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Azadirachta indica reduces black sigatoka in east african highland banana by direct antimicrobial effects against Mycosphaerella fijiensis without inducing resistance

Black Sigatoka is a major disease of East African highland cooking bananas in Uganda. Aqueous extracts of Azadirachta indica , Cinnamomum zeylanicum and Capsicum annuum have shown the potential to reduce Black Sigatoka in banana plantlets. The mechanisms by which plant extracts confer protection against plant pathogens has previously been reported to involve activation of defence and direct antimicrobial activity. In the current study, both antimicrobial activities of selected extracts were studied as well as expression of three defence-related genes using quantitative real-time PCR. Gene expression was compared in susceptible (cv. Musakala, genomic group AAA-EA) and resistant (cv. Kayinja, genomic group ABB) banana cultivars. Additionally, Musakala treated with A indica extract at 1 day before inoculation (DBI) was tested for induction of defence-related genes at 0, 10 and 20 days after inoculation (DAI). Pathogenesis-related genes ( PR-1 and PR-3 ) and non-expressor of PR-genes ( NPR1B ) were up-regulated in the resistant cultivar. The genes analysed responded at late time points to M. fijiensis inoculation in both extract-treated and control plants in the susceptible cv. Musakala. On the other hand, A. indica and C. annuum completely inhibited mycelial growth of M. fijiensis at 30% (w/v). These findings suggest that the effect of plant extracts on Black Sigatoka is strongly associated with the direct antimicrobial effects.


Introduction
Bananas and plantains constitute major tropical food crops and Black Sigatoka, caused by the fungus Mycosphaerella fijiensis, is economically the most destructive foliar disease of bananas and plantains worldwide (Friesen, 2016;Portal et al., 2011). M. fijiensis is a highly diverse hemibiotrophic fungus (Churchill, 2011), which induces foliar leaf streaks that considerably reduce the photosynthetically active leaf area. Estimates of losses to Black Sigatoka for dessert bananas and plantains are in the range of 20-80% in the absence of fungicides (Churchill, 2011;Anonymous, 2009).
In its biotrophic phase, M. fijiensis exclusively colonises the intercellular spaces between mesophyll cells and obtains nutrients from the host apoplast without formation of haustoria. The fungus stays in this phase for 3-4 weeks before any necrotic lesions appear (Lepoivre et al., 2003). When the apoplast is depleted of nutrients, the fungus switches to the necrotrophic phase in which it is predicted that toxins targeting the chloroplasts play an important role (Harelimana et al., 1997). M. fijiensis starts producing conidia in the early necrotrophic phase. The production of sexual spores, however, does not start until the final infection stage, in completely necrotic lesions (Bennett & Arneson, 2003). Knowledge derived from plant-pathogen interaction studies is highly important for the rational selection of genotypes resistant to plant pathogens (Lepoivre et al., 2003). Important aspects of the interactions between Musa spp. and M. fijiensis are still unknown (Timm et al., 2016;Portal et al., 2011). The current information in the literature regarding both fungal (Cho et al., 2008) and plant genes (Khayat, 2004) that are involved in the interaction between M. fijiensis and Musa spp. is poor. Therefore, detailed molecular studies of this host-pathogen interaction are necessary for a better understanding of the events that take place during the infection processes (Lepoivre et al., 2003).
Investigations on mechanisms of disease suppression by plant products have suggested that the active principles present in them may either act on the pathogen directly (Hubert, 2015;Amadioha, 2000) or induce resistance in the host plant, resulting in reduction of disease development (Baysal & Zeller, 2004). However, the effect depends on the type of extract. Guleria and Kumar (2006) reported that an aqueous leaf extract of neem (Azadirachta indica) provided control of the Alternaria leaf spot pathogen (Alternaria sesame) of sesame (Sesamum indicum) through induction of the enzymes phenylalanine ammonia lyase (PAL), peroxidase (POX) and phenolic compounds. In a related experiment done by Bengtsson et al. (2009), comparing the effects of a yucca extract and acibenzolar-S-methyl (ASM) on inhibition of Venturia inaequalis in apple leaves, observations suggested that the yucca extract acted mainly by a direct fungitoxic effect whereas ASM acted as a resistance inducer. Expression studies of two genes encoding the PR-proteins, PR-1 and PR-8, in apple seedlings indicated that yucca extract also activated plant defence since expression of both genes was up-regulated following yucca treatment, to a level similar to that observed after treatment with ASM. Additionally, Pal et al. (2011) reported that seed treatment of rice seeds with leaf extracts of Cymbopogan citrus and Ocimum sanctum increased accumulation of defence-related enzymes (PAL, catalase, POX, polyphenol oxidase and tyrosinase) in plants raised from the treated seeds compared to control seeds. Kagale et al. (2011) demonstrated that foliar application of aqueous leaf extracts of Zizyphus jujuba and Ipomoea carnea followed by challenge inoculation with Rhizoctonia solani induced resistance in rice. This was confirmed from the increased activities of the enzymes chitinase, β-1,3-glucanase and POX, as well as increased accumulation of defence-related compounds such as PAL and phenolic substances. This makes it clear that plant extracts can induce resistance in plants against pathogens as well as providing a direct antimicrobial effect.
The current study was designed to elucidate the mode of action of the aqueous extracts from Azadirachta indica, Cinnamomum zeylanicum and Capsicum annuum against M. fijiensis in a susceptible East African Highland banana (EAHB) cultivar Musakala (genomic group AAA-EA). Azadirachta indica, Cinnamomum zeylanicum and Capsicum annuum were selected for this study based on the results of prelimanry experiments in which 15 extracts were tested, and others rejected. Specifically, the aim was to test if direct antimicrobial effects and induced resistance were involved.

