GC-MS ANALYSIS AND ANTIFUNGAL ACTIVITY OF METHANOLIC ROOT EXTRACT OF Chenopodium album AGAINST Sclerotium rolfsii

Análise por Cromatografia Gasosa com Espectrômetro de Massa (GC-MS) e Atividade Antifúngica de Extrato Metanólico de Raiz de Chenopodium album Diante de Sclerotium rolfsii

ABSTRACT

Sclerotium rolfsii is a soil-borne fungal plant pathogen that causes diseases in more than 500 plant species. Chemical fungicides used to control this disease cause environmental pollution, therefore, plant derived compounds can be used as alternative to synthetic fungicides to reduce environmental pollution. Chenopodium album is a weed of family Chenopodiaceae that is used as food and also has medicinal importance. In the present study, antifungal activity of methanolic root extract of C. album was evaluated against S. rolfsii using six concentrations viz. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 g 100 mL-1 amended in malt extract as growth medium. All the root extract concentrations significantly reduced fungal biomass by 15-58% over control. Gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic root extract of C. album was performed. Six compounds were identified in methanolic root extract through GC-MS analysis. The most abundant compound was 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (58.56%) followed by 9-octadecenoic acid (Z)-, methyl ester (12.75%) and 9-octadecenoic acid (Z)-, methyl ester (10.27%), which might be responsible for antifungal activity of methanolic root extract of C. album.

Keywords:
biological control; natural compounds; reduction use of agrochemical

RESUMO

Sclerotium rolfsii

é um patógeno vegetal fungoso do solo que causa doenças em mais de 500 espécies de plantas. Os fungicidas químicos utilizados para controlar essa doença causam poluição ambiental. Portanto, compostos derivados de plantas podem ser usados como alternativa aos fungicidas sintéticos para reduzir a poluição ambiental. Chenopodium album é uma planta daninha da família Chenopodiaceae que é utilizada como alimento e também tem importância medicinal. No presente estudo, a atividade antifúngica do extrato metanólico de raiz C. album foi avaliada diante de S. rolfsii utilizando seis concentrações, ou seja, 0,5, 1,0, 1,5, 2,0, 2,5 e 3,0 g 100 mL-1, alteradas em extrato de malte como meio de crescimento. Todas as concentrações de extrato de raiz reduziram significativamente a biomassa fúngica em 15-58% em relação ao controle. Foi feita a análise por cromatografia gasosa com espectrômetro de massa (GCMS) do extrato metanólico de raiz de C. album. Seis compostos foram identificados em extrato metanólico de raiz por meio de análise por GC-MS. O composto mais abundante foi o ácido 1,2-benzenodicarboxílico, mono (2-etil-hexil) éster (58,56%), seguido de ácido 9-octadecenoico (Z)-, éster metílico (12,75%), e ácido 9-octadecenoico (Z)-, éster metílico (10,27%), o que pode ser responsável pela atividade antifúngica de extrato metanólico de raiz de C. album.

Palavras-chave:
fungicida natural; controle biológico; redução do uso de agroquímico

INTRODUCTION

Sclerotium rolfsii Sacc., is one of the oldest known plant pathogenic fungus that was reported for the first time in 1892 on tomatoes in Florida (Aycock, 1966). Its occurrence in warm temperate and subtropical regions of the world has increased its host range (Hameeda et al., 2010). Therefore, over 500 plant species belonging to different groups are being attacked by this fungus. The extensive host range, abundant growth, and ability to produce persistent sclerotia make it a robust fungus that has been responsible for causing massive economic losses and included it in the list of fungi that are difficult to manage through physical and cultural practices (Farooq et al., 2011; Eslami et al., 2014). In chickpea (Cicer arietinum), the collar rot disease caused by S. rolfsii is responsible for 55-95% mortality of seedlings (Gurha and Dubey, 1982). Fungicides are normally utilized to minimize the losses (Azhar et al., 2006; Lakpale et al., 2007).

