Chapte. er- 2. Screen ization

Transcription

1 Chapte er- 2 Screen ning for tifungal Properties and Dose Optimi ization 46

2 Chapter- 2 Introduction Different genera plants produce a wide range of secondary metabolites including essential oils. Essential oils are used in medicine, as preservative, fragrance and flavoring agent (Tripathi and Dubey, 24). Apart from routine uses, plants are not been fully explored for secondary metabolites, essential oils and volatile fractions (Mari and Guizzardi, 99). Essential oils are endowed with bioactive, antimicrobial, allelopathic and antioxidant properties which can be explored to control post-harvest pathogens. As essential oils can exhibit bioactivity in vapour form, they are suitable as fumigants to protect storage food commodities. Similarly plant extracts of wide variety of taxa, have been reported to contain antifungal secondary metabolites (Ramezani et al, 22; El-Mougy and Alhabeb, 29). In fact, natural products can certainly substitute harmful synthetic fungicides for plant disease control (Huang et al, 2). tifungal activity of secondary metabolites depends on the method and solvent used for extraction, its concentration and structure. With the advances in analytical instrumentation, bioassay techniques and recombinant DNA technology, the scope and use of these antimicrobial compounds for disease control has enhanced (Tripathi et al, 2). Microbial evaluation for as antifungal properties: There are a number of in vitro and in vivo reports on essential oils enhancing the shelf-life of fruits and vegetables by post-harvest treatment with essential oils to control fungal spoilage. The post-harvest decay can be prevented by spraying/dipping the fruit and vegetables in essential oils (arma and Tripathi, 26). Essential oils of many plants possess an excellent antimicrobial and antifungal activity. Essential oils can pass through cell membrane of the pathogen and enter the cytoplasm due to their lipophilic and hydrophobic tendencies. Thus they disrupt the membrane, allowing cell leakage, cytoplasmic evacuation and loss of proton motive force. Proton motive force is the proton gradient including the membrane potential (Christian and Goggi, 2). Fungal isolation: The pathogenic fungi can be isolated from the infected material on nutrient media in the laboratory and identified based on their microscopic characters including spores (Nduagu et al, 2). In the present study, fungi were isolated from the diseased fruits and vegetables on SDA medium. The isolates were used for fungal growth 47

3 inhibition assays using plant extracts. The generated data on effective antifungal properties of extracts can be used to control fungal spoilage after harvesting and during transportation to increase the self-life (Mari and Guizzardi, 99; Lee et al, 27). Primary screening: Various crude plant extracts were screened for antifungal activity at a selected concentration by Disc diffusion assay. The extracts were segregated based on the positive and negative antifungal response (Prasad et al, 2). The data was compiled as + (Positive) and (Negative). Secondary screening: The extracts with positive result for antifungal activity, were further subjected to the secondary screening and dose optimization. The extracts were tested against the test fungi to find the minimum effective concentration (Huang et al, 2). This value is defined called as MIC of extract (Minimum Inhibitory Concentration). The extract against particular fungi, is screened at various concentrations (Ogbebor and Adekunle, 2). This finding is used as optimum dose for the formulation. Dose optimization is necessary to avoid wastage of extract in the product. Formulation development and value addition: The best results in secondary screening were used for the development of formulation. Various combinations of extracts were evaluated considering their MIC obtained against selected fungal strains. Moreover, each formulation was subjected for dose optimization of fungicide for field applications. As various solvents with polar (viz water, methanol) to non-polar characteristics (chloroform, petroleum ether, hexane) were used for extraction, the antifungal metabolites get defined for its role. Similarly, the extracts having antifungal activity and well defined MIC, can be utilized for product development as fungicide least wastage, extracts and solvents. This option is for economical viability and thus adds value to the product. 4

4 Chapter- 2 Literature Review Disease development and mycotoxin production by phytopathogens: Aspergillus flavus is one of the major storage fungi of important cereals and produces aflatoxins such as aflatoxin B, B2, G, G2 (Paranagama et al, 23). Generally toxigenic Aspergillus flavus strains produce aflatoxins on a wide range of agricultural products under warm and humid climate (Pandey et al, 2). latoxins produced by fungi like Aspergillus parasiticus, Aspergillus nominus and Aspergillus flavus are biologically active secondary metabolites are known to be carcinogenic, teratogenic, hepatotoxic, immunosuppressive and inhibit several metabolic systems. Essential oils, the secondary metabolites of aromatic plants and characterized by a strong odour, are known to inhibit the aflatoxin production and the fungal growth. The antifungal efficacy of plant essential oils is thus attributable to the oil compositions and high content of active principles. The essential oils and plant extracts protect the plant cells from harmful impact of aflatoxins produced by Aspergillus parasiticus and Aspergillus flavus, by reducing DNA binding formation and by reacting with reactive oxygen species formed due to aflatoxins. Thus essential oils have a potential to be used as natural food preservatives to prevent food-borne diseases and food poisonings (Lokman, 2). latoxins are the toxic secondary metabolites and are extremely potent, naturally occurring hepatocarcinogens can be characterized in to four main types namely, B, B2, G and G2. They are found to produce by fungi like Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius and Aspergillus tamarii. Considerable attention is being focused on aflatoxin control in agricultural produce because of health risks involved in contaminated agricultural commodities consumption. Prevention of mold growth on agricultural produce offers the best means of aflatoxin control because decontamination of foods and feeds containing preformed toxin is a challenging proposition (Hajare et al, 2). Niaz and Dawar (29), have tested seed borne mycoflora of maize by using agar plate method and showed that about 7% of the maize samples had Aspergillus flavus and Aspergillus niger infections. Samples of maize kernels wound-inoculated with equivalent numbers of A. flavus and A. niger conidia showed substantial aflatoxin contamination. However, more aflatoxin was produced in kernels wound-inoculated only with A. flavus (Wicklow et al, 97). The most common species encountered from stored cotton seeds in Egypt and isolated on 2% cellulose-czapek's agar using the dilution and the seed plate methods were, A. niger, A. flavus, A. terreus, A. fumigatus, A. zonatus, Mucor racemosus and Rhizopus stolonifer. Screening of these fungal species isolated, for aflatoxin production by the fluorescence 49

