Biological Forum – An International Journal 2(2): 122-129(2010)

ISSN No. (Print): 0975-1130

Potential of appendiculatum on Inhibition of Certain Economically Important 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 , 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 several fungicidal and insecticidal seed treatment to under family . 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 (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 analyses were performed based on three from 10.64 minimum at 72 hrs in 40 µg ml-1 conc. to replicates. Values were expressed as mean ± SE. 28.25 at 24 hrs in 1000 µg ml-1 conc. In addition, ANOVA revealed level of significance at P<0.05 methanol extract ranged from 8.47 to 23.48 with among different microorganisms and different extracts minimum at 72 hrs in 40 µg ml-1 conc. and maximum at by using (JMP 5.0, SAS Institute, Cary, NC, USA) to 24 hrs in 1000 µg ml-1 conc. (Table 1). analyze the effect of each treatment separately. The treatment means were separated using Tukey HSD test.

Table 1: Percent inhibition in the growth of F. oxysporum f. sp. lycopersici with different extract of P. appendiculatum

Nature Concentration (µg ml-1) of Time (hrs.) extract 1000 500 200 40 24 48 72 24 48 72 24 48 72 24 48 72 Petroleum 28.25b 26.23b 24.45b 26.41b 23.61b 20.57b 20.31c 18.75c 14.73b 15.46d 13.90d 10.64b ether ± 0.28 ± 0.27 ± 0.40 ± 0.42 ± 0.23 ± 0.39 ± 0.32 ± 0.34 ± 0.31 ± 0.38 ± 0.30 ± 0.42 23.48d 20.12d 18.43c 20.43d 18.38d 15.27d 16.46e 12.65f 10.79d 13.83e 10.82e 8.47c Methanol ± 0.41 ± 0.24 ± 0.32 ± 0.40 ± 0.45 ± 0.30 ± 0.41 ± 0.46 ± 0.38 ± 0.31 ± 0.22 ± 0.36 21.90e 19.43d 18.49c 18.12e 15.72e 13.53e 16.81e 14.21e 12.79c 13.92e 10.87e 8.67c Chloroform ± 0.12 ± 0.41 ± 0.43 ± 0.17 ± 0.28 ± 0.42 ± 0.37 ± 0.22 ± 0.30 ± 0.36 ± 0.49 ± 0.53 33.30a 30.51a 27.48a 28.49a 25.44a 23.33a 26.46a 23.47a 19.39a 20.89a 17.77a 13.85a Ethanol ± 0.49 ± 0.44 ± 0.40 ± 0.43 ± 0.39 ± 0.33 ± 0.41 ± 0.48 ± 0.30 ± 0.80 ± 0.28 ± 0.25 25.84c 23.50c 24.46b 20.37d 18.47d 17.42c 18.39d 16.18d 14.45b 16.61c 14.77c 10.84b Acetone ± 0.24 ± 0.47 ± 0.43 ± 0.43 ± 0.41 ± 0.38 ± 0.50 ± 0.41 ± 0.27 ± 0.16 ± 0.39 ± 0.36 Chloramine 22.14e 25.26b 26.49a 23.45c 21.48c 24.28a 22.50b 20.40b 18.51a 19.46b 16.72b 13.22a T ± 0.65 ± 0.43 ± 0.35 ± 0.41 ± 0.36 ± 0.31 ± 0.39 ± 0.35 ± 0.58 ± 0.34 ± 0.48 ± 0.28 * Values are represented as mean ± SD. *The treatment means were separated using Tukey HSD at 0.05% probability level *Level not connected by same letter are significantly different in vertical columns. Out of the five extracts (organic) of P. appndiculatum, concentration (Table 2). As compared to standard the maximum percent inhibition was observed at 24 hrs antifungal petroleum ether extract shows good activity. (33.30) at 1000 µg ml-1 conc. in ethanol extract and Whereas, the extracts of P. appendiculatum showed the minimum at 72 hrs (8.47) in methanol at 40 µg ml-1 maximum percent inhibition at 24 hrs (22.73) at 1000 concentration when used against Fusarium oxysporum µg ml-1 concentration and minimum at 72 hrs (5.36) in (Table 1). Inaddition, Tilletia indica, organic extracts of methanol at 40 µg ml-1 concentration against Sclerotium P. appendiculatum, and the maximum percent rolfsii. (Table 3). inhibition was observed at 24 hrs (28.71) in petroleum ether extract at 1000 µg ml-1 concentration and minimum at 72 hrs (8.60) in chloroform at 40 µg ml-1 Singh, Srivastava and Khanna 125 Table 2: Percent inhibition in the growth of T. indica Mitra with different extract of P. appendiculatum.

Nature Concentration (µg ml-1) of Time (hrs.) extract 1000 500 200 40 24 48 72 24 48 72 24 48 72 24 48 72 Petroleum 28.71a 24.41b 20.55c 26.46a 23.80a 17.92c 23.45a 20.38b 18.46b 19.15a 14.43c 11.42b ether ± 0.43 ± 0.44 ± 0.43 ± 0.43 ± 0.46 ± 0.20 ± 0.40 ± 0.37 ± 0.36 ± 0.13 ± 0.43 ± 0.37

23.21c 20.65c 16.28e 22.38bc 18.68c 14.72d 19.78c 15.81d 12.51c 17.83b 13.50c 10.43c Methanol ± 0.18 ± 0.45 ± 0.38 ± 0.47 ± 0.31 ± 0.38 ± 0.19 ± 0.19 ± 0.49 ± 0.22 ± 0.45 ± 0.34

22.40c 25.43b 18.34d 18.38d 17.75c 12.87e 15.81d 13.46e 10.49d 12.47d 13.48c 8.60d Chloroform ± 0.94 ± 0.37 ± 0.30 ± 0.45 ± 0.36 ± 0.28 ± 0.40 ± 0.27 ± 0.38 ± 0.43 ± 0.48 ± 0.55

25.65b 23.46b 20.48c 23.45b 20.77b 17.51c 16.52d 13.51e 10.82d 13.71d 10.43d 8.79d Ethanol ± 0.38 ± 0.34 ± 0.32 ± 44 ± 0.28 ± 0.36 ± 0.49 ± 0.49 ± 0.23 ± 0.30 ± 0.40 ± 019

25.85b 24.70b 26.64a 23.38b 24.75a 23.54b 21.44b 23.49a 20.35a 15.82c 18.45a 14.59a Acetone ± 0.34 ± 0.22 ± 0.45 ± 0.43 ± 0.57 ± 0.42 ± 0.31 ± 0.35 ± 0.26 ± 0.87 ± 0.27 ± 0.34

Chloramine 25.33b 28.43a 23.42b 22.46c 20.81b 24.40a 21.47b 17.39c 19.50a 20.39a 16.34b 14.34a T ± 0.33 ± 0.30 ± 0.48 ± 0.35 ± 0.38 ± 0.30 ± 0.37 ± 0.24 ± 0.38 ± 0.34 ± 0.33 ± 0.72 *Values are represented as mean ± SD. *The treatment means were separated using Tukey HSD at 0.05% probability level. *Level not connected by same letter are significantly different in vertical columns.

Table 3: Percent inhibition in the growth of S. rolfsii with different extract of P. appendiculatum.

Nature Concentration (µg ml-1) of Time (hrs.) extract 1000 500 200 40 24 48 72 24 48 72 24 48 72 24 48 72 Petroleum 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ether 22.73a 14.42c 11.42bc 21.64a 13.79b 10.22d 15.63a 14.29a 12.23ab 11.15b 10.36bc 5.36c Methanol ± 0.36 ± 0.34 ± 0.49 ± 0.22 ± 0.43 ± 0.50 ± 0.34 ± 0.65 ± 0.97 ± 0.66 ± 0.80 ± 0.88

