GC-MS profiling and antifungal activities of Morinda citrifolia L. leaf extract against fungal pathogens of crown rot disease of banana

The increasing demand for organic agricultural products especially among the elite stimulated the search for safe and alternative means of crown rot disease control. Morinda citrifolia is one of the listed medicinal plants among the Polynesian countries. This study documented the phytochemical profile of M. citrifolia using GC-MS and their antifungal activities against crown rot pathogens. The key phytochemical constituents of the extract were Phytol 2-Hexadecen-1-ol, (Diterpene) (25.96%), Squalene (Triterpene) (15.13%), 1, 3-Propanediol (Polyphenol) (4.68%), Pyran-4-one 4H-, 9 (Flavonoid)


INTRODUCTION
Morinda citrifolia is a tree native to South-East Asia and the most commonly found variety in Malaysia is the var.citrifolia.M. citrifolia has been cultivated extensively in Malaysia and many tropical regions such as and South America, due to its economic value and health benefits (Srinivasahan & Durairaj, 2014).The fruit, juice, seed, leaf, and root have been used as sources of traditional medicines in different countries in the world to cure many diseases and ailments.Commercially, its products such as fruit juice and capsulated fruit powder extract have gained popularity in Asia, Europe and America (Nelson, 2003).All plant parts exhibited antioxidant, antimicrobial, anti-cancer and anti-inflammatory properties (Assi et al., 2017).According to the findings of McClatchey (2002), M. citrifolia is used in the treatment of approximately 2000 ailments around the world.Jayaraman et al. (2008), reported all parts of the plant possess antifungal, antibacterial, tumour suppression effects.Zin et al. (2007) also reported the ant-oxidant properties of M. citrifolia root extract.Similarly, Masuda et al. (2009) documented the inhibitory effects of M. citrifolia seeds on elastase and tyrosinase enzymes.Findings of Usha et al. (2010), reported M. citrifolia leaves are used in the treatment of minor infections and ulcerations on the skin.
Similarly, medicinal plants contain a large number of phytochemicals with antimicrobial properties which may serve as good and safe alternative biopesticides due to their low toxicity to humans and the environment (Madhumitha et al., 2012;Adefuye & Ndip, 2013).These phytochemicals are considered alternative sources of broad spectrum biopesticides derived from natural products for the management of plant diseases, due to their varied and complex mechanisms of action against pathogenic organisms (Gurjar et al., 2012;Idris et al., 2015).
The use of Gas Chromatography-Mass Spectrometry (GC-MS) for the identification and quantification of phytochemicals has been on the increase since the technique proved to be a valuable method for the identification of volatile compounds, non-polar components, lipids and fatty acids (Ganesh & Mohankumar, 2017).The GC interfaced with MS is considered a powerful tool

GC-MS profiling and antifungal activities of Morinda citrifolia L. leaf extract against fungal pathogens of crown rot disease of banana
Haruna for the detection and analysis of organic compounds (Khan et al., 2017).According to Su et al. (2005), bioactive compounds such as polyphenols, alkaloids, glycosides, polysaccharides, lignans, iridoids, morindin, anthroquinones, trisaccharide fatty acid esters, scopoletin, minerals and vitamins have been isolated from M. citrifolia fruits, leaves and roots.
Little work has been reported on GC-MS analysis of methanol extract of M. citrifolia leaves.Therefore, the present study focused on extraction, GC-MS technique analysis of bioactive components and assessment of fungicidal activities of methanol extract of M. citrifolia leaves using the poisoned food technique method.

Collection of Plant Materials
Fully matured leaves of M. citrifolia were collected from Taman Pertanian Universiti, Universiti Putra Malaysia (UPM), Selangor, Malaysia.The plant was identified and authenticated by a botanist at the Biodiversity Unit, Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), collected leaves were deposited in the Phytomedicinal Herbarium of Biodiversity Unit, IBS, Selangor with a specimen voucher No. SK 3255/17.

