Antifungal efficacy of phytoconstituents of Medicago sativa against Rhizoctonia solani

Anwaar, Amina; Jabeen, Khajista; Iqbal, Sumera; Javad, Sumera

doi:https://doi.org/10.7202/1076363ar

INTRODUCTION

Fungi are among the major crop disease agents. Pathogenic fungi employ a range of approaches to colonize crops and cause illness. Some fungi nourish on their host’s dead material (necrotrophs), while others colonize residual tissue (biotrophs) (Doehlemann et al. 2017).

Rhizoctonia solani J.G. Kühn. is a ubiquitous phyto-pathogenic fungus born in the soil. It causes massive output losses in number of major agriculturally vital crops (Paulitz et al. 2006; Verma 1996). R. solani is among the quite notorious plant pathogen that causes post-emergence and pre-emergence damping-off diseases on many essential crops (Heydari et al. 2004). Before the development of plant, seed decay and death of the radicals occurs within the soil, which is the most common form of pre-emergence damping-off (Heydari et al. 2007). R. solani damages plants by producing hyphal threads in their germinating sclerotia. The fungus could not produce any conidia or asexual spores. Thanatephorus cucumeris is perfect stage of this fungus which appears as a narrow, mildew-like structure on soil, above the leaves and ground line (Uppala and Zhou 2018).

Plant pathogens require management of plant diseases. It is mandatory because up to ten percent of the foodstuff is decayed due to these pathogens (Strange and Scott 2005). Chemical pesticides are used to enabling plant fungal diseases to be managed quickly. The use of such pesticides has, however, raised public suspicions in recent times. Because of their cytotoxic, phytotoxic, carcinogenic and persistent effects, their practices are increasingly limited (Bajwa et al. 2008).

Botanical alternatives are safer than synthetic chemicals in the environment. The antifungal activity from the plant extracts have been shown to be effective against plant pathogens and ecofriendly (Duru and Onyedineke 2010; Latha et al. 2009). As an active source for fungitoxic substances, plant secondary metabolites have inordinate potential. Numerous operations such as antifungals and antimicrobials have been demonstrated by compounds produced by plants such as hydroquinone and sesquiterpenes (cinnamodial, capsidiol), naphthoquinone (lapachol, jugllone), and alkaloids (berberine) (El-Khateeb et al. 2013).

Alfalfa and lucerne are the common names of the M. sativa. It belongs to Fabaceae pea family grown as a common forage crop in many countries. Many essential secondary metabolites (coumarines, isoflavones, naphthoquinones, alkaloids and saponins) are generated by alfalfa. Medicago spp. have reported numerous biological actions, such as haemolytic activities, nematocidal characteristics, insecticidal properties, antibacterial and antifungal influences on humans and plants pathogens (Sadowska et al. 2014).

The purpose of the current study was to discover antifungal compound/antifungal constituents through the use of M. sativa extract to suppress the growth of R. Solani.

MATERIALS AND METHODS

Test experimental material collection

Medicago sativa sample plant was obtained from the area of Kana Kacha, Lahore. The test plant was dried under sunlight after thorough washing under tap water and finely grinded to make the powder. Pure culture of target fungus R. solani was obtained from the Laboratory of Fungal Biotechnology, LCWU (Lahore College for Woman University), Lahore. Sub culturing of this culture was done ant it was preserved on 2% MEA (Malt Extract Agar) and refrigerated at 4 °C.

Antifungal activity assay

Twenty-gram dried entire test plant powder of M. sativa was immersed in 100 mL of MeOH (methanol) at room temperature (± 30 °C) for six days. The content was filtered via a sterilized muslin cloth after 6 days. After evaporating at room temperature, the 3 g final methanolic greasy mass of M. sativa was attained. Twenty percent stock solution was produced by adding 15 mL distilled water into 3 g sticky mass of M. sativa.

