Department of Mycology & Plant Pathology, Quaid-e-Azam Campus, University of the Punjab, Lahore-54590, Pakistan, www.pu.edu.pk Email anjum@mpp.pu.edu.pk
The allelopathic potential of aerial parts of chickpea (Cicer arietinum L.) was investigated in vitro for their antifungal properties as natural alternatives of plant disease control. Drechslera tetramera (Mikinney) Subram. & Jain. and Drechslera hawaiiensis (M. B. Ellis) when tested against different concentrations of aqueous extracts of aerial parts of C. arietinum in liquid malt extract medium, the crude water extract showed most significant antifungal activity even at lower concentration of 5%. In case of Dichloromethane fraction, the inhibitory effect was found to be proportional with the concentration. Cicer arietinum was found to contain antimicrobial compound(s) for the control of plant pathogenic fungi.
Key Words
Allelopathy, antifungal, chickpea, Cicer arietinum, Drechslera.
Introduction
Allelopathy has been accepted widely as an important ecological phenomenon. Due to increased awareness about the risks involved in use of pesticides, much attention is being focused on the alternative methods of pathogen control. In the past two decades, a lot of work has been done on plant-derived compounds as environmentally safe alternatives to pesticides for plant disease control. Different plant extracts have been evaluated for their antimicrobial properties. Pretorius et al. (2002) tested crude extracts from thirty nine plant species for their antifungal potential against seven plant pathogenic fungi. The most significant mycelial growth inhibition was obtained with extracts from Aristea ecklonii. Petroleum ether and methanolic extracts of nine wild plant species were tested in vitro for their antimycotic activity against eight phytopathogenic fungi and the petroleum ether extract of Origanum syriacum resulted in complete inhibition of mycelial growth of six out of eight fungi tested (Abou-Jawadah et al. 2002).
Chickpea (Cicer arietinum L.) secretes highly acidic exudates, which have pH near to 1 (Rembold 1981). These are mostly released through the trichomes located on all the plant, including pods. These exudates are reported to have a role as defence chemicals against soil pathogens (Li and Copeland 2000). The present study was designed to evaluate the antifungal potential of different extracts from aerial parts of Cicer arietinum L. against Drechslera tetramera and Drechslera hawaiiensis.
Methods
Fresh plants of C. arietinum were extracted in water making 75% w/v stock solution. The lower concentrations of 50, 25 and 5% were prepared by adding appropriate quantity of sterilized water. The extract was stored at 4°C in pre-sterilized flasks and used within 3-4 days.
Basal medium for the growth of fungus was prepared by adding Malt extract (ME) 2% in water. Chloromycetin (250mg in 100 ml of medium) was added to avoid bacterial contamination. ME (80ml) was distributed into 250ml flask. Plant extract (20ml) of each concentration was added separately to each flask in three replicates. Distilled water was added in place of extract in the control. Inoculum discs of 5mm diameter, obtained from 7-days old healthy growing fungal cultures of selected fungi were transferred to these flasks aseptically and incubated at 25±2oC.
Plant extract (75%) was partitioned with100ml of dichloromethane for the second set of experiment. The solvent from the dichloromethane and water fractions was removed under reduced pressure. The residue was re-dissolved in 100ml of sterilized water to get 75% stock solution. The further concentrations of 5, 25 and 50% were prepared by adding calculated amount of water.
For the assessment of fungal biomass yield, three harvests were designed at intervals of 5-days each. The mycelial biomass from triplicate samples for each treatment was collected on pre-weighed filter papers. Their dry weight yield was determined after 24 hours oven drying at 60°C. All the data was analysed by applying Duncan’s Multiple Range (DMR) Test to compare different treatments with one another and t-test to compare individual treatment with control.
Results
Effect of crude aqueous extract of Cicer arietinum on biomass production of Drechslera tetramera.