Preparation and Extraction of Plant Materials
Collected leaves were immediately brought to Biological Control Laboratory, at the Department of Plant Protection, Faculty of Agriculture, UPM.The leaves were washed under running tap water to get rid of dust and debris, then rinsed three times with sterile distilled water, then air dried in the laminar flow for 6 hours, then dried in an oven (Memmert, Germany) at 45 °C for 3 days.Five hundred grams of dried leaves were ground in a grinder (Retsch SK100) for 2 min to produce a powder of a uniform size (Rivera et al., 2012).Fifty grams of the powder was dissolved in 500 mL of methanol.The mixture was thoroughly agitated on orbital shaker at 150 rpm for 24 h.The mixture was filtered in three stages; first with double layered muslin cloth, second, the extract was filtered using Whatman No. 1 filter paper and thirdly using a micro (0.45 μm) syringe filter (Bhutia et al., 2016;Kurwadkar et al., 2017).Thereafter leaf filtrate was concentrated under reduced pressure of 40-45 °C using a rotary evaporator (BUCHI R-215, Switzerland) to obtain a viscous semi solid mass.Then, the semi solid mass was transferred to a beaker covered with aluminium foil and dried to powder form in an oven for 6 days at 40-45 °C.

GC-MS Analysis of M. Citrifolia Leaf Extract
GC-MS analysis was performed at the Chemistry Department, Faculty of Science, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.The analysis was performed to determine the volatile bioactive compounds present in the methanolic extract of M. citrifolia and their relative abundance following the methods described by Seotardjo et al. (2007) and Khan et al. (2017).Two hundred mg of the dried extract powder was dissolved in 2 mL of methanol in a vial.GC-MS analysis of M. citrifolia methanol leaves extract was carried out using gas chromatography coupled with a mass spectrometer (GC-MS QP-2010, Shimadzu, Japan) equipped with Zebron ZB5-MS capillary column (30 meters x 0.25 mm I.D. x 0.25μm film thickness) (Jegajeevanram et al., 2014).The capillary was set to an initial temperature of 50 °C, and maintained at this temperature for 3 min.The oven temperature was increased up to 300 °C at the end of the period and the rate of an increase of 10 °C/min and maintained for 10 min.The injection port temperature was set at 250 °C and Helium flow rate at 1.0 mL/min.The ionization voltage was set at 70eV.The samples were injected in split mode as 10:1.Mass spectral scan range was set at 35-450 (m/z).The ion source temperature was maintained at 240 °C and the interface temperature was at 300 °C.The MS start time was 3 min, and the end time was 34 min with the solvent cut time was of 2 min and 30 s. Mass spectra were taken over m/z range 35-450 atomic mass unit amm.National Institute Standard and Technology (NIST) Ver.02 MS data library was used for, comparing spectral data of the leaves sample.The mass spectrum of components obtained from GC-MS analysis, compounds' names, molecular weight and structure of the components of the test materials were also confirmed (El-Beltagi et al., 2018).

Source of Inoculum
Crown rot inciting fungi whose pathogenicity was already established in the department of Plant Protection University Putra Malaysia were isolated from naturally crown rot infected banana fruits sampled from Negeri Sembilan, Melaka and Selangor in Malaysia during the earlier study were used.Isolates were identified morphologically and confirmed molecularly using their rDNA following the method described by Karmakar et al. (2016).Generated ITS sequences were accessioned by GenBank as; Colletotrichum musae (MG386643.1),Colletotrichum asianum (MG386644.1),Lasiodiplodia theobramae (MG386642.1),and Fusarium longipes (MG386645.1).