5 technique revealed that, Aspergillus flavus, A. oryzae and A. fumigatus were found positive for aflatoxin production (El-Naghy et al, 99). Mycotoxins secreted from fungi contaminate products of vegetal origin. Therefore the study to evaluate the occurrence of toxigenic mycoflora and the extent of fungal and mycotoxin contamination in herbal plants was conducted by Alwakeel (29). The analyzed herbal and medicinal plants samples show the dominance of fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus ochraceus, Aspergillus citrinum and Penicillium spp. They are found to produce nephrotoxic and hepatoxic mycotoxins like Aspergillus ochraceus had aflatoxin B and ochratoxin A, whereas Aspergillus flavus had aflatoxin B and Aspergillus fumigatus had aflatoxin B and ochratoxin A. A natural fungicide was developed using the essential oil of lemongrass by Paranagama et al (23) against aflatoxigenic fungi, to protect stored rice. latoxigenic strain Aspergillus flavus isolated from stored rice and maintained on Potato dextrose agar (PDA). 2. mg/ml lemongrass oil in fumigant toxicity bioassay revealed complete sporulation inhibition of Aspergillus flavus and complete mycelial growth inhibition at 3. mg/ml concentration. Thus lemongrass oil can be successfully used as a safe and acceptable alternative to harmful synthetic fungicides to manage aflatoxin formation and fungal growth of Aspergillus flavus in stored rice. The pawpaw fruits in the Nigeria markets were screened for post-harvest fungal infestation. In the study, aflatoxins were detected from infected pawpaw fruits. Most common fungi found in rotten fruits were Rhizopus nigricans, Fusarium moniliforme, Aspergillus flavus and Aspergillus niger (Baiyewu et al, 27). Fungus like Aspvrgillus flavus and Aspergillus parasiticus produce toxic secondary metabolites- aflatoxins, which have been reported to contaminate a variety of foods and feeds (sari and rivastava, 99). Recent findings have demonstrated that higher plants contain fungitoxic substances. In one study, at 2 µg/ml concentration, treatments of maize with clove oil and cinnamon oil, was found to reduce toxin production compared to control maize lot. mg/kg treatment inhibited toxin production by clove oil 76% and cinnamon oil 7% (Sinha et al, 993). Decay and fruit rot are the important factors which limits the storage life of fruits and resulted in appreciable losses at whole sale, retail and consumer levels. Apricot fruit usually have short post-harvest life. Isolation of fungi from rotted fruits in Egypt yielded three genera of fungi Alternaria, Aspergillus and Rhizopus. Aspergillus spp. were more frequently from the rotted fruits.2% followed by Rhizopus spp. 23.% and Alternaria spp..26%. Aspergillus niger 4.3% was higher frequently when compared with Aspergillus parasiticus.26%. alysis of rotted apricot fruits indicated that Aspergillus parasiticus reduced significantly fresh weight, fruit quality and all chemical contents. Two isolates of Aspergillus parasiticus were found to produce aflatoxins i.e. B, B,

6 G and G. (Embaby et al, 27). The seed borne fungi of bottle gourd Lagenaria siceraria was studied by Sultana and Ghaffar (29) and showed that bottle gourd seed samples were having infection of Lasiodiplodia theobromae 33%, Fusarium semitectum 9%, Macrophomina phaseolina % and Fusarium oxysporum 66%. Fatima et al (29) have reported that, by and large post-harvest deterioration of fresh fruits, vegetables and other plant products occur between harvesting and consumption due to infection of various fungi viz., Alternaria alternata (infect apple, bell pepper, bitter gourd, bottle gourd, papaya, pear, round gourd, sponge gourd and tomato), Alternaria citri (infect sweet orange), Aspergilllus flavus (infect lemon, mango and tomato), Aspergillus niger (infect lemon, mango, round gourd and tomato), Aspergillus sp., (infect mango, pear and tomato), Cladosporium cladosporioides (infect carrot, cucumber and guava), Drechslera australiensis (infect mango, papaya and tomato), Fusarium solani (infect melon, papaya, egg plant, cucumber, sponge gourd and tomato), Fusarium sp., (infect bell pepper, bitter gourd, egg plant, okra, pear and turnip), Geotrichum candidum (infect apple, egg plant, carrot, cucumber, guava, melon, papaya, pumpkin, radish, sponge gourd and turnip), Penicillium sp., (infect grapes and pomegranate), Phytophthora capsici (infect taro, bottle gourd, eggplant, common bean, sponge gourd and tomato) and Rhizopus stolonifer (infect tomato and papaya) (Fatima et al, 29). Sclerotium hydrophilum, a sclerotium-forming fungus found on a many temperate and tropical aquatic macrophytes (Punter et al, 94). Rice sheath and stem diseases caused by Rhizoctonia and Sclerotium species are problems in rice with brownish to black lesions without distinct margins. The Sclerotium hydrophilum hyphal width was within a range of 6 to µm and binucleate condition between the septa. Also form globose, reddish brown to black coloured mature sclerotia of size mm (Aye et al, 29). Research status and advantages of botanical fungicides in agriculture: The plant world is a rich storehouse of natural chemicals with the total number of plant chemicals account more than 4,, in number including, biologically active plant Secondary Metabolites (PSMs), responsible to play a defensive role in the plants. These bio-active plant derived compounds can be formulated in to botanical fungicides to combat undesirable microorganisms and plant diseases management (Mohana and Raveesha, 27). Plants used in traditional medicines should be scientifically investigated as a potential source of novel antimicrobial compounds (Karbin et al, 29). Fungi predominantly reproduce by the production of asexual spores, which is a major source of fungal infestation and rapid proliferation (Murthy et al, 29).

