2.1 Collection of infected BananaBanana fruits at 70-80 percent maturity with typical symptoms of crown rot disease (black to brown lesions on skin) were collected from different markets in various locations of Rajshahi, Bangladesh. The collected fruits and hands were thoroughly washed in running tap water to remove dusts and other impurities.2.2Collection and Extraction of Plant MaterialSelected plant specimens were dried under shade, milled using a laboratory mill, and extracted with methanol. The extract was filtered through folded filter paper into a 500 mL round bottom flask and reduced to dryness on a rotary evaporator at 40°C water bath temperature.Fifty grams of each milled plant specimens (Datura metel, Faidherbiaalbida, Acacia catechu, Allium sativum, Solanum torvum, Solanum spp., Persicariastagninaand Azadirachtaindica) were extracted using 250ml methanol with continuous stirring for 15days using a magnetic stirrer.
2.3 Collection and Isolation of Antagonistic AgentsPure culture of Trichoderma harzianum were obtained from the institute of biological sciences central laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh. Bacilluscereuswas isolated from soil samples(rhizosphere region)by plating a dilution series on LBagar medium[9]. Colony morphology of the isolated bacteria was recorded after 16h of growth on LB agar plate at 37°C. Gram staining test(A loop full of the bacteria was spread on a glass slide and fixed by heating on a very low flame) and a series of biochemical tests including triple Sugar Iron (TSI), Simmons citrate, Kligler-Iron Agar (KIA) test, Tween 80 hydrolysis tests, Methyl red and Mannitol tests were performed for the characterization of the isolated bacteria following the company manual instructions. The chemicals of different biochemical tests were collected from Oxido Ltd. Basingstoke, Hampshire, England.2.4 Isolation of Fruit Rot FungiFirst of all, infected portions from the collected banana fruits were thoroughly washed in running tap water to remove dusts and other impurities. After that, they were surface sterilized with 0.2 percent sodium hypochlorite solution and air-dried for 6 hours at room temperature. Finally, infected portions were transferred to the surface of potato dextrose agar (PDA) plates using sterile forceps. The PDA plates were incubated at 25±1°C for seven days and the isolates obtained were purified by transferring monosporic isolates to a fresh PDA medium.
2.5 GrowthProfiling of FungiPotato Dextrose Agar (PDA), Czapek-Dox Agar (CDA), Sabouraud Dextrose Agar (SDA), Nutrient Agar (NA), SabouraudBrain Heart Infusion Agar (SBHIA) and finally Corn Meal Agar(CMA) media were used to examine the cultural characteristics of the fungal isolates. The composition and preparation of the media were obtained from Ainsworth and Bisby’s ‘Dictionary of the Fungi’ by Hawksworth et al. Cotton blue staining slide was visualized for fungal spore detection. A wet mount of the fungi was prepared by suspending a little bit of fungal culture collected using a spatula and a needle in a few drops of lactophenol cotton blue solution on a clean and sterile microscope slide and then cover with a clean coverslip and view under x40 magnification(LabomedLx400, USA). Colony morphology, spore characteristics, and other measurements were determined manually after incubation on PDA at 25 ± 1°C for 10 days. At the end of the incubation period, the resulting growth of the fungus was harvested and filtered through pre-weighed Whatman No.1 filter paper and washed thoroughly with distilled water. It was dried at 40°C for two hours in hot air oven and the dry weights were recorded. The optimum pH for mycelia growth was determined on PD broth, where pH was adjusted from 3.0to 9.0 at 2.0 intervals. The fungal discs were put on the center of all culture vessels containing PD broth media. After seven days of the experiment, colony morphology and mycelia dry weight was recorded using the previously described procedure.
