Acta Botanica 1-2017 - za web.indd 72 ACTA BOT. CROAT. 76 (1), 2017 Acta Bot. Croat. 76 (1), 72–79, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0053 ISSN 0365-0588 eISSN 1847-8476 Chemical composition and antifungal potential of medicinal plants against seedborne mycofl ora of eggplant (Solanum melongena L.) Bushra Ashiq1, Sobia Chohan1*, Rashida Perveen1, Muhammad Abid1 and Mirza Abid Mehmood2 1 Department of Plant Pathology, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan. 2 Department of Plant Pathology, Mian Nawaz Sharif University of Agriculture, Multan, Pakistan Abstract – Antifungal activities of medicinal plants were observed against seedborne mycofl ora of eggplant (Solanum melongena). The effect of ethanolic leaf extracts of Mangifera indica, Mentha spicata, Citrus li- mon, Eucalyptus camaldulensis against four isolated fungal species including Fusarium oxysporum, Asper- gillus fl avus, Rhizopus stolonifer and Penicillium digitatum was evaluated at various concentrations, by using the poisoned food technique. The impact of the extracts on seed germination and growth of eggplant was as- sessed by seed treatment and growth in a greenhouse experiment. Total fl avonoids of E. camaldulensis were analyzed through spectrophotometer, using quercetin as a standard. Physico-chemical parameters were also determined. Antifungal activity showed that maximum inhibition percentage of P. digitatum (67.78%) and F. oxysporum (64.44%) was observed at the highest concentration (80%) of C. limon and E. camaldulensis ex- tracts, respectively, followed by M. spicata extract against A. fl avus (63.33%) and R. stolonifer (52.22%). Least inhibition percentage of F. oxysporum, P. digitatum, R. stolonifer and A. fl avus was 6.67, 7.78, 14.44 and 16.67%, respectively, at the lowest (20%) concentration of M. spicata. The greenhouse experiment showed variations in seedling germination and post-germination growth. E. camaldulensis extract showed an increase in percent germination (78.98%) over untreated control (62.83%), root and shoot length and fresh and dry weight of root and shoot with the consequent reduction in disease symptoms. Phytochemical analysis depict- ed the presence of alkaloids, fl avonoids, tannins, saponins in all extracts while steroids and glycosides were absent. A fair amount (10.38 mg QE g–1DF) of fl avonoid was present in leaf extract of E. camaldulensis. Physi- co-chemical analysis showed pH of 4.6, ash content of 0.41% and weight loss on drying of 8.14%. Keywords: medicinal plants, physico-chemical parameters, secondary metabolites, seedborne mycofl ora, So- lanum melongena * Corresponding author, e-mail:sobia_mustafa2006@hotmail.com Introduction Eggplant, also commonly known as brinjal or aubergine (Solanum melongena L.), is an important Solanaceous veg- etable crop of the sub-tropics and tropics and is cultivated for its fl eshy fruits (Pandey 2010). It was later domesticated in India and is grown widely in Pakistan, India, Bangla- desh, China and the Philippines. In Pakistan it is grown for local consumption with an annual production of 88148 tones over an area of 8673 hectares (GOP 2009). This im- portant summer crop is prone to numerous diseases and pests during various stages of crop growth in all seasons in most of the tropical zones (Ali et al. 2012). The severity in any serious disease depends on the season and the region in which the crop is grown (Dhamdhere et al. 1995). Seed- borne diseases are considered important because they bring about a signifi cant reduction in yield. Different microorgan- isms affect the quality of seed by reducing its vigor, ulti- mately weakening the plant at its initial growth stage caus- ing seed rot, pre- and post-germination death of seedlings (Ellis et al. 1975). Pathogenic fungi are the largest group of plant pathogens. Seed-borne diseases caused by fungi are relatively diffi cult to control. A primary inoculum can be avoided by using disease-free seed, choosing pathogen-free seedbed soil, treating infected seed and following a three year rotation. However, some pre-emptive measures in se- lection of seed such as color and texture can be helpful in avoiding future losses. Healthy seed is always recommend- ed for plantation, to save time and labor. However, if seed is not disease free, it can be treated with pesticides or with hot ANTIFUNGAL ACTIVITY OF MEDICINAL PLANTS ACTA BOT. CROAT. 