Evaluation of Antifungal Properties of M. Citrifolia Leaf Extract
Assessment of the antifungal properties of M. citrifolia leaf extract was performed using the poisoned food technique method (Nweke, 2015).To achieve this, 100 mg of powered extract was dissolved in 1 mL of 50% Dimethyl sulfoxide DMSO to produces 100% stock solution.Then, serial dilutions of 50, 60, 70, 80, 90 and 100 mg/mL were prepared by adding 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mL of the 100% stock solution to already prepared 20 mL PDA on Petri dishes (treatments) then allow to solidify as described by Gayathri and Ramesh (2013).Using a sterilized cork borer, 5 mm plug of each purified colony mycelial disc were cut and placed on the centre of the petri dishes containing impregnated PDA, sealed and stored at room temperature of 25 °C ± 2, until the control plates were full.Controls were plates impregnated with 50% DMSO, all treatments were in triplicates (Idris et al., 2015).Antifungal activities of extract were assessed by measuring diameter of mycelial growth in each treatment, and also by taking the percentage inhibition of radial growth (% PRIG) after 72 hrs and calculated as: SEM microscopy of the antifungal activities of M. citrifolia leaf extract was performed on 7 days old C. musae growing on four different concentrations of PDA amended media; 50, 60, 70, and 80 mg/mL of leaf extracts and a control following the method described by (Kim et al., 2017).From each treatment, four pieces of 1 cm 3 fungal mycelial mat were cut and fixed in vials containing 5 mL of 25 mg/mL glutaraldehyde buffer, stored at 4 °C for 24 hours.Specimen were centrifuged at 1500 rpm, then supernatant discarded.Specimen were washed in 0.1 M sodium cacodylate buffer for 10 minutes, 3 times.Samples were further post-fixed in 1% osmium tetroxide at 4 °C for 2 hrs.Then washed 3 times in 0.1 M sodium cacodylate buffer for 10 minutes was repeated.This followed by dehydration in 35%, 50%, 75%, and 95% for 10 minutes in graded acetone series and in 100% acetone for 15 minutes.Dehydrated specimens were placed on albumin coated with aluminium foil prior to critical point drying.Thereafter, samples were transferred onto specimen baskets and kept in a critical-point drier for half an hour, then mounted onto stub by the use of double-sided tape, and gold-coated in ion sputter-coater.Specimen were then observed and photographed on a SEM (BAL-TEC, Model SCD 005 JEOL (InTouchScope, USA).

Statistical Data Analysis
Data on percentage pathogen growth inhibition was analysed using one-way ANOVA and significant means were separated by Tukey at P< 0.05 level of significance.

Assessment of Antifungal Activities of M. Citrifolia Leaf Extract
The antifungal properties of 50, 60, 70, 80, 90 and 100 mg/mL of M. citrifolia leaf extract against mycelial growth of fungal pathogens were studied.Results given in Tables 2 and 3, showed a highly significant effect of extract against mycelial growth of fungal pathogens at P<0.05.At 50 mg/mL extract concentration, radial growth (cm) recorded in L. theobroamae, C. muse, C. asianum and F. longipes were 16.00 ± 1.00, 10.33 ± 1.53, 8.33 ± 0.58 and 15.33 ± 1.53 mm respectively (Table 2), while percentage radial growth inhibition (% PIRG) caused by the treatments were 80.08 ± 1.25, 34.18 ± 9.73, 70.58 ± 5.09 and 20.67 ± 7.90 % respectively for L. theobroamae, C. muse, C. asianum and F. longipes Table 3.Under treatment with 70 mg/mL leaf extract, L. theobroamae recorded radial growth of 8.67 ± 0.73 mm, C. muse 0.00 mm, C. asianum 0.00 mm, and F. longipes showed 9.00 ± 1.00 mm radial growth respectively.From the Table 3, pathogen growth inhibition showed 90.46 ± 0.72, 100.00 ± 0.00, 100.00 ± 0.00 and 53.44 ± 5.17 % growth inhibition was recorded in L. theobromae, C. musae, C. asianum and F. longipes respectively.The higest radial growth inhibition (100%) was achieved in all pathogens at 100 mg/mL extract concentration.The trend of inhibition was concentration dependent, thus, as leaf extract concentration was increased, pathogen growth inhibition also increased (Figure 1).Results in Table 2, shows that the two Colletotrichum species were the most sensitive organisms to the extract, hence completely inhibited at 70 mg/mL extract concentration, followed by Fusarium longipes at 90 mg/mL and lastly L. theobromae at 100 mg/mL.severe hyphal distortion, cell collapsed/cell lysis and complete destruction after 7 days of incubation Figure 2 (a-d) while (e) the control remained intact.Hyphal distortion increased with an increment of extract concentration.

