Endophytic fungi of selected Nigerian plants are important sources of bioactive products with enormous potentials for the discovery of new drug molecules for drug development. Pseudomonas aeruginosa is one of the major causes of healthcare-associated bacterial infections, leading to increased mortality and morbidity. In this study, isolated endophytic fungi from Cola nitida were screened for anti-pseudomonas properties. Endophytic fungi associated with healthy leaves of C. nitida were isolated using standard methods. Fungi were identified through their morphological, cultural and microscopic characteristics. The fungi were subjected to solid-state fermentation and secondary metabolites extracted using ethyl acetate and concentrated under vacuum. The crude extracts were screened for antimicrobial activity against clinical and laboratory strains of Pseudomonas aeruginosa using the agar diffusion method. The bioactive components of the fungal extracts were identified using High-Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) analysis. Three endophytic fungi; Acremonium sp., Aspergillus sp. and Trichophyton sp. were isolated. At 1 mg/ml, extracts of the three fungi displayed antipseudomonal activity with inhibition zone diameter ranging from 6 - 4 mm. HPLC-DAD analysis revealed the presence of compounds, such as 4-hydroxyphenyl acetate. indole-3-acetic acid, and protocatechuic acid among others in the fungal extracts. The findings in this study reveal that endophytic fungi associated with C. nitida possess promising antipseudomonal properties. This finding can open new doors for the discovery of new agents against P. aeruginosa.
Endophytes refer to microorganisms that grow intercellularly and asymptomatically within living tissues establishing a mutual relationship with the host plant (1). Currently, endophytes are viewed as an outstanding source of bioactive natural products, because many of them are occupying millions of unique biological niches growing in so many unusual environments.
The mortality due to drug-resistant strains of Pseudomonas aeruginosa infections is increasing. It has become increasingly clear that resistance development in P. aeruginosa is multifactorial, with mutations in genes encoding porins, efflux pumps, penicillin-binding proteins, and chromosomal β-lactamase, all contributing to resistance to β-lactams, carbapenems, aminoglycosides, fluoroquinolones and sulphonamides (2, 3, 4, 5). Due to the emergence of MDR pathogens, it is of ultimate importance to develop new antimicrobial agents that will inhibit or completely kill this microorganism.
Almost all plant species habour one or more endophytic organisms (6). Cola nitida (Vent.) Schott & Endl. plant is popular in African traditional medicine and belongs to the family Sterculiaceae (7). They are reputed to exhibit several potent pharmacological activities such as antioxidant, antibacterial, antifungal, antiviral, and anti-inflammatory properties (8, 9).
The resistance exhibited by P. aeruginosa may be as a result of excessive antibiotic administration, leading to the accumulation of antibiotic resistance, cross-resistance between antibiotics and the appearance of multidrug-resistant P. aeruginosa. Presently, an intensive search is ongoing for newer, cheaper and more effective pharmaceutical products to deal with health disorders, and scientists believe that endophytic fungi possess novel compounds that are active pharmaceutical substances. The aim of this study, therefore, is to isolate endophytic fungi from C. nitida, extract their secondary metabolites and screen them for antipseudomonal activity.
Materials and Method
Isolation and purification of endophytic fungi
Fresh leaf samples of Cola nitida were collected from mature healthy plants from Nsukka, in Enugu State located in South-Eastern Nigeria. The plant leaves were washed thoroughly in running tap water and then cut into small fragments (about 1 cm2). The leaf fragments were surface-sterilized by immersion in 2% sodium hypochlorite solution for 2 min, 70% ethanol for nearly 2 min, before a final rinse in sterile water for 5 min. The leaf fragments were transferred into malt extract agar (MEA) plates supplemented with chloramphenicol. The Petri plates were then incubated at a temperature of 27°C and fungal growths from the leaf fragments were monitored. The efficacy of surface sterilization was tested by pressing the sterilized segments on to the surface of the MEA medium and removed immediately. The absence of growth of any fungi on the medium after incubation confirmed that the surface sterilization procedure was effective (10). Hyphal tips from several distinct colonies emerging from the leaf segments were sub-cultured on fresh MEA plates to obtain pure colonies.