Drechslera tetramera showed significant variation in dry biomass when grown in different concentrations of C. arietinum. The fungal biomass production exhibited reduction at lower extract concentrations of 5 and 25%, as compared to control at 5, 10 and 15 days incubation periods in crude aqueous extract. The inhibitory effect was found proportional with the incubation period and the percentage difference in dry biomass production from control was found highly significant at 15days incubation. Insignificant reduction in first two harvests was observed when fungus was grown in 5% extract concentration, but at the 15 days the inhibitory effect was found to be highly significant. The higher concentrations increased the fungal biomass with time of incubation. The 50% extract concentration showed negative effect on fungal dry biomass production in first harvest, but after 10 and 15 days of incubation, increase in fungal growth recorded was not very significant (Figure 1). The extract concentration of 75% markedly supported the mycelial yield and the increase in fungal biomass ranged from 6.6% after 5 days incubation to 15% after 15 days of incubation period.
Figure 1. Effect of Cicer arietinum on dry biomass production of Drechslera tetramera. Where A = crude aqueous extract, B = Dicholoromethane fraction and C = water fraction. Values with different letters show significant difference (P = 0.05) as determined by DMR Test. *, **, *** Show significant difference from control at 5, 1 and 0.1 % level of significance respectively, as determined by t-test.
Effect of Dichloromethane and aqueous fractions of Cicer arietinum extract on biomass production of Drechslera tetramera.
Dichloromethane (DCM) fraction showed the most promising antimycotic activity by reducing the fungal biomass up to 58% after 15 days of incubation. Growth reduction increased, as did the fraction concentration. There was insignificant increase in mycelial yield of 5% DCM fraction at 5 and 10 days of incubation period, but in the final phase i.e., after 15 days of incubation the mycelial growth was found significantly depressed. At 25% DCM fraction the allelopathic stress was found to be increased with increase in time of incubation ranging from insignificant decrease in first harvest to highly significant in third. The higher regimes of 50 and 75% DCM fraction concentrations were found highly significant with respect to the control at all incubation times. No particular trend was observed in case of aqueous fraction, but generally lower concentrations of 5 and 25%, decreases the fungal dry biomass production. The fungal biomass was found to be depressed after 5 days of incubation in 25 and 50% concentration. The maximum dry biomass increment was observed at 75% after 10 days of incubation (Figure 1).
Figure 2: Effect of Cicer arietinum on dry biomass production of Drechslera hawaiiensis. Where A = crude aqueous extract, B = Dicholoromethane fraction and C = water fraction. Values with different letters show significant difference (P = 0.05) as determined by DMR Test. *, **, *** Show significant difference from control at 5, 1 and 0.1 % level of significance respectively, as determined by t-test.
Effect of crude aqueous extract of Cicer arietinum on biomass production of Drechslera hawaiiensis.
The results obtained from periodic biomass assays of D. hawaiiensis in various concentrations of crude aqueous extract of C. arietinum showed significant antifungal activity in lower concentrations viz., 5-50% in comparison to control, in all the three growth phases. A nominal depression was observed after 5 and 10 days of incubation by 5% extract concentration. But at the final harvest i.e., after 15 days growth period, the mycelial yield was found to be significantly depressed as compared to control (Figure 2). The inhibitory effect decreased at 25 and 50% extract concentrations with increase in time of incubation. The maximum antimycotic activity was observed at early growth phase i.e., after 5 days of incubation. In contrast the higher concentration of 75% showed positive effects on dry biomass of D. hawaiiensis.
Effect of Dichloromethane and aqueous fractions of Cicer arietinum extract on biomass production of Drechslera hawaiiensis.
Dichloromethane fraction was found clearly superior in reducing the biomass production. The lower concentration of 5% slightly promoted the fungal growth after 5 and 15 days of growth. All the other concentrations i.e., 25-75% decreases the in vitro mycelial growth and this growth reduction was found proportional to the fraction concentration. The maximum allelopathic stress was induced by 75% concentration causing a decline of 64% after 5 days of incubation period. Statistically there was a decline in allelopathic stress with increase of incubation. Generally water fraction improved the biomass production. At initial growth stage the lower fraction concentrations (5-50%) depressed the mycelial yield up to 26%, while after 10 and 15 days of incubation period all the extract concentrations provided a considerably high boost in biomass productivity (Figure 2).