7 tifungal property of many plants has been studied earlier by many researchers in order to control plant diseases in a bio-safe way. Mostly, fungi gain entrance through natural openings and wounds created during harvesting, transporting, handling and marketing. This type of storage fungi were isolated from the rotted sweet potato Ipomoea batatas tubers namely Aspergillus flavus, Aspergillus niger, Fusarium solani, Fusarium oxysporum and Rhizopus stolonifer by Amienyo and Ataga (27). They have found an excellent antifungal property of Zingiber officinale water extract against all the fungi. Klich (94), has observed that Aspergillus flavus introduced into cotton plants through natural openings before anthesis and thus infect the seed. Ray and Majumdar (976), have reported considerable antifungal activity against pathogenic fungi from Indian plant species like Carum copticum seeds, Lawsonia inermis leaves, Pinus longifolia stem, Plumbago zeylanica and Saussurea lappa roots, Alpinia officinarum rhizome and Tamarindus indica, Terminalia belerica and Emblica officinalis fruits. Hausner and Reid (999) found that, Sclerotium hydrophilum was shown to be auxoheterotrophic for thiamine, required for initiating sclerotium production and glucose stimulates maturation. Sclerotia are an important source of Aspergillus flavus inoculum in corn. In one study by Wicklow and Horn (92), sclerotia of Aspergillus flavus formed on autoclaved artificially inoculated kernels were incubated on sterile washed sand, autoclaved soil and non-sterile garden soil. Here the treatment with light exposure resulted in a reduction in the numbers of sclerotia produced, indicating the necessity of humidity for sclerotia formation. Hasnain et al (99) have found that, the airborne Alternaria can serve a potential allergic sensitizer in susceptible individuals with symptoms of bronchial asthma and allergic rhinitis. In Japan, a new disease of peach Prunus persica, caused by fungus Alternaria alternata was found to produce phytotoxins in culture fluid, which induced necrosis on injured peach leaves (Inoue and Nasu, 2). Fusarium solani was isolated from dried seedlings of cotton variety FH-9 by Muhammad et al (2) on Potato dextrose agar (PDA) medium. Scientists have developed new technology to tackle with resistant fungal strains. Fungi such as Alternaria alternata, Fusarium avenaceum, Acremonium strictum and Rhizopus oryzae which are harmful for food industry are effectively inactivated by photosensitization technology. Photosensitization interaction in the presence of oxygen induces radical-based cytotoxic events in the microorganism which lead to the growth inhibition (Luksiene et al, 2). Many testing methods of antimicrobial activity of extracts are quite lengthy and difficult to perform routinely. Thus afi (97) has suggested an easy and instant method for MIC (Minimum inhibitory concentration) value of test sample determination called paper diffusion method. It is a simple method of incorporating the antimicrobial drug in agar. 2

8 Chapter- 2 Materials and Methods Isolation of fungi: The infected material first washed and surface sterilized with.% HgCl 2 treatment for min. The treated material was rinsed (five times) with sterilized water. The infected material ( mm) was isolated with sterilized blade and inoculated on PDA media (Table ) slants under aseptic conditions (Dube, 99). The slants were incubated (27 ± C) for 4-6 days. The experiment was performed in triplicates. For identification, the fungal growth was observed under microscope. Small portion of the hyphal growth was taken under aseptic conditions, mounted on a slide and stained with Cotton blue reagent (Table 9). The prepared slides were observed under a microscope for characteristics of mycelium and sporulation. The recorded data was compared with the characters based on the fungal identification keys (Vashishta, 2). MTCC culture activation: The lyophilized fungal culture provided by MTCC (Microbial Type Culture Collection), Chandigarh, India must be activated before use. A small amount of lyophilized culture was taken from the stock vial and transferred to ml conical flask containing 2 ml of potato-dextrose broth (media without agar) under aseptic conditions in Laminar air flow hood (Labfine, India). The flasks were incubated at room temperature for culture establishment. ter growth activation of cultures were maintained on SDA media slants (Table 7) with regular sub-cultures (4 weeks). The slant cultures were used as an inoculum to maintain broth culture for further experiments. Table. Fungal cultures used as test-fungi and growth period Fungi Stock code Incubation period (Days) Spore count ( 6 ) Alternaria alternata GUB 6.72 Aspergillus flavus GUB Aspergillus niger GUB Fusarium oxysporum MTCC Fusarium oxysporum f.sp. laginariae GUB Fusarium solani ( Potato) GUB 3.33 Fusarium solani (Tomato) GUB6 3.3 Rhizopus oryzae MTCC Rhizoctonia solani GUB7 No sporulation Sarocladium oryzae MTCC Sclerotium hydroophillum MTCC

9 Fungal spore quantification: The spore count of the culture after specific incubation period was followed through Haemocytometer (Table ). The fungal broth culture was macerated under sterile conditions. aliquote (. ml) of homogenized fungal culture transferred with micropipette to petriplate (9 9 mm) containing SDA medium (4 mm thick). The inoculum was seeded uniformly by means of sterilized cotton swabs. Such seeded plates were used for Disc diffusion assay to determine out potential antifungal activity of selected plant extracts. Paper disc preparation for the assay: Sterilized whatman paper (no ) was punched to prepare discs (6.6 mm). These discs were autoclaved. The dried discs were impregnated with the known quantity of plant extracts. The solvent was allowed to evaporate completely thereby leaving known quantity of extract on disc. The impregnated discs were stored in sterilized glass vials and stored at low temperature until further use (Parekh and Chanda, 27). Determination of in vitro antifungal properties of extracts: a. Primary screening: Homogenized fungal broth culture (. ml) (Table ) was poured on solidified SDA medium (2 ml) in each petriplate using micropipette. The culture was uniformly streaked with sterilized cotton swab under aseptic conditions. For primary screening, the impregnated paper discs ( mg/disc) were placed on the medium test extract. The plates were incubated in upside down position for 72 hr at 2 ± C (Erturk, 26; Patel and Jasrai, 2). The experiment was performed in triplicates with solvent control. The antifungal effect was observed as formation of clear zone around the extract loaded paper disc after incubation, as ZI (Zone of inhibition). This indicates antifungal property in extract and marked as + sign. The extract without any ZI was marked as sign (Table 7). Kishore et al, 27 demonstrated that paper disc diffusion assay provides qualitative information on the efficacy of test compounds. This can be used routinely to evaluate antifungal activity of extracts. b. Secondary screening and dose optimization: The secondary screening includes the dose optimization for the extracts showing positive results in primary screening. The extract from lower to higher concentrations are screened with the same disc diffusion assay to find the minimum concentration or Minimal inhibitory concentration (MIC) which is effective to inhibit the fungal growth on seeded SDA medium plate. The paper discs loaded in extracts with different concentrations (,,,, and mg/disc) were placed on fungal seeded plates under aseptic conditions. 3 to 4 discs loaded with different concentrations of extract 4