Identification of fungi isolates
For the characterization of the morphology of fungal isolates, slide preparations from cultures were stained with lactophenol cotton blue reagent and examined with a bright-field and phase-contrast microscope. Morphological identification of the fungal isolates was according to the standard taxonomic key which includes colony diameter, texture, colour, margin character and the dimensions, colony reverse and microscopic characteristics including conidiophore, the structure of hypha and conidia (11).
Fermentation and extraction of secondary metabolites
Solid-state fermentation was carried out as previously described by Okoye et al. (12) in 1000 ml Erlenmeyer flasks containing 100 g of rice media (200 ml of water was added to the rice and then autoclaved at 121oC at 15 psi for 30min). The flasks were inoculated with 3 mm diameter agar blocks containing pure fungi endophyte and incubated at 28°C for 21 days. The culture media and the mycelia were extracted in ethyl acetate and separated by filtration. The organic phase was vacuum-concentrated at 40°C under reduced pressure using a rotary vacuum evaporator to obtain the extracts.
Various clinical isolates of P. aeruginosa were obtained from orthopedic wound infection, urine, sputum, and a vaginal swab. The samples for isolation were obtained from the National Orthopedic Hospital in Enugu, Nigeria. An isolate obtained from the laboratory stock culture was also used for the study. The identities of the organisms were confirmed at the Pharmaceutical Microbiology Laboratory of Nnamdi Azikiwe University, Agulu, using standard morphological and biochemical characteristics of the organism.
Preliminary antimicrobial screening of the endophytic fungal extracts for antipseudomonal activity was carried out using the agar well diffusion assay method as described by Onyegbule et al. (13). Working concentrations (1 mg/mL) of the fungal extracts were prepared by dissolving the extracts in dimethyl sulphoxide (DMSO 100% v/v). Standardized broth cultures of test bacterial isolates (P. aeruginosa) were spread aseptically onto the surface of Mueller Hinton Agar (MHA) using sterile cotton swabs. All culture plates were allowed to dry for about 5 min and agar wells were made by using a sterile cork-borer (6 mm in diameter). These wells were respectively filled with 20 μL of the fungal extracts and controls. The plates were then kept at room temperature for 1 h to allow the agents to diffuse into the agar medium and incubated accordingly. Gentamicin (10 μg/mL) was used as the positive control, while DMSO (100% v/v) was used as the negative control. The MHA plates were then incubated at 37oC for 24 h. The inhibition zones diameters (IZDs) were measured and the size of the well (6 mm) was deducted from the values obtained to get the actual IZDs. This was conducted in triplicate and the mean IZDs were calculated and recorded.
HPLC analysis was carried on the fungal extracts as described by Eze et al. (14). A Dionex P580 HPLC system coupled to a photodiode array detector (UVD340S, Dionex Softron GmbH, Germering, Germany) was used in the analysis. The separation column (125 x 4 mm; length x internal diameter) was prefilled with Eurospher-10 C18 (Knauer, Germany) and a linear gradient of nanopore water (adjusted to pH 2 by addition of formic acid) and methanol was used as eluent. A weight of 2 mg of each fungal extract was reconstituted with 2 mL of HPLC grade methanol, and the mixture sonicated for 10 min, and thereafter centrifuged at 3000 rpm for 5 min. A volume of 100 μL of the dissolved sample was then transferred to a vial containing 500 μL of HPLC grade methanol, and the vial was put in the HPLC machine for analysis. The detection was at 235 nm. The absorption peaks of the fungal extracts were analyzed by comparing it with those in the HPLC-UV/Vis database.
Data processing and analysis
Data were expressed as mean ± standard deviation (SD) for three parallel experiments. The collection of information, analysis of data and graph was made using Microsoft Excels 2016 software. Descriptives statistics were performed using SPSS version 20.
