Materials and methods

Maintenance of fungus culture

The test fungal pathogens, Alternaria alternata (Fr.) Keissler, Aspergillus flavus Link, A. fumigatus Fres., A. niger Van Tiegham, A. parasiticus Speare, Botrytis cinerea Pers. Ex. Fr., Cladosporium cladosporioides (Fresenius) de Vries, Colletotrichum capsici (Syd.) Butler and Bisby, C. falcatum We n t., Curvularia lunata (Wakker) Boedijn, Fusarium cerealis (Cooke) Sacc., F. culmorum (W.G. Smith) Sacc., F. oxisporum Schlecht.: Fr., F. udum (Butler) Snyder and Hansen, Gloeosporium fructigenum Berk., Helminthosporium oryzae Breda de Haan, H. maydis Nisikado and Miyakel, Penicillium digi-tatum Sacc., P. expansum Link, P. italicum We hm e r, P. implicatum Biourge, P. minio-luteum Dierckx, P. variabile Sopp, Phoma violacea (Bertd) Eveleigh, Rhizopus nigricans Ehrenb (Neergaard, 1977; Samson et al., 1995) were maintained on potato dextrose agar medium (200 g scrubbed and diced potato in 1000 ml distilled water, 15 g agar, 20 g dextrose; pH ± 5.6). A seven day old culture of each fungus was used for bioactivity tests.

Isolation of active constituent(s)

The essential oil was extracted from the fresh leaves of Cymbopogon flexuosus by hydrodistillation using Clevenger’s apparatus (Clevenger, 1928). A clear light yellow green coloured oily layer was separated and dried with anhydrous sodium sulphate. The physiochemical properties of the oil were determined by the technique described by Langenau (1948).

In vitro studies

The minimum bioactive concentrations (MBCs) of the oil were determined following the poisoned food technique (PFT) of Grover and Moore (1962) with slight modification (Shahi et al., 1999). The oil was dissolved in 2 ml acetone and then added in 100 ml pre-sterilized potato dextrose agar (PDA) medium (pH 5.6). In control sets, sterilized water (in place of the oil) and 2 ml acetone were used in the medium. Mycelial discs of 5 mm diameter, cut out from the periphery of 7-day old cultures of the test pathogens, were aseptically inoculated upside down on the agar surface of the medium. Inoculated petri plates were incubated at 27 ± 1◦C and the observations were recorded on seventh day. Percentage of mycelial growth inhibition (MGI) was calculated as follows:

MGI (%) = (dc–dt) × 100 / dc where, dc = mycelial growth diameter in control sets, dt = mycelial growth diameter in treatment sets.

The nature of antifungal activity [fungistatic (temporary inhibition) / fungicidal (permanent inhibition)] of the oil was determined by the method of Garber and Houston (1959). The inhibited fungal discs (at minimum bioactive concentrations) were reinoculated upside down on plain PDA medium in petri plate. Observations were recorded on 7th day of incubation at 27 ± 1 °C. Fungal growth on 7th day indicated fungistatic action of the oil, while absence of growth indicated fungicidal action of the oil.

The effect of inoculum potentiality on bioactivity of the oil (0.3 /xl ml-1) was determined by the method of Shahi et al. (1999). Mycelial disc of 5 mm in diameter of seven day old culture were inoculated in culture tube containing 0.3 /xl ml-1 oil in liquid medium (agar free medium). In controls, sterile water was used in place of oil and run simultaneously. The number of mycelial discs in the treatment as well as control sets were increased progressively up to 30 in multiples of five. Observations were recorded after 7th day of incubation. Absence of mycelial growth in treatment sets on the seventh day exhibited the oil’s potential against heavy doses of inoculum.

Effect of temperature and expiry of toxicity during storage of the oil was evaluated according to Shahi et al. (1999). Five lots of oil were kept in small vials, each containing 5 ml of oil; these were exposed to 40, 60, 80 and 100 ◦Cin an incubator for 60 minutes. Residual activity was assayed by the poisoned food technique of Grover and Moore (1962). Expiry of toxicity of the oil was determined by storing the oil at room temperature (30 ± 4 °C). Samples were withdrawn at intervals of 60 days up to 48 months, following the activity by poisoned food technique (Grover and Moore, 1962). All the experiments were repeated twice and each contained five replicates; the data presented are the mean values.

Phytotoxic investigation

Phytotoxicity tests of the oil were carried out at different concentration (ranging from 10 to 100 /xl ml-1) on fruit skin (epicarp) of Malus pumilo. Two sets of 50 apples were maintained, one for the treatments and another for the controls. Each sample was first washed with distilled water followed by 70 % ethyl alcohol and then allowed to dry. In treatment sets, 1 ml of the different concentrations of oil was sprayed to each sample separately. In controls, sterilized water was sprayed (in place of oil). The qualitative observations have been recorded at the interval of 24 hrs up to 3 weeks.

In vivo investigation of the oil in the form of fungicidal spray (SEB-2000)

The study was designed to see the activity of the oil in the form of fungicidal spray “SEB-2000” applied on fruit skin for the control of fruit rot of Malus pumilo by different methods. For in vivo study, both pre and post inoculation treatments (fungicidal spray) were given to the fruits.

In the pre inoculation treatment, two sets were prepared. In the treatment set, fruits were sprayed in known concentrations (10–50 /xl ml–1) of oil preparation in vehicle. In controls, in place of the spray solution, the fruits were sprayed with distilled water. Thereafter, the fruits were injured with the help of sterilized needle, and the fungal inoculum of Penicillium expansum, Botrytis cinerea, Phoma violacea (5 mm diameter mycelial disc of each fungus) was placed over the injured areas. Inoculated fruits, control as well as treatments, were incubated at 26 ± 1 ◦C and the observations were recorded on seventh day.

In post inoculation treatment, fruits were first injured with the help of sterilized needle and fungal inoculum of Penicillium expansum, Botrytis cinerea, Phoma violacea (5 mm diameter mycelial disc of each fungus) was placed over the injured areas and after 24 hrs of incubation.

They were sprayed in different concentration (10–50 /xl ml–1) of oil preparation in vehicle. Incontrols, in place of the spray solution, the fruits were sprayed with distilled water in vehicle. Inoculated fruits, control as well as treatments, were incu bated at 26 ± 1 ◦C and the observations were recorded on seventh day. The data are the average of 5 replicates, repeated twice. Percentages of inhibition (I) were calculated as follows: I (%) = (Ic-It) × 100 / Ic

where,

Ic = average infected area diameter in control set, It = average infected area diameter in treatment sets

Table 1