Activity of plant extracts/essential oils against three plant pathogenic fungi and mosquito larvae: GC/MS analysis of bioactive compounds

Abstract

Certain natural products extracted from different parts of medicinal and aromatic plants were examined for their antifungal activity against three plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and Alternaria solani, and insecticidal activity against mosquito larvae (Culex pipiens). Acetone extract of Tectona grandis showed the highest antifungal activity against R. solani and A. solani with EC50 values of 118 and 294 μg/mL, respectively. The highest larvicidal activity was displayed by the essential oils of Ocimum basilicum and Eucalyptus gomphocephala with LC50 value of 22, and 30 mg/L, respectively. By gas chromatography–mass spectrometry (GC/MS) analysis 3-allylguaiacol (65.8%) and eugenol acetate (46.6%) were the main compounds in Syzygium aromaticum methanolic extract and essential oil, respectively. The main compound in T. grandis acetone extract was cyclohexylpentyl oxalate (8.7%); its water extract contained (E)-4,4-dimethyl-2-pentene (51.1%); E. gomphocephala branch oil contained p-cymene (28.8%); Euphorbia paralias leaf extract contained 1βH-romneine (26.3%); the seed extract contained α-linolenic acid, TMS (15.2%); Punica granatum extract contained furfural (32.1%); and O. basilicum essential oil contained estragole (65.9%). Thus, extracts from the tested plants can be used as natural biofungicides to manage diseases caused by F. oxysporum, R. solani, and A. solani. Additionally, these extracts show potential larvicide activities against mosquito larvae.

Full Article

Activity of Plant Extracts/Essential Oils Against Three Plant Pathogenic Fungi and Mosquito Larvae: GC/MS Analysis of Bioactive Compounds

Younis K. Hamad,^a,b Yasser Abobakr,^a,c Mohamed Z. M. Salem,^d,* Hayssam M. Ali,^e,f Ali S. Al-Sarar,^a and Ali A. Al-Zabib ^a

Certain natural products extracted from different parts of medicinal and aromatic plants were examined for their antifungal activity against three plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and Alternaria solani, and insecticidal activity against mosquito larvae (Culex pipiens). Acetone extract of Tectona grandis showed the highest antifungal activity against R. solani and A. solani with EC₅₀ values of 118 and 294 μg/mL, respectively. The highest larvicidal activity was displayed by the essential oils of Ocimum basilicum and Eucalyptus gomphocephala with LC₅₀ value of 22, and 30 mg/L, respectively. By gas chromatography–mass spectrometry (GC/MS) analysis 3-allylguaiacol (65.8%) and eugenol acetate (46.6%) were the main compounds in Syzygium aromaticum methanolic extract and essential oil, respectively. The main compound in T. grandis acetone extract was cyclohexylpentyl oxalate (8.7%); its water extract contained (E)-4,4-dimethyl-2-pentene (51.1%); E. gomphocephala branch oil contained p-cymene (28.8%); Euphorbia paralias leaf extract contained 1βH-romneine (26.3%); the seed extract contained α-linolenic acid, TMS (15.2%); Punica granatum extract contained furfural (32.1%); and O. basilicum essential oil contained estragole (65.9%). Thus, extracts from the tested plants can be used as natural biofungicides to manage diseases caused by F. oxysporum, R. solani, and A. solani. Additionally, these extracts show potential la rvicide activities against mosquito larvae.