A total of three (3) endophytic fungi were isolated from leaf segments of the C. nitida plant and labeled Cn 1-3. In Fig. 1, the three isolates exhibited different characteristic colonies and microscopic morphology on MEA. All isolates were identified based on their morphological and microscopical characters as belonging to different gene rna,amely Cn1 (Aspergillus sp.) Cn2 (Aspergillus sp.) and Cn3 (Trichophyton sp.) (Table 1).
Cultural morphology and microscopic features of the endophytic fungi isolates from C. nitida
Identification of principal secondary metabolites using HPLC
A list of the detected compounds contained in the individual fungal extracts and their previously reported biological activities is shown in Table 2. From the result obtained, four (4) known compounds were identified namely Protocatechuic acid, Indole-3-acetic acid, P-hydroxybenzoic acid, and 4-hydroxyphenyl acetate acid.
The antimicrobial activities of compounds detected in the fungal extracts
The chromatogram and structures of Cn3 fungal extracts are presented Fig. 2a and b. It reveals the peaks and structures for protocatechuic acid and P-hydroxybenzoic acid.
At 1 mg/ml, extracts the three fungi displayed varying antipseudomonal activity against the different P. aeruginosa clinical isolates, with inhibition zone diameter ranging from 6 - 3 mm (Fig. 3). Laboratory ate (Ps.a A) and urine isolates (Ps.a C) were susceptible to 100% of the fungi extracts understudy, at 1 mg/ml concentration. The extract of Cn1 exhibited the best antipseudomonal activity against 80% of the tested P. aeruginosa isolates. It was observed to be resisted by only P. aeruginosa isolated from a vaginal swab. The mean values of the zones of inhibition obtain are statistically highly significant as P < 0.001.
Endophytic fungi of selected plants are important sources of bioactive compounds with enormous potential for the discovery of new drug molecules. All fungi endophytes isolated from the plants in this study have been previously isolated as endophytes of plants. Aspergillus sp., Acremonium sp., (27) and Trichophyton sp. (28), have all been reported as endophytes isolated from various host plants. We therefore, conclude that all isolated fungi are endophytes.
The antipseudomonal activity displayed by teh endophytic fungal extracts in this study can be attributed to the antimicrobial compounds present in the extracts. A list of detected bioactive compounds present in the individual fungal extracts with antimicrobial activities is shown in Table 2. From the result obtained, four (4) known compounds were identified as Protocatechuic acid, Indole-3-acetic acid, P-hydroxybenzoic acid, and 4-hydroxyphenyl acetic acid. These compounds have been reported to be produced by fungi of endophytic origin (29, 30, 31) and reported to possess antimicrobial activities (18, 19, 20, 21, 22, 23, 24, 25, 26).
The antipseudomonal activities of the endophytic fungal extracts in this study showed a varying effect against the different P. aeruginosa clinical isolates (Fig. 3). Orthopedic wound (Ps.a B) and sputum (Ps.a D) isolates were observed to be strongly resistant to the fungi extracts of Cn2 and Cn3, being susceptible only to the extract of Cn1 ( IZD of 4±0 mm). While laboratory (Ps.a A) and urine (Ps.a C) were susceptible to 100% of the fungi extracts under study. There are several reports that P. aeruginosa wound isolates are highly resistant when compared to isolates from other sources (32, 33). This can also be evidenced by the lower IZD obtained in the case of the control gentamycin (Fig. 3). An emerging problem with P. aeruginosa infection is that this pathogenic microbe exhibits a high degree of resistance to a broad spectrum of antibiotics. One striking highlight of this study was that a higher percentage (80%) of the P. aeruginosa isolates were found to be susceptible to the extract of Cn1. This may be due to the presence of 4-hydroxyphenyl acetic acid and indole-3-acetic acid identified in the extract of Cn1, as revealed by the HPLC results (Table 2). A proper observation shows that only the extracts of Cn1 contained both 4-hydroxyphenyl acetic acid and indole-3-acetic acid, one could propose that both principal compounds combined could produce synergistic mechanism responsible for their activity against the highly resistant wound isolate. Moreover, 4-hydroxyphenyl acetic acid is a phenolic compound and phenolics have been reported to possess antimicrobial properties (22). This is an important observation and suggests the extract of Cn1 as a potential source of novel agents against multiple drug-resistant P. aeruginosa species.
