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There is a constant need for novel antibiotic and antioxidant sources due to the ever-increasing resilience of pathogens and the occurrence of chronic diseases. The natural sources of these agents have advantages due to lower production cost, structural variation, and uses of active compounds for pharmaceutical uses. The microbes living in planta termed Bendophytes^ are alternative sources of host bioactive compounds. In this study, ten endophytic fungi were isolated from Polygonum chinense L. and identified by sequencing of the internal transcribed spacer regions. The fungal strains were fermented and the ethyl acetate extracts were evaluated for antimicrobial and antioxidant capacities. Almost 80% of the endophytes showed antibacterial potency against one or more pathogenic bacteria. Among all strains, Penicillium canescens showed broad-spectrum antimicrobial activity against gram-positive and gram-negative pathogens as well as significant antioxidative and DNA protective capacities. The strain Fusarium chlamydosporum displayed significant anti-radical (126.8 ± 6.7 μg/ml) and ferric reducing (84.7 ± 2.1 mg AA/g dry extract) capacities. The bio-autography, chromatography, and mass spectroscopy analyses of P. canescens extract revealed the presence of sesquiterpene (germacrene), plasticizer (phthalic acid ester) along with phenolic acids, flavonoid (quercetin), and short chain hydrocarbons. The secondary metabolites of F. chlamydosporum were identified with phenolic acids as bioactive compounds by chromatography and mass spectroscopy. This study indicates P. chinense endophytes as potential sources of antimicrobial and antioxidant compounds for novel drug discovery. . . . . . Keywords Endophytic fungi Antimicrobial Bio-autography Germacrene Phthalic ester Antioxidant phenolics Introduction the endophytes that reside in the internal organs or cell spaces of healthy plants. Diverse fungal endophytes exist within plant Medicinal plants are investigated since long for unique and tissues and each plant can host one or more endophytes (Yu et potential metabolites. In most developing countries, it is con- al. 2010). The endophytes apparently stay in a mutualistic sidered as the source of pharmaceutical products (Kaul et al. association with the host plants and can be a novel source of 2012) and therefore are exploited rigorously. The overexploi- metabolites of pharmaceutical interest (Huang et al. 2008a). tation of medicinal plants for newer and potential pharmaceu- Endophytes are capable of producing similar secondary me- tical compounds poses threat to biodiversity. tabolites as their hosts (Alvin et al. 2014). Endophytes of medicinal plants have incited considerable The search for more novel antibiotics of modern times is interest and attention for the wide diversity of bioactive me- highly called for as the microorganisms and humans have de- tabolites (Cai et al. 2004; Strobel et al. 2004;Newman and veloped resistance to the existing antibiotics thus posing greater Cragg 2007). Medicinal plants afford unique microcosm for challenge. The fungal endophyte-derived bioactive compounds provide us with new choices of novel antibiotics which can be effectively used against infectious diseases. Many endophytic * Monnanda Somaiah Nalini fungal strains are reported to produce novel broad-spectrum nmsomaiah@gmail.com bioactive compounds belonging to alkaloids, macrolides, terpe- noids, flavonoids, glycosides, xanthones, isocoumarins, qui- Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India nones, phenylpropanoids, aliphatic metabolites, lactones, etc. (Kauletal. 2012). However, the antimicrobial compounds iso- Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India lated till date from those fungal endophytes are only a small part 596 Ann Microbiol (2018) 68:595–609 of total endophytic species (Yu et al. 2010). The endophytic necessary. Antibiotic disc and the Mueller-Hinton medium fungi produce a plethora of substances for potential use in mod- were purchased from Hi-media (Mumbai, India). ern medicine including antioxidants. The antioxidants are im- portant compounds which are potential therapeutic agents Collection of plant material against the oxidative damage caused by free radicals. Novel antioxidants have been isolated from a number of endophytic Polygonum chinense L. was collected from the Talacauvery fungi (Kaul et al. 2012). subcluster (012° 17′ to 012° 27′ N and 075° 26′ to 075° 33′ E) India is one of the 17 mega-biodiverse countries in the of Western Ghats, in Kodagu district during June 2012 world as recognized by Conservation International (Fig. 1). The plant parts (leaves and stem) were collected sep- (Mittermeier et al. 1997). The Western Ghats, being a biodi- arately, placed in zip-lock polythene bags and brought and versity hotspot in southern India, is a home for copious me- processed for isolation within 24 h of collection. A herbarium dicinal plants used ethnomedicinally. Polygonum chinense L., specimen has been deposited in the Botanical Survey of India commonly known as Chinese knotweed, belongs to the family (BSI), Western Range with the accession number 136243. Polygonaceae and is common in subtropical and warm tem- perate regions of Asia (Maharajan et al. 2012). Species of Isolation of endophytic fungi from plant parts Polygonum contain pharmaceutically important bioactive compounds (Ismail et al. 2012). The plant itself is used to treat Endophytic fungal isolations was carried out under aseptic various diseases viz., diarrhea, dyspepsia, hemorrhoid sprains, conditions according to Tejesvi et al. (2005). All plant parts tonsillitis, and poisonous snake bites (Chevallier 1996). In were subjected to surface sterilization by soaking in 70% (v/v) local health traditions, the decoction