Keywords: Antifungal activity; Larvicidal activity; Pathogenic fungi Natural extract; Essential oils

Contact information: a: Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia; b: Plant Pathology Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt; c: Plant Protection Research Institute, Sabahia Research Station, Agricultural Research Center, Alexandria, Egypt; d: Forestry and Wood Technology Department, Faculty of Agriculture (EL-Shatby), Alexandria University, Alexandria, Egypt; e: Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; f: Timber Trees Research Department, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center, Alexandria, Egypt;

* Corresponding author: zidan_forest@yahoo.com; mohamed-salem@alexu.edu.eg

INTRODUCTION

In crop plants, fungi cause more economic damage than any other group of microorganisms, with annual losses estimated at more than $200 billion (Horbach et al. 2011). Chemical control of phytopathogenic fungi efficiently reduces the negative consequences resulting from these organisms; however, field application of these chemicals is not desirable. Extensive and improper use of synthetic fungicides can lead to serious consequences on the environment and on the health of humans and animals. Moreover, the development of fungicide-resistance in phytopathogenic fungi has been reported (Hahn 2014). Therefore, the search for novel antifungal agents is needed. Plant-derived products may be an alternative approach against phytopathogenic fungi (Amadi et al. 2010; Badawy and Abdelgaleil 2014).

p-cymene and crypton were found to be the main compounds of leaf essential oil (EO) from Eucalyptus gomphocephala growing in Egypt (Salem et al. 2015). 1,8-cineole, pinene, viridiflorol, terpineol, aromadendrene and trans-pinocarveol are the abundant compounds in the leaf EO of E. procera cultivated in central Iran (Rahimi-Nasrabadi et al. 2012), while 1,8-cineole, cryptone, 4-allyloxyimino-2-carene, and 4-terpineol were found in leaf EO from E. largiflorens from the same area (Rahimi-Nasrabadi et al. 2013) with high antimicrobial activities. Also, 1,8-cineole, α-pinene, and α-terpineol are the main constituents of E. oleosa leaf EO with high antibacterial activity (Rahimi-Nasrabadi et al. 2013).

Different bioactive compounds of naphtoquinone, anthraquinone, 2-methyl anthraquinone (techtoquinone), lapachol, and deoxylapachol have been isolated from teak wood extracts (Windeisen et al. 2003; Thulasidas and Bhat 2007). Several studies have shown antifungal efficacy of plant extracts and EOs against plant and human pathogenic fungi (Mahboubi and Bidgoli 2010; Salem et al. 2016a,b; Sales et al. 2016). For example, the antifungal activity of natural extracts and EOs derived from Tectona grandis, Syzygium aromaticum, and Eucalyptus gomphocephala were tested against Fusarium moniliforme, Fusarium oxysporum, Aspergillus sp., Mucor sp., and Arthrinium phaeospermum. These compounds inhibit both the mycelial growth and sporulation of fungi (Astiti and Suprapta 2012). Moreover, the EO obtained from E. camaldulensis completely inhibits the mycelial growth of the five isolates of Fusarium spp. at a concentration range between 7 and 8 μL/mL after five days of incubation (Gakuubi et al. 2017).

Mosquitoes are insects that cause great concern for public health, as they are vectors for numerous tropical and subtropical diseases. They are an important threat for over two billion people in the world (Odalo et al. 2005). The intensive use of conventional insecticides to control mosquitoes is causing many problems such as environmental pollution, toxic hazards to mammals and non-target organisms, and the development of insecticide resistance (Sutthanont et al. 2010). These complications have become the driving force for an expeditious search for alternatives: compounds offering protection against mosquitoes suitable for both public health and the environment. Among the present alternative approaches aimed at reducing mosquito populations, the use of biopesticides based on natural plant products is now one of the most promising (Rajamma et al. 2011).

The present study investigated the antifungal activity of natural extracts and EOs obtained from six aromatic plants, T. grandis, E. gomphocephala, S. aromaticum, Euphorbia paralias, Ocimum basilicum, and Punica granatum against three plant pathogenic fungi, F. oxysporum, Rhizoctonia solani, and Alternaria solani. These extracts also were tested for their efficacy against mosquitoes. Lastly, the chemical constituents of the extracts/EOs were identified using GC/MS analysis.

