IJAAR 5 (2017) 66-75 ISSN 2053-1265 Morphological and molecular identification of pathogenic fungi of Monodora myristica Dunal kernels and their response to different phytoextracts Joseph Djeugap Fovo1,2*, Cyril Akoula Nzong1, Kyalo Martina2, Patrick Njukeng Achiangia3, Joseph Hubert Galani Yamdeu4, Jules-Roger Kuiate5 and Sita Ghimire2 1 Phytopathology and Agricultural Zoology Research Unit, Department of Plant Protection, Faculty of Agronomy and Agricultural Science, P. O. Box 222 Dschang, University of Dschang, Cameroon. 2 Biosciences Eastern and Central Africa-International Livestock Research Institute (BecA-ILRI) Hub, P. O. Box 30709-00100, Nairobi, Kenya. 3 Applied Botany Research Unit, Department of Plant Biology, Faculty of Sciences, University of Dschang, P. O. Box 67 Dschang, Cameroon. 4 Department of Agriculture and Veterinary Medicine, Université des Montagnes, P. O. Box 208, Bangangté, Cameroon. 5 Microbiology and Antimicrobials Substances Research Unit, Department of Biochemistry, Faculty of Sciences, University of Dschang, P. O. Box 67 Dschang, Cameroon. Article History Received 19 July, 2017 Received in revised form 17 August, 2017 Accepted 21 August, 2017 Keywords: Antifungal activity, ITS sequences, Monodora myristica, Pathogenicity, Plant extracts, Post-harvest fungi. Article Type: Full Length Research Article ABSTRACT Identification of fungi from calabash nutmeg kernels was based on their morphological characteristics and analysis of the internal transcribed spacer (ITS) sequences of their genomic DNA. Antifungal activity of aqueous, methanolic and ethanolic extracts of four plants species (Azadirachta indica, Citrus sinensis, Moringa oleifera and Tithonia diversifolia) was tested in vitro at 50, 75, 100 and 125 mg/ml for aqueous extracts and 40, 60, 80 and 100 mg/ml for methanolic and ethanolic extracts. Mancozeb (1 mg/ml) and distilled water were used as positive and negative controls, respectively. The most frequently isolated fungi were Cercospora purpurea (34.28%), Fusarium oxysporum (23.81%) and Aspergillus flavus (17.14%). C. purpurea and F. oxysporum isolates were more aggressive after inoculation on healthy kernels. All the extracts tested, inhibited the growth of the fungi compared to the negative control, except the aqueous extract of T. diversifolia against C. purpurea and F. oxysporum and the methanolic extract of M. oleifera against F. oxysporum at 75 mg/ml. The efficiency of aqueous extracts of M. oleifera and C. sinensis was significantly lower (P<0.05) as compared to the reference fungicide on growth of A. niger at 125 mg/ml. Antifungal activity of methanolic extracts of A. indica, C. sinensis and T. diversifolia as well as ethanolic extracts of A. indica and M. oleifera was significantly equal to mancozeb at 100 mg/ml on A. flavus. Aqueous extracts of M. oleifera and methanolic extracts of A. indica and C. sinensis could be used for protection of Monodora myristica kernels against post-harvest fungi. ©2017 BluePen Journals Ltd. All rights reserved INTRODUCTION Calabash nutmeg (Monodora myristica Dunal) is a perennial plant belonging to the family Annonaceae. It is common in the evergreen forests of West and Central Africa (Burabai et al., 2007). In Cameroon, its seeds and bark are used for food and medicinal properties. Seeds are the most important part of the tree as they have an odour and taste similar to nutmeg, and are therefore used as a popular spice in the West and Central African Int. J. Adv. Agric. Res. cuisine. It is also used as substitute for nutmeg in soups, stews and cakes. In traditional medicine, the seeds are used as a stimulant and stomachic. They are also used as rosary beads and are considered by some to have magical properties (Faleyimu and Oluwalana, 2008). The seeds are rich in alkaloids and used in the treatment of headache and as an antiseptic (N’guessan et al., 2009). Aqueous extract of M. myristica dry fruits possess cholesterol lowering potentials and protective ability (Nwozo et al., 2015). The stem bark is used in the treatments of hemorrhoids, stomach ache, fever pains and eye diseases (Uwakwe and Nwaoguikpe, 2008). Despite these tremendous nutritional and medicinal values of M. myristica kernels, no research has been carried out to study pathogenic fungi associated with kernels of this species. Observations made in the field reveal that M. myristica kernels are mostly colonized by molds that could be potentially harmful to consumers if they produce mycotoxins. Post-harvest diseases are usually controlled with chemicals, which despite their effectiveness, could be toxic to consumers and the environment. This is why the discovery of novel antimicrobial agents of plant origin, is important to preserve the environment and the health of consumers. The antifungal potential of plant extracts has long been investigated as they contain several bioactive compounds for plant disease control, such as flavonoids, polyphenols and saponins (Joshi et al., 2011; Patel et al., 2014; Riaz et