2.6.1 DNA extraction and PCR amplification after seven days of incubation, mycelium from the two pure fungal isolates (Isolate-1 and Isolate-2) was used for DNA extraction. Here, Maxwell® 16 LEV Plant DNA Kit (AS1420, Promega, USA) was used for the isolation of the genomic DNA. The isolated DNA was amplified via the polymerase Chain Reaction (PCR) technique using universal primersITS5F (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4R (5'-TCCTCCGCTTATTGATATGC-3') [14] and Hot Start Green Master Mix (Promega, USA). PCR was performed in a 50μl reaction mixture containing 25μl of Hot Start Green Master Mix (2X), 2.0 μL of each forward and reverse primer, 2.0 μL of genomic DNA and rest of the PCR water. The PCR program was as follows: pre-heat at 95°C for 2 min, followed by 32 cycles of denaturation step at 95°C for 30 sec, primer annealing at 48°C for 30 seconds, primer extension at 72°C for 45 sec. After that, the temperature of final extension was at 72°C for 10 minutes and lastly, holds at 4°C for overnight. The amplicons were separated by 1% agarose (V3125, Promega, USA) gel electrophoresis. Soil bacterium genomic DNA isolation was performed with the Cetyl-Trimethyl Ammonium Bromide (CTAB) method [15]. PCR amplification of isolated soil bacteria was performed in the same technique of fungal DNA isolation and amplification using specific primers27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R(5'-TACGGYTACCTTGTTACGACTT-3'). The quality and quantity of isolated DNA were checked by NanodropSpectrophotometer (ND2000, Thermo Scientific, USA). Finally, The PCR products were purified and used for sequencing analysis in Malaysia Ltd. via Invent Biotechnologies, Dhaka, Bangladesh. The sequence data were analyzed using similarities of nucleotide sequences between isolates through the BLAST procedure (http://blast.ncbi.nlm).
2.7 Pathogenicity Test of Isolated FungiWound inoculated and non-inoculated fruits (green banana, ladies’ finger and apple fruits) were separately subjected to pathogenicity tests. For the pathogenicity test, healthy green banana fruits, ladies finger fruits and apple fruits were surface-sterilized with 70% ethanol, and wounds were made in each fruit using the sterilized wooden rod. Each wound was inoculated with mycelia plugs (3 mm) from a 7 days old culture of each isolate, and one was treated with uncultured pure PD (Potato Dextrose) broth as a control. Inoculated fruits were covered with plastic, and incubated at 25 ± 1°C for 5 days [16].2.8Effects of Commercial FungicideTwo different conc. (25 and 50mg/disc) of fungicide (Carbendazim) and a novel antifungal agent kanamycin B (Maybe fungicidal and works by inhibiting protein synthesis) were tested against the fungal isolates for radial growth inhibition on PDA media using modified paper disc diffusion method under in vitro conditions. Myceliadiscs of 5 mm size from the actively growing culture of the isolated fungus were cut out by a sterile cork borer and one such disc was placed at the center of each agar plate. Control was maintained without adding any fungicides to the medium.The efficacy of the tested fungicide and kanamycin was expressed as percent inhibition of mycelia growth over control.
2.9Antifungal Activity ScreeningAntifungal activity screening was performed using a paper disc diffusion method. About 50mg of the MeOH extract of each plant were weighed, dissolved in 1ml of the extraction solvent and then tested for antifungal activities. The measurements were taken manually as the zone of inhibition of radial mycelia growth relative to positive control.2.10In vitro Effect of Allium sativum Extract against Conidial SuspensionVogel’s (minimal) medium [18] was used to detect the in vitro effects of garlic bulb extract against conidial suspensions of the isolates. Conidial suspensions were adjusted to 105conidia/mL using a hemacytometer.10μl of garlic bulb extract and 90μl of the conidial suspension of the fungal isolates were mixed and the mixtures were added to the surface of depression slides or group slides. The slides were then placed on a glass rod in petridish layered with moistened filter paper and incubated at 25°C for 24h. Conidial suspension mixed with an equivalent amount of the solvent served as control. After that, the treated and control samples were spread in separate petridishes containing PDA medium and incubated overnight for evaluating antifungal activity.
2.11 Determination of Different Quality Parameters after in vivo ApplicationBanana fruits were surface-sterilized by dipping into 1% sodium hypochlorite solution for 10 min, rinsed in sterile distilled water and artificially inoculated by dipping into spore suspension of both the fungus. After incubation for 15 hours, covered by a plastic sheet until conidia germinated, artificially inoculated banana fruits were dipped into methanol extracts of Allium sativum(Conc. 25% w/v), while the control fruits were dipped into methanol. Five fruits were used for each of the treatments. Standard estimation formulae were used to calculate the percentage of disease incidence and disease severity. After 10 days of experiments, fruit quality parameters including, pH (pH of banana fruit juice was determined with standard electric pH meter hy211, Hanna, USA), Total Soluble Solid (TSS was determined as °Brix by placing a drop of banana juice on an ATAGO-Automatic Refractometer Smart-1, Japan) and Total Titratable Acidity (Using one to two drops of phenolphthalein (1%) as an indicator, 5 ml of the filtrate was titrated using 0.1N NaOH to an endpoint pink)of the banana fruits were measured. Ascorbic acid was determined using a dye method and expressed as mg 100g-1of fresh fruits.