Increasing awareness of public concern regarding the deteriorating impact of the indiscriminate use of chemicals on the environment has led the researchers to seek for alternative disease management strategies for sustainability in agriculture (Rauf and Javaid, 2013). The use of natural compounds from plants could fulfill the criteria of a sustainable agriculture. Many recent studies have shown that plant crude extracts and purified compounds have great potential to be used as antifungal agents. Crude extracts, pure compounds or soil amendment with dry biomass of mango (Mangifera indica), Datura metel, Coronopus didymus, Imperata cylindrica, Withania sommnifera and radish (Raphanus sativus) have been proved very useful in the management of many soil-borne plant pathogenic fungi namely Macrophomina phaseolina, S. rolfsii and Fusarium oxysporum (Kanwal et al., 2010; Javaid and Saddique, 2011; Iqbal and Javaid, 2012; Javaid and Akhtar, 2015; Javaid and Bashir, 2015; Javaid et al., 2015).

Chenopodium album is a summer annual, fast-growing weed and is commonly used for food and it also possesses medicinal values (Singh et al., 2011). The tender shoots are eaten raw in salads or with curd, cooked as vegetables and are also used as fodder (Karwani and Sisodia, 2015). It has been found to have an antipruritic, antinociceptic (Kumar et al., 2007), and sperm immobilizing activity (Pande and Anupam, 2010). Its leaves are useful in the cure of influenza, anorexia, cough, dysentery, pneumonia, diarrhea, piles and typhoid (Cheryl et al., 2006; Singh et al., 2011). Besides, the antifungal activity of C. album has been documented against some plant pathogenic fungi. Singh’s (2005) results have revealed a strong toxic effect of leaves of C. album against S. rolfsii. Javaid and Amin (2009) have found a considerable reduction of up to 96% in growth of M. phaseolina due to leaf, stem, root and inflorescence extracts of C. album. Jabeen et al. (2014) have found a significant antifungal activity of leaf extract of C. album against Ascochyta rabiei, responsible for chickpea blight. Recently, Javaid and Rauf (2015) findings have explored the potential of soil amendment with dry leaf biomass of C. album against the management of Fusarium oxysporum f. sp. cepae. However, studies regarding antifungal activity of C. album root extract against S. rolfsii are lacking. Therefore, the aim of the present study was to investigate the antifungal potential of methanolic root extract of C. album against S. rolfsii.

MATERIALS AND METHODS

Isolation of pathogen

Chickpea (Cicer arietinum) plants infected with collar rot disease were collected from the field of district Sahiwal of province Punjab, Pakistan, during 2014. Infected portions of a basal plant part were surface sterilized with a solution of sodium hypochlorite (1%) for 1 min and then rinsed several times with sterilized distilled water. These pieces were plated on 2% malt extract agar (MEA) and incubated at 24 ^oC for 7 days. After that, culture purification was done by sub-culturing of an isolated pathogenic fungus using the single spore transfer technique. The isolated fungal species was identified as S. rolfsii on the basis of morphological characters (Watanabe, 2002). In order to check the pathogenicity of the isolated fungus, its inoculum was mixed in the pot soil and chickpea seeds were sown in these pots. The symptoms of collar rot appeared on chickpea seedling, confirming the pathogenicity of the isolated fungal species. Pure culture of the fungus was preserved at 4 ^oC for further use.

Preparations of methanolic extracts

Roots of C. album were collected from different vicinities of Lahore, Pakistan. The collected material was thoroughly washed under tap water and then dried at 45 ^oC using an electric oven. The dried material was ground to a fine powder. Powdered roots (200 g) were soaked in methanol (2 L) in a plastic jar for 7 days at room temperature. Later on, the soaked material was filtered and a rotary evaporator was used to evaporate the filtered extract at 45 ^oC under vacuum.

Antifungal assays

Bioassay experiments were done using methanolic extract of C. album root in vitro. Nine grams of methanolic root extract were dissolved in 5 mL dimethyl suloxide (DMSO) followed by the addition of sterilized distilled water to prepare 15 mL of stock solution. In a similar way, 5 mL DMSO were mixed in 10 mL distilled water to prepare a control solution. Malt extract broth medium (55 mL) was prepared in 250 mL conical flasks and then autoclaved at 121 ^oC for 30 min. After autoclaving, an antibacterial capsule (chloromycetin at 250 mg L^-1) was added in each media flask to avoid bacterial contamination. Six concentrations viz. 0, 10, 20, 30, 40, and 50 mg mL^-1 were prepared by adding 0, 1, 2, 3, 4, and 5 mL stock solution and 5, 4, 3, 2, and 1 mL control solution, respectively, to 55 mL growth medium to make the total volume of the medium 60 mL in each flask. Four replicates were made by allocating equal parts of growth medium in each replicate, i.e., 15 mL. A mycelial disc (5 mm) of S. rolfsii was transferred in each flask by a sterile borer and then incubated at 27 ^oC for 10 days. Afterwards, filtration of fungal mass was done using pre-weighed filter papers (Whatman No.1). Finally, the filtered material was dried in an electric oven at 70 ^oC to obtain a fungal dry biomass (Rauf and Javaid, 2013).