al., 2015). Due to their low toxicity, there is growing interest in using plant products (extracts, essential oils and powders) as a source of bioactive phytochemicals for antifungal properties in controlling plant diseases (Nguefack et al., 2013; Zaker, 2014). To the best of our knowledge, no study has been carried out on the identification of post-harvest pathogenic fungi of M. myristica kernels and on the assessment of antifungal properties of extracts from selected ethno-medicinal plants. The results from this study are expected to reduce post-harvest losses, increase the income of farmers and traders and enhance consumer health. MATERIALS AND METHODS Collection of plant materials Symptomatic kernels of M. myrstica were collected from Ebolowa, Yokadouma, Kumba, Dschang and Fontem markets of Cameroon, and stored at 4°C in the laboratory prior to isolation of the associated pathogens. Leaves of Mexican sunflower (T. diversifolia), neem (A. indica) and *Corresponding dschang.org. author: joseph.djeugapfovo@univ- 67 moringa (M. oleifera) and pericarp of orange fruits (C. sinensis) collected from Dschang were used to prepare phytoextracts. These plant species were choosen based on their proven antimicrobial properties against either plant or animal pathogens (Trabi et al., 2008; Nweke and Ibian, 2012; Abiamere et al., 2014). Preparation of plant extracts The plant parts collected were washed with tap water and rinsed thrice with distilled water. Subsequently, the leaves and barks were chopped into small pieces, dried in shade for a week, and powdered in an electronic blender. One hundred grams of fine powder of each sample was macerated in 500 ml of water, methanol or ethanol for 2 days then filtered through muslin cloth. The aqueous extract was oven-dried at 50°C for 7 days while ethanol and methanolic extracts were evaporated on a shaking water bath at 60 rpm at 60°C. Extracts were separately stored in small containers at room temperature for further experiments. Isolation and morphological identification of fungi Kernels of M. myristica showing disease symptoms were sliced into 2 mm² pieces and disinfected for 3 min in 5% sodium hypochlorite solution, the slices were rinsed in three successive changes of sterile distilled water and transfered on sterile potato dextrose agar medium (PDA) supplemented with chloramphenicol (1 g/l) as antibacterial, for fungal isolation and purification (Korsten et al., 1994). The pure fungal isolates obtained were identified based on morphological characteristics, that is mycelium structure and spore morphology using keys of fungi identification (Barnett and Hunter, 1972; Alexopoulos et al., 1996; Champignon, 1997). The frequency of isolation (F) of each fungus was calculated using the following formula, F = (NF÷NT) × 100, where F represents the frequency of occurrence (%) of a fungus, NF is the total number of samples from which a particular fungus was isolated and NT is the total number of samples from which isolations were carried out (Iqbal and Saeed, 2012). Molecular identification of fungi Fungal identity was confirmed using molecular method. For this purpose, fungal genomic DNA was extracted using ZR Plant/Seed DNA MiniPrep ™ kit (Zymo Research) following manufacturer’s instructions. The universal fungi species primers, ITS1 (5′TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′TCCTCCGCTTATTGATATGC-3′) were used (White et Djeugap et al. 68 al., 1990). DNA quality and quantity were checked on 1% agarose gel (w/v) and NanoDrop Spectrophotometer. The genomic DNA was adjusted to the final concentration of 20 ng/L and stored at 4°C for PCR amplification. Amplification was performed in 30 μL reaction volume containing 1 × Accu Power PCR Master Mix, 0.1 µM of each primer, and 40 ng of genomic DNA. The PCR program was as follows : initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 60s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. Purified PCR products were sent for Sanger sequencing by Macrogen (Amsterdam, Netherlands). All reactions were run following the manufacturer’s protocols. Nucleotide sequences were aligned using ClustalW program (Thompson et al., 1994), and sequences identified using NCBI-BLASTn (http://www.ncbi.nlm.nih.gov/BLAST). Pathogenicity test of fungi For each fungus isolated and identified, a 10-day-old culture in the Petri dishes was used for inoculum preparation. Healthy kernels of M. myristica were disinfected with bleach solution (5%, 2 min), rinsed in sterile water for three times and soaked during 30 min in 3 a conidial suspension calibrated at 2×10 conidia/ml using a hemacytometer. After inoculation, the kernels were removed and introduced into new Petri dishes containing moisten filter paper (Imathiu et al., 2014). All procedures were the same for the control plate but no inoculum was applied. After 7 days of incubation at 22°C, infected kernels were recorded as the presence or absence of fungal infection. In vitro efficacy of plant extracts on fungal growth Mycelia