76 (1), 2017 73 water at 50 °C (Panwar and Chand 1970). Al-Kassimand Monawar (2000) identifi ed more than 30 fungal species be- longing to 19 genera on eggplant seeds. The most prevalent seed borne fungi of brinjal were Aspergillus niger, A. fl avus, A. clavatus, Penicilliumdigitatum, Pythium sp., Rhizoctonia sp., Rhizopusarrhizus, R. stolonifer, Alternaria alternate and Fusarium oxysporum. Large quantities of fungicides have been used all over the world by farmers to control fungal diseases in their fi elds, thus increasing production costs and contaminating food and the environment with toxic substances (Bowers and Locke 2000). Consequently, indiscriminate use of fun- gicides poses a serious threat to the environment and health. Use of bio pesticides gives a hope that synthetic pesticides can be replaced.. These pesticides are made up of natural products, and are easily biodegradable. A potentially useful substitute for expensive and possibly toxic fungicides could befound in plant extracts (Joseph et al. 2008). The plant extracts of many higher plants have for centu- ries been known for their antimicrobial properties. Medici- nal plants are storehouses of secondary phytomedicines. These compounds have additive or synergistic effects on the physiological processes of single or a multiple targets. Ethno-botany surveys all over the world support the me- dicinal use of these extracts against human and plant pests (Shariff et al. 2006, Gupta et al. 2008). Recently, the anti- microbial activity of some of the higher plant products that are biodegradable and safe for human health has gained the attention of researchers working on plant defenses against microbial ingress (Koirala et al. 2005). Numerous chemical compounds are present in medicinal plants including sapo- nins, tannins, fl avonoids, alkaloids, terpenoids, steroids, glycosides and proteins. These substances are potent bioac- tive compounds and can be used for remedial purposes (So- fowora 1993). Although use of plant extracts is less laborious, more economical and more eco-friendly than that of fungicides, it is still not popular among stakeholders. The aim of this re- search was to uncover the potentials of these natural occur- ring chemicals against existing mycofl ora associated with seeds. The present study was undertaken to exploit different plant species that have antifungal properties, primarily un- der in vitro and greenhouse conditions, to control the seed- borne diseases of eggplant as an alternative to chemical fun gicide. Materials and methods Sample collection Eggplant seeds were obtained from the vegetable sec- tion of Ayub Agriculture Research Institute, Faisalabad and preserved in brown paper bags in the laboratory of Plant Pathology at Bahauddin Zakariya University (BZU) Mul- tan, Pakistan, until further investigation. Detection and isolation of fungi on seeds Two hundred seeds were taken at random, surface steril- ized with 1% (v/v) sodium hypochlorite solution and sub- jected to the standard blotter paper method for isolations of fungal mycofl ora (ISTA1993). Ten seeds were placed on three-layered moistened blotter paper in 9cm diameter petri plates. After one week of incubation, fungal colonies emerging around the seeds were isolated by the single-hy- phal isolation technique on potato dextrose agar (PDA) me- dium and incubated under a 12 h photoperiod at 27 °C for seven days for further identifi cation and purifi cation. The fungi were identifi ed according to the morphological char- acters and relevant literature (Barnet and Hunter 1972, Ellis et al. 1975, Nelson et al. 1983). Pathogenicity was con- fi rmed according to Koch’s postulates. Preparation of plant extracts A previously described method (Neycee 2012) was used for extraction procedure. Fresh healthy leaves of indige- nous medicinal plants: Mangifera indica, Citrus limon, Eu- calyptus camaldulensis and Mentha spicata were collected in the premises of Bahauddin Zakariya University, Paki- stan. Samples were thoroughly washed with tap water, fol- lowed by distilled water and fi nally with 70% ethanol (Mer- ck) to eliminate any traces of contaminants. Blot dried leaves of each sample were then dried in the oven at 50 °C for 2 h, later shade dried and homogenized to fi ne powder and stored in airtight bottles. 