of stem and roots mixed ethanol for 1 min followed by sodium hypochlorite (3.5%) for with jaggery is given to women for post-natal care. Owing to 3 min and washed three–four times in sterile distilled water. the traditional usage of this plant in providing health benefits, Dried plant parts were cut into 1.0 cm × 0.1 cm × 0.1 cm this plant was selected for the isolation of fungal endophytes from the Talacauvery subcluster of Western Ghats. The isolation of endophytic fungi from P. chinense and the characterization of bioactive compounds are limited, though the plant is reported to possess antimicrobial compounds (Ezhilan and Neelamegam 2012). Hence, the primary aim of the study was to isolate and characterize the fungal endo- phytes from the plant parts of P. chinense and characterization of secondary metabolites of fungi responsible for the antimi- crobial and antioxidant potential by chromatographic, bio-au- tography, and ESI-MS/MS techniques. To the best of our knowledge, we believe that this study is important in the search for newer sources of bioactive compounds from the endophytic sources. Materials and methods Chemicals All phenolic acids and flavonoid standards, trolox, ascorbic acid, ABTS [2, 2′-azino-bis(3- ethylbenzthiazoline-6- sulphonic acid)], DPPH (1, 1-diphenyl-2- picrylhydrazyl), and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). FolinCiocalteu’s reagent was pur- chased from SRL Pvt. Ltd. (Mumbai, India). Solvents used for HPLC analyseswere ofHPLC grade.Sodiumhypochlo- rite, potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride, sodium nitrite, aluminum chloride, potassium per- sulfate, all other general chemicals, and solvents were of ana- Fig. 1 Polygonum chinense L. collected from the Western Ghats of southern India lytical grade. Triple distilled water was used wherever Ann Microbiol (2018) 68:595–609 597 pieces under sterile conditions. A total of 400 fragments was sequences with CLUSTAL W (Thompson et al. 1994)and a plated on water agar media (2% w/v) for the isolation of en- phylogenetic tree was constructed using default program. dophytic fungi, supplemented with the antibiotic streptomycin (50 mg/l) to suppress bacterial growth. The effectiveness of Fermentation and extraction of metabolites the surface sterilization was confirmed by the disinfected plant segment imprint method on PDA (potato dextrose agar) plates The pure culture of 10-day-old isolates was inoculated into from which no growth was observed (Schulz et al. 1998). The PDB in duplicates and kept for incubation for 3 weeks at 28 plates were sealed and incubated at 28 ± 2 °C with 12 h of ± 2 °C. The fermentation broth of each endophyte was extract- light and dark cycles for 4–6 weeks. The plates were observed ed three times with ethyl acetate at room temperature and periodically. Fungal hyphae emerging from the plated frag- further concentrated by a Rotary flash evaporator (Superfit ments of tissues were cultured on PDA at 28 ± 2 °C for 10– Model, PBU-6D, India). The residue obtained was designated 15 days and maintained as the pure culture at 4 °C for further as the crude dry extract and stored in glass vials, until use. use. Detection of antibacterial activity Identification of endophytic fungi Test organisms Microscopic slides of each endophyte were prepared by stain- ing with lactophenol cotton blue and examined under a light Two gram-positive bacteria viz. Bacillus subtilis (MTCC microscope (Labovision, India) for morphological analysis by 121), Staphylococcus aureus (MTCC 7443) and four gram- the standard identification keys (Pitt 1988; Barnett and Hunter negative bacteria viz. Escherichia coli (MTCC 729), 1998; Leslie and Summerell 2006;Domschet al. 2003). Pseudomonas aeruginosa (MTCC 7093), Enterobacter Based on the mycelial characteristics and spore structure ten aerogenes (MTCC 111), and Klebsiella pneumoniae (MTCC representative endophytic fungi were selected for molecular 661) were used. The test organisms were procured from the identification and further analysis of their metabolites. Department of Studies in Microbiology, University of Mysore, Karnataka, India. Molecular characterization by DNA sequence analysis of the ITS (internally transcribed spacer) region Antibacterial activity Isolation of genomic DNA and amplification The inhibitory effect of the endophytic fungal extract was tested by disc diffusion method (Bauer et al. 1966). The crude Actively growing mycelial plugs from morphologically dif- extract of endophytic fungi (250 μg per disc) was dissolved in ferent endophytic fungi were inoculated into potato dextrose dimethyl sulfoxide (DMSO) and tested on Mueller-Hinton broth (PDB). The isolates were grown in still culture at 28 ± agar medium seeded with the test bacterium at (5 mm diame- 2°Cfor 7–10 days. The genomic DNA was extracted from ter, Whatman no. 1) concentration. Streptomycin (10 μg/disc) the freeze-dried fungal mats by cetyltrimethylammonium was used as positive control and the paper disc loaded with bromide (CTAB) method with slight modifications only DMSO as the negative control. The test plates were in- (Ausubel et al. 1994). The DNA concentration was estimated cubated for 24 h at 35 ± 2 °C and the inhibition zone was by measuring the absorbance at 260 and 280 nm (Thermo measured. Scientific Nanodrop 2000/2000c, Bangalore, India). Target regions of the rDNA ITS 1 and 2 regions and 5.8 rRNA gene Determination of minimal inhibitory and minimum were amplified using primers ITS 1 and ITS 4. The amplifi- bactericidal concentration cation was performed in a total reaction volume of 25 μL containing 200 μmol/L dNTP, 10 pmol/μL of each primer, The minimal inhibitory concentration (MIC) was determined and 50 ng template DNA. The amplification conditions by modifying the broth dilution method (Xu et al. 2008), using