disks were obtained using a cookie cutter of 5 mm diameter and taken from the margin of 10 days-old culure of A. flavus, A. niger, C. purpurea and F. oxysporum. Mycelial discs were deposited in the center of each Petri dish containing PDA medium enriched with different extracts or fungicide Mancozeb (1 mg/ml) as positive control. A negative control, non-supplemented with extract but solvent control dilution were prepared. Each treatment was conducted in four replications. The plates were incubated at 25°C and daily measurements were taken for fungal growth from the second day of the experiment. The experiment was stopped when Petri dishes of negative control were completely covered by the fungus. The radial growth of the fungi was measured daily (48 h after inoculation) and at the same time, the two perpendicular diameters along the tracks on the back of the Petri dish. The average of two perpendicular measurements of the diameter minus the diameter of explants represents the measurement of the radial growth of the fungus (Djeugap et al., 2011). The inhibition percent (IP) was calculated by the formula given by UlHaq et al. (2014): IP = (GC-GT)÷GC×100; where GC=growth in the control, GT=growth in the treated groups. An average of four replications of each test was taken for calculations. Data analyses Data on radial growth and inhibition percentage were analysed by SAS software (version 9.1) (SAS Institute, Cary, NC). Data were submitted to a one-way analysis of variance (ANOVA). Where the ANOVA was significant at 5%, means were separated using a Duncan multiple range test. RESULTS Characteristics of plant extracts and extraction yields The yields of aqueous extracts were higher than those of the methanolic and ethanolic extracts. The aqueous extract of C. sinensis gave the highest yield (32.91%) followed by A. indica (17.71%), T. diversifolia (16.40%) and M. oleifera (7.65%). For methanolic and ethanolic extracts, the yield of C. sinensis was highest (16.61 and 9.9%) while T. diversifolia (2.46 and 2.44%), respectively was the lowest (Table 1). Identification and pathogenicity of fungi isolated from M. myristica kernels Based on morphological and molecular identification, fungi associated with M. myristica kernels were A. flavus, A. niger, A. oryzae, C. purpurea, Chaetonium reflexum, Cunninghamella bainieri, F. oxysporum and Rhizopus nigricans. The most frequent fungi were C. purpurea (60.66%), F. oxysporum (22.96%), A. flavus (17.14%) and A. niger (8.57%) (Table 2). The fungal species and sequence identity of selected fungi of calabash kernels used for pathogenicity test are presented in Table 2. Among fungal isolates tested, E012_MM and K112_MM were very aggressive while D032_MM, Y046_MM, E023_MM and F181_MM were non-pathogenic isolates (Table 2). These four most frequent fungi developed symptoms at 7 days after inoculation on healthy kernels, and symptoms ranged from whitish to dark mycelium and fruiting bodies; while other isolates did not develop symptom (Figure 1). Antifungal activity of aqueous extracts Antifungal activity of plant extracts was tested against C. Int. J. Adv. Agric. Res. 69 Table 1. Physical characteristic and extraction yield of plant extracts. Plants species A. indica (leaves) Aqueous extract Methanolic extract Ethanolic extract M. oleifera (leaves) Aqueous extract Methanolic extract Ethanolic extract C. sinensis (pericarp) Aqueous extract Methanolic extract Ethanolic extract T. diversifolia (leaves) Aqueous extract Methanolic extract Ethanolic extract Physical aspect Color Yield (% of crude extracts) Powdery Thick Thick Brown Black Black 17.71 6.78 5.7 Dense Thick Dense Greenish Black Black 7.65 5.70 5.20 Thick Dense Dense Brown Brown Brown 32.91 16.61 9.9 Thick Thick Thick Greenish Black Black 16.49 2.46 2.44 Table 2. Fungal species isolated from Monodora myristica kernels with accession numbers, isolate code, maximum percent identity with Genbank sequences, isolation percentage and pathogenicity. Fungal species and accession C. purpurea (JX143676) F. oxysporum (JN230149) A. flavus (HQ340108) A. niger (KU681408) R. nigricans (KT852980) A. oryzae (AP007173) C. reflexum (U3461168) C. bainieri (KP024561) Isolate code E012_MM K112_MM E015_MM E028_MM D032_MM Y046_MM E023_MM F181_MM Max % identity 100 100 100 100 98.6 95.7 94.6 92.5 Isolation percentage (%) 34.28 23.81 17.14 8.57 7.62 3.81 2.85 1.90 Pathogenicity test* +++ +++ ++ ++ ˗ ˗ ˗ ˗ *+++, very aggressive isolates, ++, moderately aggressive isolates; -, non-pathogenic isolates. D, E, F and K are the abbreviations for Dschang, Ebolowa, Fontem and Kumba, respectively, the name of the locality of isolate origin. purpurea, F. oxysporum, A. flavus and A. niger. Based on the literature, they are among pathogenic fungi responsible for post-harvest losses in foodstuffs. All aqueous extracts significantly inhibited the growth of all the fungi tested, compared to the untreated control (Table 3). Treatment with extracts of A. indica, M. oleifera and C. sinensis, inhibited the