10 g of powdered material was extracted using Soxhlet apparatus (J. P. Selecta-Spain) with 100 mL ethanol for 48 h. The extract solutions were centrifuged at 6000 rpm for 10 min, fi ltered with fi lter pa- per (Whatman No.1) and concentrated over a water bath at 40 °C. After complete solvent evaporation extract residues were sealed in dark bottles at 4 °C for further use. Fungal growth assay (in vitro) Four out of eight isolated fungal strains including Fu- sarium oxysporum, Aspergillus fl avus, Rhizopus stolonifer and Penicillium digitatum were tested. The comparative toxicity of plant extracts on the growth of fungi was evalu- ated by the poisoned food technique (Nene and Thapilyal 2000). Prerequisite amounts of different concentrations (20, 30, 40, 60 and 80%) of plant extracts were incorporated aseptically into potato dextrose agar medium for inocula- tion of test fungi in 9 cm sterilized petri plates. Medium without any plant extract served as a control. A mycelial disk (0.6 cm) of test pathogens was placed at the center of each Petri plate and incubated for 7 days at 27 °C under a 12 h photoperiod. The experiment was carried out in tripli- cate. Mycelial growth (cm) of fungus was measured after 7 days of incubation. The inhibition percentage (P) was cal- culated according to the equation of Singhand Tripathi (1999) where dc denotes average increase in mycelial growth in control and dt denotes average increase in mycelial growth in treatment: P=(dc–dt)/dc×100 Greenhouse experiment Fresh plant extracts were prepared by the method de- scribed above with distilled water (50:50 w/v) and fi ltered ASHIQ B., CHOHAN S., PERVEEN R., ABID M. AND MEHMOOD M. A. 74 ACTA BOT. CROAT. 76 (1), 2017 through cheese cloth. Further, these extracts were diluted with distilled water to obtain an 80% concentration. Four hundred eggplant seeds per treatment were immersed in each extract solution for 30 min (ISTA 1996). Then the ex- cess extract was drained off and seeds were blot dried and kept in the open air. The pots were arranged in a random- ized complete block design with four replications. The seed rate was 10 seeds/ pot in each replication. Data regarding germination percentage, seedling height, shoot length, root length, fresh shoot weight, fresh root weight, dry root weight, dry shoot weight and average biomass parameters were recorded. Germination percentage was observed at 18 days after sowing (DAS) while other growth characters were recorded at 35 DAS. Phytochemical analysis Plant extract were subjected to qualitative phytochemi- cal screening using the methods of Sofowora (1993) and Harborne (1973). For analysis of alkaloids (Wagner’s test), a 20 mg of ethanolic extract was warmed with 2% sulphuric acid (H2SO4) for 1–2 min, fi ltered and treated with a few drops of Wagner’s reagent. Presence of reddish brown pre- cipitation or turbidity indicated the presence of alkaloids. For tannins (ferric chloride test) a 20 mg plant extract was dissolved in ethanol, a few drops of 0.1% ferric chloride were added and the extract was observed for the formation of blue black coloration. For steroids, a 2 mL of acetic an- hydride and concentrated H2SO4 were added to 50 mg etha- nolic plant extract. A blue green ring indicates the presence of steroids. For terpenoids (Salkowski Test), a 5 mL extract of each sample was mixed in chloroform (2 mL) and H2SO4 (3 mL) was added carefully to form a layer. Formation of reddish brown coloration at the interface indicates the pres- ence of terpenoids. For analysis of saponins a 20 mg pow- dered sample was boiled in 5 mL distilled water and shaken vigorously