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Endophytic fungi occurring in Moringa ovalifolia in the Tsumeb area of Namibia.

An endophytic microorganism characteristically lives within its host during a part of or throughout its entire life cycle. Plant fungal endophytes reside in or between plant cells inside the plant tissue and cause no apparent harm to the host (Kaul et al, 2012). The association between the fungal endophyte and its plant host are usually commensal or mutualistic though it can become pathogenic under certain circumstances (Photita et al, 2004). The diverse plant endophytic fungi, estimated at 1.5 million species (Hawksworth, 2004), have been gaining importance because they produce novel compounds which may potentially be of use to medicine, agriculture and industry. Fungal endophytes from medicinal plants have been identified as a potential source for novel metabolites of medicinal significance. Many bioactive compounds with antimicrobial, cytotoxic and anticancer activities have been discovered from endophytic fungi (Zhao et al., 2010).

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.


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:/ / 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.


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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:

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

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

SCAF7      bark          Leptosphaerulina      89           KU351174
                         chartarum strain

SCAF9      bark          Aspergillus niger     97           KU351175
                         strain IHEM 22432
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Article Details
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Author:Haiyambo, D.H.; Chisenga, B.; Chimwamurombe, P.M.; Mapaure, I.; Nuuyoma, P.B.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Geographic Code:6NAMI
Date:Mar 1, 2016
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