Discussion
Significant effect of chickpea extracts was found on D. hawaiiensis and D. tetramera in reducing their mycelial growth. The extracts were found relatively more effective in decreasing the mycelial growth against D. hawaiiensis. Where as D. tetramera exhibited greater resistance against allelopathic stress of C. arietinum. This difference in the susceptibility could be the cause of genetical difference in physiological and morphological characteristics of different species (Shaukat et al. 1983). Martinez et al. (2000) also reported variable effects of Sargassum filipendula extracts on Aspergillus species. Overall the general trends illustrated by both the tested species were found almost same. Greater inhibition of fungal growth was observed at lower concentrations of the crude water extract where as the higher concentrations supported the average mycelial growth rate per day. This may be because the optimal range of pH for growth of tested species lies in the acidic range. Since the exudates of C. arietinum contain several acidic compounds (Rembold 1981) the enhancement in the dry biomass production at higher concentrations may be due to low pH level of medium. These results are also supported by the fact that some allelopathic substances have variable effects when applied in different concentrations, either inhibitory or stimulatory (Purvis et al. 1985). The Dichloromethane fraction from crude aqueous extract showed stronger and broader spectrum of antimycotic activity. This activity was found proportional to the fraction concentration as the increase in concentrations decreases the biomass production significantly. The water fraction was not found much effective against selected fungal species. The fraction in general promoted mycelial growth and this positive effect increases with increase in time of incubation. In case of crude aqueous extract the highest tested concentration i.e., 75% caused a persistent positive impact on growth of both fungal species. This increase in biomass production may be due to detoxifying ability of the fungi to allelochemicals or the ability of fungal species to exploit them as nutritional source (Sicker 1998). Some allelochemicals are also known to enhance the growth at different concentrations (Mughal et al. 1996). These results are clear indication for the potential of C. arietinum to control fungal pathogens.
References
Abou-Jawdah YH and Salameh A (2002). Antimycotic activities of selected plant flora, growing wild in Lebanon, against phytopathogenic fungi. Journal of Agriculture and Food Chemistry 50, 3208-3213.
Barz W, Adamek C and Berlin J (1970). The degradation of formononetin and daidzein in Cicer arietinum and Phaseolus aureus. Phytochemistry 9, 1735-1744.
Li J and Copeland L (2000) Role of malonate in chickpea. Phytochemistry 54, 585-589.
Magama S, Pretorius JC and Zietsman PC (2003). Antimicrobial properties of extracts from Euclea crispa subsp crispa towards human pathogens. South African Journal of Botany 69, 193-198.
Martinez-Lozano SL, Garcia S and Heredia N (2000). Antifungal activity of extract of sargassum filipendula. Phyton-International Journal of Experimental Botany 66,179-182.
Mughal MA, Khan TZ and Nasir MA (1996). Antifungal activity of some plant extracts. Pakistan Journal of Phytopathology 8,46-48.
Pretorius JC, Zietsman PC and Eksteen D (2002). Fungitoxic properties of selected South African plant species against plant pathogens of economic importance in agriculture. Annals of Applied Biology 141, 117-124.
Purvis CE, Jessop RS and Ronette JV (1985). Selective regulation of germination and growth of annual weeds by crop residues. Weed Research 25, 415-421.
Rembold H (1981). Malic acid in chickpea exudates – a marker for Heliothis resistance. Chickpea Newsletter 4, 18-19.
Shaukat SS, Khan D and Ali ST (1983). Suppression of herbs by Inula grantioides Bioss in the Sindh desert, Pakistan. Pakistan Journal of Botany 15, 43-67.
Sicker D (1998). Detoxification of wheat allelochemicals. Applied Environmental Microbiology 64, 2386-2391.