Preparation of Plant Extracts and Fungal Inoculum and Cultivation of Banana
Leaves of Azadirachta indica, bark of Cinnamomum zeylanicum and fruits of Capsicum annuum were washed with running tap water to remove soil contaminations, followed by rinsing with sterile distilled water three times. The samples were placed on benches in a screen house (temperature 22-28 °C) to dry for 7 days. The dried sample of each plant species was ground thoroughly into powder using a blender (Hamilton Beach Brands, Inc., USA). Mycosphaerella fijiensis (local isolate Mak01) was grown on potato dextrose agar (Sigma-Aldrich, USA) at 25 °C. Agar plugs (3 mm in diameter) from actively growing 14-day-old cultures were aseptically transferred to malt extract agar plates (Sigma-Aldrich, USA). The inoculated plates were sealed and incubated at 25 °C in an incubator for 21 days in darkness. Mycelium from each plate was scraped off with a sterile scalpel, bulked and weighed on pre-weighed sterile filter paper. The weighed mycelium was fragmented in a blender (Amazon, USA) at full speed for 3 min. A mycelium master suspension was prepared and diluted to a final concentration of 15 mg/ml. Two month old tissue culture banana plantlets of cv. Musakala from Makerere University Agricultural Research Institute tissue culture laboratory were transplanted to individual pots containing pre-sterilized loam soil. Banana plantlets were grown in the screen house at a temperature of 22-28 °C under natural light conditions for one month. Plantlets were used for experiments when they had 4-6 fully expanded leaves.