consisted of an initial denaturation step at 95 °C for 3 min, sterile 96-well microplate (Tarsons, Kolkata, India). The wells followed by 35 cycles of 92 °C for 1 min, 50 °C for 1 min, were filled with a reaction mixture containing 90 μlbacterial 72 °C for 2 min and a final extension at 72 °C for 10 min. The suspensions (10 cfu/ml) and 10 μl of the test sample with amplified product was subjected to sequencing at Chromous different concentrations (2 mg/ml to 0.02 mg/ml). The culture Biotech Pvt. Ltd. Bangalore, India. The endophyte se- medium with 1% DMSO was used as the negative control and quences were aligned with the reference sequences using streptomycin sulfate (0.4 to 0.01 mg/ml) was a positive con- the BLAST algorithm and submitted to the NCBI GenBank trol. The microplates were incubated for 24 h at 35 ± 2 °C. nucleotide collection. The phylogenetic analysis of the en- After the incubation, 10 μl of the indicator 3-[4,5-dimethyl- dophytic strains was done by the alignment of multiple thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) 598 Ann Microbiol (2018) 68:595–609 (0.5 mg/ml phosphate buffer saline) was added to visualize the Determination of antioxidant capacity microbial growth. The lowest sample concentration at which no blue color appeared was determined as MIC. Wells con- DPPH radical scavenging activity taining MIC concentration and above were inoculated onto agar medium to check cell viability. The lowest concentration The quenching ability of DPPH was measured according to with no viable cells was determined as minimum bactericidal the procedure of Liu et al. (2007)withsome modifications. A concentration (MBC). methanolic solution of DPPH (0.001 mM) was added to the fungal extract. The absorbance was read at 517 nm spectro- photometrically, after 20 min of incubation. The scavenging Thin layer chromatography (TLC) and bio-autography activity was expressed as IC (μg/ml). Ascorbic acid was used as the standard. The scavenging ability of the DPPH The ethyl acetate extract of endophytic fungi with antibacterial radical was calculated by the formula: activity was subjected to TLC for the separation of the active compound. The solvent systems used as liquid phase was a %scavenging ¼ A –A =A 100: control sample control mixture of chloroform: ethyl acetate: formic acid in the ratio 20:16:4. The developed chromatograms were subjected to agar overlay bio-autography (Rahalison et al. 1991). Reducing power assay Identification of antibacterial compounds The reducing power was measured by the method of Oyaizu through LC-MS analysis (1986) with some modifications. The fungal extracts were mixed with phosphate buffer (0.2 M, pH 6.5) and potassium Analyses of antimicrobial compounds by LC-electrospray ferricyanide (1%, 0.5 ml). The mixture was then incubated at ionization (ESI)–MS were carried out using Synapt G2 High 50 °C for 20 min. After incubation, TCA (10% w/v, 0.5 ml) definition mass spectroscopy (Waters, Milford, MA, USA). was added and centrifuged at 3000 rpm for 10 min. To the Liquid chromatography separation was performed on a supernatant, the same volume of distilled water and ferric reversed-phase Acquity UPLC BEH C column (2.1 × chloride (0.1%, 300 μl) was added and the absorbance was measured at 700 nm. The activity was expressed as mg ascor- 50 mm) with a 1.7-μmparticle size (dp) (Waters™,Milford, MA, USA). The LC condition was as follows: solvent A bic acid (AA)/g dry extract. (0.1%) formic acid and solvent B (100%) acetonitrile. A gra- dient elution, 0–2.5 min 95% A, 5% B; 2.5–4.0 min 10% A, DNA protection assay 90% B; 4.0–5.0 min 95% A, 5% B was used with a flow rate 0.7 ml/min. The UV detector was set to an absorbance wave- DNA protection assay was conducted using calf thymus DNA length of 280 to 340 nm. The LC elute was introduced directly (Lee et al. 2002). Calf thymus DNA (5 μg) was incubated with into the ESI interface without flow splitting. The scan range of Fenton’s reagent (30 mM H O , 50 mM ascorbic acid and 2 2 ESI-MS was m/z 100–1000. The drying gas (N2) flow was 80 mM FeCl ) and fungal extracts (150 μg/ml). The mixture 500 L/h. The ESI capillary voltage was 3 kV ion mode. was analyzed by 1% agarose gel electrophoresis after 30 min of incubation at 37 °C. The positive control reaction contained calf thymus DNA and Fenton’s reagent. The negative control GC-MS analysis of antibacterial compounds consisted of the calf thymus DNA. The results were docu- mented using XR+ Molecular Imager Gel documentation sys- GC-MS (GC-17Awith QP5000 MS, Shimadzu Corp., Kyoto, tem (Bio-Rad, USA). Japan) was used to analyze the volatile and aliphatic com- pounds. An SPB-1 column (30 m × 0.32 mm with film thick- ness 0.25 μm; Sigma-Aldrich, St. Louis, MO, USA) was used Determination of total phenolic content and 2-μl samples dissolved in acetone was injected with split ratio 20:1. The analysis was carried out with oven temperature The total phenolic content of the fungal extracts was assessed programmed at 50 °C (hold 3 min) and raised to 260 °C at a according to the Folin–Ciocalteau (FC) method of Liu et al. rate of 5 °C/min. The injection port temperature was 250 °C, (2007) with some modifications. One microliter of FC reagent transfer temperature was 200 °C and the ion source tempera- and 2.0 ml of sodium carbonate (20%, w/v) was mixed with ture was 180 °C. Helium was used as the carrier gas at a flow the crude extracts. The mixture was incubated for 45 min in rate of 1 ml/min. The instrument was calibrated to scan range the dark. The absorbance was read at 765 nm (T- 60, TTL m/z 40–400. The compounds were identified by computer Technology, India). The total phenolic content of the extracts matching of their mass spectral fragmentation patterns with was expressed as mg of gallic acid equivalent (GAE)/g of the the NIST-MS