EXPERIMENTAL

Plant Materials and Their Solvent Extraction

The plant materials used in this study are presented in Table 1. The plant materials used for solvent extraction were previously air-dried under room temperature for one week and then ground to fine particles (40- to 60-mesh). Approximately 100 g of ground material was soaked in solvent (200 mL) for 3 days, filtered, and concentrated to dryness using a rotary evaporator. The essential oils (EOs) were extracted by hydrodistillation using a Clevenger-type apparatus, where 100 g of green materials was hydrodistillated for 3 h. The collected oil was stored at 4 °C prior to analysis (Salem et al. 2013).

Table 1. Plant Materials and Their Parts Used for the Extractions

Antifungal Assay

The antifungal activities of the plant extracts and/or EOs obtained from T. grandis, E. gomphocephala, S. aromaticum, E. paralias, O. basilicum, and P. granatum were evaluated on the mycelial growth of plant pathogenic fungi (Fusarium oxysporum, Rhizoctonia solani, and Alternaria solani) using the radial growth technique method (Zambonelli et al. 1996). The fungi were obtained from the Microbiological Laboratory, Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University. The plant extracts dissolved in dimethyl sulfoxide (DMSO) were added to warm PDA medium (40 to 45 °C) at different concentrations, ranging from 50 to 1000 mg/L, before immediately pouring into 9-cm Petri dishes. Each concentration was tested in triplicate. Parallel controls contained PDA mixed only with DMSO. Mycelial discs (0.5 cm in diameter) of the plant pathogenic fungi, taken from 8-day-old cultures on PDA dishes, were transferred to the center of PDA dishes supplemented with either plant extracts or only DMSO. Inoculated dishes were incubated at 25 °C in the dark. The colony growth diameter was measured when the fungal growth in the control treatments had completely covered the Petri dishes. Percentage of mycelial growth inhibition was calculated using the following equation (Pandey et al. 1982),

Mycelial growth inhibition (%) = [(DC-DT)/DC] × 100 (1)

where DC and DT are average diameters of the fungal colony of control and treatment, respectively. The concentrations of plant extract or oil that inhibited the fungi mycelial growth by 50% (EC₅₀) with an equivalent confidence limit of 95% were estimated by the probit analysis method (Finney 1971).

Mosquitoes

A laboratory colony of Culex pipiens L. (Diptera: Culicidae) obtained from the College of Food and Agriculture Sciences, King Saud University was used. Mosquitoes were maintained at 25 ± 2 °C, 60 ± 5% RH, and a 12-h photoperiod. Adult mosquitoes were provided with a 10% sucrose solution as food. A blood meal was introduced twice per week to feed the females. Larvae were reared in Cl-free water and fed daily with rabbit feed.

Larvicidal Bioassay

The larvicidal activity of the extracts or oils was evaluated on the early fourth instar of C. pipiens larvae using the standard method described by the World Health Organization (WHO 1981), with slight modification. Stock solutions of the extracts or oils were prepared in ethanol, and Tween-80 (10 ppm) was used as an emulsifier. A series of six concentrations of each extract or EO were prepared in dechlorinated tap water. Twenty mosquito larvae were put into 200 mL cups containing 100 mL of test solution. The control treatment was prepared with water containing the same concentration of ethanol and Tween-80. Three replicates were used for each concentration. Larval mortalities were recorded after 24 h of exposure. Larvae were considered dead when they did not respond to probing with a needle. Mortality data were subjected to probit analysis to calculate the median lethal concentration values (LC₅₀) of the extracts or oils (Finney 1971). Abbot’s formula (Abbott 1925) was used when necessary to correct percentage mortality in the treatments.