Tests for Direct Antimicrobial Activity of Plant Extracts
An aqueous extract concentration of 20% (w/v) was used to test for fungicidal activity under laboratory conditions. Three treatments (immersion of a 3-mm fungal plug obtained from an actively growing fungal culture on PDA separately for 1, 2 or 4 h in each extract) were randomly applied to 12 fungal plugs for each extract. A fungicide, difenoconazole at 0.1% (v/v, Syngenta, Kenya) was included for comparison. Fungal plugs were washed with sterile distilled water three times prior to inoculation on PDA plates to remove the crude extracts. Fungal blocks which were dipped in sterile distilled water for 1, 2 or 4 h were included as untreated controls. Treated fungal blocks were placed aseptically in the centre of 9 cm diameter Petri dishes, sealed and incubated at room temperature (25 °C) for 28 days. Colony growth was determined by measuring the diameter using a mathematical ruler. Percentage inhibition of mycelial growth was calculated using the formula: percentage inhibition = 100 × (1 -ab -1 ), with a = colony diameter for the treatment and b = colony diameter for the untreated control. A completely randomized design with 4 replications was adopted for the trial. The experiment was carried out twice.
Additionally, qualitative phytochemical examinations of the extracts were carried according to the procedure described by Prashanth et al. (2011) to profile the antimicrobial compounds present in the extract. To detect the presence of phenols, 1.0 ml of 10% (w/v) aqueous leaf extract of A. indica, fruit extract of C. annuum and bark extract of C. zeylanicum were treated with 3-4 drops of 1% (w/v) ferric chloride solution. Formation of bluish, black colour indicated the presence of phenols. For flavonoids, 1.0 ml 10% (w/v) extracts were treated with 3 drops of sodium hydroxide (1.2 M) solution in a test tube and formation of intense, yellow colour, which became colourless on addition of 0.1 ml of 0.1 M hydrochloric acid, indicated the presence of flavonoids. Wagner's reagent (2 g iodine and 6 g potassium iodide in 100 ml water) was used to test for alkaloids. To 1.0 ml of 10% (w/v) extract, 3 drops of Wagner`s reagent were added on the side of the test tube. Formation of brown/reddish precipitate indicated the presence of alkaloids. For tannins, 2-3 drops of ferric chloride solution (0.1 M) was added to 1.0 ml 10% (w/v) extract in a test tube. A dark green colouration indicated the presence of tannins. For saponins, 1 ml 10% (w/v) aqueous extract was added to 0.1 ml of distilled water in a test tube and shaken vigorously. A stable persistent froth for 20 min confirmed the presence of saponins. The experiment was performed twice with different batches of extract.

In Planta Tests for Effects of Plant Extracts under Controlled Conditions
Screen-house pot experiments were set up with tissue cultured banana plantlets using cooking banana cultivars Musakala, Mbwazirume, Mpologoma and Kibuzi as susceptible genotypes. In the first trial, three treatments (0, 10, 30% (w/v) of A. indica extract from the Lira district (Northern Uganda), the Mbale district (Eastern Uganda) and the Wakiso district in the central region of Uganda) were applied two days before inoculation (DBI) with M. fijiensis to test the effect of extracts made from plants harvested from different growth conditions. Treatments were randomly applied to 12 plants of Musakala for each extract growth condition. Disease was assessed at 45 DAI. To assess disease, number of lesions (severity) was counted.
In the second trial, banana cultivars Mpologoma, Mbwazirume and Kibuzi (susceptible to M. fijiensis in Uganda) were used to compare effect of the extracts on different banana cultivars. After preliminary tests using 10, 20 and 30% extract concentrations, 30% (w/v) extract was chosen for further studies since it was more efficacious than the 20% concentration used in the in vitro tests. Different spraying time points (0, 2, 4 or 6 DBI) were tested to select the optimal time of application. Disease was assessed at 45 DAI to determine how long the extract could reduce symptoms. To test whether the disease-reducing effect was dependent on induced resistance, an experiment was also performed in which treated and non-treated leaves on the same plant were inoculated. Four spraying time points (0, 2, 4 and 6 DBI) were tested and the disease was assessed at 45 DAI. For all experiments, a completely randomized design was adopted. Each experiment was performed at least twice.

Test for Effect of A. indica on Sporulation of M. fijiensis
To quantify the effect of A. indica on ascospore production of M. fijiensis, 5 cm long leaf segments were cut from banana leaves treated with A. indica (30% [w/v]) extract 0, 2, 4 and 6 DBI 45 days after inoculation. Percentage infected leaf area for each treatment was recorded. The experiment was conducted as fully randomized block experiment with three replications. Nine samples were obtained for each treatment. The leaves were observed under a stereo microscope for presence of pseudothecia. Pieces of the leaf containing three lesions with mature pseudothecia were cut and incubated in polythene bags with moist cotton wool for 48 h. The incubated samples were surface sterilised with 0.5% sodium hypochlorite for 5 min followed by 70% ethanol for 5 min. The treated leaf samples were serially rinsed in sterile distilled water, and then stapled onto sterile filter papers. The filter papers were ascetically transferred to lids of Petri dishes and left to discharge ascospores over potato dextrose broth (10 ml) for 6 h. For each sample, ascospore concentration was determined in two subsamples using a haemocytometer.