GC-MS analysis

GC-MS (gas chromatography-mass spectroscopy) analysis was done on a Perkin Elmer Turbo Mass Spectrophotometer (Norwalk, CTO 6859, USA), consisting of a 30 m length and 0.25 mm diameter capillary tube consisting of 95% dimethyl polysiloxane and a second service provider gas celled helium also used with flow amount of 0.5 mL min^-1. The obtained peak areas were measured by Turbo-Mass-OCPTVS-Demo SPL software.

Statistical analysis

All the data were subjected to one-way ANOVA. Means were compared by applying Tukey’s HSD Test at 5% level of significance using computer software Statistics 8.1. Relationship between fungal biomass and extract concentration was computed using MS Excel.

RESULTS AND DISCUSSION

Data about the effect of methanolic root extract on biomass of S. rolfsii is presented in Figure 1A-C. Different concentrations of methanolic root extract of C. album (10 to 50 mg mL^-1) significantly reduced fungal biomass by 15-58% over the negative control treatment. A linear correlation between extract concentration and biomass of S. rolfsii was recorded with R² = 0.963.

Six compounds belonging to fatty acid methyl esters, aromatic dicarboxylic ester and ketone group were identified in methanolic root extract of C. album (Figure 2, Table 1 and 2). These compounds were 2(3H)-furanone, dihydro-4,4-dimethyl (12.75%); 6-methylenebicyclo(3.2.0)hept3-en-2-one (7.21%); hexadecanoic acid, methyl ester (5.44%); 9,12-octadecadienoic acid (Z,Z)-, methyl ester (5.81%); 9-octadecenoic acid (Z)-, methyl ester (10.27%); and 1,8-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (58.50%). Names of these compounds along with their molecular formulas, molecular weights, retention time, and peak areas are presented in Table 1. Structures of these compounds are given in Figure 3. Three out of the six identified compounds (21.52%) belonged to the fatty acid methyl ester group, namely linoleic acid methyl ester, oleic acid methyl ester, and palmitic acid methyl ester (Tables 1 and 2). Fatty acid methyl esters are known to possess antifungal properties (Agoramoorthy et al., 2007). Chandrasekharan et al. (2008) have evaluated the antimicrobial activity of four halophytes of the Chenopodiaceae family and found that fatty acid methyl esters extract of Salicornia brachiata possessed the highest antifungal activity. Lima et al. (2011) have reported that fatty acid methyl esters of seeds of Annona cornifolia, mainly oleic acid methyl ester (51.5%), linoleic acid methyl ester (19.1%), and palmitic acid methyl ester (16.9%) as in the present study, have inhibited the growth of 12 strains of a clinical pathogenic fungus Paracoccidioides brasiliensis. According to Agoramoorthy et al. (2007), fatty acid methyl ester extract of Excoecaria agallocha, rich in palmitic acid methyl ester (56.02%), suppressed growth of four species of Candida namely C. albicans, C. krusei, C. tropicalis and C. parapsilosis. Similarly, 6-methylenebicyclo (3.2.0)hept-3-en-2-one is also known to exhibit a fungistatic activity (Hussein et al., 2016).

Figure 1
(A) effect of different concentrations of methanol root extract of Chenopodium album on biomass of Sclerotium rolfsii, (B) percentage reduction in fungal biomass due to different concentrations of methanolic extract over control, C: relationship between extract concentration and fungal biomass.

Figure 2
GC-MS chromatogram of methanolic root extract of Chenopodium album.