GC/MS Analysis of Extracts/Essential Oils

Samples of T. grandis (wood acetone and water extracts) and E. paralias (leaf and seed extracts) were analyzed using a derivatization process. For the derivatization process, 10 µL from the samples was added to the bottom of a 2 mL tube and dried with a gentle stream of pure nitrogen gas (99.999%). Immediately, 50 µL of N,O-Bis (trimethylsilyl, TMS) trifluoroacetamide (BSTFA) was added and vortexed. The reaction was incubated for 3 h at 70 °C. The products were dried by nitrogen blowing. After drying, hexane was added for dilution and injection into the GC-MS instrument. The injection volume was 2 µL in splitless mode. The analysis of the alteration products and external standards was carried out by GC-MS on a Hewlett- Packard 6890 GC coupled to a 5973 Mass Selective Detector using a DB-5 (J and W Scientific, Agilent, Palo Alto, CA, USA) fused silica capillary column (30 m x 0.25 mm i.d., 0.25 μm film thickness) and helium as a carrier gas. The GC was temperature programmed from 65 °C (2 min initial time) to 300 °C at 6 °C/min (isothermal for 20 min final time). The MS was operated in the electron impact mode at 70 eV ion source energy. Data were acquired and processed with a Hewlett-Packard Chemstation and compounds were identified by comparison of mass spectra with those of authentic standards, literature and library data, and characterized mixtures. Unknown compounds were characterized by interpretation of the fragmentation pattern of their mass spectra.

The chemical constituents of S. aromaticum (methanolic extract and EO), E. gomphocephala (branch oil), P. granatum (extract), and O. basilicum (essential oil) were analyzed for their chemical compositions using the previous published methods (Salem et al. 2016a,b, 2019). Identification of the chemical composition of extracts/EOs was done based on MS library searches (NIST and Wiley), as well as by comparing with the MS literature data (NIST 11. 2011; Oberacher 2011).

RESULTS AND DISCUSSION

Antifungal Activity

The antifungal activities of plant extracts/EO obtained from different plants against R. solani, A. solani, and F. oxysporum in terms of radial growth inhibition are summarized in Tables 2, 3 and 4, respectively. The acetone extract of T. grandis showed the highest antifungal activity against R. solani and A. solani with EC₅₀ values of 118 and 294 μg/mL, respectively. However, the acetone extract of T. grandis showed the lowest antifungal activity against F. oxysporum. S. aromaticum extract exhibited moderate antifungal activities against the three tested fungi. Generally, F. oxysporum was less sensitive to T. grandis and E. gomphocephala extracts compared with the other two fungi. E. gomphocephala branch EO showed weaker antifungal activity than T. grandis and S. aromaticum extracts. Extracts of E. paralias (leaves), E. paralias (fruits), O. basilicum, T. grandis (obtained from wood), and P. granatum exhibited no antifungal activity against the three tested fungi.

Table 2. In vitro Antifungal Activity of Plant Extracts/Oils against R. solani using the Mycelial Growth Inhibition Method

Table 3. In vitro Antifungal Activity of Plant Extracts/Oils against A. solani using the Mycelial Growth Inhibition Method

Table 4. In vitro Antifungal Activity of Plant Extracts/Oils against F. oxysporum using the Mycelial Growth Inhibition Method

Larvicidal Activity

The larvicidal activity results of the test plant extracts/EO against C. pipiens L. are summarized in Table 5.

Table 5. Larvicidal Activity of Plant Extracts/Oils against Culex pipiens L.

The EOs of basil O. basilicum and E. gomphocephala displayed the highest larvicidal activity with LC₅₀ values of 22 and 30.1 mg/L, respectively. Moreover, the methanolic extract of S. aromaticum showed high larvicidal activity against C. pipiens (LC₅₀ = 58.7 mg/L), while the essential oil of the same plant showed relatively lower larvicidal activity (LC₅₀ = 129 mg/L). The acetone extract of T. grandis was more toxic to the larvae than the water extract of the same plant with LC₅₀ values of 251 and 697, respectively. The fruit extract of E. paralias exhibited more larvicidal activity to the larvae (LC₅₀ = 295 mg/L) than the leaves extract (LC₅₀ = 786 mg/L). Low larvicidal activity was observed for P. granatum extract with an LC₅₀value of 955 mg/L.