Tests for Gene Expression to Verify Induction of Resistance
Screen-house pot experiments were set up with tissue culture banana plantlets using cv. Psang awak and the cooking banana cv. Musakala as resistant and susceptible genotypes, respectively. Tissue culture plants of each banana genotype were transplanted in plastic pots and allowed to establish for 8 weeks. The two-month-old seedlings were transplanted to individual pots containing pre-sterilized loam soil (pH 6.9). Plantlets were grown in the screen-house at a temperature of 22-28 °C under natural light conditions for one month and were used for experiments when they had 4-6 fully expanded leaves. A completely randomized design was adopted for the trials.
In the first trial, two treatments (inoculation with either M. fijiensis or spraying with water) were randomly applied to 24 plants of each genotype to study the expression of the target genes in a tolerant and susceptible banana without extract treatment. M. fijiensis inoculum of approximately 15 mg mycelium/ml was prepared using isolate Mak01, preserved at the Makerere University Agricultural Research Institute, Kabanyolo, Uganda. A volume of 0.5 ml of inoculum (5 mg/ml) or water was applied on the abaxial surfaces of the first and second fully unfolded leaves until run-off, using a painter's brush. A random leaf sample (10 g) of three plants for each treatment was harvested at 0, 1, 5, 9, 13, 19, 21 and 25 DPI for RNA extraction. In the second trial, four treatments (spraying with extract of A. indica, water, inoculation with M. fijiensis and spraying with extract of A. indica plus inoculation with M. fijiensis) were applied to nine plants for each treatment. Inoculation was performed as for the first trial. Random leaf samples (10 g) of three plants for each treatment was harvested at 0, 10 and 20 DPI for RNA extraction.

RNA Extraction
RNA from each sample was extracted following the CTAB nucleic acid extraction protocol (Ssekiwoko et al., 2015). All materials (centrifuge tubes, pipette tips) used were RNase free. The mortars and pestles were sterilized by soaking in 2% H 2 O 2 overnight, then in 1 M NaOH for 1 h, rinsed in distilled water, baked at 140 °C for 2 h, cooled and wiped with DEPC treated water. Generally, 0.1 g of each frozen plant tissue was crushed into a powder in a sterile and frozen mortar and 1.5 ml pre-warmed (65 °C) extraction buffer was added to a test tube, which was incubated at 65 °C for 15 min followed by centrifugation at 7,558 g for 5 min. A volume of 700µl supernatant was transferred to a new sterile tube and 700 µl chloroform: isoamyl alcohol mixture (24:1, v/v) was added and mixed by inversion and then centrifuged at 7,558 g for 5 min at 4 °C. The aqueous phase (600 µl) was transferred to a new tube and an equal volume of a mixture of phenol: chloroform: isoamyl alcohol (25:24:1, v/v) added, mixed by inversion and then centrifuged at 7558 g for 5 min at 4 °C. Finally, 500 µl of the aqueous phase was transferred to a new tube and RNA precipitated by adding 10 M lithium chloride to a final molarity of 2.25 M followed by incubation at -20 °C for 8 h. The precipitate was then collected by spinning at 7,558 for 45 min. The pellet was dissolved in water to 450 µl, then 50 µl 3 M sodium acetate (pH 5.2) was added, followed by an equal volume of cold isopropanol (-20 °C) and the tube mixed by inversion. The mixture was kept at -20 °C overnight and then spun at 7,558 g for 2 min at 4 °C. The pellet was washed twice in 500 µl 70% (v/v) ethanol by tapping and spinning at 7,558 g for 2 min at 4 °C, air dried at room temperature for 40 min followed by re-suspension in 30 µl sterile nuclease free water (Fermentas, USA). The concentration was determined using a NanoDrop 2000 spectrophotometer (Wilmington, DE, USA) and the RNA stored at -20 °C. Genomic DNA traces were removed from the extracted RNA using a DNase treatment kit (Thermo Scientific, USA) following the manufacturer's protocol and then stored at -20 °C awaiting cDNA synthesis.