radial growth of C. purpurea at 125 mg/ml and was significantly greater than the negative control. Similarly, the extract of A. indica significantly reduced the radial growth of F. oxysporum at 125 mg/ml, more than the control without fungicide. Antifungal activity of aqueous extracts of M. oleifera and C. sinensis was significantly lower as compared to Mancozeb against A. niger at 125 mg/ml. The extracts of A. indica, M. oleifera and C. sinensis significantly reduced the radial growth of A. flavus (Table 3). Antifungal activity of methanolic and ethanolic extracts All methanolic extracts significantly reduced the radial growth of the fungi compared to the negative control in all concentrations tested (Table 4). The methanolic extracts of A. indica, C. sinensis and T. diversifolia presented an antifungal activity against A. flavus, which were significantly lower to Mancozeb at 100 mg/ml. Ethanol extracts of all the tested plants significantly reduced the radial growth of C. purpurea and A. flavus, as compared to the negative control, in all concentrations Djeugap et al. 70 Figure 1. Pathogenicity test of some fungal species on kernels of M. myristica. Mycelium and fruiting bodies were visible on kernels inoculated with C. purpurea (1), F. oxysporum (2), A. flavus (3) and A. niger (4), and control kernel (0). Table 3. Diameter of radial growth of fungi in PDA medium supplemented with aqueous extracts. Concentration (mg/ml) A. indica 0 50 75 100 125 Mancozeb (1 mg/ml) 87.6±2.7 c 26.3±2.7 c 24.7±2.5 c 24.5±3.1 d 21.8±1.3 f 3.5±1.1 0 50 75 100 125 Mancozeb (1 mg/ml) 87.6±2.7 d 21.7±2.6 d 18.7±2.6 de 13.8±2.8 e 8.3±1.0 f 3.5±1.1 0 50 75 100 125 Mancozeb (1 mg/ml) 87.6±2.7 b 61.3±7.2 b 55.7±6.3 c 46.3±2.1 c 29.8±3.9 f 3.5±1.1 0 50 75 100 125 Mancozeb (1 mg/ml) 87.6±2.7 c 30.7±3.9 c 30.2±5.1 c 29.8±6.4 d 21.1±8.3 f 3.5±1.1 a a a a Radial growth of fungi (mm)* C. sinensis M. oleifera A. flavus a a 88.6±1.7 85.7±4.5 d b 22.3±1.4 23.2±1.2 d b 20.4±3.5 21.3±1.1 c b 17.8±0.2 19.1±2.3 c b 16.7±1.4 17.7±1.2 d f 2.9±1.2 3.0±0.9 A. niger a a 88.6±1.7 85.7±4.5 e d 16.5± 3.1 23.4±4.1 ef d 13.1± 1.2 19.1± 3.1 ef de 11.1± 3.2 13.3± 1.4 f e 5.3 ±1.0 6.2± 0.6 g f 2.9±1.2 3.0±0.9 C. purpurea a a 88.6±1.7 85.7±4.5 b b 57.6±6.2 55.5±5.7 b b 54.6±8.3 53.8±7.4 b c 52.3±2.7 39.5±4.3 c cd 43.1±2.0 31.2±4.9 g f 2.9±1.2 3.0±0.9 F. oxysporum a a 88.6±1.7 85.7±4.5 ef de 12.9±1.8 11.3±1.5 ef d 10.5±3.5 27.7±4.0 ef d 10.3±2.1 18.3±5.5 d c 31.2±8.1 42.5±7.3 g f 2.9±1.2 3.0±0.9 T. diversifolia a 85.8±4.1 d 29.6±2.5 d 27.9±1.4 d 31.2±1.1 d 34.8±3.7 g 3.7±1.6 a 85.8±4.1 e 21.9 ±2.7 f 15.5±2.3 f 17.3± 1.8 e 21.7± 2.5 g 3.7±1.6 a 85.8±4.1 c 57.3±5.5 c 63.2±9.7 c 68.7±5.4 b 76.2±6.5 g 3.7±1.6 a 85.8±4.1 c 51.6±7.3 e 21.9±5.2 cd 46.2±8.6 cd 48.1±9.9 g 3.7±1.6 *Means follow by the same letter in the column are not significantly different based on Duncan multiple range test at 5%. Int. J. Adv. Agric. Res. 71 Table 4. Diameter of radial growth of fungi in methanolic extracts. Radial growth of fungi (mm)* Concentration (mg/ml) Azadirachta indica 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 e 18.6±2.2 f 14.4±1.2 f 14.1±1.5 f 12.8±2.1 g 3.5±1.1 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 e 20.2±1.2 e 19.1±1.5 e 17.4±2.9 f 13.6±6,1 g 3.5±1.1 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 b 58.7 ±4.5 b 57.2± 3.1 b 55.7± 4.9 c 46.3± 4.8 g 3.5±1.1 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 d 33.3±6.3 d 35.4±8.7 d 36.5±3.1 c 42.3±4.1 g 3.5±1.1 C. sinensis M. oleifera T. diversifolia A. flavus a a a a a 88.6±1.7 f 15.5±3.6 f 14.3±0.7 e 19.2±4.2 e 19.4±4.6 g 2.9±1.2 a 85.7±4.5 d 15.6±5.8 d 14.4±1.5 de 12.6±1.2 de 11.8±0.7 f 3.0±0.9 A. niger a a 88.6±1.7 85.7±4.5 e d 22.4±1.5 19.9±2.7 e d 22.3±1.6 17.4±1.2 e d 20.8±2.7 16.8±2.1 d d 19.7±3.8 14.1±3.5 g e 2.9±1.2 3.0±0.9 C. purpurea a a 88.6±1.7 85.7±4.5 b b 60.6 ±5.4 61.3± 3.2 c b 55.2 ±4.7 59.5 ±5.3 c b 49.3± 2.1 59.4 ±2.1 c b 46.8 ±3.5 57.3± 4.4 g e 2.9±1.2 3.0±0.9 F. oxysporum a a 88.6±1.7 85.7±4.5 cd c 41.4±5.9 35.3±5.6 cd c 38.5±0.9 38.5±6.3 d c 34.2±5.9 34.2±4.5 e c 22.3±1.8 37.7±3.9 g e 2.9±1.2 3.0±0.9 a 85.8±4.1 e 19.9±1.5 f 15.7±1.1 f 15.5±1.3 f 14.2±0.8 g 3.7±1.6 a 85.8±4.1 ef 22.3±2.8 ef 20.6±2.4 ef 20.3±2.4 g 16.3±2.5 h 3.7±1.6 a 85.8±4.1 b 75.7± 6.9 c 65.5±4.2 c 61.6±8.4 d 55.7± 1.2 h 3.7±1.6 a 85.8±4.1 e 36,7±9,3 e 31.5±4.6 e 30.3±3.2 ef 23.6±2.2 h 3.7±1.6 *Means follow by the same letter in the column are not significantly different based on Duncan multiple range test at 5%. tested. On the other hand, the extract of M. oleifera presented an antifungal activity significantly lower to the negative control at 40 mg/ml with respect to F. oxysporum. It was the same for the extract of C. sinensis at concentrations of 40, 60 and 80 mg/ml against A. niger. Extracts of A. indica and M. oleifera have shown antifungal activity against A. flavus comparable to mancozeb at the concentration of 100 mg/ml (Table 5). DISCUSSION Yields of plant extracts The variability of extraction yields from one plant species to