10 were placed at an equal distance from each other in an anticlockwise manner. Each plate was replicated thrice. The plates were then incubated upside down for 72 hr and the ZI was recorded by tibiotic Zone Reader (Labfine, India) (Fig ) (Dawar et al, 2). In case the fungal growth is not in regular circle, the mean diameter (average of the longest and shortest diameter of the same colony) was calculated. Formulation and dose optimization preparation: The plant extracts and oils with lower MIC in the secondary screening were used in combination for formulation development. The formulation ingredients were mixed in an appropriate concentration. Total eleven formulations (Table 6) were designed based on the selected plant extracts in defined MIC. The designed formulations were tested through same disc diffusion assay using concentration range of,, 2, 3, 4, and 6 mg/disc, against the test fungi in triplicate. Bavistin- a synthetic fungicide as positive control was tested at, 2, 3, and mg/disc concentration. The ZI was recorded after 72 hr of incubation with tibiotic Zone Reader. The results of formulation dose optimization will help to define the efficacy of designed formulations and in value addition of fungicide product. Table 6. Composition of formulations based on secondary screening Ingredients Formulation (gm) Nature of Extract Cinnamomum tamala HX Cinnamomum zeylanicum HX Cymbopogon citrates Oil Elettaria cardamomum HX Eucalyptus globules Oil Gaultheria procumbens Oil Illicium verum HX Piper betel ME Syzygium aromaticum Oil Trachyspermum ammi HX

11 Chapter- 2 Results and Discussion Primary antifungal screening: The primary screening for antifungal activity using disc diffusion assay on SDA media (Table 7) revealed excellent results. The screening clearly and demonstrated the effectiveness of twenty three extracts and four essential oils as potential antifungal agents. The obtained data signifies the use of selected plants as herbal and safe bio-protectant. Additionally, antifungal reports on Cassia fistula, Piper betel, Sphaeranthus indicus, ethum sowa, Cymbopogon caesius, Illicium verum extracts and Gaultheria procumbens oil have not been published earlier. Present studies report the antifungal potential of the selected plants. A broad spectrum antifungal activity of Piper betel (CH extract), Cinnamomum tamala, Cinnamomum zeylanicum, Trachyspermum ammi (HX extracts) and Gaultheria procumbens and Syzygium aromaticum (oil) were found to inhibit all the eleven fungal strains used in the study. While Piper betel (ME extract), Citrus sinensis, Illicium verum (HX extracts) and Cymbopogon citrates (oil) successfully inhibited ten fungi. Such broad spectrum effect was also observed with Elettaria cardamomum (HX extract) and Eucalyptus globules (oil) causing inhibition of eight test-fungi. This proved the broad-spectrum antifungal effect of the selected plants for the control of fungal diseases. This clearly signifies the role of these plants as potent natural antifungal agent and can be further utilized to develop effective herbal formulation. Overall, primary screening experiment demonstrated, plant from Utility plants category, 2 plants from Medicinal plants, Aromatic herbs/spices and 4 plants from Essential oils category as effective antifungal agent at mg/disc concentration. In primary screening, 27 extracts were found to possess antifungal potential out of total extracts/oils (Table 7). No visible growth inhibition of fungi was observed for other selected plants at any of the studied concentration. 6

12 Table 7. Primary screening for antifungal potential of extracts (mg/disc) Plant category Utility plants Medicinal plants Aromatic herbs/ spices Essential oils Plants Ex Test fungi Fo Fl Fp Ft Rs CH Sphaeranthus indicus ME PE Cassia fistula CH PE CH Piper betel ME PE Curcuma longa PE ethum sowa HX Cinnamomum tamala HX Cinnamomum zeylanicum HX Citrus sinensis HX Cuminum cyminum HX Cymbopogon caesius HX Elettaria cardamomum HX Foeniculum vulgare HX Illicium verum HX Mentha piperita HX Myristica fragrans HX Ocimum sanctum HX Santalum album HX Trachyspermum ammi HX Cymbopogon citrates Oil Eucalyptus globules Oil Gaultheria procumbens Oil Syzygium aromaticum Oil [Note: (+) = indicates antifungal activity, ( ) = indicates no antifungal activity, = Alternaria alternata, = Aspergillus flavus, = A. niger, Fo = Fusarium oxysporum, Fl = F. oxysporum f.sp. laginariae, Fp = F. solani (Potato), Ft = F. solani (Tomato), Rs = Rhizoctonia solani, = Rhizopus oryzae, = Sarocladium oryzae, = Sclerotium hydrophilum, Ex =extract, ME =methanol extract, CH =chloroform extract, PE =petroleum-ether extract and HX= hexane extract] Many authors have reported potential antifungal activity from broad range of plants and different solvent extracts, various in vitro screening methods/techniques and artificial nutrient media. A remarkable antifungal activity was reported from Azadirachta indica plant extracts. Azadirachta indica seeds n-hexane extract at.% concentration inhibited seed borne fungi, Aspergillus niger, Aspergillus flavus, Fusarium oxysporum and Alternaria alternata in agar diffusion plate method on PDA media (Sitara et al, 2). Similarly, neem oil at.2% concentration inhibited Aspergillus niger (Niaz et al, 2). Study by Mondal et al (29) demonstrated that, ethanol and water extract of neem leaves inhibited seed 7

