Molecular characterization of fungal endophyte diversity isolated from Aconitum heterophyllum: a critically endangered medicinal plant of Kashmir Himalaya.

Aconitum heterophyllum is a rare perennial herb from Kashmir Himalayas. Due to its threatened status and dependence on its environment, the plant was examined for any potential endophytes, which is of utmost importance for bioprospection. In the current study, endophytic fungal diversity associated with A. heterophyllum was examined, and 328 fungal isolates were found in the plant's leaf, stem, and root tissues. Twelve (12) endophytic fungal species were identified utilizing, molecular analysis of the nuclear ribosomal DNA Internal Transcribes Spacer (ITS), rLSU, and rSSU sequences. Maximum likelihood analysis was used to determine the phylogenetic connection between each isolate. The genera Arthrinium, Chaetomium, Purpureocillium, Alternaria, Penicillium, Aspergillus, Cladosporium, and Bjerkandera species dominated the ascomycete and basidiomycete fungal endophytes.

Patterns of floristic and functional diversity in two treeline ecotone sites of Kashmir Himalaya.

Globally, the treelines at higher elevations in mountains are reported to be advancing up-slope in response to recent climate warming. However, little is known about the treeline advancement in the Himalaya due to paucity of baseline vegetation data with which to compare, thus making their assessment and monitoring challenging. To fill this knowledge gap, the present study documented floristic and functional diversity of two treeline ecotone sites in Kashmir Himalaya. At each site, we conducted field sampling by laying five 20-m2 plots, with one at the highest limit (T0 plot), two plots below and two above the treeline and two nested subplots of 5-m2 for shrubs and five 1-m2 for herbs in each plot. We recorded 97 plant species belonging to 33 families from the two sites. We observed a considerable difference in species composition and distribution along the treeline ecotone. Majority of the species reported were perennial herbs. We observed a significant association of growth forms with the particular plots along the treeline ecotone. At both the sites, we recorded highest species richness at the T0 plot which was correlated well with the functional traits, thus indicating convergence of floristic and functional diversity at this transition zone. Interestingly, the T0 plot at both the sites showed maximum overlap of species with the plots above and below the treeline. In an era of climate warming, our study provides crucial baseline data that will facilitate assessment and monitoring of the Himalayan treelines.

Development and validation of a reverse phase HPLC-DAD method for separation, detection & quantification of rutin and quercetin in buckwheat (Fagopyrum spp.).

Buckwheat has tremendous nutraceutical potential owing to its rutin and quercetin content. The aim of this study was to optimise and validate an analytical method for separating and quantifying these two flavonoids from it. Factors, such as range, linearity, precision, accuracy, limit of detection and limit of quantification, were evaluated for the two compounds using high performance liquid chromatography. On the basis of resolution and symmetry, mobile phase consisting of methanol and methanol:water:acetic acid in the ratio of (100:150:5), flow rate 1.3 ml/min and column temperature 30 °C were found to be optimal analytical conditions. Calibration curves exhibited good linearity with correlation coefficient of 0.995 & 0.9907 over the range 60-180 µg/ml & 2-10 µg/ml for rutin and quercetin respectively. LOD and LOQ values for rutin and quercetin were 6.36, 0.58 and 19.28, 1.77 µg/ml respectively. Recovery values of 96-100.8% confirmed that the method was accurate for rutin and quercetin analysis. This validated method was successfully used to analyse rutin and quercetin in leaves and seeds of buckwheat plant.

Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.).

Saffron (Crocus sativus L) is a triploid (2n = 3x = 24), sterile geophyte which can only be propagated by means of underground vegetative corms. Since corm multiplication does not induce genome variations, therefore, the entire saffron population is expected to have a similar genetic makeup. Keeping in view the economic importance of the plant and the factors responsible for its low yield, the present investigation has been undertaken to establish an in vitro ethyl methanesulfonate (EMS) mutagenesis protocol followed by characterization of the induced variability in the advanced generations. The present report is limited to standardization of in vitro mutagenesis protocol only. Among the mutagenic treatments tested, concentrations ranging from 0.1 to 0.5% EMS showed a varied survival of explants. Based on various growth parameters, the LD50 was calculated to be 0.3% EMS for 3 h. Among the two types of explants analyzed, the corm explant gave better results for in vitro survival and the growth parameters than callus explant. An average of 57.33 and 92.00 daughter cormlets in all EMS treatments as compared to 47.67 and 57.67 daughter cormlets in control, obtained from callus and corm explants respectively, were transferred to the field. The maximum, average daughter cormlet weight was obtained in control (3.01 g, corm explant) followed by 0.1% EMS (2.8 g, corm explant). In general, the growth parameters showed decreasing trend with an increase in EMS concentration in both the explants. The present study has been a significant achievement in the sense that the first mutagenesis protocol for C. sativus has been standardized.

Assessment of species richness in Lake Dal, Kashmir, based on classical approach, physiological approach and rDNA ITS sequences from isolates.

As a first description to document the species richness in Dal Lake, a freshwater lake ecosystem in Kashmir valley, an extensive network of sixteen sampling stations with distinguishing features was sampled seasonally for two years. The identification process yielded fifty-one species probably first and new records for this area to date. The taxonomic groups observed were those with species from Ascomycetes (inclusive of yeasts), Basidiomycetes, Blastocladiomycetes, Zygomycetes, and Peronosporomycetes. Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In the filamentous fungal group, family Trichocomaceae was dominant followed by Saccharomycetaceae, Mucoraceae, Nectriaceae, Tremellaceae and Hypocreaceae. However, in the group of zoosporic & fungal like eukaryotes, family Saprolegniaceae was most dominant followed by Blastocladiaceae and Pythiaceae. A dramatic decrease in fungal load was observed in different seasons with highest colonial load in the summer season and lowest in the winter season. The observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) as well as zoosporic fungi & fungal like eukaryotes (p < 0.05). In order to assess biodiversity patterns of fungi more accurately, it is necessary to repeat such investigations in other areas and to improve the tools for taxonomic identification of these highly diverse but mostly microscopic organisms.

Dynamics of bacterial class Bacilli in the deepest valley lake of Kashmir-the Manasbal Lake.

In recognition of the importance of bacteria as ecological indicators of the aquatic systems a comprehensive and systematic analysis was carried out on Manasbal Lake, the deepest spring fed valley lake of Kashmir. The main objective envisaged was to analyze bacterial community composition (BCC) and for this purpose systematic and regular sampling of waters from ten different sampling stations, predetermined in the Lake according to differences in degree of human interference and also as zones of special ecological interests were selected. The isolated species were identified according to Bergey's Manual specification by examining their micro and macro morphological characteristics and biochemical characteristics on different culture media. Further confirmation was done by sequencing the 16s rRNA gene by using universal bacterial primers 27F and 1429R. From all the sampling stations the class Bacilli showed a maximum relative abundance with a contribution of 16 bacterial species. The whole process resulted in the identification of Bacillus aerius, