Biological Forum – An International Journal 2(2): 122-129(2010) ISSN No. (Print): 0975-1130 Potential of Plagiochasma appendiculatum on Inhibition of Certain Economically Important Plant Pathogens Shivom Singh*, Kajal Srivastava** and Dev Raj Khanna* *Department of Zoology and Environmental Science, Gurukula Kangari Vishwavidyalaya, Haridwar, Uttarakhand, India. **Department of Biological Sciences, CBSH, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India. ABSTRACT: Present study was carried out to evaluate the antimicrobial properties of Plagiochasma appendiculatum (liverwort) extract used against selected test fungus and bacteria for antimicrobial assay. Effect of aqueous and different organic extracts viz. petroleum ether, ethanol, chloroform, acetone, methanol and aqueous extract of Plagiochasma appendiculatum, at different concentrations (1000, 500, 200 and 40 µg ml-1 and at different time intervals i.e. 24, 48 and 72 hrs) were examined for their bioactivity against certain important fungal species (Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysporum and Tilletia indica) and some important bacterial pathogens (Xanthomonas oryzae pv. oryzae, Salmonella enterica, Pasteurella multocida, and Melissococcus plutonius). All organic extracts of P. appendiculatum failed to inhibit growth of Xanthomonas oryzae and S. enterica. Most of the extracts showed maximum activity against the R. solani with MIC of 2.50 and MFC value of 5.00 in petroleum ether extract. MBC value was found maximum in M. plutonius with value of 5.00 extracted in petroleum ether. Key Words: Plagiochasma appendiculatum, liverwort, antimicrobial properties, Plant Pathogen. INTRODUCTION Subramonian (2005) screened the extracts of Pallavicinia leyelli and evaluated that it posses All bryophytes contain cocktails of different active antifunagal activity. Iwashina (2003) reported that compounds, mainly terpenoids to protect themselves flavanoid compounds are widely distributed in against infections of fungi and bacteria, with which bryophytes and posses many biological activities they live in direct contact. These effects of bryoflora against plants, fungi and other microorganism. were thus not developed by chance but are a necessity In particular, liverworts contain pinguisane-type for their survival. The anti-microbial effects of sesquiterpenoids, sacculatane-type diterpenoids and bis bryophytes go back on an inhibition of cell division, not (bi-benzyl) aromatic compounds which are not found in only of bacteria and fungi but also of mammal cells. higher plants (Asakawa, 1995; Nagashima et al., 2002). Other effects include anti-feeding effects against They show higher antifungal properties than the beetles, slug and snail or fish killing effects. Several mosses. It is due to the presence of lunularic acid, an insect anti-feedents have now been found in diverse aging hormone found in liverworts but not in mosses bryophytes making this group of plants a useful source (Pryce, 1972). Degree of antibiotic activity in a given of insecticides and insect repellant. species may depend upon the age of the gametophyte, Mekuria et al. (1999) studied the effect of moss extracts season of collection and other such ecological against phytopathogenic fungi and showed that parameters (Banerjee and Sen, 1979). alcoholic extract of mosss was active against candida Plagiochasma appendiculatum is one of the important albicans. Keyhanian et al. (2002) studied the effect of Indian liverwort belongs to the order Marchantiales several fungicidal and insecticidal seed treatment to under family Aytoniaceae. Plagiochasma is a thalloid control rapeseed seedling damaging plants and liverwort represented by 30 species (Bischler, 1978) but observed that fungicidal and insecticidal compounds in India only 10 species have been reported. were active against tested pathogen. Subhisha and Singh, Srivastava and Khanna 123 Plagiochasma appendiculatum is widely distributed in (40, 200, 500 and 1 000 μg/mL) were prepared and western, eastern Himalayas, central India and south used for further study. India and generally grow to an altitude upto 3000-8000 ft from sea level. This species in known from the east D. Antimicrobial assay part of the Central and South African continent Eritrea, Disc diffusion assay was used for evaluation of Ethiopia, Kenya, Tanzania to Rhodesia, Zimbabwe and antimicrobial activity (Basri and Fan, 2005). In assay South Africa (Perold, 1999; Wigginton, 2002). for antibacterial activity, the nutrient agar plates of Plagiochasma appendiculatum is significant taxon bacteria treated with organic extracts (40 μL into each which possesses antimicrobial property (Banerjee, disc) of different concentrations were incubated at (37 ± 2000). In India, it