Integrated disease management is a non-separable part of all the eco-friendly stable agricultural programs. Plant extracts have played significant role in the inhibition of pathogens and in the improvement of crop production. Cuscuta reflexa Roxb. is a parasitic plant. Agriculturalists consider Cuscuta spp is only a destructive weed and attempt to eradicate it. The main purpose of this study was to assess its significance in developing plant based formulations for fungal disease management. In vitro, antifungal potential of C. reflexa (parasitizing Bougainvillea glabra L.) extract was evaluated against five different pathogenic fungi namely, Alternaria alternate, Aspergillus niger, Fusarium solani, Fusarium oxysporium and Macrophomina phaseolina. Different aqueous concentrations (0, 10, 20, and 30 %) of C. reflexa were prepared. Aqueous concentrations of C. reflexa were evaluated against fungal isolate by well diffusion method. The fungitoxicity of extract in terms of inhibition zone diameter was calculated. Results indicated that fungal growth inhibition was directly proportional to the concentration of C. reflexa extract. Cuscuta extract exhibited significant antifungal activity against all test fungal isolates. However, extract was highly effective against F. solani, F. oxysporium and M. phaseolina, and was least effective against A. niger. It was also found that 30% (w/v) concentration was significantly effective in reducing the mycelial growth of fungal isolates after 6 days of incubation. Further investigations however are required to analyze nature of antifungal compounds in C. reflexa and their stability.
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Key words: Cuscuta reflexa, parasitic plants, antifungal extract, pathogenic fungi, parasitic weeds
Cuscuta reflexa Roxb. is an angiospermic hustorial advance, obligate, hemiparasite belonging to family cuscutaceae. It parasitizes wild and cultivated plants, and is especially destructive to such commercially valuable crops as flax, alfalfa, beans, and potatoes. It also grows on common ornamental plants though the plans of Pakistan (Bhattacharya, 1976; Malik et al., 1980). Agriculturalists consider Cuscuta species a destructive weed and attempt to eradicate it. However on biological aspects, strong antifungal and antibacterial compounds have been extracted from C. reflexa (Loffler et al., 1997; Da-Nian Qin et al., 2000). Its therapeutic properties such as anticancer, antidiabetic, antiviral, and anti-inflammatory are also well documented (Awasthi, 1981; Poudel, 2002). Studies have also proved that the presences of some stable phytochemicals in Cuscuta species are irrespective of host plants and locations (Loffler et al., 1997). Plant metabolites and plant-based pesticides appear to be one of the better alternatives as they are known to have minimal environmental impact and in the improvement of crop production (Varma and Dubey, 1999; Nwachukwu and Umechuruba, 2001). Conventional breeding of disease-resistant cultivars and plant protection based on extensive use of agrochemicals represent still common strategies in the contest between human approaches to combat microbial food competitors and the evolutionary adaptability of bacterial and fungal phytopathogens. These are responsible for enormous crop losses worldwide and therefore threaten human nutrition (Osusky et al., 2000). Integrated disease management is a non-separable part of all the eco-friendly stable agricultural programs. Biological control of plant diseases and plant pathogens is of great significance in forestry and agriculture (Inderjit, 2006). Therefore, new strategies to fight phytopathogens have to be explored (Moffat, 2001). Keeping in view the strong phytochemical potential of C. reflexa, this study has been carried out to evaluate antifungal activity against important pathogenic fungal species with the ultimate aim of developing plant based formulations for plant disease management.
Material and Methods
Plant material and extraction
Fresh plant material of C. reflexa (parasitizing Bougainvillea glabra L.), growing in the premises of University of the Punjab, was collected. Plant material was washed thoroughly under running tap water, dried with blotting paper and cut into small pieces. A 50% (w/v) aqueous extract was prepared by homogenizing plant material (50 g) in a blender with 100ml sterile distilled water for 5 min and allowed to stand for 24 hours. The mixture was filtered through muslin cloth and then centrifuged at 4000 g for 30 min; the supernatant was filtered through Whatman No.1 filter paper to remove the debris. Three concentrations (10, 20 and 30 %) were prepared by dilution of stock solution. Each concentration was simmered at 40o C for 15 minutes, cooled and stored at 4Â° C. To avoid contamination and prospective chemical change, the extracts were used within 2-3 days.
Pathogenic fungal isolates namely, Alternaria alternate, Aspergillus niger, Fusarium solani, Fusarium oxysporium and Macrophomina phaseolina were obtained from the First Fungal Culture Bank of Pakistan, University of Punjab, Lahore, Pakistan. Fungal cultures were maintained on 2% Malt extract agar (MEA) medium at 25Â±2o C for seven days.
Antifungal Assay- Well Diffusion Technique
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Three concentrations (10, 20 and 30 %) were evaluated against five different fungal pathogens. Antifungal activity of C. reflexa extract was assayed by well diffusion method (Rani et al., 2008) with some modification. For antifungal assay, inoculum disc (10mm) was prepared aseptically from seven days old culture. Wells (10mm in diameter) were made on 2% MEA plates using a sterile glass tube. Each plate contained three wells, evenly distributed around the inoculum disc of test fungi that was placed aseptically at the center. For each test concentration, 100ÂµL extract was poured into each well. Each treatment was replicated thrice. For control, sterile distilled water was filled in wells. The plates were incubated at 25Â±2o C for 6 days. The clear zone surrounding each well indicated inhibition activity of C. reflexa. The actual diameter of zone of inhibition was got after subtracting the diameter of the well. All the data was analyzed statistically.
Results and Discussions
Antifungal activity of C. reflexa was analyzed by well diffusion assay against five pathogenic fungi. The results are shown in Fig1 and Fig 2. The size of the zone of inhibition is usually related to the level of antimicrobial activity present in the sample or product - a larger zone of inhibition usually means that the antimicrobial is more potent. Results showed that C. reflexa extract was significantly effective against all test fungi. The C. reflexa extract exhibited variable degree of inhibition with respect to each test fungi. C. reflexa extract was more active against M. phaseolina, F. solani and F. oxysporium and was least effective A. niger (Fig 1). It had been observed that at 10% (w/v), C. reflexa extract showed least anifungal activity. C. reflexa extract at 20% and 30%, showed significant inhibition potential against M. phaseolina, F. solani and F. oxysporium but least effective against A. alternate and A. niger. Results also indicated that size of the zone of inhibition was directly proportional to the concentration of C. reflexa extract. Studies have reported growing interest in antimicrobial potential of C. reflexa (Loffler et al., 1997; Da-Nian Qin et al., 2000). Antifungal (Mohammad et al., 1984) and insecticidal (Chavan et al., 1982) effect of C. reflexa has been studied. The methanol extract of C. reflexa and Corchorus olitorius showed a broad spectrum of antibacterial activity (Pal et al., 2006). However, in case of F. moniliforme, C. reflexa extract inhibit fungal growth only up to 25% (Yasmin et al., 2008). This study showed that C. reflexa contains strong antifungal potential, exerting strong inhibition against the growth of some pathogenic fungi. It is concluded that C. reflexa extract can be helpful in developing a strong plant-based formulations for plant disease management. The documentation of the antifungal value of this notorious and rapidly growing plant brings a lot of relief to the plant conservation community, which is concerned about its effect on other biodiversity. In effect, this plant has potential of being exploited for its antifungal potential and hence controlling its ground cover in the world at large.