Essential oils (Eos) may be defined as volatile oils that may be obtained from plant materials by steam distillation (Guenther, 1949). In the last few years there has been an increasing interest in Eos as substitutes for conventional synthetic pesticides. This has been due, in part, to concerns over pollution, the development of resistance to conventional pesticides (Holmes and Eckert, 2009), and to the needs of producers of organic agricultural products.

Certain parts of various plant species harbour secondary metabolites, which show a variety of chemical structures (Bell and Charlwood, 1980). Their roles are mostly unknown though many of them have been found to exhibit anti-fungal properties (Patel and Jasrai, 2009; Sujatha, 2010). Various types of anti-fungal chemicals such as saponins, unsaturated lactones, cyanogenic glycosides, oils and phenolic compounds are found to be present in relatively large quantities in tissues of some plant species. Their occurrence, distribution and possible functions have been reviewed by Schlosser (1988). Anti-fungal action of Eos and other chemical plant components has been reported by several scientists (Mann and Markhan, 2006; Deena and Thopil, 2008; Demirci et al., 2009; Mathpal et al., 2005 and Simic et al., 2004).

Considering the environmental pollution, we should exploit different plant volatile Eos for disease management in organic system (Barman et al., 2015). Keeping the value of synthetic pesticide free organic commodity, in vitro study was carried out. In this study, the antifungal potency of four different plant volatile Eos from eucalyptus (Eucalyptus globulus), citronella (Cymbopogon citrate), karanj (Pongamia pinnata) and neem (Azadirachta indica) has been tested against ten selected plant pathogenic fungal isolates and results achieved are presented.

This experiment was conducted during the year 2012 to 2013. Distilled Eos (Upshaw Aromatics Private Ltd. Hyderabad) of four plant species, namely eucalyptus, citronella, karanj and neem were purchased from the local market, and were screened for in-vitro antifungal activity against the following 10 phytopathogenic fungi (Table 1) isolated from infected material: Fusarium equiseti, Colletotrichum gloeosporioides, Alternaria alternata, Pestalotiopsis theae, Aspergillus flavus, Fusarium solani, Alternaria solani, Bipolaris oryzae, Erysiphe pisi, and Cercospora nicotianae grown in Sabouraud Dextrose Agar (SDA) medium and maintained in the laboratory at 25±1°C.

The pathogens were subjected to Koch’s postulates for verification of the diseases. Thereafter, they were incubated at 4°C. After seven days of incubation, the hyphal tip of the fungus radiating from the infected tissue was transferred onto SDA slants. Freshly prepared sterile SDA slants were used for the maintenance of the fungal cultures by sub-culturing periodically. Pathogens grown on sterile SDA media were stored in two different conditions, viz. at low temperature in refrigerator (at 4°C) and in incubator at 27±1°C. At the interval of one week, subculture was done taking sample from incubator at 27±1°C for preparation of inoculums for different experiments.

To avoid loss of virulence, fresh isolations were made when required. The chemical compounds present in the selected plant volatile Eos are listed in Table 2. The antifungal screening was performed through Disk diffusion assay method (Patel and Jasrai, 2011).

Paper disc preparation for the assay

Sterilized Whatman paper (No.1) discs (6.5 mm in diameter) were impregnated with the known quantity of plant volatile EO at 0.5 to 8 mg/disc, and then air dried. The impregnated discs were used to conduct bioefficacy study against the above mentioned fungal isolates.

Inoculum preparation of test fungi

50 µl of each fungal culture from SDA slant was transferred and established into a 150 ml conical flask containing 25 ml of SDA medium for bioassay study. The inoculated flasks were successively incubated for a specific time period at room temperature (25±1°C). The number of all fungal colonies/flask was recorded by means of a haemocytometer spore count (Table 1). Then the fungal colonies formed in each single flask were homogenized in sterile conditions and the relative suspensions were used in the bio-assay study (Patel and Jasrai, 2011).