another can be explained by the difference in plant species used, and the stage of the vegetative cycle of the plant during harvest. Svoboda and Hampson (1999) and Smallfield (2001) also reported that environmental conditions, the harvest period and the age of the plant material, can influence the extraction yields. In addition, the yeild may depends on the botanical family to which the species belongs (Valnet, 1980; Djeugap et al., 2011). Inventory of fungal species associated with M. myristica kernels Several known post-harvest fungi were associated with M. myristica kernels. They included A. flavus, A. oryzae, A. niger, C. purpurea, F. oxysporum and R. nigricans. Djeugap et al. 72 Table 5. Diameter of radial growth of fungi in ethanolic extracts. Concentration (mg/ml) A. indica a Radial growth of fungi (mm)* C. sinensis M. oleifera A. flavus a a 88.6±1.7 85.7±4.5 de e 27.5±3.2 21.4±2.8 de f 25.5±1.3 16.5±1.1 e f 22.5±4.1 15.2±2.6 e g 22.7±4.5 11.3±2.8 f h 2.9±1.2 3.0±0.9 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 d 22.3±3.6 d 19.1±2.4 de 17.4±1.5 e 14.5±2.1 f 3.5±1.1 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 d 21.5±2.1 d 20.5±1.8 d 21.7±2.6 d 19.3±2.2 f 3.5±1.1 a 88.6±1.7 de 30.5±2.7 de 28.5±2.1 de 28.2±2.3 e 23.5±1.2 f 2.9±1.2 0 40 60 80 100 Mancozeb (1 mg/ml) a 87.6±2.7 b 60.1±1.4 b 59.3±0.7 c 55.1±3.2 c 53.6±1.1 f 3.5±1.1 a 0 40 60 80 100 Mancozeb (1 mg/ml) 87.6±2.7 c 52.6±3.4 cd 48.1±4.9 cd 47.6±5.4 cd 45.4±7.1 f 3.5±1.1 T. diversifolia a 85.8±4.1 f 24.8±1.3 f 20.6±3.7 f 18.2±1.4 f 17.6±2.3 g 3.7±1.6 A. niger a a 85.8±4.1 f 25.3±3.1 f 24.6±1.2 f 22.1±1.5 f 21.8±2.6 g 3.7±1.6 85.7±4.5 b 60.4±2.3 b 60.1±3.7 c 55.5±3.9 c 53.7±4.8 h 3.0±0.9 a 85.8±4.1 b 61.5±2.8 c 54.8±2.5 d 46.4±3.4 d 43.7±2.1 g 3.7±1.6 F. oxysporum a a 88.6±1.7 85.7±4.5 d c 34.6±2.6 56.3±2.5 de d 30.5±3.1 45.6±4.2 de d 30.4±3.2 44.5±8.6 de d 27.6±6.3 40.2±6.1 f h 2.9±1.2 3.0±0.9 85.8±4.1 d 45.8±3.1 d 44.3±3.5 e 37.7±3.6 e 34.8±2.2 g 3.7±1.6 85.7±4.5 e 24.7±3.1 ef 19.8±1.5 ef 18.5±3.4 f 16.3±2.1 h 3.0±0.9 a C. purpurea a 88.6±1.7 b 55.3±4.6 b 50.4±3.5 c 45.8±3.4 c 42.3±3.3 f 2.9±1.2 a a *Means follow by the same letter are not significantly different based on Duncan test at 5%. Most of these fungi have been isolated from other edible non-timber forest products and other crop products. They are generally the cause of the post-harvest damage in certain fruits and foodstuffs, with high losses (El-Guilli et al., 2009; Enyiukwu et al., 2014; Onuorah and Orji, 2015). This is the case of C. purpurea which was also isolated on Persea americana fruit with a high occurrence frequency (Erute and Oyibo, 2008; Djeugap et al., 2015). F. oxysporum, isolated from M. myristica kernels was also reported on P. americana, but at a low frequency (Erute and Oyibo, 2008). Gaikwad et al. (2014) showed that F. oxysporum was pathogenic on onion (Allium cepa L.) during storage. It was also found that A. niger was responsible for post-harvest losses on Psidium guajava and P. americana with a high isolation frequency (Erute and Oyibo, 2008; Amadi et al., 2014). Djeugap et al. (2009) showed that R. nigricans and other fungal species were associated with post-harvest pathology of mangoes. A higher isolation frequency of F. oxysporum was also reported by Ebele (2011) on papaya fruit (Carica papaya L.); however, that of A. niger was high in papaya compared to M. myristica. Marin et al. (1996), during studies carried out in Mexico, Costa Rica and Ecuador showed that Fusarium spp. and Penicillium spp. were identified as the main causes of bananas crown rot. The high occurrence frequencies of C. purpurea, F. oxysporum and A. flavus obtained from M. myristica kernels could be explained by the fact that these fungi are polyphagous and cosmopolitan. The poor storage conditions of the kernels (in air tight bags for a long period of time) could also favor their presence in the kernels. To the best of our knowledge, this is the first time Int. J. Adv. Agric. Res. these fungi are reported in M. myristica kernels. Evaluation of the antifungal efficiency of plant extracts In vitro effectiveness of plant extracts on fungal growth differs from one fungus to another and from one plant extract to another. The plant extracts tested showed a higher suppressive effect on the radial growth of the four fungi tested, compared to the negative control. Aqueous extracts of M. oleifera and C. sinensis showed a suppressive effect which was comparable to the reference fungicide used. Aspergillus niger was the most sensitive fungus to the aqueous extracts, compared to A. flavus, C. purpurea and F. oxysporum. Onyeani et al. (2012) obtained similar results with the aqueous extracts of Acalypha ciliata, Aloe vera and Vernonia amygdalina on radial growth of A. flavus and Penicillium expansum. The efficacy of the aqueous extracts of A. indica is similar to results obtained by Nweke and Ibian (2012) and Tsopmbeng et al. (2014) against Colletotrichum gloeoporioides and Phytophthora colocasiae, respectively. However, Oluma