Table 1
Compounds identified from methanolic root extract of Chenopodium album through GC-MS analysis

Comp. No.	Names of compounds	Molecular formula	Molecular weight	Retention time (min)	Peak area (%)
1	2(3H)-Furanone, dihydro-4,4-dimethyl	C6H10O2	114	9.881	12.75
2	6-Methylenebicyclo(3.2.0)hept-3-en-2-one	C8H8O	120	10.773	7.21
3	Hexadecanoic acid, methyl ester	C17H34O2	270	18.716	5.44
4	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	C19H34O2	294	20.355	5.81
5	9-Octadecenoic acid (Z)-, methyl ester	C19H36O2	296	20.406	10.27
6	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	C16H22O4	278	24.238	58.50

Table 2
Nature and properties of compounds identified from methanolic root extract of Chenopodium album through GC-MS analysis

Comp . No.	Names of compounds	Nature	Property	Reference
1	2(3H)-Furanone, dihydro-4,4-dimethyl	Ketone	No activity reported
2	6-Methylenebicyclo(3.2.0)hept-3-en-2-one	ketone	Bacteriostatic, fungistatic, antiparasitic	Hussein et al. (2016)
3	Hexadecanoic acid, methyl ester	Palmitic acid methyl ester (fatty acid methyl ester)	Antifungal, Antioxidant, Antibacterial	Agoramoorthy et al. (2007)
4	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	Linoleic acid methyl ester (fatty acid methyl ester)	Antibacterial	Lima et al. (2011)
5	9-Octadecenoic acid (Z)-, methyl ester	Oleic acid methyl ester (fatty acid methyl ester)	Antimicrobial, Nematicidal	Chandrasekharan et al. (2008); Lima et al. (2011)
6	1,2-Benzenedicarboxylic acid, mono(2ethylhexyl) ester	Aromatic dicarboxylic ester	Antimicrobial, Cytotoxicity, Antioxidant, Antiinflammatory, Antiviral	Govindappa et al. (2014); Krishnan et al. (2014)

The most abundant compound 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester has been previously identified from marine Streptomyces sp., Alternaria sp. and metabolites of a cyanobcterium Oscillatoria terebriformis. This compound is known to have a number of biological properties, including cytotoxicity, antioxidant, anti-inflammatory, antimicrobial, and antiviral activity (Govindappa et al., 2014; Krishnan et al., 2014; Mukund et al., 2014). Since it is a biologically active compound and also found as the major compound in the present study (58.05%), therefore, there is possibility that it might have had a role in the antifungal activity of methanolic root extract of C. album in the present study.

Figure 3
Structures of compounds identified in methanolic root extract of Chenopodium album through GC-MS analysis.

The present study concludes that S. rolfsii can be managed by the methanolic root extract of C. album. The antifungal activity of this extract is possibly because of various fatty acid methyl esters and 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester.

