Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L.)

Ahmadu, T.; Ahmad, K.; Ismail, S. I.; Rashed, O.; Asib, N.; Omar, D.

doi:10.1590/1519-6984.233173

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Original Article • Braz. J. Biol. 81 (4) • Oct-Dec 2021 • https://doi.org/10.1590/1519-6984.233173 copy

Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L.)

Eficácia antifúngica de extratos de folha e de semente de Moringa oleifera contra Botrytis cinerea causador da doença do molde cinza de tomate (Solanum lycopersicum L.)

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Drawbacks associated with the use of chemical fungicides to control plant pathogenic fungi such as Botrytis cinerea stimulate the need for alternatives. Therefore, the present study was carried out to determine the antifungal potentials of Moringa oleifera extracts against B. cinerea. Phytochemical analysis using qualitative chemical tests revealed the presence of huge amount of crucial phytochemicals compounds like phenolic compounds, alkaloids and saponins in the M. oleifera leaf extract. Antifungal bioassay of the crude extracts indicated better mycelial growth inhibition by methanol leaf extract (99%). The minimum inhibitory concentration (MIC) was 5 mg/ml with 100% spore germination inhibition and minimum fungicidal concentration (MFC) was 10 mg/ml with 98.10% mycelial growth inhibition using broth micro dilution and poisoned food techniques. Gas chromatography–mass spectrometry (GC-MS) analysis led to the identification of 67 volatile chemical compounds in the leaf extract with 6-decenoic acid (Z)- (19.87%) was the predominant compound. Further chemical elucidation of the crude extracts performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) showed the presence of non-volatile chemical compounds, mostly flavones, flavonoids and phenolic acids (i.e. quercetin and kaempferol). Scanning electron microscopy and transmission electron microscopy analysis showed positive effect of M. oleifera leaf extract on the treated conidia and mycelium of B. cinerea. Findings revealed that irreversible surface and ultra-structural changes with severe detrimental effects on conidia and mycelium morphology compared to control treatment. Overall findings suggested that M. oleifera leaf extract is a promising candidate for biological control of fungal pathogens, thus limiting overdependence on chemical fungicides.

Keywords:
antifungal activity; Botrytis cinerea; Moringa oleifera; phytochemicals

