive compounds of the most active ethyl acetate (50 ml) was added to each filtrate endophyte were identified and quantified.
and centrifuged for 10 min at 1500 rpm.mg/ml) and 0.1ml sterile distilled water (0 mg/ml extract) served as positive control and negative Isolation of endophytic fungi: The method control respectively.The plates were incubated described by Suryanarayanan et al. (2003) with o for 24-48 hr at 37 C.The clearance zones around some modifications was employed for the the wells were measured in millimetre and used endophytic fungi isolation.The leaves were as indicator of antibacterial activity.carefully washed with running tap water, cut with sterile precautions into small fragments (0.5-1 Molecular identification of endophytic fungi: DNA cm) and surface sterilized with 1% sodium extraction was performed using Norgen's hypochlorite for 1 minute and then 75% ethanol Yeast/Fungi Genomic DNA Isolation Kit.Genomic for 30 seconds.Sodium hypochlorite and alcohol  Staphylococcus aureus (a gram positive concentrations of 80-100 mg/ml showed bacterium).antibacterial activity against at least one of the tested pathogens.However, the different Molecular characteristics of the most active concentrations (20-100 mg/ml) of ethyl acetate endophytic fungus: The fungal isolate (WB2) extracts of WB2 (Fusarium sp.) were active which was the most active fungus was identified against all the tested pathogens.The zones of as Fusarium phyllophilum KU350622.1.The inhibitions obtained were between 8 and 17.67 phylogenetic tree is presented in Figure 1 DNA was proficiently extracted from the cells Gas chromatography-mass spectrometry (GCaccording to the method employed by Zhang et MS) analysis: Agilent 7890A-5975C GC-MS al. (2010).Spin column chromatography was system (Tao et al., 2011) was employed.Exactly used for purification.The purified genomic DNA 0.5 µl of the most active fungal extract was was completely digestible with restriction injected into the GCMS system with injector o enzymes.DNA quantification was carried out temperature of 250 C. Nitrogen was used as a using DNA standard and the absorbance carrier gas during the compounds separation measured at 450 nm.Polymerase chain reaction which was carried out on a 60m HP-INNOWAX (PCR) master mix from Norgenbiotek Canada capillary column (0.25 mm).The flow of the was employed for PCR analysis which was carrier gas was 1ml/min with a split ratio of 10:1.agarose gel.DNA ladder (100 bp) was 280 C at 5 C/min and left for 9 min and ionization employed as DNA molecular weight marker.energy of 70 eV was employed.The mass Electrophoresis was carried out at 80V for 1½ h.spectra of the unidentified bioactive compounds The gel was stained with ethidium bromide and were related with mass spectra of identified viewed using UV light.The sequence was compounds in the National Institute of observed by the use of Chromaslite for base Standards and Technology (NIST) Database.calling.Then, BioEdit was employed for The molecular weight, name and peak area (%) sequence editing.A Basic Local Alignment of the bioactive compounds were determined.Search Tool (BLAST) was performed using National Centre for Biotechnology Information Results ( N C B I ) d a t a b a s e Bacterial isolation: A total of seven (7) ( ). endophytic fungi were obtained from black Related sequences were aligned with Cluster W mangrove (Avicennia africana) leaves.The after downloading.The phylogenetic tree was fungal genera isolated were: Penicillium, constructed with Molecular Evolutionary Fusarium, Collectotrichum, Rhizopus, Genetics Analysis (MEGA) version 6 (Tamura et Aspergillus, Epicoccum and Phomopsis ( . mm.Furthermore, gram negative bacteria Fusarium phyllophilum PEN6 (KR909206.1)Fusarium phyllophilum CBS 216.76 (AB587006.1)Fusarium phyllophilum MRC7543 (KR909430.1)Fusarium phyllophilum BCCM/IHEM:10241 (KJ126553.1)Fusarium phyllophilum MRC7543 (KR909359.1)Fusarium phyllophilum CBS 216.76 (AB586912.1)Fusarium phyllophilum CBS 239.56 (AY526489.2) Fusarium phyllophilum CBS 137.35 (HQ232206.1structuresand peak area Bioactive compounds in the ethyl acetate extract percentage of each compound identified in the of WB2: The bioactive compounds, their extract are presented in Table 2. retention time, molecular weight, molecular Gas chromatogram of bioactive compounds in the ethyl acetate extract of WB2 (Fusarium phyllophilum KU350622.1) is presented in Figure 2.

Figure 3 :
Figure 3: Mass spectrum of the major bioactive compound (Dibutyl phthalate) in the ethyl acetate extract of Fusarium phyllophilum KU350622.1 from black mangrove leaves

Table 1 :
Antimicrobial activity of ethyl acetate extract of black mangrove leaf -endophytic fungi against pathogenic bacteria Akinduyite and Ariole / Nig.J. Biotech.35 (2) : 35 -42 (Dec.2018)Antibacterialactivity:Theresult of the (Salmonella sp. and Shigella sp.) were more antibacterial assay presented in Table1revealed resistant to the fungal extracts than that all the endophytic fungi extracts at