cDNA Synthesis
cDNA was synthesized using the RevertAid TM first strand cDNA synthesis Kit (Fermentas Life Sciences), according to manufacturer's direction, using 2.5 ng/ml random hexamers for priming. To 8 µl nuclease free water, 3 µl pure RNA sample and 1 µl random hexamer primers (0.2 µg/µl) were added. This mixture was incubated at 65 °C for 5 min, then immediately chilled on ice for 5 min and the content collected by a brief spinning. During reverse transcription, 4 µl 5 × reaction buffer, 1 µl Ribolock RNase inhibitor (20 U/µl), 2 µl 10 mMdNTP mix, and 1 µl reverse transcriptase (200 U/µl) was added to the primer annealing reaction mixture. This mixture was incubated at 25 °C for 5 min followed by incubation at 42 °C for 1 h and heating at 70 °C for 5 min to inactivate the reverse transcriptase. The cDNA was then chilled on ice, collected by spinning and quantified using a NanoDrop 2000 spectrophotometer, followed by dilution to 0.1 µg/µl using sterile nuclease free water before storage at -20 °C. The quality of the cDNA was verified by amplifying a 200 bp actin fragment with banana actin (house keeping gene) forward 5'-ACCGAAGCCCCTCTTAACCC-3' and reverse 5'-GTATGGCTGACACCAT CACC-3' primers.

Real-Time RT-PCR Primers
The expression profile of three banana defence-related genes was analysed in cDNA samples obtained from banana leaves. Primers for the genes are seen in Table 1. One of these genes, PR-1, was previously reported to be up-regulated in a Cavendish selection (genomic group AAA) tolerant to Fusarium oxysporum f. sp. cubense (Foc) (Van den Berg et al., 2007). PR-3, was found to be up-regulated following Foc challenge of cv. Lady Finger (genomic group AAB) and cv. Cavendish plants previously inoculated with non-pathogenic F. oxysporum (Forsyth, 2006). An endogenous gene, Musa 25s (Van den Berg et al., 2007), was used as a house-keeping gene (Endah et al., 2010). Primer sequences for the amplification of PR-1, PR-3 and Musa 25s were obtained from Van den Berg et al. (2007), whereas for non-expressor of pathogenesis-related protein (NPR1B), primer sequences came from Endah et al. (2010). MNPR1B expression is associated with increased PR gene expression in disease tolerant banana cultivar GCTCV-218 (Endah et al., 2010). All primers were synthesized by Bioneer Corporation (Munpyeongseo-ro, South Korea).

Quantitative RT-PCR
Gene expression analysis was performed in two technical replications for each of three biological replications, using a 7500 Applied Biosystem qPCR system with 150 ng cDNA and 10 ml of AccuPower® 2X Greenstar qPCR Master Mix (Bioneer Corporation, South Korea). Each PCR reaction consisted of 5 µl diluted template (30 ng/µl), 0.2 μM primers, and 10 µl AccuPower® 2X Greenstar qPCR Master Mix. The reaction volume was adjusted to 20 µl with nuclease-free water. Non-template control (NTC) reactions contained water instead of cDNA as template. Cycling consisted of an initial denaturation phase of 7 min at 95 °C, an amplification phase of 30 cycles each consisting of a denaturation step at 95 °C for 10 s, annealing at 63 °C for 5 s and extension at 72 °C for 30 s and data acquisition at 95 °C. Analysis of melting curves was carried out at the end of each run to evaluate undesired amplifications. Expression of the Musa 25s rRNA gene that was previously evaluated to be constitutively and constantly expressed in Musa spp. (Endah et al., 2010) was used as to normalise target gene data.

Data Analysis
The data on the effect of treatments on the growth of pathogens and Black Sigatoka severity scores, were analysed by analysis of variance assuming a normal distribution. Variances were stabilised by appropriate transformation of data if necessary. Data were analysed by PC-SAS (release 9.2, SAS Institute, Cary, NC). For the gene expression studies, temporal expression of each selected gene at all-time points were compared to its expression at 3 h (day 0) using the 2 -ΔΔCT relative gene expression method (Livak & Schmittgen, 2001). Hypotheses were rejected at P ≤ 0.05.

