Endophytic fungi occurring in Moringa ovalifolia in the Tsumeb area of Namibia.
Moringa species (moringa) are angiosperms that are native to India, Pakistan, Arabia, South Asia and some parts of Africa (Anwar et al., 2007). There are about thirteen species in the family Moringaceae, of which M. oleifera is the most widely known. M. oleifera is cultivated throughout the tropics, subtropics and many African countries (Mbikay, 2012) and has an extensive number of uses. The leaves and flowers are used for culinary purposes while powder from the leaves is used as a food supplement to prevent malnutrition in children and pregnant women. Moringa oleifera seeds are fried and eaten, used to prepare sauces and used in water purification whereas the seed oil can be used in cosmetics (Oliveira et al., 1999; Bosch, 2004). Moringa spp are known to have medicinal properties and is used as an anodyne, antidiabetic, antihypertensive, circulatory tonic, aphrodisiac, antihelmintic, and treatment for asthma, muscle disease as well as dental caries (Bosch, 2004; Anwar et al., 2007; Cheenpracha et al., 2010; Atawodi et al., 2014). However, M. ovalifolia (which occurs only in Namibia and southern Angola) is reported to be mainly used as food by locals (Van Wyk and Van Wyk, 1997). It has not received much research attention compared to M. oleifera.
The multitude of beneficial uses that moringa species possess provides an opportunity to investigate the novel fungal endophytes associated with the M. ovalifolia population in the Tsumeb area of Namibia. Considering the medicinal and nutritional importance and a paltry knowledge about the microbiota of moringa plants in Namibia, the present study aimed to isolate and identify the fungal endophytes inhabiting the stem and the leaves of M. ovalifolia.
MATERIALS AND METHODS
Collection of plant material
Fresh leaves and stems of healthy Moringa ovalifolia were collected from mature and healthy plants located in the Tsumeb area, Northern Namibia. The plant materials were placed in sterile bags and transported to the laboratory. The leaves and stems were washed with sterile distilled water and moved to the laminar air flow for processing.
Isolation of fungi
Leaves were surface-sterilized by submerging in 80% ethanol for 5 minutes and rinsed five times with sterile distilled water. The leaves were then sliced across the length of the lamina with a sterile blade and aseptically placed onto potato dextrose agar (PDA) media. A piece of a M. ovalifolia stem was surface-sterilized by submerging in 99% ethanol for 7 minutes and rinsed five times with sterile distilled water. A piece of the bark was carved off with a sterile blade and scalpel set and aseptically transferred onto PDA medium with the epidermis facing up. The surface sterilized piece of stem was dissected and pieces of its pith were transferred onto PDA media with flamed tweezers. The PDA plates were then incubated at 35[+ or -]2[degrees]C for 14 days in dark conditions. Young sections from distinct fungi growing from the M. ovalifolia plant parts were subcultured onto fresh PDA medium and incubated for up to 10 days at 35[+ or -]2[degrees]C.
PCR amplification of Internal Transcribed Spacer
The Internal transcribed spacer (ITS) region of the 18S rRNA gene was amplified with primers ITS-1 (52 -TCCGTAGGTGAACCTGCGG32 ) and ITS-4 (52 -TCCTCCGCTTATTGATATGC32 ) (White et al., 1990). Genomic DNA of one week old fungal cultures were isolated using the Zymo Research Fungal/Bacterial DNA MiniPrep[TM] kit and used as the template for amplification. The polymerase chain reaction (PCR) mixture consisted of 12.5il Dream Taq[TM] Green PCR Master Mix 2x, 0.5iM each primer, 6il nuclease free water and 4il genomic DNA. The reaction was carried out in a Bio-Rad MyCycler[TM] thermal cycler Thermal Cycler. The PCR cycle steps were as follows: initial denaturation at 95[degrees]C for 5 min, 35 cycles of denaturation at 95[degrees]C for 45 sec, annealing at 51 [degrees]C for 30 s, extension at 72 [degrees]C for 2 min, final extension at 72 [degrees]C for 6 min and hold at 4[degrees]C. The products were separated by electrophoresis in a 1.2% agarose gel stained with ethidium bromide and visualized under UV light. The PCR fragments were purified and sequenced at Inqaba biotechnical Industries (Pty) Ltd. (Pretoria, South Africa). The Basic Local Alignment Search Tool program (http:/ /www.ncbi.nlm.nih.gov/BLAST) was used to identify the fungal isolates by searching for the organisms with the highest similarity to the sequences of the isolates.