In 250 mL flask, malt extract (1.2 g) and agar (1.2 g) were inserted in 60 mL of DW (distilled water) to attain 2% MEA and autoclaved at 121 °C for 30 min. Five test plant material’s concentrations (1%, 2%, 3%, 4% and 5%) of methanolic extract were formed by combining 0.3, 0.6, 0.9, 1.2 and 1.5 mL of stock solution into 59.7, 59.4, 59.1, 58.8 and 58.5 mL of DW (distilled water), respectively. In this way, total volume (60 mL) for each concentration was made. No plant extract was applied to any control group. Vibramycin capsule (100 mg) was introduced to each concentration as well as in control flask to prevent bacterial contamination. Using sterilized cork borer, 5 mm mycelial disks were prepared from one-week-old fungal culture and putted in the middle of all Petri dishes. Each treatment was made in triplicate manner. For one week, the whole setup was incubated at 25 ± 2 °C (Jabeen and Javaid 2010). The fungal growth diameter was calculated in centimeters after one week of fungal growth. Percentage decrease in each colony diameter size was calculated by given formula:

Bioassays with fractions of organic solvents

Various organic solvents were used to fractionate the M. sativa methanolic extract. For this, 40 g of plant material powder was immersed into 200 mL of MeOH (methanol) for seven days to acquire a methanolic extract of 20%. The acquired extract was then put to evaporate. For partitioning the extract, five organic solvents including n-hexane (100 mL), chloroform (CHCl₃) (100 mL), acetone (100 mL), ethyl acetate (EtOAc) (100 mL) and n-butanol (100 mL) were separated by using a separating funnel. Such samples were evaporated at room temperature to get their gummy masses (Bashir et al. 2019). For each separate fraction and Mancozeb (synthetic fungicide), 20% stock solution was formed. For all five organic fractions, three concentrations were made. Antibiotic (vibromycin capsules) of 50 mg was also used to prevent bacterial impurities at each concentration. In control treatments there was no plant extract. Three replicates were created for each concentration. In each flask, five-millimeter disk of R. solani was inserted (Hanif et al. 2017). Antifungal activity was detected after one week and findings had been noted down.

GC-MS analysis

Ethyl acetate fraction of methanolic extract of M. sativa was preceded in further bioassay to identify potential antifungal compounds. M. sativa plant material (50 g) was soaked in EtOAc (250 mL) and rapidly shaken for 48 hours on incubator shaker for the full incorporation of metabolites into the organic solvent. Nylon membrane filter paper of diameter 47 mm was used with 0.22 µm of pore size to purify this extract. The extracted filtrate was then utilized for GC-MS (Gas Chromatography-Mass Spectrometry).

The test plant was examined using a GC-MS QP chromatograph and BD-5 (30 m, 0.25 mm, 0.25 μm) capillary column was used for isolation by implementing the temperature of 50 °C, using He (helium) (99.999%) as carrier gas (mobile phase). Temperatures for the injector and detector were set at 200 °C and 250 °C correspondingly. The parameters of the mass detector were 70 eV ionization voltages with 55-950 Da m Z^-1 mass scanning range. Via peak areas of the GC, the volatile compounds proportion was estimated. Retention times (RT), mass spectra and indices were compared with earlier provided literature data provided by NIST Library 2010 word software for qualitative evaluation (Waheed et al. 2016).

Statistical analysis

Entire record was collected to examine on Statistix 8.1 software by applying the LSD (least significant difference) test. All the data were analyzed at significance level of P ≤ 0.05.

RESULTS AND DISCUSSION

In present study, the Medicago sativa methanolic extract was tested against R. solani. Several methanolic extract concentrations (1% to 5%) of M. sativa inhibited R. solani development. The most efficient concentrations were 5% and 4%, since they restricted the R. solani growth up to 77% and 75% respectively (Fig. 1). Previously (Kagale et al. 2004), Datura metel’s antifungal efficacy was evaluated to retard growth R. solani and X. oryzae by using its methanolic and aqueous leaf extracts. Methanolic extract has been found to be considerably more efficient. The antifungal attributes are attributable to chemical substances formed by plants as phytochemicals such as polyphenols, which are involved in the mechanism for plants’ protection against bacteria and fungi (Castillo et al. 2010).