20.82b 16.50b 10.58c 20.79b 9.83c 12.78b 14.81ab 12.67b 12.69ab 12.61a 11.48b 7.94b Chloroform ± 0.33 ± 0.44 ± 0.49 ± 0.44 ± 0.49 ± 0.44 ± 0.74 ± 0.69 ± 0.46 ± 0.31 ± 0.49 ± 0.20

19.02c 16.46b 12.41b 16.66c 14.29b 11.43c 13.44c 12.18b 11.07b 10.94b 9.72c 9.30b Ethanol ± 0.13 ± 0.43 ± 0.32 ± 0.41 ± 0.55 ± 0.43 ± 0.36 ± 0.55 ± 0.83 ± 0.37 ± 0.54 ± 0.76 Acetone 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Chloramine 19.79d 21.49a 18.61a 16.29c 18.67a 15.31a 14.45bc 15.13a 13.27a 12.42a 14.41a 11.30a T ± 0.39 ± 0.16 ± 0.55 ± 0.21 ± 0.62 ± 0.56 ± 0.47 ± 0.41 ± 0.83 ± 0.18 ± 0.44 ± 0.47 *Values are represented as mean ± SD. *The treatment means were separated using Tukey HSD at 0.05% probability level. *Level not connected by same letter are significantly different in vertical columns. Singh, Srivastava and Khanna 126 Table 4: Percent inhibition in the growth of R. solanii with different extract of P. appendiculatum.

Nature Concentration (µg ml-1) of Time (hrs.) extract 1000 500 200 40 24 48 72 24 48 72 24 48 72 24 48 72 Petroleum 6.49f 5.37d 3.04f 7.31d 7.33c 2.43e 5.28e 9.14c 2.45d 6.31e 3.32d 0.72d ether ± 0.46 ± 0.35 ± 0.19 ± 0.47 ± 0.43 ± 0.40 ± 0.34 ± 0.36 ± 0.48 ± 0.31 ± 0.43 ± 0.25 15.14c 17.60a 15.23d 17.34a 14.09b 12.33b 19.30a 8.47cd 7.25c 10.32c 11.38a 10.37b Methanol ± 0.31 ± 0.51 ± 0.43 ± 0.37 ± 0.18 ± 0.18 ± 0.36 ± 0.32 ± 0.33 ± 0.27 ± 0.36 ± 0.40 16.24b 17.07a 16.17c 16.97a 14.52b 12.38b 14.37b 12.42a 10.74ab 12.68b 9.54b 6.28c Chloroform ± 0.45 ± 0.21 ± 0.27 ± 0.30 ± 0.36 ± 0.52 ± 0.37 ± 0.32 ± 0.24 ± 0.40 ± 0.49 ± 0.35 11.06e 10.71c 14.18e 13.61c 7.94c 5.63d 10.25d 7.49d 3.39d 8.29 d 2.36e 0.65d Ethanol ± 0.10 ± 0.46 ± 0.12 ± 0.32 ± 0.12 ± 0.30 ± 0.68 ± 0.43 ± 0.52 ± 0.31 ± 0.30 ± 0.29 17.38 14.23b 17.74b 15.36b 14.45b 10.70c 12.30c 11.32b 9.66b 10.80c 8.31c 6.97c Acetone a ± ± 0.25 ± 0.44 ± 0.27 ± 0.43 ± 0.50 ± 0.37 ± 0.54 ± 0.48 ± 0.42 ± 0.43 ± 0.30 0.47 12.30d 14.10b 19.07a 15.97b 16.95a 14.28a 15.23b 13.19a 11.21a 14.34a 12.17a 13.27a Chloramine T ± 0.43 ± 0.28 ± 0.13 ± 0.36 ± 0.23 ± 0.52 ± 0.46 ± 0.33 ± 0.48 ± 0.24 ± 0.16 ± 0.34 *Values are represented as mean ± SD. *The treatment means were separated using Tukey HSD at 0.05% probability level. *Level not connected by same letter are significantly different in vertical columns.

