World Journal of Microbiology & Biotechnology 20: 317321, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Evaluation of some essential oils as botanical fungitoxicants in management of post-harvest rotting of citrus fruits
Pramila Tripathi1, N.K. Dubey1,*, R. Banerji2 and J.P.N. Chansouria3 1 Department of Botany, Banaras Hindu University, Varanasi 221005, India 2 Lipid Metabolism Laboratory NBRI, Lucknow, India 3 Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India *Author for correspondence: Tel.: +91-542-2307147, Fax:+91-542-368174, E-mail: nkdubey@banaras.ernet.in
Received 3 June 2003; accepted 26 November 2003

Keywords: Essential oils, fungitoxicity, Penicillium italicum, post-harvest diseases

Summary During screening of some essential oils against Penicillium italicum, the oils of Mentha arvensis, Ocimum canum and Zingiber ocinale were found to exhibit absolute fungitoxic activity against the test fungus. The oils were subsequently standardized through physico-chemical and fungitoxic properties. Practical applicability of the essential oils was observed in control of blue mould rot of oranges and lime fruits caused by P. italicum during storage. The Mentha oil-treated oranges and lime fruits showed enhancement of storage life of 6 and 8 days, respectively.The storage life of Ocimum oil-treated oranges and lime fruits was found to be enhanced by 6 days while in the case of Zingiber oil, it was 4 and 8 days enhancement of shelf life of oranges and lime fruits, respectively.

Introduction Post-harvest losses particularly due to fungal invasions are much more signicant for highly perishable fresh fruits and vegetables than for cereals and other eld crops. Because of their high moisture content, fresh fruits and vegetables are highly susceptible to attack by dierent pathogenic fungi. Maximum research eorts have been directed towards chemical control of postharvest diseases of fruits and a large number of synthetic chemicals are used. However, due to the development of new physiological races of pathogens, many of these synthetic chemicals are gradually becoming ineective (Delp 1980; Spotts & Cervantes 1986). Further, the use of synthetic chemicals to control post-harvest deterioration of food commodities is restricted, due to their possible carcinogenicity, teratogenicity, high and acute toxicity, long degradation periods, environmental pollution and their eects on human beings (Lingk 1991). In recent years, the study and use of chemicals from natural sources (particularly of plant resources) which are non-toxic and specic in their action are gaining attention as alternative chemical control measures. Higher plants contain a wide spectrum of secondary substances viz. phenols, avonoids, quinones, tannins, essential oils, alkaloids, saponins and sterols. Such plant chemicals may be exploited for their dierent biological properties (Wain 1977; Mahadevan 1982). Biologicals,

because of their natural origin, are biodegradable and mostly do not leave toxic residues or byproducts to contaminate the environment. Some phytochemicals of plant origin viz. azadirachtin, pyrethroids, carvone (trade name TALENT) have been formulated as botanical pesticides and are used successfully in integrated pest management programme as botanical pesticides. Unlike the prevalent fumigants, the problem of development of resistant strains of fungi and insects may be solved by the use of essential oils as fumigants in management of storage pests, because of synergism between dierent components of the oils. The essential oils produced by dierent plant species are in many cases biologically active and have antimicrobial, allelopathic, antioxidant and bioregulatory properties (Vaughn & Spencer 1991; Caccioni & Guizzardi 1994). The antimicrobial eects of essential oils or their constituents on plant pathogens have been quite extensively studied on articial media during in vitro trials (Bishop & Thornton 1997). There are, however, not many reports based on in vivo experiments comprising a complete host-pathogen system. It is not always possible to correlate the practical applicability of a substance as a fungicide based on in vitro trials only. Many volatiles probably aect the host as well as the pathogen. In vitro testing of a plant product as an antifungal agent alone will not be sucient to attract agrochemical rms for their formulation as botanical pesticides. The practical