Overall, results obtained suggest that C. nitida harbor many species of endophytic fungi which are potential sources of novel antibacterial compounds. The presence of bioactive compounds has been reported to confer resistance to plants against bacteria, fungi, and pests (34), and therefore explains the antibacterial activity demonstrated by the endophytic fungi extracts in this study. Also, compounds responsible for the observed antimicrobial activity may have a structural analogue to previously established drugs known to show such effective anti-Pseudomonas activity (35). This can be ascertained through the structural elucidation of the identified compounds, and on further purification and characterization, it may be considered as promising compounds for the development of an anti-Pseudomonas drug.
Endophytic fungal extracts of C. nitida exhibited potential for anti- Pseudomonas activity. This could be attributed to the presence of principal compounds identified in the fungal extracts in this study. This plant and its associated endophytic fungi could be a promising source of bioactive compounds of natural origin for the treatment of infections caused by different strains of Pseudomonas aeruginosa.
Anyasi RO, Atagana HI. Endophyte: understanding the microbes and its applications. Pakistan Journal of Biological. Science. 2019; 22(4): 154 – 167.AnyasiROAtaganaHIEndophyte: understanding the microbes and its applications. Pakistan Journal of Biological2019224154167Search in Google Scholar
Ozer B, Tatman-Otkun M, Memis D, Otkun M. Characteristics of Pseudomonas aeruginosa Isolates from Intensive Care Unit. Central European Journal of Medicine. 2009; 4(2): 156-163.OzerBTatman-OtkunMMemisDOtkunMCharacteristics of Pseudomonas aeruginosa Isolates from Intensive Care Unit20094215616310.2478/s11536-008-0090-2Search in Google Scholar
Porras-Gómez M, Vega-Baudrit J, Núñez-Corrales S. Overview of Multidrug-Resistant Pseudomonas aeruginosa and Novel Therapeutic Approaches. Journal of Biomaterials and Nanobiotechnology 2012; 3: 519-527.Porras-GómezMVega-BaudritJNúñez-CorralesSOverview of Multidrug-Resistant Pseudomonas aeruginosa and Novel Therapeutic Approaches2012351952710.4236/jbnb.2012.324053Search in Google Scholar
Atkin SD, Abid S, Foster M, Bose M, Keller A, Hollaway R, Sader HS, Greenberg DE, Finklea JD, Castanheira M, Jain R. Multidrug-resistant Pseudomonas aeruginosa from sputum of patients with cystic fibrosis demonstrates a high rate of susceptibility to ceftazidime-avibactam. Infection and Drug Resistance. 2018; 11: 1499 – 1510.AtkinSDAbidSFosterMBoseMKellerAHollawayRSaderHSGreenbergDEFinkleaJDCastanheiraMJainRMultidrug-resistant Pseudomonas aeruginosa from sputum of patients with cystic fibrosis demonstrates a high rate of susceptibility to ceftazidime-avibactam2018111499151010.2147/IDR.S173804Search in Google Scholar
Egbujor MC, Nwobodo DC, Egwuatu PI, Abu IP, Ezeagu CU. Sulphonamide drugs and Pseudomonas aeruginosa resistance: A review International Journal of Modern Pharmaceutical Research. 2020; 4(1): 78-83.EgbujorMCNwobodoDCEgwuatuPIAbuIPEzeaguCUSulphonamide drugs and Pseudomonas aeruginosa resistance: A review2020417883Search in Google Scholar
Strobel G. The emergence of endophytic microbes and their biological promise Journal of Fungi. 2018; 4(2): 57.StrobelG2018425710.3390/jof4020057Search in Google Scholar