DISCUSSION
GC-MS chromatogram of M. citrifolia leaf extract revealed that the extract is a mixture of several volatile bioactive compounds.
(2017), documented over 200 phytochemicals isolated and identified from various parts of M. citrifolia plant, and that M. citrifolia leaf extract was antimicrobial, anticancer, larvicidal and antioxidant in nature.According to Kakad et al. (2015), phenol, tannin, alkaloid, glycosides, flavonoid, terpenoids and steroids were isolated from M. citrifolia leaf extract.Bharathy et al. (2012) reported phytol, as diterpene with significantly strong antimicrobial activities against many bacterial and fungal strains.Zin et al. (2007) reported existence of phenolic and flavonoids components of M. citrifolia leaves constitute 1,095 ± 0,241 mg/g GAE and 0.0483 mg/g EQ respectively.However, Assi et al. (2017) reported the strongest inhibitory effect of methanol extract of M. citrifolia leaf (79.8 %) against different fungal pathogens.Wang and Su (2001), Kurniawan (2018) and Setyani and Setyowati (2018) and reported the major phytochemicals in M. citrifolia are phenolic compounds, organic acids, and alkaloids.
Based on the findings of the present study, researcher reports the significant inhibitory effect of the methanolic extract of M. citrifolia leaf (P< 0.05) on fungal growth, which was concentration dependent.Furthermore, the results on the antimicrobial properties of M. citrifolia in this study were in agreement with the report of Kakad et al. (2015) who reported the antifungal activities of methanol-ethanol leaf extract of M. citrifolia against Daedalea flavida, Candida albicans and Aspergillus niger.

CONCLUSION
Seventeen major volatile bioactive constituents were identified from the extract of M. citrifolia.The identified compounds were found to have strong antifungal properties against crown rot inciting pathogens; L. theobromae, C. musae, C. asianum, and F. longipes on PDA media.The presence of tannins, steroids, saponins, flavonoids and alkaloids in the leaf extract established through GC-MS analysis of the extract further confirmed the presence of strong potent bioactive compounds, principal among them were diterpene, triterpene, alkaloids, phenolic compounds with their derivatives, flavonoids, steroids, tannins, organic acids, and some vitamins.Hence, this study has evidently indicated the strong antifungal properties of M. citrifolia properties that can be exploited and used as safe alternatives to the use of synthetic fungicide for the control of crown rot inciting pathogens.
PIRG = percentage inhibition of radial growth R 1 = radius of fungi colony in control plate R 2 = radius of fungi colony in treated plate Scanning Electron Microscopic (SEM) of the Antifungal Activities of M. Citrifolia Leaf Extract Against C. Musae on Different Concentrations of Extract Amended PDA Media.
Sundrarajan et al. (2017) also found a superior antimicrobial properties of M. citrifolia leaf extract against human pathogens such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillus niger and Candida albicans.

Furthermore
, previous studies byJagtap et al. (2009),Idris et al. (2015) andBhutia et al. (2016) reported a highly inhibitory effect of plant extract and essential oils on mycelial growth of Colletotrichum musae and further stated that the inhibition was directly dependent on the quantity of extract added to the growth medium.However, each fungal isolate in the present study showed different levels of susceptibility to the extract, hence resulting in different MIC for each fungus.Implying Colletotrichum species were the most susceptible fungal species to the extract followed by F. longipes and L. theobromae.

Figure 1 :
Figure 1: Names and structures of the identified principal phytochemicals compounds that constitutes 50.91% of the total volatile bioactive constituents of M. citrifolia using GC-MS technique

Figure 2 :
Figure 2: SEM images of antifungal activities of different concentrations (a) 50, (b) 60, (c) 70, and (d) 80 mg/mL of M. citrifolia leaf extract against C. musae and (e) C. musae grown on plate without leaf extract (control).Photos were taken 7 days after the treatment at 1,500 magnification

Antifungal Activities of 50, 60, 70, and 80 mg/mL Concentration M. Citrifolia Leaf Extract in PDA Media Against C. Musae
SEM image of C. musae isolates growing on 50, 60, 70, and 80 mg/mL of M. citrifolia extract amended PDA showed severe distortion of fungal hyphae.Microscopic observation showed Haruna

Table 1 : GC-MS analysis of M. citrifolia leaf phytochemical compounds with their biological activities
Key: RT=Retention time, MW=Molecular weight, MF=Molecular formula