From 18 sections (6 sections each from leaves, bark and pith) of M. ovalifolia, five fungi were isolated and identified as Emericella nidulans SCAF2, Alternaria alternata SCAF4, Aspergillus sp. SCAF9, Leptosphaerulina sp. SCAF7 and Peyronellaea glomerata SCAF6 (Table 1). The endophyte A. alternata SCAF4 was isolated from the leaves whilst E. nidulans SCAF2 was the sole isolate from moringa stem pith in this particular instance (Fig. 1). Aspergillus sp. Leptosphaerulina sp. SCAF7 and Peyronellaea glomerata SCAF6 were all obtained from the bark segments of M. ovalifolia stem.
The isolation of a variety of fungal endophytes from different parts of the medicinal M. ovalifolia is crucial for identifying novel fungal isolates which further increases the potential to discover new bioactive compounds. Emericella is the sexual form of the genus Aspergillus which has been described to produce many bioactive compounds that are antimicrobial, anti-Hepatitis C and cytotoxic to liver cancer cell lines (Hawas et al., 2012). An endophyte from a Mediterranean green alga Emericella nidulans var. acristata produced antitumor benzophenone polyketide compounds against 36 human tumor cell lines (Krajl et al., 2006). Given the data presented in this paper, it is understandable to imagine that the medicinal effects by Moringa could actually be from endophytic fungi and not only from the plant's secondary metabolites. However, this line of argument needs to be tested by further experiments.
Alternaria spp. are common occurrence of plant diseases but are known to be epiphytic and endophytic (Osono, 2002). The dematiaceous nature of Alternaria spp. allows them to withstand sunlight and desiccation conditions that the leaf surroundings consist of (Butler and Day, 1998). Studies by Huang et al. (2011) and Sadananda et al. (2011) described two isolates (Alternaria sp. ZJ9-6B and A. alternata) from Aegiceras corniculatum and Tabebuia argentea which produce quinones alterporriol K and lapachol, respectively. Polyketides and bicyclic acid derivatives from an Alternaria sp. endophyte of the medicinal Polygonum senegalense exhibited cytotoxic activity against L5178Y mouse lymphoma cells (Aly et al, 2008). Endophytes belonging from the genera Alternaria and Aspergillus have been isolated from Moringa spp. in previous studies (Dhanalakshmi et al., 2013; Khan et al. 2014). Endophytic A. niger isolates are known to produce quinone and cytotoxic pyrone compounds (Sadananda et al., 2011; Li et al. 2013).
The fungus P. glomerata has been isolated as an endophyte of a pine (Pinus koraiensis) and two medicinal plants, specifically Salvia miltiorrhiza and Macleaya cordata (Deng et al., 2011; Shan et al., 2012; Lou et al., 2013). Extracts from the Peyronellaeae sp. isolated from Macleaya cordata showed antimicrobial activity against Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Pseudomonas lachrymans, Staphylococcus aureus, Staphylococcus haemolyticus, Salmonella typhimurium and Xanthomonas vesicatoria. Known to cause leaf diseases in cereal grains Pithomyces chartarum), L. chartarum has been isolated as an endophyte from the medicinal Tephrosiapurpurea (Suryanarayanan and Murali, 2006; Toth et al, 2007; Verma et al., 2008; Sharma et al., 2013). Leptosphaerulina spp. are regarded as saprophytes and latent pathogens as demonstrated by Suryanarayanan and Murali (2006) when a Leptosphaerulina isolate from the leaves of peanut could not be regarded as pathogenic after infection studies proved otherwise. Irrespective, an endophytic Leptosphaerulina chartarum MA164 exhibited moderate antimicrobial activity against Cryptococcus neoformans ATCC90112 and Microsporum gypseum MU-SH4 (Sakayaroj et al., 2012).
For the present study five fungal endophytes from M. ovalifolia of the Tsumeb area in Namibia were successfully isolated and identified. To our knowledge this is a first report on the endophytic fungi cultured from M. ovalifolia plants of Namibia. The BLAST results indicate that the isolates SCAF7 is a novel endophytic species isolated from M. ovalifolia. The genera to which the isolates that are reported in the study belong are documented to be endophytic constituents of many medicinal plants. The metabolites that are produced by the endophytic fungi could be of pharmaceutical significance and if such is the case the medicinal compounds can be sourced from microbial culture rather than plant extraction thus shortening the time of drug production.