Indabawa II, Arzai AH. Antibacterial Activity of Garcinia kola and Cola nitida Seed Extracts. Bayero Journal of Pure and Applied Sciences. 2011; 4(1): 52 – 55.IndabawaIIArzaiAHAntibacterial Activity of Garcinia kola and Cola nitida Seed Extracts201141525510.4314/bajopas.v4i1.11Search in Google Scholar
Adegboye MF, Akinpelu DA, Okoh AI. The bioactive and phytochemical properties of Garcinia kola (Heckel) seed extracts on some pathogens. African Journal of Biotechnology. 2008; 7(21):3934-3938.AdegboyeMFAkinpeluDAOkohAIThe bioactive and phytochemical properties of Garcinia kola (Heckel) seed extracts on some pathogens200872139343938Search in Google Scholar
Tende JA, Ezekiel I, Dare SS, Okpanachi AO, Kemuma SO, Goji AD. Study of the Effect of Aqueous Extract of Kolanut Cola nitida on Gastric Acid Secretion and Ulcer in White Wistar Rats. British Journal of Pharmacology and Toxicology. 2011; 2(3): 132-134.TendeJAEzekielIDareSSOkpanachiAOKemumaSOGojiADStudy of the Effect of Aqueous Extract of Kolanut Cola nitida on Gastric Acid Secretion and Ulcer in White Wistar Rats201123132134Search in Google Scholar
Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA: Are tropical fungal endophytes hyperdiverse? Ecology Letters. 2000; 3: 267-274.ArnoldAEMaynardZGilbertGSColeyPDKursarTAAre tropical fungal endophytes hyperdiverse?2000326727410.1046/j.1461-0248.2000.00159.xSearch in Google Scholar
Schulz B, Wanke U, Draeger S: Endophytes from herbaceous and shrubs: effectiveness of surface sterilization methods. Mycology Research. 1993; 97:1447-1450.SchulzBWankeUDraegerSEndophytes from herbaceous and shrubs: effectiveness of surface sterilization methods1993971447145010.1016/S0953-7562(09)80215-3Search in Google Scholar
Okoye FBC, Lu S, Nworu CS, Abdessamad D. Depsidone and diaryl ether derivatives from the fungus Corynespora cassiicola an endophyte of Gongronema latifolium. Tetrahedron Letter 2013; 54:4210–4214.OkoyeFBCLuSNworuCSAbdessamadDDepsidone and diaryl ether derivatives from the fungus Corynespora cassiicola an endophyte of Gongronema latifolium2013544210421410.1016/j.tetlet.2013.05.117Search in Google Scholar
Onyegbule FA, Ilouno IO, Eze PM, Abba CC, Chigozie VU: Evaluation of the analgesic, anti-inflammatory and antimicrobial activities of leaf extracts of breynia nivosa. Chemical Science Review Letter. 2014; 3(12):1126-1134.OnyegbuleFAIlounoIOEzePMAbbaCCChigozieVUEvaluation of the analgesic, anti-inflammatory and antimicrobial activities of leaf extracts of breynia nivosa20143121126113410.1055/s-0034-1395078Search in Google Scholar
Eze PM, Nnanna JC, Okezie U, Buzugbe HS, Abba CC, Chukwunwejim CR, Okoye FBC, Esimone CO. Screening of metabolites from endophytic fungi of some Nigerian medicinal plants for antimicrobial activities. The EuroBiotech Journal. 2019; 3(1): 10 -19.EzePMNnannaJCOkezieUBuzugbeHSAbbaCCChukwunwejimCROkoyeFBCEsimoneCOScreening of metabolites from endophytic fungi of some Nigerian medicinal plants for antimicrobial activities201931101910.2478/ebtj-2019-0002Search in Google Scholar
American Type Culture Collection. Sarociadium strictum (ATCC® 62827™). American Type Culture Collection, USA. 2018.American Type Culture Collection, USA2018Search in Google Scholar
Ülhan S, Demürel R, Asan A, Bay C, Kinaci E. Colonial and Morphological Characteristics of Some Microfungal Species Isolated from Agricultural Soils in Eskişehir Province (Turkey). Turkish Journal of Botany. 2006; 30: 95-104ÜlhanSDemürelRAsanABayCKinaciEColonial and Morphological Characteristics of Some Microfungal Species Isolated from Agricultural Soils in Eskişehir Province (Turkey)20063095104Search in Google Scholar