13 borne saprophytic fungi Aspergillus. Ethanol and ethyl acetate leaf extracts of neem leaves at 2% concentration inhibited Fusarium oxysporum using poison food technique (Hassanein et al, 2). Cold aqueous extracts of Azadirachta indica at 7% concentration inhibited Fusarium oxysporium mycelium on PDA medium after 7 days of incubation at room temperature (2 ± 3 C) (Taiga et al, 2). Mukhtar (27) found that, Azadirachta indica aqueous extracts at % concentration inhibited Fusarium oxysporum f. sp. ciceri and reduced fungal colony growth. Similarly, Azadirachta indica fresh leaf aqueous extract inhibited Fusarium oxysporum f.sp. cumini with 62.2% control of radial growth in the poisoned food technique (arma and Trivedi, 22). Siva et al (2) found that, water, ethanol and acetone extracts of neem at % concentration showed 9% control of radial growth of Fusarium oxysporum f. sp. melongenae. Moreover, Azadirachta indica extract at 2% concentration inhibited Fusarium solani f. sp. melongenae (Joseph et al, 2). Amadioha (2) and Mondal et al (29) demonstrated that, Azadirachta indica leaf (ethanol and water extract) inhibited Rhizopus oryzae (- % inhibition) in poison food technique on PDA medium. However, in the present study, the WT, ME, CH and PE extracts of Azadirachta were found to be ineffective up to mg/disc concentration in disc diffusion assay. The plant secondary metabolites can combat pathogens by varying mode of action. Lowsonia inermis can inhibit catalase production by interference with the metabolism leading to cell death (Khan and Nasreen, 2). It was demonstrated that, Lowsonia inermis and Datura stramonium methanolic leaf extract (%) inhibited (6- %) Alternaria alternata, Fusarium oxysporum and Rhizoctonia solani in food-poisoned technique on PDA medium. Similarly, fresh leaf extracts (aqueous) of Datura stramonium, Calotropis procera and Lawsonia inermis inhibited Fusarium oxysporum f.sp. cumini in the poisoned food technique (arma and Trivedi, 22). In Datura stramonium methanolic extracts of root and flower exhibited inhibition of Rhizoctonia solani with 2 and 6 mm inhibition, respectively due to presence of atropine alkaloid (Iranbakhsh et al, 2). In another study with Acacia nilotica, Datura stramonium, Eucalyptus globules, Lawsonia inermis, Polyalthia longifolia aqueous, methanol, ethanol, chloroform, benzene and petroleum ether extracts (2%) showed more than % inhibition of Aspergillus flavus and Aspergillus niger by poisoned food technique (Satish et al, 27).

14 According to Boughalleb et al (2), the antifungal activity of Lantana camara is due to the presence of terpenes such as sabinene, caryophyllene, cineole, bicyclogemacrene, humilene and free flavonoids in essential oil. Lantana camara leaf oil inhibited Alternaria solani, Fusarium solani f. sp. cucurbitae, Fusarium oxysporum f. sp. niveum and Rhizoctonia solani at mg/l concentration of oil incorporated in PDA medium. Similarly, arma and Kumar (29) showed that Lantana camara flowers and other plant parts % ethanol extracts at mg/ml concentration inhibited Fusarium oxysporum spore germination on PDA medium. However in the present study, Datura, Calotropis, Lawsonia and Lantana WT, ME, CH and PE extracts did not show any significant antifungal activity against Fusarium. Ethanol and chloroform extracts of Calotropis procera leaves inhibited Aspergillus flavus and Aspergillus niger in agar well diffusion and paper disc methods (Kareem et al, 2). Methanolic extract of Acacia nilotica bark ( µg/ml) inhibited Aspergillus flavus by disc diffusion assay (Mahesh and Satish, 2). Satish et al (29) and Satish et al (2) demonstrated that Polyalthia longifolia aqueous, petroleum ether, benzene, chloroform, methanol and ethanol extracts (- %) inhibited Fusarium equiseti, F. graminearum, F. lateritium, F. moniliforme, F. oxysporum, F. proliferatum, F. semitectum, and F. solani, Aspergillus candidus, A. columnaris, A. flavipes, A. flavus, A. fumigatus, A. niger, A. ochraceus, A. tamari, A. terreus and A. versicolor, Penicillium chrysogenum, P. griseofulvum and P. oxalicum, Drechslera halodes and D. tetramera and Alternaria alternata by poisoned food technique. Chanda and Nair (2) reported that Polyalthia longifolia leaves,4-dioxan extract (2μl extract/disc) inhibited Aspergillus niger (ATCC 627) in agar disc diffusion. In the present study, Acacia nilotica, Azadirachta indica, Calotropis procera, Datura stramonium, Lantana camara, Lawsonia inermis and Pongamia longifolia WT, ME, CH and PE extract, Acacia nilotica WT and ME extracts were ineffective to control selected fungal strains in paper disc diffusion assay. 9