is used by Gaddi tribes in Himachal 2°C) for 24 h. Antibacterial activity of the plant extracts Pradesh for the treatment of cuts, wounds and burns was determined by measuring the zone of inhibition (Kumar et al., 2001; Singh et al., 2006). (ZI) in mm against all bacteria. The antibiotics as Keeping this view, present work will provide a positive controls (streptomycin and ampicillin) were comparative study of sensitivity of bacteria and fungi used for comparison with the extracts regarding against P. appendiculatum which indicates that the antibacterial activity; fungicides (Chloramine T for F. liverworts are rich store house of antibacterial and oxysporum f. sp. lycopersci, T. indica, S. rolfsii and R. antifungal substances. The observations of this work solanii) were used as positive control for antifungal will play a key role for biological control of plants activity, and respective solvents as negative control. In pathogens. assay for antifungal activity, potato dextrose agar was poured aseptically in the plates and kept for MATERIAL AND METHODS solidification at (28 ± 2) °C for 72 h. Four discs, two A. Collection of bryophytes treated with plant extracts and two controls along with Bryophytes were collected from walls, roofs and natural the test fungus were kept in same Petri plate. rocks where nearly no overhanging vegetation or tree Percentage (%) Inhibition of fungal growth was canopy was present. They were collected only from calculated by the following formula: over approx. 1.5 m above ground level, to avoid road- % Inhibition = Mycelial growth (control) - Mycelial water splashes and areas rich with domestic wastes. growth (treatment)/Mycelial growth (control) × 100 The survey was made in urban, sub-urban and rural where, mycelial growth was determined by measuring sites of Kumaon hills. For the present study, only the diameter of the fungus both in control and mature plants were collected and brownish or pale treatment. yellow and dried plants were rejected. E. Determination of minimum inhibitory concentration B. Identification and Taxonomy (MIC) and minimum bactericidal concentration (MBC) Collection of bryophytes was made during 2009-2010 or minimum fungicidal concentration (MFC) of organic from different parts of Kumaon hills. They were extracts brought to the laboratory in plastic bags and identified Micro broth dilution assay was done to determine both on the basis of morphological examination and with the inhibitory and bactericidal/fungicidal concentration of help of various available literatures (Chopra, 1975; organic extracts (Janovská et al., 2003). Freshly Smith, 1978; Haji, 1984; Gangulee, 1969; Saxena et al., prepared nutrient broth for bacteria and potato dextrose 2008). broth for fungi were used as diluents. Fresh and revived C. Preparation of plant extract for antimicrobial culture of test microorganisms were diluted 100 folds in activity broth (100 μL of microorganism in 10 mL broth). For The plant material was carefully cleaned from attached inoculation of culture, CFU was determined and was litter and dead material, under running tap water and found to be 1 × 106 CFU/mL for bacteria, while it was 1 finally with sterile distilled water, shade-dried and then × 109 CFU/mL for fungi by taking optical density at finely powdered (100 g) with the help of a grinder. The 620 nm using UV-visible spectrophotometer. powdered plant material was then Soxhlet extracted Decreasing concentrations of the plant extract (1000 to with 500 ml of different organic solvents in different 0.98 μg/mL) in two fold dilution series were added to solvents such as petroleum ether, chloroform, acetone, the test tubes containing the fresh microorganism ethanol, methanol. The extracts were filtered through cultures. All tubes with bacterial and fungal organisms muslin cloth and kept at room temperature till complete were incubated at 37°C for 24 h and 28°C for 72 h, evaporation. Different concentrations of crude extract respectively. Singh, Srivastava and Khanna 124 Visible turbidity and optical density of cultures were RESULTS AND DISCUSSION determined at 620 nm by using UV visible The results obtained showed that most of the test spectrophotometer. The lowest concentration that microorganisms were sensitive to the organic extracts inhibited visible growth of tested organisms was of P. appendiculatum in dose dependent manner. recorded as MIC, and that caused no visible microbial All organic extracts of P. appendiculatum showed growth was considered as MBC. inhibition at different conc. (1000, 500, 200 and 40 µg F. Data Analysis ml-1) and at different time intervals i.e. 24, 48 and 72 The statistical evaluation of complete data was done hrs. Percent inhibition in petroleum ether extract ranged and all