Phytochemical compound	Test type	Procedure	Reference
Tannins	Ferric chloride test	-About 0.5 g of methanolic extract was dissolved into 10ml of distilled water and filtered.	(Harbone, 2001HARBONE, J.B., 2001. Phytochemical methods. London: Chapman and Hall Ltd, 113 pp.)
Tannins	Ferric chloride test	- 4-6 drops of 10% ferric chloride was then added into the filtrate.
Alkaloids	Dragendorff’s test	- Methanolic extract (0.2 g) was put into a conical flask, followed by the addition of 20 ml of sulphuric acid diluted in methanol.	(Ghani, 1998GHANI, A., 1998. Medicinal plants of Bangladesh. Dhaka: Asiatic Society of Bangladesh, pp. 78-83.; Harbone, 2001HARBONE, J.B., 2001. Phytochemical methods. London: Chapman and Hall Ltd, 113 pp.).
Alkaloids	Dragendorff’s test	- The mixture was then filtered and treated with 2 drops of Dragendorff’s reagents (Potassium bismuth iodide solution).
Flavonoids	Ammonium test	-About 10 ml of ethyl acetate was added to 0.2 g of the extract and heated in water bath for 4 min.	(Harbone, 2001HARBONE, J.B., 2001. Phytochemical methods. London: Chapman and Hall Ltd, 113 pp.; Sofowora, 2005SOFOWORA, A., 2005. Medicinal plants and traditional medicine in Africa. Ibadan, Nigeria: Spectrum Books Ltd, 289 pp.)’
		-The mixture was allowed to be cooled and then filtered.
		- 1 ml of diluted ammonia was shaken with 4 ml of the filtrate and the layers were allowed to separate.
Saponnins	Froth test	-About 20 ml of water was added to 0.25 g of the extract in 100 ml beaker, boiled and then filtered.	(Harbone, 2001HARBONE, J.B., 2001. Phytochemical methods. London: Chapman and Hall Ltd, 113 pp.; Khalil et al., 2013KHALIL, A.S., RAHIM, A.A., TAHA, K.K. and ABDALLAH, K.B., 2013. Characterization of methanolic extracts of agarwood leaves. Journal of Applied and Industrial Sciences, vol. 1, no. 3, pp. 78-88.; Sofowora, 2005SOFOWORA, A., 2005. Medicinal plants and traditional medicine in Africa. Ibadan, Nigeria: Spectrum Books Ltd, 289 pp.).
		-The filtrate (5 ml) was diluted with 20ml of distilled water and shaken vigorously.
		-This was the allowed to stand.
Steroids/ Triterpenoids	Liebermann-Burchardt test.	-Methanolic extract (1 ml) was boiled with 2 ml of chloroform and allowed to cool.	(Khalil et al., 2013KHALIL, A.S., RAHIM, A.A., TAHA, K.K. and ABDALLAH, K.B., 2013. Characterization of methanolic extracts of agarwood leaves. Journal of Applied and Industrial Sciences, vol. 1, no. 3, pp. 78-88.).
Steroids/ Triterpenoids	Liebermann-Burchardt test.	-Then 1-3 drops of concentrated sulphuric acid were added, shaken and allowed to stand.
Phenolic compounds	Ferric chloride test	-3 drops of 1% ferric chloride was added to 3ml portion of methanolic extract.	(Harbone, 2001HARBONE, J.B., 2001. Phytochemical methods. London: Chapman and Hall Ltd, 113 pp.: Khalil et al., 2013KHALIL, A.S., RAHIM, A.A., TAHA, K.K. and ABDALLAH, K.B., 2013. Characterization of methanolic extracts of agarwood leaves. Journal of Applied and Industrial Sciences, vol. 1, no. 3, pp. 78-88.).
Anthraquinone	Borntrager’s test	-10ml of benzene was added to 4 g of the methanol extract, shaken and filtered.	(Kadhim and Al-Shammaa, 2014KADHIM, E.J. and AL-SHAMMAA, D.A., 2014. Phytochemical characterisation using GC-MS analysis of methanolic extract of Moringa oleifera (Moringaceae) plant cultivated in Iraq. Chemistry and Materials Research, vol. 6, no. 5, pp. 9-26.).
Anthraquinone	Borntrager’s test	-Then 5 ml of 10% ammonia solution was added to the filtrate, shaken and allowed to stand.
Terpenoids	Salkowski test	-The extract (2ml) was dissolved in chloroform (2ml) and then evaporated to dryness.	(Kadhim and Al-Shammaa, 2014KADHIM, E.J. and AL-SHAMMAA, D.A., 2014. Phytochemical characterisation using GC-MS analysis of methanolic extract of Moringa oleifera (Moringaceae) plant cultivated in Iraq. Chemistry and Materials Research, vol. 6, no. 5, pp. 9-26.; Khalil et al., 2013KHALIL, A.S., RAHIM, A.A., TAHA, K.K. and ABDALLAH, K.B., 2013. Characterization of methanolic extracts of agarwood leaves. Journal of Applied and Industrial Sciences, vol. 1, no. 3, pp. 78-88.).
Terpenoids	Salkowski test	-Sulphuric acid (2ml) was added and boiled for 2 minutes.

Part of plant	Solvents	Percentage yield (%).
Leaf	Methanol	14.16
	Ethanol	10.23
	Ethyl acetate	5.24
	Hexane	2.3
	Distilled water	1.71
Seed	Methanol	11.54
	Ethanol	6.41
	Ethyl acetate	3.43
	Hexane	1.27
	Distilled water	2.83

Part of plant	Solvent	BCH02		BCH07
Part of plant	Solvent	MIC	MFC	MIC	MFC
Leaf	Methanol	5.00	10.00	5.00	10.00
	Ethanol	10.00	20.00	15.00	30.00
Seed	Methanol	10.00	10.00	10.00	10.00
	Ethanol	15.00	20.00	15.00	30.00