for a stable persistent froth. Three drops of olive oil were mixed vigorously with the frothing and observed for the formation of emulsion. For fl avonoids analysis, a powdered sample (20 mg) was heated with 10 mL of ethyl acetate for 3 min and fi ltered. The fi ltrate (4 mL) was mixed with 1 mL of dilute ammonia solution. A yellow coloration that disappears on addition of concentrated HCl indicated the presence of fl avonoids. Estimation of total fl avonoid contents Total fl avonoid contents were determined following the procedure of Chang et al. (2002) with a slight modifi cation. Quercetin was used as a standard to make the calibration curve. Plant extract (0.5 mL) was mixed with 1.5 mL meth- anol, 10% (w/v) aluminum chloride (0.1 mL), 1 M potassi- um acetate (0.1 mL) and distilled water (2.8 mL). It was kept at room temperature for 30 min. Absorbance was mea- sured at 415 nm by spectrophotometer (UV-3000).The cali- bration curve was prepared using quercetin standard solu- tions of 25, 50 and 100 mg L–1 in ethanol. Total fl avonoid values are expressed in terms of mg quercetin equivalent per g of dried fraction (mg QE g–1DF). The experiment was run in triplicate. Physico-chemical analysis Total ash contents, pH values and weight loss on drying were determined for ethanolic extract of E. camaldulensis, following the methods of Vaghasiya et al. (2008). Statistical analysis The data obtained were subjected to analysis of vari- ance (ANOVA) and treatment means were compared with Fisher’s least signifi cance difference (LSD) test at 5% level of signifi cance (Steel and Torrie 1980) by using the statisti- cal software Sigma plot 11. Results Eight fungal species belonging to 5 genera were isolat- ed by the standard blotter method from seeds of eggplant. The highest percentage frequency was recorded by Penicil- lium digitatum (49.67%) followed by Aspergillus fl avus, Fusarium oxysporum, Rhizopus stolonifer,Alternaria alter- nata, Curvularia lunata, Aspergillus niger and Fusarium solani with 47.33, 38.00, 33.00, 15.00, 13.67, 8.33 and 5.67% recovery percentage, respectively (Fig. 1). Out of re- covered fungi, C. lunata, A. alternata, A. niger and F. so- lani were found in minimum incidence percentages, hence only predominant fungi, viz. F. oxysporum, A. fl avus, P. digitatum and R. stolonifer were purifi ed and subjected to antifungal assay with four plant extracts at fi ve different concentrations (20, 30, 40, 60 and 80%). All plant extracts revealed varied inhibitory effects on the mycelial growth of test fungi, by affecting their normal growth. The data regarding mean mycelial growth inhibi- tion of four fungal species and ethanolic leaf extract of four extracts with different concentrations was statistically sig- nifi cant (Fig. 2). Extract of M. spicata at 20% concentration was found less effective against A. fl avus, R. stolonifer, P. digitatum and F. oxysporum with 7.5, 7.7, 8.3 and 8.4 cm growth, respectively. At 30% concentration, mycelial growth of P. digitatum (7.9 cm), F. oxysporum (7.7 cm), A. fl avus and R. stolonifer (6.8 cm) was observed. Less myce- Fig.1. Recovery percentage (%±SD) of fungi isolated from egg- plant seeds by using the standard blotter method. ANTIFUNGAL ACTIVITY OF MEDICINAL PLANTS ACTA BOT. CROAT. 76 (1), 2017 75 lial growth, of 5.8 and 5.9 cm, was found for A. fl avus and R. stolonifer, while more growth, of 7.2 and 7.5 cm, was observed for F. oxysporum and P. digitatum respectively at 40% concentration of M. spicata extract. Maximum myce- lial growth at 60% concentration of M. spicata extract was of F. oxysporum (6.7 cm) and P. digitatum (6.6 cm) and minimum growth was of R. stolonifer (5.1 cm) and A. fl a- vus (4.7 cm). At 80% concentration of M. spicata extract, the mycelial growth of A. fl avus was best inhibited with only 3.3 cm growth followed by R. stolonifer (4.3 cm), P. digitatum (6.1 cm) and F. oxysporum (6.4 cm) compared to control fungi with 9.0 cm growth (Fig. 2A). At 20% concentration of C. limon, the diagonal growth of F. oxysporum (7.9 cm), A. fl avus (7.3 cm), P. digitatum (7.1 cm) and R. stolonifer (6.9 