This was a study was funded by a research grant URPC/2015/221 from the Research and Publications Office at University of Namibia.
(1.) Aly AH, Edrada-Ebel R, Indriani ID, Wray V, Muller WE, Totzke Fand Ebel R. Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense. Journal of Natural Products 2008; 71(6): 972-980.
(2.) Anwar F, Latif S, Ashraf M, & Gilani AH., Moringa oleifera: a food plant with multiple medicinal purposes. Phytotherapy Research, 2007; 21(1): 17-25.
(3.) Atawodi SE, Olowoniyi OD, Adejo GO, Liman ML and Dubey NK., Review of the antioxidant potential of African medicinal and food plants. Plants as a Source of Natural Antioxidants, 2014; 34.
(4.) Bosch CH., Moringa oleifera Lam. In: Grubben GJH and Denton OA (Eds). PROTA 2: Vegetables/ Legumes. [CD-Rom] PROTA Wageningen, Netherlands. Retrieved from http:/ /database.prota.org/PROTAhtml/ Moringa_oleifera_En.htm, 2004.
(5.) Butler MJ and Day AW., Fungal melanins: a review. Canadian Journal of Microbiology 1998; 44: 1115-1136.
(6.) Cheenpracha S, Park EJ, Yoshida WY, Barit C, Wall M, Pezzuto JM, and Chang LC. Potential anti-inflammatory phenolic glycosides from the medicinal plant Moringa oleifera fruits. Bioorganic & Medicinal Chemistry, 2010; 18(17): 6598-6602.
(7.) Deng JX, Paul NC, Li MJ, Seo EY, Sung GH, and Yu SH., Molecular characterization and morphology of two endophytic Peyronellaeae species from Pinus koraiensis in Korea. Mycobiology, 2011; 38(4): 266-271.
(8.) Dhanalakshmi R, Umamaheswari S, Sugandhi P and Prasanth DA., Biodiversity of the Endophytic Fungi isolated from Moringa oleifera of Yercaud hills. International Journal of Pharmaceutical Sciences and Research 2013; 4(3): 1064-1068.
(9.) Hawas, UW, El-Kassem LT, Ahmed, EF, and Emam M, In-vitro bioassays on the metabolites of the fungus Emericella nidulans isolated from the Egyptian Red Sea algae. Egypt Pharmacuetical Journal 2012; 11: 124-128.
(10.) Hawksworth DL., Fungal diversity and its implications for genetic resource collections. Studies inMycology 2004; 9-18, 50.
(11.) Huang CH, Pan JH, Chen B, Yu M, Huang HB, Zhu X, Lu YJ, She ZG, and Lin YC., Three bianthroquinone derivatives from the mangrove endophytic fungus Alternaria sp. ZJ9-6B from South China Sea. Mar Drugs 2011; 9: 832-843.
(12.) Kaul S, Gupta S, Ahmed M, and Dhar MK., Endophytic fungi from medicinal plants: a treasure hunt for bioactive metabolites. Phytochemstry Reviews, 2012; 11(4): 487-505.
(13.) Khan AL, Waqas M,, Hussain J, Al-Harrasi A, Al-Rawahi A, Al-Hosni K, Kim MJ, Adnan M, & Lee IJ., Endophytes Aspergillus caespitosus LK12 and Phoma sp. LK13 of Moringa peregrina produce gibberellins and improve rice plant growth, Journal of Plant Interactions, 2014; 9(1): 731-737.
(14.) Krajl A, Kehrarus S, Krick A, Eguereva E, Kelter G, Maurer M, Wortmann A, Fiebig HH, and Konig GM., Arugosins G and H: prenylated polyketides from marine-derived fungus Emericella nidulans var acristata. Journal of Natural Products 2006; 69: 995-1000.
(15.) Li XB, Xie F, Liu SS, Li Y, Zhou JC, Liu TQand Lou HX . Naphtho-y-pyrones from endophyte Aspergillus niger occurring in the liverwort Heteroscyphus tener (Steph.) Schiffn. Chemistry and Biodiversity, 2013; 10(7): 1193-1201.
(16.) Lou J, Fu L, Luo R, Wang X, Luo H and Zhou L., Endophytic fungi from medicinal herb Salvia miltiorrhiza Bunge and their antimicrobial activity. African Journal of Microbiology Research, 2013; 7(47): 5343-5349.
(17.) Mbikay M. Therapeutic potential of Moringa oleifera leaves in chronic hyperglycemia and dyslipidemia: a review. Frontiers in Pharmacology, 2012; 3.