Phytochemical compound	Chemical/colour tests	Results/observations	Inferences
Tannins	Ferric chloride test	-Greenish black colour	+
Alkaloids	Dragendorff’s test	-Development of an orange colour	+
Flavonoids	Ammonium test	-Yellow colour in bottom layer	+
Saponnins	Froth test	-Stable froth on standing	+
Steroids/ Triterpenoids	Liebermann-Burchardt test.	-Red colour top and yellow colour on bottom	+
Phenolic compounds	Ferric chloride test	- Development of violet or dark blue colour	+
Anthraquinone	Borntrager’s test	- Development of red colour	+
Terpenoids	Salkowski test	-Formation of red colour at the lower layer indicates the presence of Steroids	+

Peak No.	RT (min)	Height (%)	Chemical compound	Molecular weight (g/mol)	Molecular formula	Chemical structure
8	7.317	2.11	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	144.12	C₆H₈O₄
9	7.967	2.14	2-Furancarboxaldehyde	188.18	C₁₀H₈N₂O₃
17	9.700	3.77	1,3-Propanediol	76.09	C₃H₈O₂
18	9.758	3.13	Benzeneacetonitrile	117.15	C₈H₇N
23	10.725	2.38	1,2,3,5-Cyclohexanetetrol	148.16	C₆H₁₂O₄
31	12.467	5.87	n-Hexadecanoic acid, methyl ester	270.45	C₁₇H₃₄O₂
38	13.333	19.87	6-decenoic acid (Z)-	170.25	C₁₀H₁₈O₂
39	13.433	2.95	Octadecanoic acid/Stearic acid	284.5	C₁₈H₃₆O₂
47	14.808	2.20	Cyclohexanecarboxylic acid	128.17	C₇H₁₂O₂
48	14.900	11.90	2-Dimethyl(trimethylsilylmethyl) silyloxymethyltetrahydrofuran	246.49	C₁₁H₂₆O₂	-
50	15.058	6.22	Malonic acid	104.06	C₃H₄O₄
51	15.150	2.24	Docosanoic acid	340.6	C₂₂H₄₄O₂
52	15.192	11.07	beta.-l-Rhamnofuranoside, 5-O-acetyl-thio-octyl-	334.5	C₁₆H₃₀O₅

Peak No	Chemical compound	Chemical group	Biological activity
8	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	New compound	No activity reported
9	2-Furancarboxaldehyde		Antimicrobial, Antifungal, Antioxidant
17	1,3-Propanediol	-	Antimicrobial
18	Benzeneacetonitrile		Cytotoxicity.
23	1,2,3,5-Cyclohexanetetrol	-	Cytotoxicity, Antineuropathatic
31	n-Hexadecanoic acid	Palmitic acid	Antimicrobial, Antibacterial Antifungal. Antioxidant, Nematicide, Antiandrogenic
38	6-decanoic acid (Z)-	Stearic acid	Antioxidant, insectifuge, Cancer preventive.
39	Octadecanoic acid/Stearic acid	Stearic acid	Antimicrobial, Antifungal.
47	Cyclohexanecarboxylic acid	Aromatic compound	Antimicrobial
48	2-Dimethyl(trimethylsilylmethyl) silyloxymethyltetrahydrofuran	New compound	No activity reported
50	Malonic acid	Dicarboxylic acid	Antimicrobial Anticancer
51	Docosanoic acid	Saturated fatty acid	Antioxidant, Antimicrobial
52	beta.-l-Rhamnofuranoside, 5-O-acetyl-thio-octyl-	Glycoside	Antimicrobial

Peak No.	tR/min	[M-H] (Error (ppm))	Compound name	Molecular formula	Molecular weight (g/mol)
1	0.927	174.93	Phenylvaleric acid	C₁₁H₁₄O₂	178.23
2	1.210	377.25	Caffeic acid	C₉H₈O₄	180.16
3	1.290	191.55	Quinic acid	C₇H₁₂O₆	192.17
10	3.433	592.61	Apigenin-6,8-di-C-β-D-glucopyranoside (Vicenin II)	C₂₇H₃₀O₁₅	594.52
11	3.998	431.60	Apigenin 6 C glucoside	C₂₇H₃₀O₁₅	594.52
12	4.159	928.25	Quercetin-3-O-glucoside dimer	C₂₁H₂₀O₁₂	464.38
14	4.524	896.31	Kaempferol 3-o glucoside dimer	C₂₁H₂₀O₁₁	286.24

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