Evaluation of antifungal activity

Fungitoxic spectrum of the selected plant volatile Eos was determined at various concentrations (0.5 to 8 mg/disc) using the standardized protocol of the Disk diffusion assay (Patel and Jasrai, 2011) under axenic conditions. For this, an aliquot 0.1 ml fungal culture of known (spore count) unit forming colonies (UFC) was aseptically transferred with micropipette in each Petri plate containing SDA medium (15 ml in a 3.5 cm thick layer), and uniformly seeded on its surface with sterilized cotton swabs (Himedia). At the same time, extract loaded single Whatman paper discs were placed and slightly pressed on the media surface with sterile forceps to ascertain a firm contact. Then the plates were incubated in upside-down position for 72 h at 25±1°C. The experiment was performed in triplicate with untreated controls. The Zone of inhibition (ZI) indicating the EO antifungal effectiveness was measured (in mm) by the antibiotic Zone reader (Labfine) (Patel and Jasrai, 2011). The experiment was carried at five different concentration viz. 0.5, 1.0, 2.0, 3.5, 5.0 and 8.0 mg/disc. Three replications were maintained for each pathogen with CRD design. The data were subjected to statistical analysis using INDOSTAT package developed by Indostat service Hyderabad, India.

Results of this study showed that the four tested plant volatile Eos had excellent broad- spectrum antifungal activity against the ten selected plant fungal pathogens. The inhibition of fungal phytopathogens by the tested Eos can be due to presence of complex mixture of secondary metabolites containing different volatile compounds such as phenylpropanes, various terpenoids and their oxygenated derivatives. The fungi toxic spectrum or MIC (minimum inhibitory concentration) values of the tested Eos, determined in terms of zone of inhibition (ZI), is presented in Tables 4, 5, 6 and 7. The antifungal screening for all the four Eos clearly indicates the effective lowest concentration to control the fungal growth. The more lower the MIC value, the better antifungal potency of the relative plant volatile EO. The lowest MIC value, 0.5 mg/disc, was recorded for EO from C. citrate. P. pinnata and A. indica EOs showed the maximum MIC values. A. indica EO showed minimum inhibition of fungal pathogen at certain MIC level (Table 3).

In the present study, E. globulus EO exhibited highest zone of inhibition against F. solani (14.45 mm) followed by A. flavus (13.88 mm). Its lowest MIC, 0.5 mg/disc, was recorded against F. solani followed by inhibition of Aspergillus flavus. The same EO at only 0.5 mg/disc was able to control F. solani. C. citrate EO showed maximum ZI against A. alternata (13.84 mm). P. theae was inhibited only at concentration of 1 mg/disc (Table 5). In contrast, P. pinnata EO successfully inhibited all tested fungi and exhibited its maximum ZI against A. alternata (12.98 mm) followed by C. gloeosporioides (11.67 mm).

In addition, the assay revealed that P. pinnata restricted the growth of all tested fungi at MIC of 5.0 mg/disc (Table 6). A. indica EO demonstrated highest ZI against A. alternata (12.98 mm) followed by C. gloeosporioides (11.85 mm), A. flavus (10.95 mm). But A. indica EO did not performe well in comparison to the other Eos (Table 7). All the selected fungi were inhibited with all the Eos even if at different performance levels. Fungal growth inhibition by Eos often involves prevention of hyphal growth and sporulation, interruption in nutrient uptake and metabolism, plasma membrane disruption, mitochondrial structure disorganization and interference with respiratory enzymatic reactions of the mitochondrial membrane (Patel and Jasrai, 2011).

Locally available plant volatile Eos may play a great role in controlling major plant disease. This may encourage the farmers to produce organic commodities to generate more revenue. As EOs antifungal activity is very probably due to the synergistic action of chemical compound mixtures, there would be a negligible chance of resistance development in fungal pathogens. Disease control through such natural available volatile substances would also be an important tool for integrated disease management in organic farming. 

The authors have not declared any conflict of interests.