REFERENCES

• Agoramoorthy G. et al. Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from India. Braz J Microbiol. 2007;38:739-742, 2007.
• Aycock R. Stem rot and other diseases caused by Sclerotium rolfsii. Raleigh: North Carolina Agriculture. Experiment. Station, 1966. (Technical Bulletin, 174).
• Azhar H. et al. Factors affecting development of collar rot disease in chickpea. Pak J Bot. 2006;38:211-6.
• Chandrasekharan M., Kannathasan K., Venkatesalu V. Antimicrobial activity of fatty acid methyl esters of some members of chenopodiaceae. Z Naturforsch C. 2008;63:331-6.
• Cheryl L. et al. Ethnoveterinary medicines used for horses in Trinidad and in British Columbia, Canada. J Ethnobiol Ethnomed. 2006;31:2-31.
• Eslami A.A. et al. Evaluation of the virulence of Sclerotium rolfsii isolates on Arachis hypogaea and screening for resistant genotypes in greenhouse conditions. Hellenic Plant Prot J. 2014;8:1-11.
• Farooq M.A. et al. Evaluation of sugar beet (Beta vulgaris L.) genotypes for resistance against root rot caused by Sclerotium rolfsii. Mycopath. 2011;9:13-15.
• Govindappa M. et al. Chemical composition of methanol extract of endophytic fungi, Alternaria sp. of Tebebuia argentea and their antimicrobial and antioxidant activity. Int J Biol Pharm Res. 2014;5:861-9.
• Gurha S.N., Dubey R.S. Occurrence of possible sources of resistance in chickpea (Cicer arietinum L.) against Sclerotium rolfsii Sacc. Madras Agric J. 1982;70:63-4.
• Hameeda B. et al. Biological control of chickpea collar rot by co-inoculation of antagonistic bacteria and compatible rhizobi. Indian J Microbiol. 2010;50:419-24.
• Hussein H.J., Hadi M.Y., Hameed I.H. Study of chemical composition of Foeniculum vulgare using Fourier transform infrared spectrophotometer and gas chromatography - mass spectrometry. J Pharm Phytother. 2016;8:60-89.
• Iqbal D., Javaid A. Bioassays guided fractionation of Coronopus didymus for its antifungal activity against Sclerotium rolfsii. Nat Prod Res. 2012:26:1638-44.
• Jabeen K., Sherazi A.Z., Iqbal S. Antifungal potential of Chenopodium album L. against Chickpea Blight. J Agric Sci Technol. 2014;4:69-75.
• Javaid A., Amin M. Antifungal activity of methanol and n-hexane extracts of three Chenopodium species against Macrophomina phaseolina. Nat Prod Res. 2009;23:1120-7.
• Javaid A., Saddique A. Management of Macrophomina root rot of mungbean using dry leaves manure of Datura metel as soil amendment. Span J Agric Res. 2011;9:901-5.
• Javaid A., Rauf A. Antifungal activity of methanolic root extract of Withania sommnifera against pathogen of basal rot disease of onion. Afr J Trad Complemen Alter Med. 2015;12:22-7.
• Javaid A., Bashir A. Radish extracts as natural fungicides for management of Fusarium oxysporum f. sp. lycopersici, the cause of tomato wilt. Pak J Bot. 2015;47:321-4.
• Javaid A. et al. Management of Macrophomina phaseolina by extracts of an allelopathic grass Imperata cylindrica. Pak J Agric Sci. 2015;15:37-41.
• Kanwal Q. et al. Antifungal activity of flavonoids isolated from mango (Mangifera indica L.) leaves. Nat Prod Res. 2010;24:1907-14.
• Karwani G., Sisodia S.S. Chenopodium album Linn. (bathua): a review of potential therapeutic applications. Int J Chem Pharm Anal. 2015;2:1-5.
• Krishnan K., Mani A., Jasmine S. Cytotoxic activity of bioactive compound 1, 2- benzene dicarboxylic acid, mono 2- ethylhexyl ester extracted from a marine derived Streptomyces sp. VITSJK8. Int J Mol Cell Med. 2014;3:246-54.
• Kumar S. et al. Chenopodium album seed extract: a potent sperm immobilizing agent both in vitro and in vivo. Contraception. 2007;75:71-8.
• Lakpale N., Khare N., Thrimurty V.S. Suppression of Sclerotium rolfsii Sacc. an integrated approach. Soil Crop. 2007;17:241-5.
• Lima L.A.R. et al. In vitro antifungal activity of fatty acid methyl esters of the seeds of Annona cornifolia A.St.-Hil. (Annonaceae) against pathogenic fungus Paracoccidioides brasiliensis. Rev Soc Bras Med Trop. 2011;44:777-80.
• Mukund S. et al. GC-MS and FT-IR analysis of some bioactive constituents from Oscillatoria terebriformis. Indian J Pharm Sci Res. 2014;4:102-7.
• Pande M., Anupam P. Preliminary pharmacognostic evaluations and phytochemical studies on leaf of Chenopodium album. Asian J Exp Biol Sci. 2010;1:91-5.
• Rauf S., Javaid A. Antifungal activity of different extracts of Chenopodium album against Fusarium oxysporum f. sp. cepae, the cause of onion basal rot. Int J Agric Biol. 2013;15:367-71.
• Singh R.K. Fungitoxicity of some higher plants and synergistic activity of their essential oils against Sclerotium rolfsii sacc. causing foot-rot disease of barley. Hindustan Antibiot Bull. 2005;47-48:45-51.
• Singh K.P., Abishek, K.D., Gunjan D. Evaluation of antibacterial activities of Chenopodium album L. Int J Agric Technol. 2011;2:398-401.
• Watanabe T. Pictorial atlas of soil and seed fungi, morphologies of cultured fungi and key to species. 2^nd ed. London: CRC Press, 2002.

Publication Dates

• Publication in this collection
  2017

History

• Received
  01 June 2016
• Accepted
  01 July 2016