library. extract. Ann Microbiol (2018) 68:595–609 599 Determination of total flavonoid content Data reported as the mean ± standard deviation (SD) of three independent replicates. Comparison of means was ana- The total flavonoid was determined according to the method lyzed with one-way ANOVA and Tukey-Kramer multiple of Barros et al. (2007). The fungal extract was mixed with comparisons tests using Graph Pad InStat 3.0. Any two data sodium nitrite (5%, 75 μl). After 5 min aluminum chloride were considered statistically significant at p < 0.05 and denot- (10%, 150 μl) and sodium hydroxide (1 M, 500 μl) were ed by different superscripts. added. The absorbance was measured at 510 nm. The content of flavonoid was calculated using a calibration curve of cate- chin and the results were expressed as mg of catechin equiv- Results alent (CE)/g of the extract. Isolation and identification of fungal endophytes Identification of antioxidative phenolics through HPLC analysis Endophytic fungi were isolated from the stem and leaf parts of P. chinense L. A total of 264 isolates distributed in 10 endo- Phenolic acids and flavonoids were separated by the reverse phytic species were recovered from 400 plant fragments. The phase analytical HPLC using Shimadzu LC-8A (Shimadzu isolates were identified with their spore morphology as well as Corporation, Tokyo, Japan) HPLC fitted with C column by analyzing the DNA sequence of the ITS region. The iden- (25 cm × 4 mm length, 5 μm, Kromasil, India) and a diode tified strains with their GenBank accession numbers, isolation array detector. An isocratic mobile phase consisting of number and colonization frequency (%CF) is depicted in water:formic acid:acetonitrile:(78:2:20 v/v) was delivered at Table 1. The result indicated that the stem part had number a flow rate of 1 ml/min to elute phenolic acids. The absorbance of isolates (141) than leaves (123). The ten endophytic species of phenolic acids was recorded at 280 nm. Flavonoids were belonged to seven genera. Three different species of Fusarium eluted with a gradient of solvent A (water adjusted with acetic and two of Alternaria were recovered. Fusarium solani acid to pH 2.8) and solvent B (acetonitrile) as described by showed the highest %CF (15.9) followed by Fusarium Das and Singh (2016). The gradient was linear to 10% B in chlamydosporum (15.2). The least %CF was recorded for 5 min, 23% B in 31 min, and 35% B in 43 min. The column Alternaria alternata. The phylogenetic relationships of isolat- was washed with 100% B for 6 min and equilibrated for 6 min ed endophytic strains were deduced by constructing a phylo- at 100% A to start the next sample. The absorbance was re- genetic tree using ITS sequences (Fig. 2). The isolates were corded at 260 nm. The peaks were identified by comparison clustered into five major clades, each of which was further with the standards. The HPLC water was purified by a Milli-Q divided into subclades except the clade of Cladosporium System. The LC fragments of all individual peaks were col- tenuissimum. lected in separate vials and used for ESI-MS/MS analysis. ESI-MS/MS analysis of antioxidative phenolics Antibacterial activity ESI-MS and MS/MS were performed using quadrupole time The antibacterial activity of strains was tested against six path- of flight (Q-TOF) mass spectrometer (Micromass Waters, ogenic bacteria and the appeared area of inhibition zone is Milford, MA, USA) with electrospray ionization (ESI). The presented in Table 2. The strains were further tested for MIC instrument was calibrated through a mass range of 100–1000 and MBC. The results are depicted in Table 3. Penicillium and operatedat negativemode [M-H] . The capillary voltage canescens (Fig. 3) exhibited the highest inhibition zone was 3 kV; source and desolvation temperatures were 120 and against all the pathogens except B. subtilis followed by F. 300 °C, respectively; cone gas (argon) and desolvation gas chlamydosporum. The inhibition zone formed by P. canescens (nitrogen) flow rates were 50 L/h and 500 L/h, respectively. is represented in Fig. 4. Bacillus subtilis was found to be MS/MS spectra were acquired using collision energy of 20 V. resistant against all the extracts. Alternaria longipes did not The m/z ratio, as well as fragmentation patterns, was used for show any activity. Curvularia geniculata did not depict any the confirmation of the phenolic acids and flavonoids. inhibition zone at 250 μg but showed inhibition at higher concentration against a gram-positive (P. aeruginosa)and a Data and statistical analysis gram-negative (E. coli) bacteria. All the endophytic fungal extracts showed inhibitory activity against E. coli, the concen- The colonization frequency (CF) was calculated by the fol- tration ranged from 2 to 0.06 mg/ml. Emericella nidulans lowing formula: % CF = [N /N ] × 100, where, N is the showed an inhibition zone only against E. coli. col t col number of tissue segments colonized by a fungus; N is a total Penicillium canescens showed good antibacterial activity number of tissue segments plated (Nalini et al. 2014). against all the pathogens. Therefore, the extract of P. 600 Ann Microbiol (2018) 68:595–609 Table 1 Taxonomic Endophytic fungi Code Accession no. Leaf* Stem* Total %CF identification and percent colonization frequency (%CF) of I %CF I %CF the fungal endophytes isolated from P. chinense Bipolaris sorokiniana PC-WG-01 KY024401 - - 11 5.5 2.8 Penicillum canescens PC-WG-02 KY052774 - - 14 7.0 3.6 Fusarium chlamydosporum PC-WG-03 KY072925 27 13.5 34 17.0 15.2 Cladosporium tenuissimum PC-WG-04 KY039168 - - 27 13.5 6.9 Fusarium solani PC-WG-05 KY039169 25 12.5 39 19.5 15.9 Alternaria alternata PC-WG-06 KY039171 11 5.5 - - 2.5 Curvularia geniculata PC-WG-07 KY052771 26 13.0 - - 6.2 Fusarium incarnatum PC-WG-08 KY052774 5 2.5 7 3.5 2.8 Emericella