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Annals of West University of Timişoara, ser. Biology, 2023, vol. 26 (2), pp.123-130123FUNGICIDAL EFFECT OF TEAK (TECTONA GRANDIS L.)LEAF EXTRACTS ON FUSARIUM OXYSPORUMGaniyu Shittu OLAHAN1*, Ibrahim AJADI 1, Patience Olayinka BEN-UWABOR 21Department of Plant Biology, University of Ilorin, Nigeria.2 Department of Plant and Environmental Biology, Kwara State University, Malete, KwaraState.*Corresponding author’s e-mail: olahan.gs@unilorin.edu.ngReceived 14 December 2023; accepted 29 December 2023ABSTRACTFusarium oxysporum f. sp. lycopersici is the pathogen of tomato wilt, a disease of greateconomic importance worldwide. Although application of synthetic fungicides such asthiophanate methyl and mancozeb can prevent the occurrence of this disease, theireffects on the physical environment especially, is a limiting factor. Botanicals, i.e. plant-based fungicides are now being preffered for controlling fungal pathogens because theyhave minimal environmental impact and are less dangerous to consumers in contrast tosynthetic fungicides. Teak leaves have been reported to possess a very greatantimicrobial activity because of their high content of phytochemicals. In an effort todevelop eco-friendly chemical strategy for control of Fusarium wilt disease of tomatoplants, in vitro effect of 10, 30 and 50% (w/v) concentrations of the aqueous andethanolic leaf extracts of teak (Tectona grandis L.) on the radial growth of the myceliaof Fusarium oxysporum f. sp. lycopersici was investigated using the pour plate method.Results of the study revealed that both extracts retarded the radial growth of mycelia ofF. oxysporum f. sp. lycopersici compared to that of the control, with the ethanolic extracthaving a greater effect at the concentrations tested in this study. It is thereforerecommended that an in vivo study of effects of the same leaf extracts on wilt – infectedtomato plants be conducted.KEY WORDS: botanicals, in vitro, pathogen, teak, tomato, wilt.INTRODUCTIONThe tomato plant, Solanum lycopersicum L. is susceptible to a number ofbacterial and fungal diseases, which severely reduce its yield, one of which is Fusariumwilt disease caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) (Ma et al., 2023).Symptoms of the disease include wilting as well as droopiness and yellowing of theaffected plant (Koo et al., 2023). Although synthetic pesticides are available forprotection of crop plants from pathogens and prevent disease occurences, the use of plantmaterials such as fresh extracts from various medicinal plant parts generally referred to
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OLAHAN et al: Fungicidal effect of teak (Tectona grandis l.) leaf extracts on Fusarium oxysporum124as botanicals, is becoming very popular and better alternatives in recent time due to thehigh cost of some of the synthetic pesticides and their potentially dangerous side effectson the environment and living things (Boboescu et al, 2020; Drăgoi & Ianovici, 2021;Almășan & Ianovici, 2022; Ayilara et al., 2023).Some medicinal plants have been reported to possess antimicrobial activityagainst some plant pathogens (Lee et al., 2022). The antimicrobial activity is attributedto the presence of phytochemicals (secondary metabolites) such as flavonoids, alkaloids,terpenoids, tannins, phenols, etc in the medicinal plants (Alexan & Ianovici, 2018). Theirextracts can, therefore be utilized to create new, environmentally friendly, and safer nonsynthetic pesticides for controlling plant pathogens as well as the diseases they cause.Popular amongs these medicinal plants are neem, pawpaw, mango, guava, bitterleaf,khaya, mahogany, aloe vera, teak, orange, teak, etc.(Asdaq et al., 2022). The volume ofstudies conducted on in vitro antimicrobial activity of teak leaf extracts is less comparedto the other medicinal plants listed above, hence the justification of this study, whichwas aimed at determining the in vitro antifungal aactivity of teak leaf extracts againstFusarium oxysporum f. sp. lycopersici.MATERIALS AND METHODSFusarium oxyspororum f. sp. Lycopersici (CRIN/PPL/FO/08-03-23), the testorganism was acquired from the Plant Pathology Laboratory of Cocoa Research Instituteof Nigeria (CRIN), Ibadan, Nigeria. The organism was kept alive on streptomycin –amended Potato Dextrose Agar (PDA) media in bottle slants. The plant materials usedwere fresh teak leaves which were also collected from a teak tree within the CocoaResearch Institute of Nigeria (CRIN), Ibadan, Oyo State, Nigeria.Preparation of the Aqueous and Ethanolic Teak Leaf Extracts. Aqueous andethanolic (70% v/v) teak leaf extracts were prepared in 10, 30, and 50% (w/v)concentrations following the methods of Ekhuemelo & Eigege (2017). The extracts werefilter-sterilized, and kept at 25oC in a refrigerator until they were ready for use.Preparation of Potato Dextrose Agar (PDA) Culture Medium. PotatoDextrose Agar (PDA) culture medium was prepared following the Manufacturer’sinstructions. Nineteen grams of the powder was weighed into 500 ml of sterile distilledwater inside a conical flask. It was allowed to dissolve on a hot plate and then autoclavedfor 15 minutes at 121 °C and 15 lb/inch2 of pressure. After cooling, Streptomycin wasadded to it at 1 ml to prevent the growth of bacteria.Effects of the Leaf Extracts on the Test Organism. The in vitro effects of theleaf extracts on the test organism were assessed using the pour plate method as described