and Elaigwe (2006) reported that the aqueous extracts of A. indica have no inhibitory effect on the growth of Macrophomina phaseolina and Rhizoctonia solani. The efficiency of the plants tested in this study against foodborne fungi was previously established against other plant and human pathogens. Maragathavalli et al. (2012) have demonstrated that maximum growth inhibition of Bacillus pumilus, Pseudomonas aeruginosa and Staphylococcus was obtained with methanol and ethanol extract of A. indica leaves, compared to antibiotics gentamycin (200 mg) and gentamycin (10 mg). Leaves and bark extract of the same plant was also efficient against Escherichia coli and Staphylococcus aureus. Madhuri et al. (2014) showed that the peel extract of Citrus fruits was efficient against Colletotrichum capsici, which causes anthracnose of chili. Antifungal properties of T. diversifolia extracts have been previously established against plant pathogenic fungal species, such as Alternaria alternata, A. solani, P. expansum and P. italicum and human pathogenic bacteria such as Enterococcus faecalis, E. coli, P. aeruginosa and S. aureus (Linthoingambi and Singh, 2013). Therefore, the plants tested have molecules with antifungal properties. Phytochemical screening of extracts of some of these plants had been carried out showing that they possess many organic compounds that could be responsible for their antimicrobial activities. It is the case of T. diversifolia leave extracts contain alkaloids, saponins, tannins, cardiac glycosides and volatile oils (Dewole and Oni, 2013); M. oleifera petroleum leaves extracts are rich in tannins, phenols, alkaloids, flavonoids, oxalates and saponins, which forms bioactive components against 73 Streptococcus spp. growth (Ndhlala et al., 2014; Ajayi and Fadeyi, 2015). The peel of C. sinensis fruit contains limonene, linalool and α-pinene which have antifungal activities against foodborne pathogens such as A. paraciticus (Abdel-Fattah et al., 2015; Shalu et al., 2015). Nevertheless, in some cases there is an increase in radial growth of fungi with increase of plant extracts concentration. Bonzi (2007) showed that, depending on the fungus species and the maceration time, plant extracts could have opposite effects. For example, aqueous extracts of Cymbopogon citratus macerated during 6 and 12 h stimulate the radial growth of Phoma sorghina, whereas those macerated during 24 and 48 h completely inhibit the growth of that pathogen. In this study T. diversifolia aqueous extract increase radial growth of all the fungi tested (A. flavus, A. niger, C. purpurea and F. oxysporum) from 75 to 125 mg/ml. This could be due to the high nitrogen content of T. diversifolia leaves (Kaho et al., 2011; Kiye et al., 2013). Nitrogen is one of the key elements that enhance growth and increase microbial biomass (Wang et al., 2008; Ramirez et al., 2012). Conclusion The most frequent fungi associated with M. myristica kernels are A. flavus, A. niger, C. purpurea and F. oxysporum. C. purpurea and F. oxysporum isolates were more aggressive in inoculation studies. Aqueous extracts of C. sinensis and M. oleifera showed high antifungal activity at 125 mg/ml against A. niger which was comparable to Mancozeb. Methanolic extracts of A. indica, C. sinensis and T. diversifolia showed antifungal activity at 100 mg/ml against A. flavus which was comparable to the Mancozeb. However, the aqueous extract of T. diversifolia favored the growth of the tested fungi from the concentration of 75 mg/ml. A. indica, C. sinensis, M. oleifera and T. diversifolia extracts indeed possessed valuable products which could be exploited to control post-harvest diseases on M. myristica kernels. Aspergillus isolates should be tested for their ability to produce aflatoxin and in vivo tests should be carried out to confirm the efficiency of these plant extracts in managing fungi of M. myristica kernels. ACKNOWLEDGEMENTS The molecular part of this project was supported by the BecA-ILRI Hub through the Africa Biosciences Challenge Fund (ABCF) program. The ABCF Program is funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Djeugap et al. 74 Department for International Development (DFID) and; the Swedish International Development Cooperation Agency (Sida). The collaboration of researchers from the Phytopathology and Agricultural Zoology Research Unit and Microbiology and Antimicrobial Substances Research Unit of the University of Dschang is highly appreciated. REFERENCES Abdel-Fattah S. M., Yehia H. A., Fouzy A. S. M., Manal M. & Nooh M. (2015). Antifungal efficacy and chemical composition of essential oil from the Egyptian sweet orange peel (Citrus sinensis L.). Int. J. Adv. Res. 3(10):1257-1269. Abiamere C. O., Nweke C. N., Ogbadu L. J., Onyia O. C. & John C. O. (2014). Evaluation of Moringa oleifera, Zingiber officinale and Chromolena odorata plant extracts against seed borne fungi of cowpea. J. Pharm. Biol. Sci. 9(6):66-70. Ajayi A. O. & Fadeyi T. E. (2015). Antimicrobial activities and phytochemical analysis of Moringa oleifera leaves on Staphylococus aureus and Streptococcus species. Am. J. Phytomed. Clin. Ther. 3(10):643-653. Alexopoulos C. J., Mims C. W. & Blackwell M. (1996). Introductory mycology. 