Chemical Composition of the Extracts/Essential Oils

3-Allylguaiacol (65.8%) and eugenol acetate (23.4%) were the main compounds in S. aromaticum methanolic extract (Table 6), while eugenol acetate (46.6%), isoeugenol (21.5%), trans-caryophyllene (15.8%) and α-humulene (9.0%) were the main compounds in the EO (Table 7).

Table 6. Chemical Constituents of S. aromaticum Methanolic Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Table 7. Chemical Constituents of S. aromaticum Essential Oil

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Table 8. Chemical Constituents of T. grandis Wood Acetone Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Cyclohexylpentyl oxalate (8.7%), 1,2-bis(TMS-oxy)-cyclooctene (8.6%), techtoquinone (8.5%), 3-(1,3-benzodioxol-5-yl)-5-hydroxy-4-nitrocyclohexanone (7.2%), and 2-thiobarbituric acid, TMS (6.6%) were the main compounds in Tectona grandis wood acetone extract (Table 8). The main compounds isolated from water extract of T. grandis were (E)-4,4-dimethyl-2-pentene (51.14%), vinylather (15.7%), and divinyl carbinol (7.6%) (Table 9).

Table 9. Chemical Constituents of T. grandis Wood Water Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Table 10 presents the chemical compounds of Eucalyptus gomphocephala branch EO where the main compounds were p-cymene (28.8%), (+)spathulenol (13.0%), ∆3-carene (7.5%), 2-methyl-3-phenylpropanal (3.9%), and 1,8-cineole (3%).

Table 11 presents the chemical constituents of E. paralias leaf extract where the main compounds were 1βH-romneine (26.3%), β-amyrin, TMS derivative (8.3%), 8-bromo-neoisolongifolene (10.0%), 3,5,6,7,8,8α-hexahydro-4,8α-dimethyl-6-(1-methyl-ethenyl)-2(1H)-naphthalenone (5.7%) and 24-methylene-cycloartenol, acetylated (4.7%).

Table 10. Chemical Constituents of E. gomphocephala Branch Essential Oil

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Table 11. Chemical Constituents of E. paralias Leaf Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

In E. paralias seed extract (Table 12) the main compounds were α-linolenic acid, TMS (15.2%), 2-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl)-N-(4-fluorophenyl) acetamide (8.3%), 8-bromo-neoisolongifolene (7.5%), gallic acid, 4TMS (5.7%), α-D-glucopyranose, 5TMS derivative (5.4%), 24-methylene-cycloartenol, acetylated (5.3%), palmitic acid, TMS (4.0%), cyclohexylpentyl oxalate (3.3%), shikimic acid (4TMS) (3.2%) and β-amyrin-TMS (3.1%).

Table 12. Chemical Constituents of E. paralias Seeds Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index.

Table 13 shows chemical composition of peels of P. granatum extract: furfural (32.1%), orotyl amide (11.0%), D-allose (9.2%), n-capric acid (6.4%), 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one (6.1%), 2,5-dimethylfurane (4.6%), ethyl α-D-gluco-pyranoside (4.3%), cis-isoeugenol (3.9%), and 1,6-anhydro-β-D-glucofuranose (3.7%).

Table 13. Chemical Constituents of P. granatum Extract

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index

Table 14 presents the chemical composition of O. basilicum essential oil where the main compounds were estragole (65.9%), eucalyptol (5.1%), linalool (4.9%), trans-4-methoxycinnamaldehyde (3.8%), and fenchyl acetate (2.2%).

Table 14. Chemical Constituents of O. basilicum Essential Oil

^aRT, Retention Time (min.).

^bSI, Standard Index.