15 Secondary antifungal screening: Based on the results obtained with primary screening, the plant extracts were subjected to secondary screening for dose optimization. The fungitoxic spectrum or the MIC value (Minimum Inhibitory Concentration) of the extracts/oils against specific test fungi was determined in terms of ZI (zone of inhibition). The extracts were tested in the selected range ( to mg/disc) of concentration in the paper disc diffusion assay. The assay was carried out on SDA medium (Table 7) with three replicates for each extract. ZI (clear zone showing absence of fungal growth) was recorded as the diameter (mm) of complete growth inhibition. Inhibition at low MIC value indicates very effective potential. Therefore prevention of fungal growth at, and mg/disc concentration is highly important for fungicide formulation with excellent outcome. In the present studies, Gaultheria procumbens oil was found to inhibit all eleven fungi at MIC value of mg/disc; Cinnamomum tamala (HX extract) at MIC - mg/disc, followed by Illicium verum (HX extract) with inhibition of seven fungi at mg/disc concentration. Cinnamomum zeylanicum (HX extract) inhibited six fungi namely, Alternaria alternata, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, Sarocladium oryzae, Sclerotium hydrophilum at lowest MIC ( mg/disc), followed by Syzygium aromaticum (oil) with inhibition of five fungi Aspergillus niger, Fusarium oxysporum, Fusarium solani (Potato), Rhizopus oryzae, Sarocladium oryzae inhibition, Piper betel (CH and ME extracts) with inhibition of Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, Rhizoctonia solani and Aspergillus niger, Fusarium oxysporum, Fusarium solani (Potato), Sarocladium oryzae respectively (Table ). Simultaneously, a narrow spectrum of inhibitory activity was demonstrated by Piper betel (PE extract) along with Foeniculum vulgare, Myristica fragrans and Santalum album (HX extracts) for a single fungi. Sphaeranthus indicus (PE extract), ethum sowa and Ocimum sanctum (HX extracts) were found to inhibit two fungi. intermediate activity was observed with Sphaeranthus indicus (CH and ME extracts), Cassia fistula and Curcuma longa (PE extracts), Cymbopogon caesius and Mentha piperita (HX extracts) for three fungi. In the present study, all four essential oils revealed excellent and broad-spectrum antifungal activity against the selected pathogens. The antifungal activity was effective at lowest concentration. Lower the MIC value, indicates the better antifungal potency. The lowest MIC value ( mg/disc) was recorded with Cymbopogon citrates, Gaultheria procumbens and Syzygium aromaticum oils. Syzygium aromaticum (oil) demonstrated inhibition of maximum fungi. The inhibition of fungi can be attributed to the complex mixture of secondary metabolites, volatile compounds and as phenylpropanes, terpenoids 6

16 and their oxygenated derivatives. Cymbopogon citrates (oil) in the present study exhibited highest ZI against Fusarium oxysporum f.sp. laginariae followed by Fusarium oxysporum. Cymbopogon (oil) could inhibit all fungi except Rhizopus oryzae at the tested concentrations. Table. MIC value overview obtained with plant extracts Plant category Plants Ex MIC Utility plants Medicinal plants Aromatic herbs/ spices Aromatic oils Sphaeranthus indicus CH Fo, ME Fo - -, - - PE Fo, Cassia fistula CH - -, Fo, - - PE Fo, CH,, Fo, Rs -,, Fl, Fp, Ft - Piper betel ME, Fo, Fp,, Rs, Fl, Ft, PE ethum sowa HX Cinnamomum tamala HX, Fo,,, Ft, Fp Fl, Rs - Cinnamomum HX,,, Fp, Ft Fl, Rs, zeylanicum Fo,, Citrus sinensis HX,,, - Fo, Fl, Ft, - - Fp, Cuminum cyminum HX - - Fp, Ft Curcuma longa PE Cymbopogon caesius HX - - Elettaria cardamomum HX,, Fl Ft, - Fo Foeniculum vulgare HX Illicium verum HX Fo -,,, Fl, Fp, - - Ft, Rs, Mentha piperita HX - - -, - Myristica fragrans HX Ocimum sanctum HX Santalum album HX Trachyspermum ammi HX -, Fo,, Fp, Ft,, Fl, - - Rs, Cymbopogon citrates Oil Fo, Fl,, Fp, Ft, Rs, - - Eucalyptus globules Oil - Fo,, Rs, Fp Ft Gaultheria procumbens Oil - - -,,, Fo, - - Fl, Fp, Ft, Rs,,, Syzygium aromaticum Oil, Fo, Fp,,, Fl, Rs, - - -, Ft [Note: = Alternaria alternata, = Aspergillus flavus, = A. niger, Fo = Fusarium oxysporum, Fl = F. oxysporum f.sp. laginariae, Fp = F. solani (Potato), Ft = F. solani (Tomato), Rs = Rhizoctonia solani, = Rhizopus oryzae, = Sarocladium oryzae, = Sclerotium hydroophilum] 6

17 The lowest MIC ( mg/disc) was recorded against Fusarium oxysporum followed by Aspergillus niger, Fusarium oxysporum f sp. laginariae and Sarocladium oryzae at mg /disc concentration. Eucalyptus globules oil showed highest ZI against Sarocladium oryzae followed by Fusarium oxysporum (Table ). The oil was ineffective to control Alternaria alternata, Fusarium oxysporum f sp. laginariae and Rhizopus oryzae at the tested concentrations. Sarocladium oryzae was inhibited at mg/disc while other fungi were found to get inhibited in the range of - mg/disc concentration of plant extracts. In contrast, Gaultheria procumbens oil successfully inhibited all the test-fungus in the study, with maximum ZI exhibited against Rhizopus oryzae followed by Alternaria alternata. In addition, the assay revealed that Gaultheria procumbens restricted the growth of all the testfungi at mg/disc concentration. Syzygium aromaticum (oil) in the present study demonstrated largest ZI against Alternaria alternata followed by Sarocladium oryzae, Fusarium solani (Potato) and Fusarium oxysporum f.sp. laginariae. In the study, fungus Sarocladium oryzae was found most susceptible, as it was found to get inhibited by many extracts, while Rhizopus oryzae was found most resistant fungal strain. Conversely, this is the first report on the control of Sarocladium oryzae and Sclerotium hydrophilum and Rhizopus oryzae with selected plants in the present study. In fact, very little work has been conducted on botanical controls for these highly destructive fungal strains. As these fungi damage the important crops and affect the present yield, the findings will contribute to control the fungi. In this context, present work was carried out with utility, medicinal and aromatic plants. The dose optimization of extracts through secondary screening helps to find the effective dose of extract to prevent fungal growth. The CH extract of Cassia fistula in the present study, exhibited largest ZI (2.42 ±.3 mm) against fungi Sarocladium oryzae at mg/disc concentration. The extract inhibited Aspergillus niger (6. ±. mm ZI) at mg/disc MIC. While, other fungi were found to get inhibit at higher MIC value (Fig 6, ). Cassia fistula (PE extract) exhibited largest ZI (.43 ±.24 mm) against Fusarium oxysporum at mg/disc concentration (Fig 6, 9). This extract was found effective against fungi at mg/disc MIC. Sphaeranthus indicus (CH extract) demonstrated inhibition of Fusarium oxysporum and Sarocladium oryzae at lowest MIC value ( mg/disc) with ZI being 4.92 ±.3 and 9.2 ±.2 mm respectively. Sclerotium hydrophilum was successfully inhibited (6.64 ±.4 mm ZI) at mg/disc MIC. A largest zone was documented for Sphaeranthus indicus (CH extract) against Fusarium oxysporum (23.7 ±. mm) and Sarocladium oryzae (22.2 ±.3 mm) at mg/disc concentration (Fig 7, 6). 62