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Annals of West University of Timişoara, ser. Biology, 2023, vol. 26 (2), pp.123-130125by Gonelimali et al. (2018). In a laminar air-flow chamber, 7 disposable, sterile Petridishes were set up (Fig. 1). One milliliter (1 ml) of different concentrations of theaqueous and ethanolic teak leaf extracts was pipetted into 6 of the Petri dishes as labeledin Fig. 1, after which 10 ml of the streptomycin amended molten PDA was asepticallypoured into each of the labeled Petri dishes and the 7th Petri dish labeled as control. Eachplate was gently swirled to allow the teak leaf extracts and the culture medium mix upuniformly. At the centre of the cover of each of plate, two parallel lines were drawn,crossing each other. Seven inoculi (of 1m mm diameter each) were cut out from theactively growing edge of the test organism (F. oxysporum f. sp. lycopersici(CRIN/PPL/FO/08-03-23)) using a sterile 10 mm cork borer into a sterile Petri dish.Each of these was inoculated at the centre of each plate using a sterile forcep. The aboveset up was replicated three times within the laminar air – flow chamber. The plates wereincubated at 25°C for five days, with radial growth of the inoculim (D) being measuredaccording to Olahan et al. (2022), using the formula:D(mm) = dx+dy2where dx is the diameter of the mycelium along the horizontal line and dy is the diameterof the mycelium along the vertical line on each of the cover of the plates.The Precentage of Growth Inhibition by the teak leaf extracts was determinedusing the formula proposed by Okigbo and Nneka (2013), i.e.PGI (%) = DC − DTDC × 100Where, PGI (%) = Percentage Growth Inhibition;DC = Diameter of the test organism on the Control plates; andDT = Diameter of the test organism on the treated plates.Analysis of Variance of the mean percentage growth inhibition of the leafextracts at p<0.05 and the differences between the means were determined using Fisher’sLSD test.Aqueous Extracts10%(w/v)50%(w/v)30%(w/v)
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OLAHAN et al: Fungicidal effect of teak (Tectona grandis l.) leaf extracts on Fusarium oxysporum126Ethanolic ExtractsControlFIG. 1: Experimental Design (Olahan and Amadi, 2006)RESULTS AND DISCUSSIONSThe results obtained revealed that the three concentrations of the extracts usedin this study retarded the radial growth of Fusarium oxysporum f.sp. lycopersicum onPDA plates compared to the control. At 24 Hours After Incubation (HAI), the PercentageGrowth Inhibition (PGI) induced by the 10, 30 and 50%(w/v) concentrations of theaqueous leaf extracts were 33.54, 36.16 and 33.88 respectively, while the PGI for the 3concentrations of the ethanolic leaf extracts was the same (30.38). The ALE at the 3concentrations were more potent than the same concentrations of the ELE. Results of theanalysis of variance showed that there was significant difference between the PGIsinduced by 10 and 50% of ALE, and no significant difference between the 3concentrations of ELE at (P<0.05) (Table 1).At 48 HAI, the PGI for 10% (w/v) ALE was 38.08, while those for the 30 and50% (w/v) were 32.16 and 32.29 respectively. There was no significant differencebetween the PGI for the 3 concentrations of ALE at (P<0.05) (Table 1). The scenarioswere the same for the 3 concentrations of the ELE, with the PGIs being 61.23 for10%(w/v), 58.33 for 30% (w/v) and 55.44 for 55% (w/v). The ELE was more potentthan the ALE, in contrast to what was observed at 24 HAI. At 72 HAI, there was nosignificant difference at (P<0.05) between the PGIs induced by 30 and 50% (w/v)concentrations of the ALE (25.04 and 22.23 respectively), but there exist a significantbetween these PGIs and the PGI induced by 10% (w/v) concentration (36.71) at (P<0.05)(Table 1). The ELE was again more potent than the ALE. There was no significant10%(w/v) 50%(w/v)30%(w/v)Plate withoutextract(Control)