(18.) Oliveira JTA, Silveira SB, Vasconcelos IM, Cavada BS, and Moreira, RA., Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck. Journal of the Science of Food and Agriculture, 1999; 79(6): 815-820.
(19.) Osono T., Phyllosphere fungi on leaf litter of Fagus crenata: occurrence, colonization, and succession. Canadian Journal of Botany 2002; 80(5): 460-469.
(20.) Photita W, Lumyong S, Lumyong P, McKencie EH and Hyde, KD., Are some endophytes of Musa acuminate latent pathogens. Fungal Diversity 2004; 16: 131-140.
(21.) Sadananda, TS, Nirupama R, Chaithra K, Govindappa M, Chandrappa CP, Vinay Raghavendra B. Antimicrobial and antioxidant activities of endophytes from Tabebuia argentea and identification of anticancer agent (lapachol). Journal of Medicinal Plants Research, 2011; 5(16): 3643-3652.
(22.) Sakayaroj J, Preedanon S, Phongpaichit S, Buatong J, Chaowalit P and Rukachaisirikul V, Diversity of endophytic and marine derived fungi associated with marine plants and animals. In: Jones EBG & Pang K-L (Eds.), Marine Fungi and Fungal-like Organisms. Walter de Gruyter, Berlin, 2012; 291-328.
(23.) Shan T, Sun W, Lou J, Gao S, Mou Y and Zhou L., Antibacterial activity of the endophytic fungi from medicinal herb, Macleaya cordata. African Journal of Biotechnology 2012; 11 (19): 4354-4359.
(24.) Sharma R, Mehan S, Kalra S, and Khanna D., Tephrosia purpurea--a magical herb with blessings in human biological system. International Journal of Recent Advances in Pharmaceutical Research, 2013; 3(3): 12-22.
(25.) Suryanarayanan TS and Murali, TS., Incidence ofLeptosphaerulina crassiasca in symptomless leaves of peanut in southern India. J. Basic Microbiol. 2006; 46: 305-309.
(26.) Toth B, Csosz M, Dijksterhuis J, Frisvad JC, and Varga J., Pithomyces chartarum as a pathogen of wheat. Journal of Plant Pathology. 2007; 89(3): 406-408.
(27.) Van Wyk, B and Van Wyk, P., Field Guide to trees of southern Africa. Struik Publishers, Cape Town, 1997.
(28.) Verma, OP, Gupta RBL and Shivpuri A., A new host for Pithomyces chartarum, the cause of a leaf spot disease On Withania somnifera. Plant Pathology 2008; 57: 385.
(29.) Zhao J, Zhou L, Wang J, Shan T, Zhong L, Liu X and Gao X., Endophytic fungi for producing bioactive compounds originally from their host plants. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology 2010; 1: 567-576.
D.H. Haiyambo, B. Chisenga, P.M. Chimwamurombe *, I. Mapaure and P.B. Nuuyoma
Department of Biological Sciences, University of Namibia, 340 Mandume Ndemufayo Ave Pioneerspark, Windhoek, Namibia.
(Received: 04 December 2015; accepted: 30 January 2016)
* To whom all correspondence should be addressed. E-mail: email@example.com
Caption: Fig. 1. A Fungal endophytes isolated from M. ovalifolia stem. B Single isolate Alternaria alternata SCAF4 from leaves of M. ovalifolia
Table 1. Fungi isolated from M. ovalifolia Isolate M. Organism with % Accession ovalifolia highest similarity to code part isolate Similarity SCAF2 leaf Emericella nidulans 100 KU351171 strain DHMJ15 SCAF4 pith Alternaria sp. 100 KU351172 BSO7 ITS 1 SCAF6 bark Peyronellaea 99 KU351173 glomerata strain ZG-4-2-1 SCAF7 bark Leptosphaerulina 89 KU351174 chartarum strain TPL10 SCAF9 bark Aspergillus niger 97 KU351175 strain IHEM 22432
|Printer friendly Cite/link Email Feedback|
|Author:||Haiyambo, D.H.; Chisenga, B.; Chimwamurombe, P.M.; Mapaure, I.; Nuuyoma, P.B.|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2016|
|Previous Article:||Optimization of fermentation conditions for production of bioactive metabolites effective against Staphylococcus epidermidis by a newly isolated...|
|Next Article:||Microalgae as a renewable energy source for biofuel production.|