nidulans PC-WG-10 KY039167 16 8.0 - - 4.1 Alternaria longipes PC-WG-11 KY039170 22 11 9 4.5 7.7 *200 fragments were plated from leaf and stem fragments respectively. I number of isolates. B-^ indicates the absence of the endophytic fungi in the respective plant part canescens was further characterized for the identification of aliphatic compounds. LC-MS result revealed the presence of antimicrobial compounds by TLC and bio-autography. quercetin in the TLC fraction of the extracts. GC-MS of the TLC fraction revealed four peaks corresponding to the pres- ence of 1,7-dimethyl-4-(1-methyl ethyl) cyclodecane TLC and bio-autography (germacrene), phthalic acid ester, Cyclopentane, 1,1,3,3- tetramethyl-and 1-Nitrododecane (Table 4). The chromato- The presence of antimicrobial compounds in P. canescens gram of liquid and gas chromatography with mass spectra is extract was detected by TLC and bio-autography methods. represented in Fig. 6. This aided direct visualization of an active compound with an inhibition zone at Rf − 0.88 on the chromatogram (Fig. 5a). The antibacterial activity was confirmed by the for- Antioxidant capacity mation of halo around the chromatogram post-spray with 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium DPPH radical scavenging capacity bromide (MTT) (Fig. 5b), the reliable indicator cellular meta- bolic activity. The compound was collected carefully and fur- The DPPH radical is almost stable and extensively used for ther characterized by liquid chromatography (LC), gas chro- the antioxidant activity. The radical scavenging activity is pre- matography (GC), and mass spectral analysis. sented as 50% scavenging activity (IC ) in Table 5. The re- sults showed that the IC value of the fungal extracts varied Identification of antimicrobial compounds from 73.6 ± 3.3 to 1114.9 ± 71.1 μg/ml. Penicillium through LC-MS and GC-MS analysis canescens showed the highest scavenging activity with 73.6 ±3.3 μg/ml, followed by F. chlamydosporum (126.8 ± 6.7 μg/ The identification of phenolic compound was done by LC- ml). Bipolaris sorokiniana and A. alternata exhibited the IC MS, while GC-MS was employed to identify volatile and value of 319.2 ± 40.2 and 366.2 ± 15.8 μg/ml respectively. Fig. 2 Phylogenetic tree based on ITS sequences of the endophytic fungal isolates. The left side indicates the sequence of the nearest strain. The scale bar indicates 0.1 substitutions per nucleotide position Ann Microbiol (2018) 68:595–609 601 Table 2 Antibacterial activity of fungal endophytes isolated from P. chinense against six pathogenic bacteria Fungal strains Pseudomonas Bacillus Staphylococcus Escherichia Enterobacter Klebsiella extract aeruginosa subtilis aureus coli aerogenes pneumoniae P. canescens 19.5 ± 0.9 - 18.6 ± 1.0 21.9 ± 2.1 23.4 ± 0.7 15 ± 1.1 F. chlamydosporum 12.8 ± 1.1 - 14.0 ± 0.5 16.8 ± 0.6 21.2 ± 0.1 13.5 ± 0.9 A. alternata 11.3 ± 0.2 - 9.9 ± 0.3 13.8 ± 0.3 8.9 ± 0.2 11.2 ± 0.1 B. sorokiniana 14.4 ± 1.3 - 10.5 ± 0.4 12.2 ± 1.4 9.2 ± 0.1 10.2 ± 0.5 F. solani 14.6 ± 0.2 - 10.6 ± 0.4 11.6 ± 1.0 - 11.7 ± 0.5 C. geniculata -- - - - - C. tenuissimum 11.7 ± 0.9 - 9.5 ± 0.7 - - 10.5 ± 0.2 E. nidulans - - - 7.3 ± 0.1 - - F. incarnatum - - - 9.0 ± 0.1 8.5 8.3 ± 0.3 A. longipes -- - - - - Streptomycin* 33 ± 0.3 32 ± 0.1 31.5 ± 0.5 20 ± 0.1 22 ± 0.1 30 ± 0.2 Data are reported as mean ± SD of three independent analyses (n = 3). *Streptomycin 10 μg disc used; B-^ indicates the absence of inhibition zone by the endophytic fungal extract in the disc diffusion method Reducing power assay Among the endophytic fungal strains, P. canescens and F. chlamydosporum exhibited high total phenolic content and The reducing power of ferric ion to ferrous ion by the fungal antioxidative capacities. Hence the phenolic compounds pres- extracts is represented in terms of ascorbic acid equivalent ent in the crude extracts were further characterized by HPLC (Table 5). The values of reducing power assay ranged from and ESI-MS/MS techniques. 12.8 ± 0.2 to 95.8 ± 2.5 mg AA/g dry extract. Penicillium canescens showed the highest activity (95.8 ± 2.5 mg AA/g dry extract) followed by F. chlamydosporum (84.7 ± Phenolic and flavonoid content 2.1mgAA/gdry extract)and A. alternata (48.2 ± 2.0 mg AA/g dry extract). The total phenolic content (TPC) of the extracts is represented in Table 5. TPC of the extracts ranged from 9.9 ± 0.7 to 78.9 ± 2.5 mg GAE/g dry extract. Penicillium canescens extract DNA protection assay showed the highest total phenolic content (78.9 ± 2.5 mg GAE/g dry extract) followed by F. chlamydosporum extracts Among the five isolates, P. canescens extract visibly showed (50.3 ± 0.5 GAE/g dry extract respectively). A. longipes ex- the DNA protection ability by inhibiting its fragmentation hibited the least phenolic content (9.9 ± 0.7 5 mg GAE/g dry through the scavenging of –OH radicals generated by extract). Fenton’s reagent (Fig. 7). Table 3 Minimal inhibitory Endophytic fungal P. aeruginosa S. aureus E. coli E. aerogenes K. (MIC mg/ml) concentration and strain extract pneumoniae minimum bactericidal concentration (MBC mg/ml) of MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC fungal endophytes P. canescens 0.1 0.12 0.1 0.12 0.08 0.1 0.06 0.08 0.14 0.16 F. chlamydosporum 0.14 0.16 0.18 0.2 0.12 0.14 0.08 0.1 0.18 0.2 A. alternata 0.18 0.2 0.2 0.4 0.14 0.16 0.4 0.6 0.18 0.2 B. sorokiniana 0.12 0.14 0.18 0.2 0.18 0.2 0.4 0.6 0.2 0.2 F. solani 0.18 0.2 0.18 0.18 0.18 0.2 - - 0.16 0.18 C. geniculata 1.8 2 - - 1.6 1.6 - - - - C. tenuissimum 0.16 0.18 0.4 0.6 1.4 1.6 - - 0.2 0.4 E. nidulans -- - - 1.2 1.4 -- 2.0 - F. incarnatum 2.0 - - - 0.4 0.6 0.6 0.8 0.6 0.8 A. longipes -- - - 2 - -- -- B-^ indicates the absence of inhibition of endophytic fungal extracts in the MIC and MBC assay 602 Ann Microbiol (2018) 68:595–609 Fig. 3 Penicillium canescens isolated