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Annals of West University of Timişoara, ser. Biology, 2023, vol. 26 (2), pp.123-130127difference at (P<0.05) between the PGIs induced by the 10 and 30% (w/v) concentrationsof ALE (27.36 and 27.03, respectively) at 96 HAI, while there exist a significantdifference at (P<0.05) between the above PGIs and the PGI for 50% (w/v) concentration(17.84) for the ALE (Table 1). For the ELE, there was no significant difference at(P<0.05) between the 3 concentrations tested, i.e. 52.25 by the 10% (w/v), 58.26 by the30% (w/v) and 59.16 by the 50% (w/v) (Table 1). The ELE was again more potent thanthe ALE.At 120 HAI, the scenario observed for ALE were the same as reported for ALEat 96 HAI (Table 1). For the ELE, there was a significant difference at (P<0.05) betweenthe PGIs induced by the 3 concentrations tested in this study, with the 50% (w/v)concentration inducing the highest PGI (61.80), followed by the 30% (w/v) with PGI of58.39 and the 10% (w/v) with PGI of 53.53 (Table 1). The PGI for ALE were 33.09,37.69 and 16.79, respectively at the 10, 30 and 50% (w/v) concentrations.These scenarioare the same with what was observed at 96 HAI (Table 1). The ELE was again, morepotent than the ALE at all the concentrations tested.TABLE 1: Percentage of Growth Inhibition inhibition of Teak leaf extracts on the radial growth of Fusarium oxysporumf.sp. lycopersicum at different concentrations after 120 hours of incubation and are statistically significant (p<0.05).Concentrations of Percentage growth Inhibition (%)The Extracts 24HAI 48HAI 72HAI 96HAI 120HAI(%w/v)ALE10 33.54ab 38.08b 36.71c 27.36b 33.093c30 36.16a 32.16b 25.04d 27.03b 37.69c50 33.88ab 32.29b 22.23d 17.84bc 16.79dELE10 30.38b 61.23a 56.79b 52.25a 53.53b30 30.38b 58.33a 61.36a 58.26a 58.39ab50 30.38b 55.44a 55.973b 59.16a 61.797aMean values with same letters in the same column are not significantly different at P<0.05 using Fisher's LSD Test.Keys: ALE: Aqueous Leaf Extract; ELE: Ethanolic Leaf Extract; HAI: Hours After Incubation and PGI: PercentageGrowth Inhibition.This study was conducted to test the in vitro antifungal activity of aqueous andethanolic leaf extracts of teak at 10, 30 aand 50% (w/v) using Pour Plate Method on F.
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OLAHAN et al: Fungicidal effect of teak (Tectona grandis l.) leaf extracts on Fusarium oxysporum128oxysporum f.sp. lycopersici. This was because over the years, there has been a lot ofinterest in the possibility of using plant-derived compounds as effective agents forchemical control of plant diseases (Lahlali et al., 2022). The importance of Tectonagrandis (teak) leaves as a therapeutic herb containing bioactive/antimicrobial substanceshave earlier been reported by Vaou et al. (2021) and Asdaq et al. (2022). Findings fromthis study showed that both the ethanolic and aqueous teak leaf extracts inhibited theradial growth of F. oxysporum f.sp. lycopersici, with the ethanolic extract being morepotent. This may be partly due to the effectiveness of ethanol for extraction as well as achemical sterilant (Olahan & Amadi, 2006). According to Altemimi et al. (2017),extracts made with alcohol increase the presence of bioactive molecules that possessstronger antifungal activity. Stéphane et al. (2022) and Mogana et al. (2020) differentlyreported that alcohol-based extractants have higher volatility water, and tend to extractmore kinds of antimicrobial substances from plant materials. These reports seem toexplain why the ethanolic and aqueous leaf extracts of Tectona grandis had differentantimicrobial effects on F. oxysporum f.sp. lycopersici in this study.Olahan and Amadi (2006) investigated in-vitro effect of various concentrationsof pawpaw (Carica papaya L.) leaf extracts on the radial growth of Fusariumverticilloides and reported that the aqueous and ethanolic leaf extracts retarded radialgrowth of the test microorganism compared to the control at the tested concentrations.Similarly, the aqueous extracts of Polystichum squarrosum, Adiantum venustum,Parthenium hysterophorus, Urtica dioeca and Cannabis sativa leaves exhibitedantifungal activity against R. solani, F. oxysporum and A. solani but with a lowereffectiveness compared to E. hirta ethanolic extracts (Tapwall et al., 2011). Also,Mekam et al. (2019) reported that ethanolic extracts of Oxalis barrelieri L.(Oxalidaceae), Stachytarpheta cayennensis L. (Verbenaceae) and Euphorbia hirta L.(Euphorbiaceae) had a higher antifungal activity compared to the aqueous extracts onthe cultures of Fusarium solani, Rhizoctonia solani and Alternaria solani, using the pourplate and agar diffusion techniques. The aqueous extract of Picralima nitida fruitsproved less effective than its ethanolic extract at suppressing the growth of Fusariumoxysporum (Akabassi et al., 2022). Also, Olahan et al. (2020) reported the effectivess ofethanolic extracts against Colletotrichum falcatum compared to aqueous extract.CONCLUSIONSThe aqueous and ethanolic leaf extracts of Tectona grandis have in vitroantifungal efficacy against F. oxysporum f.sp lycopersici, with the ethanolic leaf beingmore potent at the concentrations tested.
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