Table 5: Activity Index of the growth of M. plutonius (MP) and P. multocida (PM) with different extract of P. appendiculatum.

Nature Concentration (µg ml-1) of 1000 500 200 40 extract MP PM MP PM MP PM MP PM 8.33d 10.17c 6.19d 9.18d 7.21d 5.23d Petroleum ether 0.0 0.0 ±0.28 ±0.24 ±0.29 ±0.25 ±0.27 ±0.37

10.26c 13.22b 9.25c 11.24c 8.37c 9.19c 6.28c 6.27c Methanol ±0.31 ±0.33 ±0.27 ±0.28 ±0.22 ±0.27 ±0.28 ±0.34

15.23a 15.24a 13.24a 13.21a 11.22a 11.18a 9.16a 9.20a Chloroform ±0.29 ±0.29 ±0.28 ±0.32 ±0.24 ±0.25 ±0.25 ±0.24

Ethanol 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Acetone 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

15.23a 15.20a 13.22a 13.56a 11.22a 11.24a 9.20a 9.20a Ampicillin ±0.29 ±0.28 ±0.29 ±0.52 ±0.26 ±0.28 ±0.28 ±.23

13.22b 15.27a 11.23b 12.22b 10.23b 10.24b 8.22b 8.19b Streptomycin ±0.22 ±0.30 ±0.36 ±0.35 ±0.35 ±0.28 ±0.34 ±0.28 *Values are represented as mean ± SD. *The treatment means were separated using Tukey HSD at 0.05% probability level. *Level not connected by same letter are significantly different invertical columns. Singh, Srivastava and Khanna 127 For Rhizoctonia solani, out of all the organic extracts Non significant values were observed in chloroform at of P. appendiculatum, the maximum percent inhibition all concentrations over to both the positive controls i.e. was observed at 72 hrs (17.74) in acetone extract at ampicillin and streptomycin. In the present finding, 1000 µg ml-1 conc. and minimum at 72 hrs (0.65) in M. plutonius was found to be most sensitive bacteria ethanol at 40 µg ml-1 concentration (Table 4). while F. oxysporum f. sp. lycopersici and T. indica All the organic extracts of P. appndiculatum showed Mitra were most sensitive fungi. This broad spectrum strong and broad spectrum inhibition against F. antimicrobial activity of the liverwort extract is because oxysporum, T. indica, S. rolfsii, R. solanii, M. plutonius of the presence of phinolics, flavonoids, and other and P. multocida. Earlier reports also suggested good antimicrobial substances. antimicrobial activity in organic extracts of liverwort. Bodade et al. (2008) indicated that the extracts of Wolter (1964) studied antifungal activities of 18 species different bryophytes were found to be active against of bryophytes. Out of which, Diplophyllum albicans, different microorganisms. Ethanolic, acetone and Plagiothecium denticulatun and Pogonatum aloides chloroform extracts were found to be more effective on showed remarkable antifungal activity. Number of Escherichia coli and Staphylococcus aureus. Among studies were carried out in past many year. Dubey et al. the fungi Aspergillus niger was most sensitive to the (2002) screened acetone extract of Conocephalum ethanolic extract of Plagiochasma appendiculatum and conicum, Marchantia polymorpha and P. Bryum argentium. In the present study, ethanol extract appendiculatum against two fungi (Aspergillus niger was also most effective against the Salmonella enterica; and Candida albicans) and two Gram-negative bacteria acetone extract was most effective against the (Escherichia coli and Salmonella typhi) and found that Melissococcus plutonius and chloroform extract was acetone soluble extract of all the bryophytes showed most effective against the Xanthomonas oryzae. The inhibitory effect against the pathogens. In the present MBC and MFC values were found to be highest for study only the acetone extract of P. appendiculatum acetone extracts than their MIC value (Table 6). This showed potent activity against the fungus Rhizoctonia may be due to impure form of bioactive compounds. In solani. Later on, Singh et al., (2006) reported that the some cases, the MIC and MBC/MFC values were same alcoholic and aqueous extract of P. appendiculatum which may be due to the presence of specific group of used ethno medically by Gaddi tribe in Kangra valley compounds as also indicated in other studies. for treating skin diseases and showed significant The MIC values were greater than their respective antibacterial and antifungal activity with MIC value of MFC/MBC values. This may be due to impure form of 2.5 μg/disc. Two year later, Bodade et al., (2008) tested the bioactive compounds while in some cases these the antimicrobial activity of P. appendiculatum and values were equal to their respective MFC/MBC values some other bryophytes and showed that among the which may be due to presence of a specific group of different tested fungi A. niger, Fusarium monili, compounds. Mewari et al., (2008) also found that the Rhizoctonia bataticola and Fusarium moniliforme were MBC/MFC values were higher in case of methanol most sensitive to the ethanolic extract of P. extract than their MIC values. While free flavonoid appendiculatum. For Melissococcus plutonius, out of extract showed the same MBC/MFC and MIC values. the five organic extracts of P. appendiculatum, the An overview of bioactivity data obtained from the activity was maximum 15.23 with chloroform extract present investigation indicates higher antifungal activity and minimum 6.19 with petroleum ether extract (Table of methanol, acetone and chloroform extracts of P. 5). On comparing chloroform with ampicillin (positive appediculatum of liverwort than the other extracts. A control) non significant values were obtained at all perusal of different extracts of organic, from P. concentrations. appediculatum with different concentrations (1000, All the extracts of P. appendiculatum failed to inhibit 500, 200 and 40 µg ml-1) and at different time intervals growth of Xanthomonas oryzae and S. enterica. In case (24, 48 and 72 hrs) indicated maximum inhibition on of Pasteurella multocida out of the five extracts of P. the growth with ethanol extract of P. appendiculatum at appendiculatum, the activity was maximum 15.24 with 1000 µg ml-1 concentrations. chloroform extract and minimum 5.23 in petroleum ether extract (Table 5). Singh, Srivastava and Khanna 128 Table 6: MIC, MFC and MBC of different extract of P. appendiculatum different pathogens (µg/ml).