as an endophyte from the stem of P. chinense L. a Velutinous colonies on PDA medium b.Light microscopy view (× 40 magnification) with biverticillate branching conidiophores with phialides and metulae bearing chains of globose roughened conidia Flavonoid was detected in seven endophytic strains Identification of antioxidative phenolics (Table 5). The total flavonoid content (TFC) ranged through HPLC and ESI-MS/MS analysis from 5.9 ± 0.4 to 33.6 ± 1.0 mg CE/g dry extract. Penicillium canescens exhibited high flavonoid content The extracts of P. canescens and F. chlamydosporum were (33.6 ± 1.0 mg CE/g dry extract). TFC of B. sorokiniana characterized for the phenolic compounds. HPLC analysis and A. alternata was documented as 17.5 ± 0.5 and 14.6 revealed that P. canescens extract contained phenolic acids ± 0.2 mg CE/g dry extract respectively. The lowest TFC as well as flavonoids whereas, F. chlamydosporum contained was recorded for A. longipes. Flavonoid was not detect- solely phenolic acids (Fig. 8). The compounds were isolated ed in C. geniculata, Cladosporium tenuissimum,and individually and subjected to ESI-MS and MS/MS analyses Fusarium incarnatum extracts. for the confirmation of their presence. The concentrations of Fig. 4 Inhibition zone formed by P. canescens extract. Penicillium canescens extract (250 μg/ml) was tested for the antibacterial potential against the test bacteria seeded onto Mueller-Hinton agar (MH) by the disc diffusion assay. The plates were incubated for 24 h at 35+ °C and the presence of or absence of the inhibitory zone around the disc was measured and represented. S streptomycin disc, C negative control, P P. canescens extract, A A. longipes extract Staphylococcus aureus Ann Microbiol (2018) 68:595–609 603 Fig. 5 Bio-autography of P. canescens extract. Penicillium canescens extract was subjected to TLC. The chromatogram was placed on MH agar plate and molten agar seeded with the bacterial suspension of 1.5 × 10 cfu/ml was poured on the chromatogram. Post- solidification, the plates were place at 4 °C for 3 h and observed for the halo around the chromatogram (a). The antibacterial activity was confirmed by the formation of halo around the chromatogram post-spray with MTT (b) individual phenolic compounds in both the extracts and MS/ biodiversity. Many researchers in the recent past have ex- MS fragmentation patterns are depicted in are presented in plored the richness of endophytes instead of using the plants Table 6. Caffeic acid (CA) was found in both the extracts. per se (Nalini et al. 2014; Uzmaa et al. 2016; Das et al. 2017). Vanillic acid (VA) was present only in P. canescens,whereas The endophytes are rich sources of bioactive compounds. ferulic (FA) in F. chlamydosporum. Penicillium canescens Hence, the present study deals with the isolation of endophytic also contained a flavonoid compound, quercetin (6.9 ± fungi and their potential bioactive compounds. A total of ten 0.3 mg/of extract). endophytic fungal strains distributed in seven genera viz., The peak at the retention time (RT) 5.3 ± 0.3 min with a Fusarium, Alternaria, Penicillium, Curvularia, Emericella, major ion 179 m/z and a fragment of 135 m/z [M-H-44] due to Cladosporium,and Bipolaris were identified. In order to iden- the loss of a –COO group, i.e., decarboxylation was confirmed tify the fungal strains to the species level, both morphological as CA. The peak at RT 6.0 ± 0.3 min had a major ion of 167 m/ and phylogenetic analyses using molecular tools were used. z and a fragment of 152 m/z due to the loss of a CH moiety, The taxonomic identification of dark septate endophytes is i.e., demethylation [M-H-15] on the aromatic benzene ring, difficult due to the lack of clearly defined sexual stages, the thus confirmed as VA. FAwas confirmed at RT 11.3 ± 0.1 with absence of asexual spores and lack of identifiable morpholog- major ion of 193 m/z and 3 fragments of 178 m/z (demethyl- ical traits (Samaga and Rai 2016). Hence, sequence analysis of ation), 149 m/z (decarboxylation) and 134 m/z (both demeth- the strains is essential. The blast result of ITS region showed ylation and decarboxylation). The flavonoid molecule querce- 98–100% similarity and almost all the fungi were previously tin was identified at RT 42.8 ± 3.1, produced fragments of 151 reported as endophytes. Polygonum chinense was previously and 179 m/z as a result of cleavage of the heterocyclic C-ring reported to host 15 endophytic fungal taxa with Fusarium by Retro-Diels-Alder rearrangement (Sun et al. 2007). exhibiting high fungal diversity (Huang et al. 2008a). In the present study, ten morphologically distinct taxa with high col- onization frequency was recorded for two Fusarium species, which again substantiates the dominance of Fusarium. Apart Discussion from Fusarium, A. longipes recorded high CF (7.7%) while other species of Alternaria showed lowest CF (2.5%). Polygonum chinense L. is a medicinal plant, found growing in Alternaria sp. were previously isolated as common endophyt- the Western Ghats region of India. Being a biodiversity ic fungi from tropical, subtropical, and temperate regions hotspot, this region attracts researchers to unfold the treasure (Guo et al. 2004; Jordaan et al. 2006; Aly et al. 2007). In house of mother nature, causing irreparable damage to Table 4 Compounds identified Peak Retention time Molecular Chemical Compound name % through GC-MS from P. (min) weight structure area canescens extract 127.09 126 C H Cyclopentane, 1,1,3,3-tetramethyl- 1.507 9 18 230.79 306 C H O Phthalic acid, butyl hexyl ester 0.909 18 26 4 3 31.1 210 C H 1,7-Dimethyl-4-(1-methyl 1.36 15 30 ethyl)cyclodecane 434.77 215 C H NO 1-Nitrododecane 0.679 12 25 2 604 Ann Microbiol (2018) 68:595–609 Fig. 6 Mass spectra of the antibacterial compound. a Quercetin. b Phthalic acid, butyl hexyl ester. c