cm) was observed. R. sto- lonifer, P.digitatum, A. fl avus and F. oxysporum showed 6.2, 6.4, 6.6 and 7.5 cm mycelial growth respectively at 30% concentration of C. limon extract. The minimum growth (5.7 cm) was of P. digitatum after R. stolonifer (5.8 cm), A. fl avus (6.1 cm) and F. oxysporum (7.1 cm) at 40% concentration of C. limon extract. Maximum mycelial growth (6.3 cm) was of F. oxysporum followed by A. fl avus (5.5 cm), R. stolonifer (4.9 cm) and P. digitatum (4.1 cm) at 60% concentration of C. limon extract. On the other hand, 80% concentration of C. limon extract was found to be the most effective and illustrated maximum growth inhibition of P. digitatum (2.9 cm) and minimum inhibition in R. sto- lonifer, A. fl avus, F. oxysporum, with 4.7, 5.2 and 5.9 cm growth respectively compared to control fungi with 9 cm growth (Fig. 2B). The mycelial growth of P. digitatum, F. oxysporum, R. stolonifer and A. fl avus on agar plate containing different concentrations of ethanolic leaf extract of E. camaldulensis was highest with 7.9, 7.8, 7.7 and 7.4 cm at 20% concentra- tion. At 30% concentration, the growth of P. digitatum and R. stolonifer was 7.3 and 7.4, whereas in F. oxysporum and A. fl avus it was 6.9 and 6.6 cm respectively. Maximum my- celial growth (6.9 cm) was of R. stolonifer followed by P. digitatum (6.4 cm), A. fl avus (5.8 cm) and F. oxysporum (5.4 cm) at 40% concentration of E. camaldulensis extract. Mycelial growth of F. oxysporum (4.3 cm), A. fl avus (4.9 cm), P. digitatum (5.1 cm) and R. stolonifer (5.8 cm) de- creased at 60% concentration E. camaldulensis extract. Minimum mycelial growth of 3.2, 3.6, 3.9 and 4.3 cm was observed in F. oxysporum, P. digitatum, A. fl avus and R. stolonifer respectively, at 80% concentration of E. camald- ulensis extract compared to control fungi with 9 cm growth (Fig. 2C). At 20% concentration of ethanolic leaf extract of M. in- dica, mycelial growth of F. oxysporum (8.3 cm) was greater than in P. digitatum (8.1 cm), R. stolonifer (7.8 cm) and A. fl avus (7.4 cm). At a 30% concentration, the mycelial growths of A. fl avus (6.9 cm), R. stolonifer (7.2 cm), P. digi- tatum (7.6 cm) and F. oxysporum (7.7 cm) were observed. Growth of mycelia was 6.4, 6.7, 6.8 and 7 cm in A. fl avus, R. stolonifer, P. digitatum and F. oxysporum respectively at a 40% concentration of M. indica extract. At a 60% concen- tration, maximum growth (6.6 cm) was seen for F. oxyspo- rum followed by R. stolonifer (6.3 cm), P. digitatum (6.2 cm) and A. fl avus (5.7 cm). Minimum mycelial growth (4.8 Fig. 2. Mycelial growth (mean±SE) of four tested fungi after using different concentrations of ethanolic leaf extract of four plants: A) Mentha spicata, B) Citrus limon, C) Eucalyptus camaldulensis, D) Mangifera indica. ASHIQ B., CHOHAN S., PERVEEN R., ABID M. AND MEHMOOD M. A. 76 ACTA BOT. CROAT. 76 (1), 2017 cm) was observed in A. fl avus after R. stolonifer (5.4 cm), P. digitatum (5.7 cm) and F. oxysporum (6.1 cm) at 80% con- centration of M. indica extract, compared to control fungi with 9 cm growth (Fig. 2D). M. indica at 20% concentration showed minimum my- celial growth inhibition (13.33%) while maximum inhibi- tion (52.22%) was observed for M. spicata and E. camaldu- lensis extracts at 80% concentration against R. stolonifer. Growth of A. fl avus was inhibited by 16.67% and 63.33% at 20% and 80% concentration of M. spicata extract, respec- tively. Mean mycelial growth inhibition of F. oxysporum was lower (6.67%) at 20% concentration but maximum inhi bition of 64.44% was observed at 80% concentration of E. camaldulensis. A maximum inhibition percentage (67.78%) of P. digitatum was observed at 80% concentra- tion of C. limon with, while a minimum inhibition (7.78%) was observed at a 20% concentration of M. spicata extract (Fig 3A–D). Results of variance analysis for the germination experi- ment showed that germination percentage and growth char- acters of eggplant were statistically signifi cant except for dry root weight. Maximum germination was observed after treatment with E. camaldulensis extract (78.98%) followed by extracts of C. limon (73.05%), M. spicata (71.00%) and M. indica (64.10%), and control untreated plants (62.83%), respectively. Maximum seedling height was observed in seeds treated with E. camaldulensis (18.57 cm) and C. li- mon extracts (18.27 cm) and a minimum in control plants (12.87 cm). Shoot length was greater (15.25 cm) after treat- ment with E. camaldulensis extract than in control plants Table 1. Effect of different plant extracts on eggplant seed germination and growth parameters. Means sharing different letters are statisti- cally different at 5% level of signifi cance. Values are means of four replications. T1 – Eucalyptus camaldulensis, T2 – Mangifera indica, T3 – Citrus limon, T4 – Mentha spicata, T5 – negative control; LSD – least signifi cant difference. Treatments Germination (%) Seedling height (cm) Shoot length (cm) Root length (cm) Fresh shoot weight (g) Fresh root weight (g) Dry shoot weight (g) Dry root weight (g) Biomass (g) T1 78.98 a 18.575 a 15.25 a 4.05 a 0.825 a 0.0723 a 0.0805 a 0.0080 a 1.015 a T2 64.10 c 14.175 c 11.95 bc 2.73 c 0.550 c 0.0463 d 0.0588 d 0.0053 a 0.685 cd T3 73.05 b 18.275 a 14.85 a 3.85 ab 0.900 ab 0.0660 b 0.0730 b 0.0078 a 0.898 b T4 71.00 b 16.125 b 12.90 b 3.40 b 0.750 abc 0.0593 c 0.0660 c 0.0065 a 0.738 c T5 62.83 c 12.875 d 11.53 c 2.80 c 0.625 bc 0.0478 d 0.0565 d 0.0070 a 0.648 d LSD P=0.05 2.129 1.113 1.323 0.514 0.205 0.005 0.006 0.0028 0.075 Fig. 3. Mycelial inhibition (mean±SE) by four different plant extracts at different concentrations: A) Mentha spicata, B) Citrus limon, C) Eucalyptus camaldulensis, D) Mangifera indica. ANTIFUNGAL ACTIVITY OF MEDICINAL PLANTS ACTA BOT. CROAT. 76 (1), 2017 77 (11.53 cm). Minimum root length was observed in seeds treated with M. indica extract (2.73 cm) and maximum in seeds treated with E. camaldulensis extract (4.05 cm). Fresh shoot weight was greater after seed treatment with C. limon extract (0.90 g) than after treatment with M. indica extract (0.55 g). Highest fresh root weight was after seed treatment with E. camaldulensis extract (0.072 g) and the lowest after treatment with M. indica extract (0.046 g). Greatest dry shoot weight and biomass was observed in seeds treated with E. camaldulensis (0.08 g and 1.015 g) and lowest in control (0.05 g and 0.64 g), respectively (Tab. 1). Ethanol leaf extract of E. camaldulensis was found rich in fl avonoids, alkaloids, tannins and saponins while steroids and glycosides were absent. Quantitative analysis of etha- nol extract resulted in 10.38 mg QE g–1 fl avonoid contents in Eucalyptus leaves. The results of physico-chemical anal- ysis revealed pH value of 4.6, ash content of 0.41% and weight loss on drying of 8.14%. Discussion Seedborne microorganisms play an important role in af- fecting the quality of seed, causing signifi cant crop losses. Predominant fungi associated with seed samples of egg- plant were identifi ed as Fusarium oxysporum, Aspergillus fl avus, Penicillium digitatum and Rhizopus stolonifer, caus- ing quantitative and qualitative losses to seeds during stor- age (Neergard 1977). Opportunistic pathogens such as As- pergillus sp. and Penicillum sp. were found associated with seed infection and caused severe damage to both quality and quantity of seed production (Kumar et al. 2005). Tradi- tionally, seeds have been treated with fungicides to kill mi- croorganisms. However, the World Health Organization (WHO) banned many agriculturally important pesticides due to their toxicity against non-target organisms including humans, and because they are known to cause pollution problem (Barnard et al. 1977). Many plant extracts and oils, such as tea tree and clove had been used as contemporary antiseptics, or had been reported to have antimicrobial properties (Hoffman 1987, Rice 2012). Extracts from dif- ferent plants have been reported to have antifungal, antibac- terial and antioxidant activities. The antifungal activity of diverse plants against different fungi has been reported (Farrag and Moharam 2012, Vidua-Martos et al. 2008). Results in the present study showed that four fungal species were isolated from eggplant seeds. Their