4th Ed. Wiley, New York. Amadi J. E., Nwaokike P., Olahau G. S. & Guraba T. (2014). Isolation and identification of fungi involved in the post-harvest spoilage of Guava (Psidium guajava) in Awka Metropolis. Int. J. Eng. Appl. Sci. 4(10):7-12. Barnett H. L. & Hunter B. B. (1972). Illustrated genera of imperfect fungi. 3rd Edition. Burgess Publishing Company. 200p. Bonzi S. (2007). Efficacité des extraits aqueux de quatre plantes dans la lutte contre les champingnons transmis par les semences de sorgho (Sorghum bicolor (L) Moench): cas particulier de Colletotrichum graminicola (Ces.) Wilson et Phoma sorghina (Sacc.) Bocrema. Burabai W., Akor A. J., Igoni A. H. & Puyate Y. T. (2007). Fracture resistance of African Nutmeg (Monodora myristica) compressive loading. Electron J. Environ. Agric. Food Chem. 6(10):2434-2442. Champignon R. (1997). Identifier les champignons transmis par les semences. Edition INRA France. 397p. Dewole O. O. & Oni S. O. (2013). Phytochemical and antimicrobial studies of extracts from the leaves of Tithonia diversifolia for pharmaceutical importance. J. Pharm. Biol. Sci. 6(4):21-25. Djeugap F. J., Kuiate J. R. & Fontem D. A. (2009). Etat sanitaire postrécolte de la mangue commercialisée dans la ville de Dschang et efficacité in vitro des huiles essentielles contre Colletotrichum gloeosporioides Penz., agent causal de l’anthracnose. Annales de la 9ème Conférence Internationale des maladies des plants. Pp. 77-91. Djeugap F. J., Fontem D. A. & Tapondjou A. L. (2011). Efficacité in vitro des extraits de plantes contre le mildiou (Phytophthora infestans) de la morelle noire. Int. J. Biol. Chem. Sci. 5(6):2205-2213. Djeugap F. J., Tsopmbeng N. G., Keuete K. E., Yaouba A. & Serferbe S. (2015). Isolation and identification of fungi associated with Avocado fruits from local markets of the West Region of Cameroon. Int. J. Agric. Biosci. 4(2):64-68. Ebele M. I. (2011). Evaluation of some aqueous plant extracts used in the control of pawpaw fruit (Carica papaya L.) rot fungi. J. Appl. Biosci. 37:2419-2424. El-Guilli M., Achbani E., Fahad & Jijakli H. (2009). Biopesticides, alternatives à la lutte chimique? Partie 4: Culture, itinéraire technique et productivité. Symposium international Agriculture durable en région Méditerranéenne. Pp. 266-280. Enyiukwu D. N., Awurum A. N. & Nwaneri J. A. (2014). Efficacy of plant derived pesticides in controlof myco-induced post-harvest rots of tubers and agricultural products: a review. J. Agric. Sci. 2(1):30-46. Erute M. O. & Oyibo A. E. (2008). Effects of three plant extracts (Ocimum gratissimum, Acalypha wilkesiana and Acalypha macrostachya) on post harvest pathogen of Persea americana. J. Med. Plants Res. 2(11):311-314. Faleyimu O. I. & Oluwalana S. A. (2008). Proximate analysis ofMonodora myristica (Gaertn.) Dunal (Africa Nutmeg) in Ogun state, Nigerian. World J. Biol. Res. 1:1-7. Gaikwad K. N., Jadhav S. U. & Kakulte V. R. (2014). Management of fungal diseases of onion (Allium cepa L.) by using plant extract. Int. J. Life Sci. Pharm. Res. 4(2):28-30. Imathiu S. M., Edwards S. G., Ray R. V. & Back M. (2014). Artificial inoculum and inoculation techniques commonly used in the investigation of Fusarium head blight in cereals. Acta Phytopathologica et Entomologica Hungarica. 49(2):129-139. Iqbal N. & Saeed S. (2012). Isolation of mango quick decline fungi from mango bark beetle, Hypocryphalus mangiferae S. (Coleoptera: Scolytidae). The J. Anim. Sci. 22:644-648. Joshi B., Sah G. P., Basnet B. B., Bhatt M. R., Sharma D., Subedi K., Pandey J. & Malla R. (2011). Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentate (Datiwan) and Azadirachta indica (Neem). J. Microbiol. Antimicrob. 3(1):1-7. Kaho F., Yemefack M., Teguefouet P. F. & Tchantchaouang J. C. (2011). Effet combiné des feuilles de Tithonia diversifolia et desengrais inorganiques sur les rendements du maïs et lespropriétés d’un sol ferralitique au Centre Cameroun. Tropicultura. 29(1):39-45. Kiye N. M. N., Ayingwe L., Luyindula N. L. & Babelangi A. (2013). Amendement des sols: Influence des fertilisants pour l’amélioration de laculture de Vigna unguiculata (L) Walp. Int. J. Biol. Chem. Sci. 7(5):2029-2039. Korsten L., Sanders G. M. & Grosse-Weischede E. (1994). Isolation and pathogenicity of avocado post-harvest pathogens from Westfalia and other avocado producing areas. South African Avocado Growers’ Association Year book. 17: 46-48. Linthoingambi W. & Singh M. S. (2013). Antimicrobial activities of different solvent extracts of Tithonia diversifolia (Hemsely) A. Gray. Asian J. Plant Sci. Res. 3(5):50-54. Madhuri S., Hedge A. U., Srilakshmi N. S. & Kekuda P. T. R. (2014). Antimicrobial activity of Citrus sinensis and Citrus aurantium peel extracts. J. Pharm. Sci. Innov. 