318 applicability of the product (in vivo trials) is also required. With these facts in mind it was thought desirable to nd out the potency of some plant products (essential oils) in protection of oranges and lime fruits from post-harvest fungal deterioration caused by Penicillium italicum Whemer. Material and methods Isolation of essential oils Some locally available aromatic angiospermic taxa belonging to dierent families were selected randomly for the extraction of essential oils. Leaves of the plants were used for extraction of the essential oils. In the case of Zingiber ocinale rhizomes and Citrus reticulata peels were used for essential oil extraction. 250 g of fresh parts of the plants were cut into small pieces and then thoroughly washed with sterilized water. The essential oils were isolated by hydrodistillation through Clevengers apparatus. Screening of essential oils against P. italicum Fungitoxic activity of the extracted oils was tested by the poisoned food technique of Perrucci et al. (1994) using potato-dextrose-agar (PDA) medium against the test fungus P. italicum at 500 ppm (lg/l). The concentration of the essential oils were prepared by dissolving the requisite amounts in 0.5 ml of 0.1% Tween-80 and then mixing with 9.5 ml of potato-dextrose-agar (PDA) medium. The control sets were prepared similarly using equal amounts of sterilized water in place of the oil. The prepared plates were inoculated aseptically with assay discs of the test fungus and incubated for 6 days. The observations were recorded on the seventh day and the percentage mycelial inhibition was calculated by the following formula: Percent mycelial inhibition dc dt 100 dc

P. Tripathi et al. ments were carried out by the above mentioned poisoned food technique using graded concentrations of essential oils below 500 ppm. The nature of the toxicity (fungistatic/fungicidal) of the oils against the test fungus was determined following Thompson (1989). The inhibited fungal discs of the oil-treated sets were reinoculated into fresh medium and revival of their growth was observed. Fungitoxic properties of the essential oils of M. arvensis, O. canum and Z. ocinale The eect of increased inoculum density of the test fungus on fungitoxicity of the oils was studied following Moleyar & Pattisapu (1987) at their respective minimum inhibitory concentrations. The eect of storage and temperature on the fungitoxicity of the essential oil was evaluated according to Tripathi et al. (1978). The range of fungitoxicity of the oils was determined at their respective toxic and hypertoxic concentrations by the poisoned food technique. Comparison of the fungitoxicity of the essential oils with some prevalent synthetic fungicides The ecacy of the oils was compared with some fungicides, viz benzimidazole (Benomyl), diphenylamine, phenylmercuric acetate (Ceresan) and zinc dimethyl dithiocarbamate (Ziram) by the usual poisoned food technique. In vivo applicability of the oils of M. arvensis, O. canum and Z. ocinale in control of post harvest fungal decay of citrus fruits The oranges and limes were treated with the essential oils by the standard techniques followed by Chandra (1984) and Sharma & Yadav (1996) in order to nd out the ecacy of the oils against blue mould rot diseases caused by P. italicum. Mature healthy oranges and lime fruits of medium size were used for the experiment. The fresh oranges and lime fruits of control as well as of treatment sets were washed in running water and were surface sterilized with 0.1% sodium hypochlorite solution and were then washed with distilled water. The pathogenicity of the fungus was tested following Garcha et al. (1980). The fruits were inoculated by 1 ml of the standard spore suspension of P. italicum. For fruit inoculation spores from a 7 day old culture were suspended in sterile distilled water and 0.03% Tween80. Fruits were wounded on two sides to a depth of 1.5 mm by puncturing them with a pin. Each wound site was then inoculated with 40 ll of spore suspension (105 spores/ml) of P. italicum. The inoculated fruits were kept in desiccators (four fruits per desiccator). In treatment sets the requisite amount of M. arvensis, Z. ocinale and O. canum and oils were introduced separately in to the desiccator by soaking in a piece of cotton so as to give concentrations of 100, 200 and 500 ppm (v/v), respectively. The initiation of rotting of

where, dc mean colony diameter of control sets, dt mean colony diameter of treatment sets. Physico-chemical properties of essential oils of Mentha arvensis, Ocimum canum and Zingiber ocinale The oils were standardized through GLC and physicochemical properties viz. specic gravity, refractive index, acid number, saponication value, ester value, phenolic content and GLC following Chowdhury & Kapoor (2000). Minimum inhibitory concentration and Nature of toxicity of essential oils of M. arvensis, O. canum and Z. ocinale To nd out the minimum inhibitory concentration at which the oils showed absolute fungitoxicity, experi-

Essential oils as post-harvest fungitoxicants the fruits were observed. Six replicates were kept for treatment and control sets.