growth was best during February, March and confi rms the results of Pandey (2010) who detected fi ve fungi on brinjal viz. F. solani, Helminthosporium spiciferum, Phomopsis vexan, C. lunata, Trichothecium roseum. The growth of these fungi was particularly luxurious from October to November, and February to April. Different concentrations of different plant extracts exhibited different activities. In this study fi ve extract concentrations (20, 30, 40, 60 and 80%) were used against test fungi and illustrated varying results. Some concentrations were weak while few were effective in con- trolling mycelial growth of test fungi. Similarly, Joseph et al. (2008) used different concentrations, i.e., 5, 10, 15 and 20% of plant extracts. Among the different extracts 20% of Azardiachta indica was found most effective followed by Rheum emodi, Eucalyptus globulus, Artemessia annua and Ocimum sanctum. In our study, in the case of R. stolonifer and A. fl avus the best inhibition was exhibited by E. camal- dulensis and M. spicata extracts, respectively, at 80% con- centration. Similar results were also reported by different scientists at different times (Satish et al. 2007). On the other hand, Tzortzakis and Economakis (2007) found lemongrass (Cympopogon citratus) extract to be effective against R. stolonifer and A. niger. In our study, F. oxysporum was best inhibited by E. camaldulensis extract at 80% concentration. The results correlated with previous fi ndings that aqueous leaf extracts of E. citriodora, M. indica, Accacia nilotica, A. indica and Syzygium cumini signifi cantly reduced the in- cidence of the two most frequent seed-borne fungi, viz., F. solani, and A. alternata (Shafi que et al. 2007, Marzoug et al. 2011). P. digitatum was best inhibited at higher concen- tration by C. limon extract in our study. These fi ndings are in line with the results of Kanan et al. (2008), who evaluat- ed the effect of different plant extracts and liquid fractions against citrus post-harvest disease agent P. digitatum. Simi- larly, Ragab et al. (2012) found that mint, thyme, pepper- mint and clove had signifi cant inhibitory effects on fungal mycelia. Fungal mycelial growth decreased signifi cantly as the concentrations of extracts increased. Mango leaf extract moderately reduce the growth of pathogenic fungi. In the same way Suvarna and Patil (2009) showed that extract of M. indica had moderate activity against the human patho- genic fungi Candida albicans. Seed germination and other growth parameters of egg- plant were greater in seeds treated with E. camaldulensis extract as compared to control treatment. Conversely E. ca- maldulensis signifi cantly reduced seed germination of sor- ghum (Mohamadi and Rajaie 2009). M. indica extract was found least effective among all the extracts used. Present results are in harmony with the fi ndings of Kumar et al. (2005) and Alberts et al. (2006). They reported that pre- and post-harvest bio-deterioration of crop seeds are mainly due to seed infestation by microorganisms and insects, causing up to 100% losses. Contrary to our results, leaf extract of Moringa oleifera increased seed germination of eggplant up to 92% over control treatment (Kuri et al. 2011). E. camaldulensis extract was found most effective in re- ducing the fungal growth of seedborne mycofl ora, eventu- ally increasing the germination of eggplant seed. Similarly E. camaldulensis exhibited pronounced antifungal activity, among different species of eucalyptus (May and Ash 1990, Rukhsana. 2005, Babayi et al. 2004, Ghalem and Mohamed 2008) and this might be due to the presence of secondary compounds. Thus the phytochemical and physico-chemical parameters of E. camaldulensis were studied. Phytochemi- cal screening of leaf extract of E. camaldulensis extract re- vealed the presence of tannins, fl avonoid, alkaloids and sa- ponins, while steroids and glycosides were absent, and these results are similar to earlier fi ndings (Vaghasiya et al. 2008). ASHIQ B., CHOHAN S., PERVEEN R., ABID M. AND MEHMOOD M. A. 78 ACTA BOT. 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