Direct Antimicrobial Activity of Plant Extracts against M. fijiensis
The fungicidal activities of extracts of Azadirachta indica leaves, Capsicum annuum fruits and Cinnamomum zeylanicum bark were tested to understand their ability to inhibit the vegetative forms of Mycosphaerella fijiensis, which was immersed in extract solutions (20% w/v) for different time periods (1, 2 and 4 h) (

Effect of Foliar Spraying with Azadirachta indica Leaf Extract on Black Sigatoka
Leaf extracts of Azadirachta indica grown at different locations in Uganda was used to verify that the protective effect was not dependent on the geographical origin of plant material ( Figure 1A). Concentrations of 0, 10 and 30% of the extracts were tested by spraying at 2 DBI. Mean lesion numbers were reduced compared with the control treatment using water at 45 DAI for 30% extract made from plants grown in the North (35.1%), East (30.1%) and 31.9% Central Uganda. Extracts from the three locations did not differ in ability to confer protection at 30% (w/v) extract concentration (P ≤ 0.110). Within each location, there was a significant effect of extract concentration (P ≤ 0.010, P ≤ 0.022 and P ≤ 0.010 for North, East and Central Uganda, respectively). The effect was subsequently verified in the three EABH cultivars Mpologoma, Mbwazirume and Kibuzi ( Figure 1C), using 0, 10 and 30% (w/v) extract from plants grown in northern Uganda. For 30 % extract, significant reductions of mean lesion numbers compared with the control treatment were observed in all the cultivars (35.9% for cv. Mpologoma, 37% for cv. Mbwazirume and 42.2% for cv. Kibuzi) (P ≤ 0.010).To find the optimal spraying time point and to study how long the extract could reduce symptoms of the disease, four spraying time points (0, 2, 4, and 6 DBI) were tested and the disease was assessed at 45 DAI ( Figure 1D). Based on previous results, 30% leaf extract was chosen for this experiment. Spraying time points of 0, 2 and 4 DBI significantly reduced disease (65.8, 41.4 and, 19.0%, respectively) whereas there was no effect for 6 DBI (2.6%).

Local and Systemic Effects of Azadirachta indica on Infection by M. fijiensis
To test whether the disease-reducing effect was systemic, an experiment was also performed in which extract treated and non-extract treated leaves on the same plant were inoculated with M. fijiensis. Four spraying time points (0, 2, 4, and 6 DBI) were tested and the disease was assessed at 45 DAI ( Figure 1B). Treatment of the youngest fully opened leaves with A. indica extract reduced the average infection level of M. fijiensis by 70.3, 38.8, 17.6 and 8.6% for pre-extract treatment timing of 0, 2, 4, 6 DBI respectively (Figure1B). In contrast, no systemic disease-reducing effect was observed on the non-extract primed leaves on the same test plants. No differences were seen between non-extract treated leaves on the test plants and control plants.

Effect of A. indica on Sporulation of M. fijiensis
Application of A. indica extract (30% w/v) reduced the sporulation of M. fijiensis (Table 3.). The efficacy depended on the timing of the application of A. indica. Three A. indica treatments (0, 2 and 4 DBI) reduced the average production of M. fijiensis ascospores, with the least reduction when A. indica was applied 4 DBI with M. fijiensis. The treatment of at 6 DBI did not reduce the mean ascospores production. Note. Means followed by the same letter within a column are not significantly different (P ≤ 0.05).

Qualitative Phytochemical Analysis of the Aqueous Extracts of Plant Extracts
The results of qualitative phytochemical analysis of A. indica leaf extract, C. annuum fruit extract and C. zeylanicum bark extract are given in Table 4. There were high levels of tannins and flavonoids and moderate levels of phenolic compounds and alkaloids in A. indica. In contrast, C. annuum and C. zeylanicum extracts only showed moderate level of flavonoids and tannins, respectively. Azadirachta indica and C. annuum were from the Northern part Uganda and C.zeylanicum from the Central part Uganda. Note. + = Trace, ++ = Moderate, +++ = High, -= Absent.

Expression of Defence-Related Genes (PR-Proteins) in Response to M. fijiensis Infection
The Note. Means marked with the same letters are not significantly different at P ≤ 0.05.

Expression of Defence-Related Genes of Banana Treated with Azadirachta indica Extract
Expression of the PR-proteins PR-3 and PR-1 (Figure 3) was studied in cv. Musakala after treatment with A. indica extract. Whereas the extract alone did not result in any alteration of expression of either gene, inoculation with the pathogen, and pre-treatment with the extract followed by pathogen inoculation, resulted in increased expression at 10 and 20 DAI.