Impact of M. sativa plant methanolic extract on in vitro development of R. solani — Figure 1

Impact of *M. sativa* plant methanolic extract on *in vitro* development of *R. solani*

Impact of M. sativa extract of various concentrations on in vitro development of R. solani — Figure 2

Impact of *M. sativa* extract of various concentrations on *in vitro* development of *R. solani*

GC-MS chromatogram of the M. sativa ethyl acetate extract — Figure 3

**GC-MS chromatogram of the *M. sativa* ethyl acetate extract**

Compounds analyzed by GC-MS of M. sativa ethyl acetate extract — Table 1

**Compounds analyzed by GC-MS of *M. sativa* ethyl acetate extract**

Partitioning of M. sativa methanolic extract was carried out using five organic fractions. The fraction of EtOAc (ethyl acetate) showed highest antifungal activity at concentrations of 1-2% as it suppressed fungal growth of 61-67% respectively. Treatment of control with synthetic fungicide also recorded 61-67% efficacy against test fungus R. solani at the concen-trations of 1-2%, respectively, the same as the fraction of ethyl acetate (Fig. 2). Other fractions also hindered growth of the test fungus. Previously, Seema et al. (2011) checked various organic solvent extracts of L. inermis, P. betel, P. longifolia and P. graveolens against R. solani and ethyl acetate fraction was registered the most substantial for antifungal activity testing against R. solani, as compared with other extracts.

The chromatograph of ethyl acetate extract of M. sativa revealed nine peaks of nine specific compounds after GC-MS analysis (Fig. 3). The leading constituents listed in the chromatogram of extract were phytol (RT: 20.84) 19.7%, 1,2,3-Propanetriol, monoacetate (RT: 12.47) 16.1%, Z,Z-3, 15-Octadecadien-1-olacetate (RT: 21.75) 15.9%, Hexadecanoic acid, ethyl ester (RT: 19.72) 13.3%, Benzene, nitro- (RT: 10.20) 11.7% and Ethanol, 2-(9-octadecenyloxy)-, (Z)- (RT: 23.17) 9.6% (Table 1). The compounds like Octadecane, 3-ethyl-5-(2-ethylbutyl)- (RT: 22.74) 7.7%, 1,2,3-Propanetriol, 1-acetate- (RT: 10.60) 5.3% and 1-Dodecanol, 3,7,11 -trimethyl- (RT: 14.66) 0.8% were present in lesser amount (Table 1). Earlier, Ponmathi et al. (2017) found the phytol in B. courtallica leaf extract which had shown notable antimicrobial activities. Previously, phytol recorded in Ipomoea staphylina ethanolic extract had shown great antimicrobial potential (Padmashree et al. 2018). Pejin et al. (2014) investigated the phytol’s anti-microbial assessment towards eight bacterial and fungal pathogens, and phytol was strong antimicrobial agent against all tested microorganisms. The earlier findings assisted our research work that the phytol present in EtOAc extract of Medicago sativa could have characteristic antimicrobial attributes. According to previous research, high concentrations of Benzene, nitro- had significant antifungal efficacy to retard the growths of M. Verrucaria and T. mentagrophytes (Gershon et al. 1971). Zekeya et al. (2014) founded that Hexadecanoic acid, methyl ester was present in root bark and Ethanol, 2-(9-octadecenyloxy)-, (Z)- was present in leaves of Bersama abyssinica methanolic extract which showed strong antimicrobial efficacy.

CONCLUSION

The current research came to the conclusion that M. sativa had substantial antifungal activity against R. solani and ethyl acetate fraction was found highly successful in combating target fungi and it might be due to the presence of various natural phytoconstituents examined in M. sativa via analysis of GC-MS.

Antifungal efficacy of phytoconstituents of Medicago sativa against Rhizoctonia solaniEfficacité antifongique des phytoconstituants de Medicago sativa contre Rhizoctonia solani

Abstract

Résumé

INTRODUCTION