18 2 Cassia fistula CH extract mg/disc Fo Fl Fp Fungi Ft Rss Fo Cassia fistula PEE extract Fl Fp Ft Fungi Rs mg/disc Fo Curcuma longaa PE extract Fl Fp Fungi Ft Rs mg/disc Fo Piper betel CH extract Fl Fp Fungi Ft Rs (mg/disc) Fig 6. ZI graphs of plantt extracts (CH, PE) 63

19 Sphaeranthus indicus (ME extract) at the lowest MIC ( mg/disc), found to prevent the growth of fungi Fusarium oxysporum (7. ± mm ZI). Moreover, the extract at mg/disc concentration, exhibited largest ZI (3. ±.9 mm) against Fusarium oxysporum (Fig 7, ). Fusarium oxysporum and Sarocladium oryzae were also found to get inhibited through Sphaeranthus indicus (PE extract) at MIC of mg/disc with 3. ± and.7 ±.2 mm respective ZI. Besides, Sphaeranthus indicus (PE extract) in the study, demonstrated largest ZI against Sarocladium oryzae (23.67 ±.29 mm) followed by Fusarium oxysporum (23.63 ±. mm) at MIC mg/disc (Fig 7, 7). In the present study, Curcuma longa (PE extract) had effectively prevented the growth of Sarocladium oryzae at mg/disc and Aspergillus flavus at mg/disc MIC with respective ZI 6.7 ±.4 and 6.79 ±.3 mm. the extract showed 7. ± mm ZI against Rhizopus oryzae at mg/disc concentration. The Curcuma longa (PE extract) exhibited largest ZI (9.7 ±.4 mm) against Rhizopus oryzae at mg/disc concentration (Fig 6, 4). Lee et al (23) in their study had demonstrated inhibition of Rhizoctonia solani in a whole plant bioassay using Curcuma longa rhizome methanol extract (2, ppm concentration). In the present study, fungi Rhizoctonia solani however doesn t showed inhibition through Curcuma longa at tested concentration range. Cho et al (26) had also reported antifungal potency of Curcuma longa rhizome methanol extract and purified curcuminoids (demethoxycurcumin, curcumin and bisdemethoxycurcumin) against Magnaporthe grisea and Phytophthora infestans. While Curcuma longa hexane and ethyl acetate extract ( mg/l) inhibited Erysiphe graminis, Phytophthora infestans and Rhizoctonia solani (Kim et al, 23). Piper betel (CH extract) in the present study had exhibited biggest ZI against fungi Sarocladium oryzae (34.9 ±.46 mm) followed by Alternaria alternata (27.2 ±.34 mm) at mg/disc concentration. At mg/disc MIC value, Piper betel (CH extract) found to restrict the growth of Aspergillus flavus, Aspergillus niger, Fusarium oxysporum and Rhizoctonia solani with their respective ZI 7 ±; 6.9 ±.;.43 ±.27 and 6.7 ±.6 mm. The Piper betel (CH extract) had also demonstrated inhibition of fungi Sarocladium oryzae and Sclerotium hydrophilum at mg/disc MIC with 2. ±.46 and 6.63 ±.4 mm respective ZI in the study (Fig 6, 2). Piper betel ethanol extract is been reported by Begum et al (27) to inhibit Alternaria alternata. In the present study, Piper betel CH and ME both extracts were found to effectively inhibit Alternaria alternata respectively at mg/disc concentration (ZI 9. ±.39 mm) and at mg/disc concentration (4. ±.27 mm ZI). 64

20 Fo Piper betel ME extract Fl Fp Fungi Ft Rs mg/disc) Sphaeranthus indicuss ME extract Fo Fl Fp Ft Rs (mg/disc) Fungi Sphaeranthus indicus CH extract mg/ /disc Fo Fl Fp Fungii Ft Rs 3 2 Sphaeranthus indicuss PE extract mg/disc 2 Fo Fl Fp Fungi Ft Rs Fig 7. ZI graphs of plant extracts (CH, ME, PE) 6