Discussion
In the present study, aqueous extract of Azadirachta indica was found to possess the ability to protect banana against Black Sigatoka caused by Mycosphaerella fijiensis under controlled conditions in Uganda in the three East African Highland banana cultivars, cvs. Mbwazirume, Mpologoma and Kibuzi. Leaf extract of A. indica and fruit extract of Capsicum annuum completely inhibited the mycelial growth of M. fijiensis under in vitro conditions at a concentration of 30% (w/v). Extracts of A. indica from dry leaves grown in different regions in Uganda were able to significantly reduce disease. Plants of the same species grown under different conditions could vary, for example, in secondary metabolite production and therefore, in their ability to control the disease, but in the present study, the active components were maintained in plants grown under different conditions. Additionally, both fresh (data not presented) and dried leaves of A. indica were able to reduce the disease, showing that the active components of the extract still maintained their effect after drying. Although A. indica is a widely grown tree in Uganda whose fresh leaves could be easily obtained, using dried leaves potentially represents a useful solution for farmers in areas where the plant is not abundant.
Management of several fungal (Alternaria solani, Fusarium oxysporum, Hassanein et al., 2008;Sallam, 2011), bacterial (Xanthomonas campestris, Reddy, 2012) and nematode diseases (Meloidogyne javanica, Meloidogyne incognita) (Javed et al., 2008;Hadian et al., 2011) in greenhouse and field grown tomato plants by the application of extracts from various parts of A. indica have been reported (Goel et al., 2016). Additionally, Nahunnaro (2008) enumerated the antifungal effect of Azadirachta indica seed oil against yam rot pathogen (Rhizopus stolonifer), Joseph et al. (2008) and Nwogbaga and Utobo (2012) reported significant control of wilt and leaf spot diseases in Brinjal (Solanum melongena) by application of aqueous, ethanol and acetone extracts of A. indica leaves and seeds. The diverse findings clearly suggest that the use of A. indica in plant disease management has a vast future.
We observed a direct inhibitory effect of A. indica extract on M. fijiensis. Previously, it has been well documented that antimicrobial compounds are abundant in medicinal plants (Sangeetha et al., 2013;Ghosh et al., 2002;Fiori et al., 2000;Gayatri & Rajani, 2014) and these compounds could be involved in the defence of plants against pathogens. Interestingly, when two leaves on the same plant, one treated with extract and the other untreated, were inoculated with M. fijiensis, a disease reduction was observed only on the treated leaf. Furthermore, when the extract was soil drenched, the plants could not be protected when they were inoculated with the pathogen at 45 DAI. These observations indicate that protection was due to a direct effect and that no systemic effects were present. Indeed, several plant extracts have been reported to be capable of reducing diseases in plants through direct antimicrobial effects on the causal organisms (Guleria & Kumar, 2006;Konstantinidou-Doltsinis et al., 2006). Thousands of diverse natural products are produced by plants and many of these are involved in plant defence. The phytochemical diversity of antimicrobial compounds include terpenoids, saponins, phenolics and phenyl propanoids, pterocarpans, stilbenes, alkaloids, glucosinolates, hydrogen cyanide, indole and also elemental   -Guzmán et al., 2011;Snehlata et al., 2014). In this study, the phytochemical analysis of the aqueous extract of C. zeylanicum, A. indica and C. annuum (Table 4) showed the presence of different groups of secondary metabolites such as flavonoids, tannins, alkaloids and phenolics which are of medicinal importance. The result of our present study is in agreement with the results published by the other research groups (Prashanth & Krishnaiah, 2014;Susmitha et al., 2013).
Timing of the A. indica application was crucial for reduction in the M. fijiensis lesion number, as A. indica extract applied 6 DBI had no effect on lesion development, i.e., this extract acted as a protectant. The production of M. fijiensis ascospores was reduced depending on the timing of extract application. A. indica extract applied 6 DBI had no effect on ascospore production. When comparing ascospore production per 3 lesions (approximately 3mm in diameter) in the infected leaf area, reductions of 36.1-65.7% (P < 0.001) were also seen and this indicates that in the lesions, the pathogen was weakened and therefore produced fewer ascospores per lesion area. This implies that A. indica also influenced pathogen development after penetration, although this was not confirmed using microscopy. Such an observation could have a significant epidemiological impact, as the amount of inoculum available would be reduced since ascospore is the main mode of spread of M. fijiensis.