^cRSI, Reverse Standard index

The results showed that the EOs extracted in acetone of T. grandis, S. aromaticum, and E. gomphocephala have remarkable antifungal activities against the three tested pathogenic fungi, A. solani, F. oxysporum, and R. solani. The acetone extract of T. grandis showed the highest antifungal activity against R. solani and A. solani with EC₅₀ values of 118 and 294 μg/mL, respectively. However, T. grandis acetone extract showed lower antifungal activity against F. oxysporum. The other plant extracts from S. aromaticum and E. gomphocephala exhibited moderate antifungal effects against the three tested fungi. These results confirmed the antifungal activity of different plant extracts against three plant pathogenic fungi.

The major compounds (eugenol acetate, eugenol and caryophyllene (Nassar et al. 2007) of the EOs from S. aromaticum have been known to possess various antibacterial and antifungal properties (Fu et al. 2007; Singh 2018). Eugenol, eugenol acetate, caryophellene, acetyle, eugenol, sesquiterpene ester, phenyl propanoid were the main compounds in the ethanolic extract from dried flower buds of S. aromaticum (Ghelardini et al. 2001; Miyazawa and Hisama 2003; Sumalatha et al. 2010). A 20% concentration of flower bud aqueous extract of S. aromaticum showed 100% inhibition of mycelial growth of Aspergillus niger (Avasthi et al. 2010). The antifungal effect of S. aromaticum was found on Aspergillus spp. and Penicilliumspp. (Garg and Siddiqui 1992; Vazquez et al. 2001).

There was an increase in mycelial growth over time except for 50 μL/20 mL of PDA, where no mycelial growth was detected. In addition, the present results agree with other studies, where essential oils of S. aromaticum were used against various common fungal pathogens of plants namely, F. moniliforme, F. oxysporum, Aspergillus sp., Mucor sp., Trichophyton rubrum and Microsporum gypseum (Pinto et al. 2009; Rana et al. 2011; Sharma et al. 2016).

Extracts from almost every part of T. grandis were composed of chemical compounds from different classes such as flavonoids, steroidal compounds, glycosides, quinones, and phenolic acids (Ohmura et al. 2000), with remarkable antifungal and antitermitic effects (Healey and Gara 2003; Thulasidas and Bhat 2007; Shalini and Rachana 2009; Florence et al. 2012; Guerrero-Vásquez et al. 2013). Lapachol is a naphthoquinone and lapachonone, found in T. grandis wood and bark (Goel et al. 1987; Sumthong et al. 2006). Bis (2-ethylhexyl) phthalate has been isolated from wood extracts of T. grandis and is a good repellent to termites (Alabi and Oyeku 2007). Heartwood extract from T. grandis reduces the weight loss caused by white rots (Pleurotus squarrosulus and Lentinus subnudus) in two hardwood species (Triplochiton scleroxylon and Gmelina arborea) (Adegeye et al. 2009).

Naphtoquinone, anthraquinone (Thulasidas and Bhat 2007), and 2-methyl anthraquinone (techtoquinone) (Windeisen et al. 2003) that inhibited the growth of of Coniophora puteana, a brown rot fungus (Haupt et al., 2003). Lapachol and deoxylapachol are naphtoquinone derivatives which also reported as biologically active compound (Windeisen et al. 2003). Plant extracts containing naphtoquinones (chromatic pigments) have been used for cancer and rheumatoid arthritis treatment (Babula et al. 2006). Lapachol is a natural quinone has been isolated from heartwood of Asian and South American Bignoniaceae (Tabebuia, Taigu, and Tecoma) (Steinert et al. 1995).

The sawdust of T. grandis contains the active components deoxylapachol and tectoquinnone, which inhibited the growth of A. niger (Neamatallah et al. 2005; Sumthong et al. 2006; Hussain et al. 2007). Furthermore, its leaf extract significantly suppressed the growth of Arthrinium phaeospermum (Astiti and Suprapta 2012). Leaf and bark extracts of T. grandis also have antifungal activity against A. niger, Trichoderma viride, and A. flavus (Lanka and Parimala 2017).