21 In the study, Piper betel (ME extract) found to inhibit ten fungi at lower MIC value, indicating its great antifungal potency. At the lowest tested MIC value ( mg/disc), the Piper betel (ME extract) prevented the growth of Aspergillus niger, Fusarium oxysporum, Fusarium solani (Potato) and Sarocladium oryzae with their respective ZI 7.42 ±.;.2 ±.3; 7.7 ±. and 4.37 ±4 mm. Alternaria alternata, Aspergillus flavus and Rhizoctonia solani get inhibited at mg/disc MIC and Fusarium oxysporum f sp. laginariae, Fusarium solani (Tomato) and Sclerotium hydrophilum at mg/disc MIC and their respective ZI documented in order was 4. ±.27; 7.33 ±.;.42 ±.4; 6.72 ±.2; 6.92 ±. and 6.9 ±. mm. Piper betel (ME extract) inhibited and demonstrated largest ZI against Sarocladium oryzae (3. ±.22 mm) followed by fungi Fusarium oxysporum (29.3 ±.2 mm) and Alternaria alternata (2.3 ±.33 mm) (Fig 7, 3). ethun sowa (HX extract) was found effective against Rhizopus oryzae and Sarocladium oryzae at higher MIC - mg/disc. In the present study, largest ZI for ethun sowa (HX extract) was documented against fungi Rhizopus oryzae.32 ±. mm (Fig, 2). Cinnamomum tamala had excellently controlled the growth of seven fungi at lower MIC value. In the study, Cinnamomum tamala (HX extract) exhibited inhibition of Aspergillus niger, Fusarium oxysporum and Sarocladium oryzae at the lowest tested MIC ( mg/disc) with their respective ZI recorded in order 6.69 ±.2;.33 ±.6 and 7.2 ±. mm (Fig, 2). While the fungi Alternaria alternata, Fusarium solani (Tomato) and Sclerotium hydrophilum were had documented the MIC value of mg/disc with.97 ±.7; 6.63 ±. and 6.62 ±.6 mm respective ZI. Fusarium solani (Potato) get inhibited (4.9 ±.37 mm ZI) at mg/disc concentration of Cinnamomum tamala (HX extract). Study by Dawar et al (2) demonstrated that, in paper disc method, Cinnamomum tamala, Coriandrum sativum, Cuminum cyminum, Curcuma longa, Foeniculum vulgare and Myristica fragrans (% aqueous extract concentration) inhibited Fusarium solani and Rhizoctonia solani after - 6 days of incubation on PDA containing penicillin (2, unit/l) and streptomycin (2 mg/l). In present study also, Cinnamomum tamala (HX extract) had successfully inhibited Fusarium solani and Rhizoctonia solani at MIC and mg/disc respectively. However, Cuminum, Coriandrum, Foeniculum, Myristica and Curcuma longa extracts were found ineffective to control Rhizoctonia solani in paper disc method on SDA media up to mg/disc concentration. The Cinnamomum tamala (HX extract) had exhibited largest ZI against fungi Alternaria alternata (32.2 ±.29 mm) followed by Sarocladium oryzae (3.2 ± mm) and Fusarium solani (Potato) (24.7 ±.29 mm) in the present study. 66

22 6 4 ethun sowa HX extract mg/disc 2 Fo Fl Fp Fungii Ft Rss Cinnamomum tamala HX extract Fo Fl Fp Fungi Ft Rs mg/disc Cinnamomum zeylanicum HX extract Fo Fl Fp Fungi Ft Rss mg/disc Fo Citrus sinensis HX extract Fl Fp Ft Rs Fungii mg/disc Fig. ZI graphs of plant extracts (HX) 67

23 Cinnamomum zeylanicum (HX extract) found to effectively inhibit all eleven fungal strains used in the study. At mg/disc MIC, Cinnamomum zeylanicum (HX extract) found to prevent the growth of six fungi namely Alternaria alternata (6. ±.4), Aspergillus flavus (7.92 ±. mm), Aspergillus niger (.32 ±.6 mm) Fusarium oxysporum (.47 ±.3 mm), Sarocladium oryzae (9. ±.49 mm) and Sclerotium hydrophilum (6.7 ±.3 mm ZI). While inhibition of Fusarium solani (Potato) (.4 ±.23 mm) and Fusarium solani (Tomato) (6.66 ±.2 mm) was recorded at mg/disc concentration of Cinnamomum zeylanicum (HX extract). Whereas rest three fungi Fusarium oxysporum f.sp. laginariae (.73 ±.4 mm), Rhizopus oryzae (7.7 ±.9 mm) and Rhizoctonia solani (9.67 ±.3mm ZI) were found to get inhibit at mg/disc concentration of Cinnamomum zeylanicum (HX extract) in the study (Fig, 22). Cinnamomum zeylanicum (HX extract) in the present study had demonstrated largest ZI against Alternaria alternata (32.77 ±.29 mm) followed by, Rhizoctonia solani (2.72 ±.3 mm) and Fusarium solani (Tomato) (2.3 ±4 mm). According to Fawzi et al (29), the antifungal activity of Cinnamomum zeylanicum and Syzygium aromaticum is due to its active principle cinnamaldehyde and eugenol respectively. Cinnamomum zeylanicum cold distilled water extracts (2% concentration) inhibited 46.% growth of Alternaria alternata f. sp. lycopersici and 4.4% growth of Fusarium oxysporum f. sp. lycopersici. In disc diffusion method Xing et al (2) showed that, after 72 hr of incubation, cinnamon oil inhibited Aspergillus flavus ( µl of inoculum/plate of 4 - conidia/ml) on PDA media at MIC value.6%. In another study by Kishore et al (27) showed that, cinnamon and clove oil (.% concentration) inhibited Alternaria alternata and Aspergillus flavus using paper disc agar diffusion assay. Cinnamomum zeylanicum leaf, bark oils and Syzygium aromaticum oil (.3-.% concentration) in liquid bioassay inhibited Colletotrichum musae, Lasiodiplodia theobromae and Fusarium proliferatum (Ranasinghe et al, 22). In the present study, Cinnamomum zeylanicum (HX extract) found most effective to inhibit Alternaria alternata and Aspergillus flavus at lowest MIC value ( mg/disc) using paper disc diffusion assay. Similar results were obtained with Syzygium aromaticum (oil) and inhibited Alternaria alternata and Aspergillus flavus at mg/disc MIC. A study by El-Baroty et al (2) shows that, Cinnamomum zeylanicum bark essential oil ( μg/ml concentration) inhibited Aspergillus niger, Fusarium oxysporum and Rhizoctonia solani ( 6 cfu/ml spore count) with % fungal growth inhibition in dry weight method on potato dextrose broth (PDB) medium. Thompson (96) in his study showed, cinnamon bark, cinnamon leaf and clove bud food-grade quality essential oils inhibited Aspergillus flavus. 6

24 Cuminum cyminum HX extract Fo Fl Fp Ft Rss mg/disc Fungi Cymbopogon caesius HX extract Fo Fl Fp Ft Rs Fungii mg/disc 3 2 Elettariaa cardamomum HX extract mg/disc 2 Fo Fl Fp Fungii Ft Rs Illicium verum HX extract Fo Fl Fp Ft Fungii Rs mg/disc Fig 9. ZI graphs of plant extracts (HX) 69