Francisca HH, Patricia MG, Erika CM, Luis-Javier MT, Rubén LM, Beatriz GA, Rocío OT, Cerbón MA. Morphological varieties of Trichophyton rubrum clinical isolates. Revista Mexicana de Micología. 2007; 25: 9-14FranciscaHHPatriciaMGErikaCMLuis-JavierMTRubénLMBeatrizGARocíoOTCerbónMAMorphological varieties of Trichophyton rubrum clinical isolates200725914Search in Google Scholar
Zuo WJ, Jin PF, Dong WH, Dai HF, Mei WL. Metabolites from the endophytic fungus HP-1 of Chinese eaglewood. Chinese Journal of Natural Medicines. 2014; 12(2):151-153.ZuoWJJinPFDongWHDaiHFMeiWLMetabolites from the endophytic fungus HP-1 of Chinese eaglewood201412215115310.1016/S1875-5364(14)60025-XSearch in Google Scholar
Chapla VM, Zeraik ML, Leptokaraydis IH, Silva GH, Balzani VS, Young MCM, Pfenning LH, Araùjo AR. Antifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic fungus from Micheliachanpaca. Molecules. 2014; 19:19243-19252.ChaplaVMZeraikMLLeptokaraydisIHSilvaGHBalzaniVSYoungMCMPfenningLHAraùjoARAntifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic fungus from Micheliachanpaca201419192431925210.3390/molecules191119243Search in Google Scholar
Arnao MB, Sanchez-Bravo J, Acosta M. Indole-3-carbinol as a scavenger of free radicals. Biochemistry and Molecular Biology International. 1996; 39(6): 1125-1134.ArnaoMBSanchez-BravoJAcostaMIndole-3-carbinol as a scavenger of free radicals19963961125113410.1080/15216549600201302Search in Google Scholar
Trinagaraju K, Prasad AV, Venkateswara RP, Prasad PS. Synthesis, Characterization and Biological activity of 3- Indole Acetic acid. International Journal of Novel Trends in Pharmaceutical Sciences. 2015; 5(1): 21-24.TrinagarajuKPrasadAVVenkateswaraRPPrasadPSSynthesis, Characterization and Biological activity of 3- Indole Acetic acid2015512124Search in Google Scholar
Merkl R, Hrádková I, Filip V, Šmi drkal J. Antimicrobial and Antioxidant Properties of Phenolic Acids Alkyl Esters Czech Journal of Food Science. 2010; 28( 4): 275-279.MerklRHrádkováIFilipVŠmi drkal J. Antimicrobial and Antioxidant Properties of Phenolic Acids Alkyl Esters201028 427527910.17221/132/2010-CJFSSearch in Google Scholar
Manuja R, Sachdeva S, Jain A, Chaudhary J. A Comprehensive Review on Biological Activities of P-Hydroxy Benzoic Acid and Its Derivatives. International Journal of Pharmaceutical Science Review Research. 2013; 22(2): 109-115ManujaRSachdevaSJainAChaudharyJA Comprehensive Review on Biological Activities of P-Hydroxy Benzoic Acid and Its Derivatives2013222109115Search in Google Scholar
Ciftci O, Disli OM, Timurkaan N. Protective effects of protocatechuic acid on TCDD-induced oxidative and histopathological damage in the heart tissue of rats. Toxicology and Industrial Health. 2013; 29(9):806-811.CiftciODisliOMTimurkaanNProtective effects of protocatechuic acid on TCDD-induced oxidative and histopathological damage in the heart tissue of rats201329980681110.1177/0748233712442735Search in Google Scholar
Kakkar S, Bais S. A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacology. 2014; ID: 952943.KakkarSBaisSA review on protocatechuic acid and its pharmacological potential2014ID: 95294310.1155/2014/952943Search in Google Scholar
Nguyen XH, Naing KW, Lee YS, Moon JH, Lee JH, Kim KY. Isolation and characteristics of protocatechuic acid from Paenibacillus elgii HOA73 against Botrytis cinerea on strawberry fruits. Journal of Basic Microbiology. 2015; 55(5):625-34.NguyenXHNaingKWLeeYSMoonJHLeeJHKimKYIsolation and characteristics of protocatechuic acid from Paenibacillus elgii HOA73 against Botrytis cinerea on strawberry fruits20155556253410.1002/jobm.201400041Search in Google Scholar