Previous studies have shown that n-hentriacontane, n-nonacosane, n-triacontane, n-dotriacontane, n-tritriacontane, and n-pentatriacontane, hexacosanol, octacosanol, cycloartenol, methylenecycloartenol, β-sitosterol, stigmasterol, campesterol, cholesterol, oleanolic, betulin and β-amyrin were isolated from extracts of E. paralias (Rizk et al. 1974; Shi et al. 2008; Noori et al. 2009) and have potential antimicrobial activities (Jassbi 2006; Shi et al. 2008; Noori et al. 2009).

Many previous studies reported that the EO of Eucalyptus sp. completely inhibited mycelial growth of plant pathogens. p-cymene (17.2%) and crypton (8.9%) were the main compounds in E. gomphocephala leaf essential oil (Salem et al. 2015(. The EOs of extracts from different parts of E. gomphocephala have been reported to have potential antimicrobial activities (Salem et al. 2015; Elansary et al. 2017). EOs of Eucalyptus sp. have been shown to have antibacterial and antifungal activities (Bendaoud et al. 2009; Bachheti et al. 2011). The antimicrobial/antifungal activities of essential oils are generally due to the presence of components such as 1,8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, and linalool (Nezhad et al. 2009).

Alternaria alternata, Stemphylium botryosum, and Fusarium spp. were significantly inhibited by the aqueous extracts of pomegranate peels (Glazer et al. 2012). Methanolic extracts of pomegranate showed inhibitory activity against A. niger, Penicillium citrinum, Rhizopus oryzae, and Trichoderma reesei (Dahham et al. 2010).

Phenolic compounds such as punicalagin, punicalin, granatins A and B, gallagyldilacton, tellimagrandin I, pedunculagin, and corilagin were isolated from peel extract of Punica granatumwere responsible for the antimicrobial activity (Fetrow and Avila 2000; Catão et al. 2006; Dudonné et al. 2009). Tannins, flavonoids, and alkaloids presented in peel aqueous extract of P. granatum were observed positive tests against diarrhea (Qnais et al. 2007). P. granatum peel methanolic extract showed antifungal activity against Candidia albicans (Höfling et al. 2010). Alcoholic and hot water extracts of P. granatum peel with high amount of gallotanic acid showed good antifungal activities against C. albicans, C. tropicalis, A. fumegatus, and A. nigar(Shaokat et al. 2007). Many tactics have been developed to control the threat of mosquito-borne disease. One such tactic is to kill mosquito larvae, using a strategy based on synthetic insecticides. Although they are effective, they have generated many problems such as insecticide resistance, environmental pollution, and adverse effects on human beings and livestock (Liu et al. 2005; Lixin et al. 2006).

The present study revealed that the EO of O. basilicum has a potent larvicidal activity against C. pipiens larvae. In line with our results, the larvicidal activity of O. basilicum EO was reported against four other mosquito species Culex tritaeniorhynchus, Aedes albopictus, Anopheles subpictus, and Aedes aegypti with LC₅₀ values of 14.01, 11.97, 9.75, and 75.35 ppm, respectively (Govindarajan et al. 2013; Manzoor et al. 2013). Furthermore, the EO of O. basilicum displayed the highest larvicidal activity against the larvae of the lymphatic filariasis vector (Culex quinquefasciatus), in comparison to O. sanctum and O. gratissimum (Rajamma et al. 2011). Diverse species of Ocimum from different countries displayed a potent larvicidal activity against mosquito larvae such as O. sanctum L. from India and Nigeria (Pathak et al. 2000; Gbolade and Lockwood 2008), O. americanum L. and O. gratissimum L. from Brazil (Cavalcanti et al. 2004), and O. lamiifolium Hochst./O. suave Willd from Ethiopia (Massebo et al. 2009). This activity could be related to the presence of important chemical compounds in the EOs. The main compounds found in the EO of O. basilicum were stragole and eucalyptol. Previously, the major EO constituents of O. basilicum plants included methyl chavicol (estragole), linalool, eugenol, and 1,8-cineole (eucalyptol) (Sajjadi 2006; Telci et al. 2006; Chalchat and Özcan 2008; Pripdeevech et al. 2010).