To test whether induced resistance was involved in the protection observed, expression of PR-protein genes during the infection process in the susceptible banana cultivar cv. Musakala and the resistant cv. Psang awak was investigated. The resistant cultivar was included as a positive control to achieve a better insight into the expression of the target genes during an incompatible interaction. PR-1 and PR-3 as well as NPR1 were differentially up-regulated in cv. Psang awak. This is in contrast to an earlier report where PR-3 and NPR1B transcription was not induced in cultivated banana (GCTCV-218) in response to Fusarium oxysporum (Endah et al., 2008). NPR1B expression is related to the expression of the two PR-related genes, PR-1 and PR-3, included in the study. The non-expressor of pathogenesis-related gene 1 (NPR1) is an essential positive regulator of systemic acquired resistance (SAR) (Endah et al., 2008) and induced systemic resistance (ISR) (Pieters et al., 1998). Being a hemibiotrophic fungus, M. fijiensis acts mainly as a necrotroph at the late stages of disease progression, which was part of the timing selected for the study of the expression of the target genes. Our findings suggest that SA signalling transduction pathways, which are generally correlated with resistance against necrotrophic pathogens (Glazebrook, 2005), are active during late stages of this incompatible interaction. Unlike necrotrophic pathogens, biotrophs and hemi-biotrophs, have been reported to induce predominantly a SA-mediated pathogen response (Spoel et al., 2007;Tao et al., 2009) and SA-elicitation has been shown to induce NPR1B transcription (Endah et al., 2008;Yocgo, 2011). Timely recognition of the pathogen and rapid, appropriate expression of defence responses has been reported to make a key difference between susceptible and resistant plants (McDowell & Dangl, 2000;Umemura et al., 2003;Bennett et al., 2005). Black Sigatoka development in the susceptible cultivar cv. Musakala is probably because defence mechanisms were induced too late.
The activity of natural plant products on the host tissue may involve direct interaction with the pathogen or induction of host resistance (Sangeetha et al., 2013;Capdeville et al., 2002). Previously, several authors have reported the association between higher levels of chitinase and greater disease resistance against postharvest diseases. In the present study, expression of PR-3 and PR-1 were up-regulated early during infection in the resistant cv. Psang awak, indicating an early induction in resistant plants. The levels of the two genes were substantially increased during the late stages of infection, further indicating high induction of these genes as the pathogen switched to the necrotic stage. The expression of NPR1 was consistent with the expression of PR-3 and PR-1 in cv. Psang awak plants challenge inoculated with M. fijiensis. To examine further whether A. indica could induce resistance against M. fijiensis, quantitative RT-PCR of the defence response genes PR-1, and chitinase (PR-3) were performed. These genes were selected because PR-proteins have previously been shown to be involved in the defence against M. fijiensis (Yocgo et al., 2011;Endah et al., 2008). However, none of the genes showed elevated expression when comparing plants treated with A. indica + M. fijiensis with plants treated with M. fijiensis alone. Collectively, our data therefore suggest that induced resistance is not responsible for the reduction in M. fijiensis severity by foliar application of A. indica extract. Of course, it is not possible to conclusively state that induced resistance is not part of the protection on the sole basis of the absence of expression of very specific defence responses, as other defence responses may be enhanced. Induced resistance as a mechanism, does not rely on the expression of specific, single defence genes. Therefore, the A. indica-mediated reduction in disease was most likely related to the direct inhibitory effect on the pathogen.

Conclusion
The finding that A. Indica leaf extract reduced the mycelia growth of of M. fijiensis implies that application of the extract should be conducted as a preventive treatment to reduce Black Sigatoka disease severity. However, A.
indica leaf extract may also have an impact on the epidemiological development of the disease in terms of reduced sporulation potential (ascospore formation) of the pathogen, as we observed. Ascospores are the main mode of spread of M. fijiensis. These issues need to be verified in field trials. Moreover, formulation of A. Indica extract with surfactants such as alkylbenzene sulfonates that improves coverage over an area may enhance the feasibility of the A. indica extract for spray applications by retaining the extract longer on the target area, slowing evaporation and inhibiting degradation by sunlight. The prospects of developing A. Indica extract into a commercial product are therefore promising, especially as alternative control options are anticipated to be a major part of future sustainable crop protection strategies.