Germacrene [1,7- dimethyl-4-(1-methyl ethyl) cyclodecane] addition, Aly et al. (2008) reported an endophytic Alternaria array of metabolites synthesized by the endophytes confers sp. from another species Polygonum (P. senegalense). The plants with more resistance. Plants that inhabit specific endo- differences in the diverse endophytic taxa isolated from P. phytes are often able to grow faster due to the production of chinense sampled from distinct regions can be attributed to phytohormones enabling them to dominate in a particular en- the geographical location of the plant. vironment (Kaul et al. 2012). A variety of relationships exist between the fungal endo- Polygonum chinense is traditionally used to treat various phytes and its host plant. It can be symbiotic, mutualistic, diseases. Huang et al. (2008b) reported the plant extract to antagonistic, or slightly pathogenic (Huang et al. 2008a). exhibit strong antibacterial activity against Bacillus cereus Due to this interaction with the host plant, the endophytic (MIC, 2.50 mg/ml), Listeria monocytogenes (MIC, 5.00 mg/ fungi can produce a plethora of substances for potential use ml), and S. aureus (MIC, 5.00 mg/ml). Maharajan et al. (2012) in modern medicine, agriculture, and industry (Mitchell et al. reported that P. chinense extract possess strong antimicrobial 2008). The endophytes are synergistic to their host. The en- activity against B. subtilis, S. aureus, P. aeruginosa, E. coli, dophytes, against their nutritional benefits, release metabolic and Aspergillus niger. In the present study, the isolated endo- substances beneficial to the host plant in resisting the abiotic phytes were tested for the antibacterial activity, where 80% of stresses and attacks from herbivore pathogens and insects. The them showed antibacterial potency against one or more Ann Microbiol (2018) 68:595–609 605 Table 5 Total phenolic content Fungal strains/ TPC (mg GAE/g TFC (mg CE/g Reducing power DPPH radical (TPC), total flavonoid content extracts dry extract) dry extract) (mg AA/g scavenging capacity (TFC), and antioxidant capacity dry extract) (IC μg/mL) of fungal endophytes from P. 50 chinense a a a b P. canescens 78.9 ± 2.5 33.6 ± 1.0 95.8 ± 2.5 73.6 ± 3.3 b d b c F. 50.3 ± 0.5 12.5 ± 0.5 84.7 ± 2.1 126.8 ± 6.7 chlamydospo- rum c c c d A. alternata 24.9 ± 1.1 14.6 ± 0.2 48.2 ± 2.0 319.2 ± 40.2 d b d d B. sorokiniana 22.7 ± 0.8 17.5 ± 0.5 44.3 ± 0.8 366.2 ± 15.8 e e e e F. solani 21.4 ± 1.9d 10.1 ± 0.1 40.0 ± 1.1 440.5 ± 40.8 e e e C. geniculata 19.3 ± 0.8 - 37.6 ± 1.1 472.5 ± 8.7 f g f C. tenuissimum 17.8 ± 1.6 - 25.6 ± 0.8 542.6 ± 44.0 f f f f E. nidulans 16.5 ± 0.9 7.5 ± 0.3 30.1 ± 1.1 581.1 ± 35.2 g h g F. incarnatum 13.6 ± 0.6 - 18.8 ± 1.0 766.9 ± 33.1 h g i h A. longipes 9.9 ± 0.7 5.9 ± 0.4 12.8 ± 0.2 1114.9 ± 71.1 Ascorbic acid 7.7 ± 0.02 Data are reported as mean ± SD of three independent analyses (n = 3). Mean with the different superscript within a column are significantly different (p < 0.05) by one-way ANOVA test. B-^ indicates the absence of activity in the respective assays by the endophytic fungal extracts pathogenic bacteria. Unlike plant extracts, the endophytes The endophytic P. canescens was previously reported to demonstrated activity against B. subtilis. Penicillium exhibit antibacterial activity against E. coli, B. subtilis,and canescens extract exhibited strong antibacterial activity S. aureus (Malhadas et al. 2017) without determining the against E. coli (MIC, 0.06 mg/ml), P. aeruginosa (MIC, MIC of the extract. In order to determine the exact potency 0.08 mg/ml), S. aureus (MIC, 0.1 mg/ml), and K. pneumoniae of the extract, the MIC is an important parameter to perform (MIC, 0.1 mg/ml). Fusarium chlamydosporum, A. alternata, and the present study demonstrates the same. and B. sorokiniana also showed antibacterial potency against AliteraturesurveybyYuetal. (2010) reveals species of all pathogens employed except B. subtilis (MIC, 0.2–0.08 mg/ Penicillium produce antimicrobial compounds. However, ml). The results clearly showed that the endophytes are might- most authors have reported the activities of endophytic P. ier antibacterial agents than their host plant. canescens against phytopathogenic fungi (Bertinetti et al. 2009; Nicoletti et al. 2014). In the present study, we have attempted the characterization of compounds responsible D+F PC FC BS AA for antibacterial activities. Since P. canescens showed strong activity against the bacterial pathogens, the active compounds of this endophyte were isolated through bio- autography and identified through LC-MS and GC-MS. A flavonoid compound-quercetin along with germacrene and phthalic acid ester (1,2-benzene dicarboxylic acid, butyl hexyl ester) were identified. Endophytes are chemical syn- thesizers inside plants (Owen and Hundley 2004). In other words, they play aroleasaselectionsystemfor microbes to produce low toxic bioactive substances for higher organ- isms (Strobel 2003). Alvin et al. (2014) reported that en- dophytes are able to produce a similar secondary metabo- lite as their host. In previous studies, the plant extract of P. chinense was also found to contain quercetin-3-glucoside Fig. 7 DNA protection assay of endophytic fungal extracts. Calf thymus and quercetin-3-galactoside (Huang et al. 2008b). DNA (5 μg) was incubated with Fenton’s reagent and the endophytic Quercetin has previously been reported to exert antibacte- fungal extracts (150 μg/ml). After the incubation at 37 °C for 30 min, the mixture was run through agarose gel (1%) electrophoresis. The results rial activity against S. aureus and S. epidermidis (Hirai et were documented using XR+ Molecular Imager Gel documentation al. 2010). The plasticizer compound 1,2-benzene dicarbox- system (Bio-Rad, USA). D calf thymus DNA, (D + F) DNA + Fenton’s ylic acid ester has also been reported from P. chinense plant reagent, PC P. canescens,FC F. chlamydosporum,BS B. sorokiniana, AA A. alternata by GC-MS analysis (Ezhilan and Neelamegam 2012). Both 606 Ann Microbiol (2018) 68:595–609 b e Fig. 8 HPLC chromatograms of phenolic compounds. a Separation of phenolic acids in the standard mixture (at 280 nm). b P. canescens. c F. chlamydosporum. d Separation of quercetin in a standard mixture (at 260 nm). e P. canescens. 