The Eucalyptus genus (Family: Mitraceae) includes more than 700 species and is one of the world’s most important widely planted genera (Menut et al. 1995). In the present study, E. gomphocephala EO showed a remarkable larvicidal activity (LC₅₀ = 30.07 mg/L) against C. pipiens. Confirming the present results, similar larvicidal activity of E. camaldulensis EO was reported with an LC₅₀ value of 31 µg/mL against A. aegypti (Cheng et al. 2009). S. aromaticum is an evergreen tree in the family Myrtaceae. Its aromatic flower buds are widely used in traditional medicine (Alqareer et al. 2012). The methanolic extract of S. aromaticum displayed higher larvicidal activity (LC₅₀= 58.73 mg/L) against C. pipiens compared with its EO (LC₅₀= 128.92 mg/L). A very similar result of the larvicidal activity of the EO from S. aromaticum was reported against A. aegypti (LC₅₀ = 124.69 ppm) and C. quinquefasciatus (LC₅₀ = 124.42 mg/L) (Sutthanont et al. 2010; Fayemiwo et al. 2014).

CONCLUSIONS

1. The essential oils extracted from T. grandis (acetone extract), S. aromaticum (methanolic extract), and E. gomphocephala (branch oil) had remarkable antifungal activities against the three tested pathogenic fungi, A. solani, F. oxysporum, and R. solani.
2. The essential oils from O. basilicum, E. gomphocephala, and S. aromaticum as well as the methanolic extract of S. aromaticum have remarkable larvicidal effects.
3. By GC/MS analysis, the most abundant compounds identified in S. aromaticum methanolic extract were 3-allylguaiacol and eugenol acetate; in S. aromaticum essential oil were eugenol acetate, isoeugenol, trans-caryophyllene and α-humulene; in Tectona grandis wood acetone extract were cyclohexylpentyl oxalate, 1,2-bis(TMS-oxy)-cyclooctene, techtoquinone, 3-(1,3-benzodioxol-5-yl)-5-hydroxy-4-nitrocyclo-hexanone, and 2-thiobarbituric acid, TMS; in T. grandis water extract were (E)-4,4-dimethyl-2-pentene, vinylather, and divinyl carbinol; in E. gomphocephala branch oil were p-cymene, (+)spathulenol, ∆3-carene, 2-methyl-3-phenylpropanal, and 1,8-cineole; in E. paralias leaf extract were 1βH-romneine , β-amyrin, TMS derivative, and 8-bromo-neoisolongifolene; in E. paralias seed extract were α-linolenic acid, TMS, 2-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl)-N-(4-fluorophenyl)acetamide, 8-bromo-neoisolongifolene, gallic acid, 4TMS, α-D-glucopyranose, 5TMS derivative, 24-methylene-cycloartenol, acetylated, palmitic acid, TMS, cyclohexyl pentyl oxalate, Shikimic acid (4TMS), and β-amyrin-TMS, and in P. granatum peel extract were furfural, orotyl amide, D-allose, n-capric acid, 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one, 2,5-dimethylfurane, ethyl α-D-glucopyranoside, and cis-isoeugenol; in O. basilicum essential oil were estragole, eucalyptol, linalool, trans-4-methoxycinnamaldehyde and fenchyl acetate.
4. These results support the idea of using natural plant extracts or essential oils as alternative antifungal compounds to control phytopathogenic fungi and the mosquito population.
5. This application could reduce the use of synthetic fungicides and insecticides.

ACKNOWLEDGMENTS

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No. (RG- 1440-028). The authors thank the RSSU at King Saud University for their technical support.

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Article submitted: February 27, 2019; Peer review completed: April 12, 2019; Revised version received and accepted: April 18, 2019; Published: April 22, 2019.

DOI: 10.15376/biores.14.2.4489-4511