Pathogen Petroleum ether Methanol Chloroform Etahnol Acetone STANDARDS

MBC/ MBC/ MBC/ MBC/ MBC/ MIC Fungi MIC MIC MIC MIC MIC MFC MFC MFC MFC MFC (MBC/MFC) S. rolfsii - - 1.25 1.75 1.25 1.75 1.25 1.75 - - 0.50 (0.80) R. solani 2.50 5.00 1.25 1.75 2.50 2.50 2.50 4.50 2.50 2.50 1.00 (1.25) T. indica 1.25 1.85 0.65 1.00 1.25 2.00 1.75 2.00 0.65 1.25 0.50 (0.60) F. oxysporium 1.25 1.75 1.25 1.85 1.25 1.75 0.65 0.65 1.25 1.25 0.50 (0.60) Bacteria P. multocida 1.25 1.65 1.25 2.50 0.65 1.25 - - - - 0.50 (0.70) S. enterica ------0.25 (0.25) M. plutonius 2.50 5.00 1.25 2.50 0.65 1.25 - - - - 0.65 (0.80) X. oryzae ------0.65 (0.75)

Chloramines T and Ampicillin are used as standards for fungi and bacteria respectively. CONCLUSION ACKNOWLEDGMENTS The present investigation clearly indicates that bryophytes are The authors are thankful to Dr. Anil Kumar Sharma and Dr. rich storehouse of unknown botanicals, justifying a more Rashmi Srivastava, Pantnagar, for their support at various extensive and critical analysis of bioactivity for this group. stages. Help rendered by G. B. Pant University of Agriculture From this study, it may be concluded that P. appendiculatum and Technology, Pantnagar, Uttarakhand, India is gratefully have a vast potential as botanical pesticides and can serve as acknowledged. new source of fungicide and antibiotics in near future. Singh, Srivastava and Khanna 129

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