319 erate fungitoxicity (<100% but >50%). Poor fungitoxicity (<50%) was observed by the oils of Callistemon lanceolatus, Citrus reticulata (peel), Lippia alba and Nardostachys jatamanasi. The oils of Ocimum canum, Mentha arvensis and Zingiber ocinale were selected for further studies in the present investigation. The GLC of M. arvensis oil indicated it to be a mixture of four major and thirty minor components. The oil of O. canum was a mixture of 2 major and 15 minor components. The GLC of Zingiber oil showed it to be a mixture of 8 major and 21 minor components. The specic gravity of the Mentha oil, Ocimum oil and Zingiber oil was found to be 0.9150, 0.9670 and 0.8900, respectively. The phenolic contents were present in the Mentha and Zingiber oil while the phenolic content was not recorded in the Ocimum oil (Table 2).

Results It is evident from Table 1 that out of twenty essential oils tested against P. italicum, thirteen oils viz. Artemisia nilagirica, Caesulia axillaris, Chenopodium ambrosioides, Elettaria cordamomum, Eucalyptus citriodora, Eupatorium cannabinum, Mentha arvensis, Ocimum canum, Ocimum gratissimum, Pogostemon patchouli, Prunus persica, Salvia ocinalis, and Zingiber ocinale, showed absolute toxicity (100% mycelial growth inhibition) against the test fungus at 500 ppm. The essential oils of Aegle mormelos and Nepeta hindostana exhibited mod-

Table 1. Screening of essential oils of higher plants for fungitoxicity against Penicillium italicum Wehmer. Essential oils Family Percent inhibition of growth at 500 lg/l 76 80 100 100 37 100 48 100 100 100 32 100 23 70 100 100 100 100 100 100

Ageratum conyzoides (Linn.) (leaf) Aegle mormelos (Linn.) (leaf) Artemisia nilagirica (Clarke) Pamp. (leaf) Caesulia axillaris Roxb. (leaf) Callistemon lanceolatus DC. (leaf) Chenopodium ambrosioides (Linn.) (leaf) Citrus reticulata Blanco. (peel) Elettaria cordamomum Maton (leaf) Eucalyptus citriodora Hook. (leaf) Eupatorium cannabinum (Linn.) (leaf) Lippia alba Mill N. B.Br (leaf) Mentha arvensis (Linn.) (leaf) Nardotachys jatamansi DC. (leaf) Nepeta hindostana Roth Haines (leaf) Ocimum canum Sims (leaf) Ocimum gratissimum (Linn.) (leaf) Pogostemon patchouli Hook. (leaf) Prunus persica (L) Stackes (leaf) Salvia ocinalis (Linn) (leaf) Zingiber ocinale Rosc. (rhizome)

Asteraceae Rutaceae Asteraceae Asteraceae Myrtaceae Chenopodiaceae Rutaceae Zingiberaceae Myrtaceae Asteraceae Verbenaceae Lamiaceae Valeriaceae Lamiaceae Lamiaceae Lamiaceae Lamiaceae Rosaceae Lamiaceae Zingiberaceae

Table 2. Physico-chemical properties of essential oils of Mentha arvensis, Ocimum canum and Zingiber ocinale. Parameters Colour Odour Specic gravity Optical rotation Refractive index Solubility Acetone Absolute alcohol 90% alcohol Ethyl acetate Benzene Chloroform Hexane Methanol Acid number Saponication value Ester value Phenolic content Mentha arvensis oil Pale yellow Pungent 0.9150 ())25 1.4620 (at 24 C) Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 2.4 mg 37.475 mg 35.235 mg Present conc) conc) conc) conc) conc) conc) conc) conc) Ocimum canum oil Yellow green Pungent 0.967 ()7.60 1.4780 (at 24 C) Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Nil 25.45 mg 25.45 mg Nil conc) conc) conc) conc) conc) conc) conc) conc) Zingiber ocinale oil Yellow Aromatic pleasant 0.8900 ())5 1.4840 (at 24 C) Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 Soluble(1:1 4.48 mg 23.84 mg 19.36 mg Present conc) conc) conc) conc) conc) conc) conc) conc)

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Table 3. Eect of increased inoculum density of test fungus on the fungal toxicity of the oils. Number of fungal discs Approximate number of spores Growth of the test fungus Mentha oil Treatment 1 2 4 8 16 32 64 2358 47174 94348 188696 377392 754784 1509568 103 103 103 103 103 103 103 ) ) ) ) ) ) ) Control + + + + + + + Ocimum oil Treatment ) ) ) ) ) ) ) Control + + + + + + +

P. Tripathi et al.

Zingiber oil Treatment ) ) ) ) ) ) ) Control + + + + + + +

)indicates no growth of test fungus. +Indicates growth of test fungus.