3(4):366-368. Maragathavalli S., Brindha S., Kaviyarasi N. S., Annadurai B. & Gangwar S. K. (2012). Antimicrobial activity in leaf extract of neem (Azadirachta indica Linn.). Int. J. Sci. Nat. 3(1):110-113. Marin D. H., Sutton T. B., Blankenship S. M. & Swallow W. H. (1996). Pathogenicity of fungi associated with crown rot of bananas in Latin America on Grande Naine and disease-resistant hybrid bananas. Plant Dis. 80:525-528. N’guessan K., Kadja B., Guédé N. Z., Traoré D. & Aké-Assi L. (2009). Screening phytochimique de quelques plantes médicinales ivoiriennes utilisées en pays Krobou (Agboville, Côte d’Ivoire). Sciences et Nature. 6(1):1-15. Ndhlala A. R., Mulaudzi R., Ncube B., Abdelgadir H. A., Plooy C. P. & Staden J. V. (2014). Antioxidant, antimicrobial and phytochemical variations in thirteen Moringa oleifera Lam. cultivars. Molecules. 19:10480-10494. Nguefack J., Wulff G. E., Dongmo J. B. L., Fouelefack F. R., Fotio D., Mbo J. & Torp J. (2013). Effect of plant extracts and essential oil on the control of brown spot disease, tillering, number of panicles and yield increase in rice. Eur. J. Plant Pathol. 137:871-882. Nweke C. N. & Ibian O. F. A. (2012). Effect of extracts from leaves, bark and root of Azadirachta indica L. on the vegetative growth of Colletrichum gloeosporoides: field soft rot pathogen of soursop (Anona muricata L.) fruit. Agric. Boil. J. N. Am. 3(12):481-485. Nwozo S. O., Kasumu T. F. & Oyinloye B. E. (2015). African Nutmeg (Monodora Myristica) Lowers Cholesterol and Modulates Lipid Peroxidation in Experimentally Induced Hypercholesterolemic Male Wistar Rats. Int. J. Biomed. Sci. 11(2):86-92. Oluma H. O. A. & Elaigwe M. (2006). Antifungal activity of extracts of some medicinal plants against Macrophomina phaseolina. J. Bot. 19(1):121-128. Onuorah S. & Orji M. U. (2015). Fungi associated with the spoilage of Int. J. Adv. Agric. Res. post-harvest tomato fruits sold in major markets in Awka, Nigeria. Univ. J. Microbiol. Res. 3(2):11-16. Onyeani C. A., Osunlaja S. O., Oworu O. O. & Joda A. O. (2012). Evaluation of effect of aqueous plant extract in the control of storage fungi. Int. J. Sci. Technol. Res. 1:76-79. Patel P., Patel N., Patel D., Desai S. & Meshram D. (2014). Phytochemical analysis and antifungal activity of Moringa oleifera. Int. J. Pharm. Pharm. Sci. 6(5):144-147. Ramirez K. S., Craine J. M. & Fierer N. (2012). Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes. Global Change Biology 18:1918-1927. Riaz H., Begum A., Raza S. A., Khan Z. M., Yousaf H. & Tariq A. (2015). Antimicrobial property and phytochemical study of ginger found in local area of Punjab, Pakistan. Int. Curr. Pharm. J. 4(7):405409. SAS Institute Inc., SAS 9.1.3 Help and Documentation, Cary, NC: SAS Institute Inc. 240p. Shalu H. S., Geeta I. & Ashok W. (2015). Antimicrobial activity of Citrus sinensis (Orange), Citrus limetta (Sweet Lime) and Citrus limon (lemon) peel oil on selected foodborne pathogens. Int. J. Life Sci. Res. 3(3):35-39. Smallfield B. (2001). Introduction to growing herbs for essential oils, medicinal and culinary purposes. Crop and Food Research. 45:4. Svoboda K. P. & Hampson J. B. (1999). Bioactivity of essential oils of selected temperate aromatic plants: antibacterial, antioxidant, antiinflammatory and other related pharmacological activities. http://www.csl.gov.uv/ienica/seminars. Thompson J. D., Higgins D. G. & Gibson T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Resources. 22:4673-4680. Trabi F. H., Irié G. M., N’Gaman K. C. C. & Mohou C. H. B. (2008). Études de quelques plantes thérapeutiques utilisées dans le 75 traitement de l’hypertension artérielle et du diabète: deux maladies émergentes en Côte d’Ivoire. Sci. Nat. 5(1):39-48. Tsopmbeng N. G., Megatche C. J. P., Lienou J. A., Yaouba A., Djeugap F. J. & Fontem D. A. (2014). Evaluation des activités antifongiques des extraits de plantes contre Phytophthora colocasiae, agent causal du mildiou du taro [Colocasia esculenta (L.) Schott]. J. Appl. Biosci. 81:7221-7232. Ul-Haq S., Hasan S. S., Dhar A., Mital V. & Sahaf K. A. (2014). Antifungal properties of phytoextracts of certain medicinal plants against leaf spot disease of mulberry, Morus spp. J. Plant Pathol. Microb. 5: 224. Uwakwe A. A. & Nwaoguikpe R. N. (2008). In-vitro antisickling effects of Xylopia aethiopica and Monodora myristica. Med. Plant Res. 2(6):119-124. Valnet J. (1980). Aromathérapie: traitement des maladies par les essences de plantes. 9e Ed. Maloine. 510p. Wang H., Mo J. M., Lu X. K., Xue J. H. & Li J. (2008). Effects of elevated nitrogen deposition on soil microbial biomass carbon in the main subtropical forests of southern China. Acta Ecologica Sinica. 28: 470-478. White T. J., Bruns T., Lee S. & Taylor J. (1990). PCR Protocols. In: A. Innis, Gelfand D.H. and J.J. Sninsky (eds.), Academic Press, San Diego, USA. Pp. 315-322. Zaker M. (2014). Antifungal Evaluation of some plant extracts in controling Fusarium solani, the causal agent of potato dry rot In vitro and In vivo. International Journal of Agriculture and Biosciences. 3(4): 190-195.