Zhao J, Mou Y, Shan T, Li Y, Zhou L, Wang M, Wang J. Antimicrobial metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris Polyphylla var. Yunnanensis Molecules. 2010; 15:7961-7970.ZhaoJMouYShanTLiYZhouLWangMWangJAntimicrobial metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris Polyphylla var. Yunnanensis2010157961797010.3390/molecules15117961Search in Google Scholar
Rocha R, Eleuterio da Luz D, Engels C, Pileggi S, Matiello RR, Pileggi M. Selection of endophytic fungi from comfrey symphytum officinale L) for in vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.) Brazil Journal of Microbiology. 2009; 40(1): 73-78.RochaREleuterioda Luz DEngelsCPileggiSMatielloRRPileggiMSelection of endophytic fungi from comfrey symphytum officinale L) for in vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.)2009401737810.1590/S1517-83822009000100011Search in Google Scholar
Lu H, Zou W, Meng JC, Hu J, Tan RX. New bioactive compound produced by collectotrichum sp., an endophytic fungus of Artemisia annua Plant science. 2000; 151(1): 67-73.LuHZouWMengJCHuJTanRXNew bioactive compound produced by collectotrichum sp., an endophytic fungus of Artemisia annua20001511677310.1016/S0168-9452(99)00199-5Search in Google Scholar
Hasan S, Ansari MI, Ahmad A, Mishra M. Major bioactive metabolitea from marine fungi: A review. Bioinformation. 2015; 11(4): 176-181HasanSAnsariMIAhmadAMishraMMajor bioactive metabolitea from marine fungi: A review201511417618110.6026/97320630011176447905726124556Search in Google Scholar
Nwobodo DC, Ihekwereme CP, Ugwu MC, Okoye FBC: Screening of endophytic fungal secondary metabolites from Garcinia kola and Cola nitida for antioxidant properties. Open Access Journal of Pharmaceutical Research. 2017; 1(6): 000136.NwobodoDCIhekweremeCPUgwuMCOkoyeFBCScreening of endophytic fungal secondary metabolites from Garcinia kola and Cola nitida for antioxidant properties20171600013610.23880/OAJPR-16000134Search in Google Scholar
Brown PD, Izundu A. Antibiotic resistnce in clinical isolates of P.aruginosa in Jamaica. Rev. Panam Salud Publica. 2004; 16(2): 125-130.BrownPDIzunduAAntibiotic resistnce in clinical isolates of P.aruginosa in Jamaica. Rev2004162125130Search in Google Scholar
Garba I, Lusa YH, Bawa E, Tijjani MB, Aliyu MS, Zango UU, Raji MI. Antibiotics Susceptibility Pattern of Pseudomonas aeruginosa Isolated from Wounds in Patients Attending Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. Nigerian Journal of Basic and Applied Science. 2012; 20(1): 32-34.GarbaILusaYHBawaETijjaniMBAliyuMSZangoUURajiMIAntibiotics Susceptibility Pattern of Pseudomonas aeruginosa Isolated from Wounds in Patients Attending Ahmadu Bello University Teaching Hospital, Zaria, Nigeria20122013234Search in Google Scholar
Webber J. A natural control of Dutch elm disease. Nature, London. 1981; 292:449-451.WebberJA natural control of Dutch elm disease198129244945110.1038/292449a0Search in Google Scholar
Nwobodo DC, Ihekwereme CP, Ikem CJ, Okoye FBC: The anti-pseudomonal potentials of metabolites from some endophytic fungi isolated from Garcinia kola leaves. Novel Research in Microbiology Journal. 2020; 4(3): 845-855.NwobodoDCIhekweremeCPIkemCJOkoyeFBCThe anti-pseudomonal potentials of metabolites from some endophytic fungi isolated from Garcinia kola leaves20204384585510.21608/nrmj.2020.95326Search in Google Scholar