The MIC of the essential oils of M. arvensis, O. canum and Z. ocinale were found to be 100, 500 and 200 ppm, respectively. It was found that all these essential oils were fungicidal at their respective MIC. It has been observed that the oils inhibited the fungal growth of the treatment sets containing even 64 discs of the test fungus indicating the potency of the essential oils to withstand high inoculum density of test fungus (Table 3). It was found that these oils have long shelf lives. The oil of M. arvensis remained active for 360 days while that of Ocimum canum and Zingiber ocinale could retain their toxicity up to 330 and 270 days, respectively. The oils remained fungitoxic in nature at dierent temperatures between 5 and 80 C showing the thermostable nature of their fungitoxicity. The MIC of synthetic fungicides viz. benzimidazole, diphenylamine, phenylmercuric acetate and zincdimethyl dithiocarbamate was found to be 3000, 1000, 1000 and 5000 ppm, respectively which was higher than the essential oils tested (Table 4). Thus, the oils have been found to be more ecacious than the synthetic fungicides. The oils were found to exhibit a broad fungitoxic spectrum. The Mentha and Ocimum oil inhibited all the 10 fungi at their respective toxic and hypertoxic concentrations. The Zingiber oil was found to be active only against three fungi viz. A. niger, F. oxysporium and P. aphanidermatum at toxic concentration while at hypertoxic

Table 5. Ecacy of the oils against blue mould rot orange and lime fruits. Initiation of rotting of fruits Orange Control Mentha oil Ocimum oil Zingiber oil 3 9 9 7 Lime 3 11 9 11 Enhancement of storage life (in days) Orange Lime

6 6 4

8 6 8

concentration it was active against four fungi viz A. niger, F. oxysporum, P. aphanidermatum and R. solani. The Mentha oil-treated oranges and lime fruits showed enhancement of storage life of 6 and 8 days, respectively. The storage life of Ocimum oil-treated oranges and lime fruits was found to be enhanced by 6 days. In case of Zingiber oil, there was 4 and 6 days enhancement of shelf life of oranges and lime fruits (Table 5). Thus the oils showed their antifungal ecacy in control of storage rotting of fruits during in vivo trials.

Discussion In the present piece of work an attempt has been made to standardize the essential oils of Mentha, Ocimum and Zingiber based on their fungitoxic properties and practical ecacy in controlling the rotting of some fruits as well as enhancing their market life. Such studies are lacking with most of the fungitoxic plant products, but are required before recommending them to agrochemical rms. As the quality of the essential oil varies in dierent sets of environment conditions, the Mentha, Ocimum and Zingiber oils were standardized for physico-chemical properties. The superiority of the oils over the active moieties of some synthetic fungicides further strengthens their exploitation as economical source of fungitoxicants. The oils as such may be exploited for their fungitoxic potency because of the synergistic activity of their dierent compounds. The specic compounds isolated from the oils may be non-fungitoxic

Table 4. Comparative ecacy of the oils with prevalent synthetic fungicides. Fungicides/oils Minimum inhibitory concentration against Penicillium italicum (ppm) 3000 1000 1000 5000

Benzimidazole (Benomyl) Diphenylamine Phenylmercuric acetate (Ceresan) Zincdimethyldithiocarbamate (Ziram) Oils Ocimum canum Mentha arvensis Zingiber ocinale

500 100 200

Essential oils as post-harvest fungitoxicants in nature. Dierent components of the oils as such may also check development of races of fungi during their application due to more than one sites of action. Fungi can easily develop resistant races against a single component due to its specic mode of action. Secondly, the exploitation of the essential oils as such would be more economical than a single component as fungitoxicant. The ecacy of the oils in controlling the rotting of the fruits during in vivo conditions clearly indicates their practical applicability as storage fungitoxicants of plant origin (botanical pesticides) for fruits and enhancing their market life for appreciable period. Acknowledgements Authors are grateful to the Head of the Department of Botany, Banaras Hindu University, Varanasi, India and CSIR, New Delhi for nancial assistance. References
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