1 caffeic acid, 2 vanillic acid, 3 ferulic acid, 4 quercetin bioactive compounds are produced by the endophytic P. These pieces of evidence substantiate endophytes as alter- canescens,isolatedfrom P. chinense in the present study. native sources of plant metabolites. Phthalic acid ester Table 6 Concentration and ESI- Source fungal RT of m/z [M- MS/MS Compound Concentration (mg/g of MS/MS analysis of phenolic strain peak H] fragment identified extract) compounds of endophytic fungal extracts P. canescens 5.3 ± 0.3 179 134 Caffeic acid 4.3 ± 0.1 F. 5.5 ± 0.3 179 134 Caffeic acid 3.3 ± 0.02 chlamydospo- rum P. canescens 6.3 ± 0.3 167 152, 123 Vanillic acid 2.7 ± 0.07 F. 11.3 ± 0.1 193 178, 149, Ferulic acid 0.95 ± 0.001 chlamydospo- and 134 rum P. canescens 42.8 ± 3.1 300 151, 179 Quercetin 6.9 ± 0.3 Data are reported as mean ± SD of two independent analyses (n =2) Ann Microbiol (2018) 68:595–609 607 reported in the present study was found to be antimicrobial Conclusions by previous researchers (Barakat and Beltagy 2015). In addition, Al-Bari et al. (2006) isolated phthalate derivative In order to cater to the need of constant development of novel from Streptomyces bangladeshiensis andreportedto have antibiotics against the drug-resistant pathogens, the studies on good potency against B. subtilis and S. aureus,whereas bio-prospecting while selecting the most suitable sources are Uddin et al. (2013) reported the compound to possess an- keys to success. The Western Ghats of India, popularly known tiviral activity against dengue and chikungunya viruses. as biodiversity hotspot, is a source of many medicinal plants Germacrene identified in this study is a sesquiterpene. which are yet to be explored. Based on the background that Germacrene B and germacrene D are previously reported many plant bioactive compounds are actually produced by from Guatteria australis leaf-essential-oil showed anti- their microbial symbionts, exploration of the endophytes from bacterial activity against S. aureus and E. coli (MIC these medicinal plants will assist in isolating and producing 250 mg/ml) and demonstrated antioxidant activity their active components. The present study explores various (Siqueira et al. 2015). Terpenes also have unique antiox- fungi isolated as endophytes from the plant P. chinense.Tothe idant activity in their interaction with free radicals best of our knowledge, these endophytes were not fully pro- (Dillard and German 2000). Penicillium canescens is re- filed for their bioactive metabolites. The strain, P. canescens ported to have antioxidant capacity against DPPH radical was found to have high antibacterial properties against gram- scavenging (IC 73.6 ± 3.3 μg/ml) and reducing ability of positive and gram-negative pathogens as well as significant 3+ 2+ Fe to Fe . The antioxidative capacity is consistent with antioxidative capacities. The strain F. chlamydosporum, the total phenolic content. displayed significant anti-radical and ferric reducing capaci- The oxidative stress causes DNA damage and interrup- ties. The secondary metabolites characterized by chromatog- tion of the cell cycle (Pizarro et al. 2009) which can lead raphy and mass spectroscopy revealed the presence of to other chronic diseases. Penicillium canescens alone ex- germacrene, phthalic acid esters, short-chain hydrocarbons, hibited DNA protection ability against –OH-induced and phenols. The bioactivities of these compounds are well DNA damage and was ascertained to be a potential recognized in the literature. Hence, our findings encourage the DNA protective agent. exploration of these fungi for bioprospecting in search of nov- Flavonoid was detected in seven endophytic strains among el compounds which are safe and efficacious for human use. the ten strains. The extract of P. canescens exhibited highest Therefore, the potential of drug discovery using endophytes TFC (33.6 ± 1.0 mg CE/g). Flavonoids were reported as im- from traditional medicinal plants is immense. portant bioactive constituents of Polygonum species (Huang et Funding information This work was supported by the grant from The al. 2008b) hence rationalizes the presence of high flavonoid University Grants Commission–Major Research Project (Grant number content in endophytes. F.No. 40-307/2011 (SR) dt. 30.06.2011), from the Government of India. HPLC analysis of P. canescens and F. chlamydosporum The Institution of Excellence, University of Mysore is thankfully ac- extracts revealed that these isolates contained various phe- knowledged for the facilities provided. nolic acids. The presence of bulky side chains (Jing et al. 2012) and the number of hydroxyl moieties attached to Compliance with ethical standards the aromatic ring of the benzoic or cinnamic acid mole- Conflict of interest The authors declare that there are no conflicts of cules (Karamac et al. 2005) was favorable for the DPPH interest. radical scavenging activity of phenolic acids. Both the extracts contained CA that have two –OH groups and a bulky side chain (CH=CHCOOH) may be responsible for References the high scavenging activity of the extracts. Other than CA, P. canescens contained VA (2.7 ± 0.07 mg/g of ex- Al-Bari MAA, Sayeed MA, Rahman MS, Mossadik M. 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Annals of Microbiology – Springer Journals
Published: Aug 24, 2018
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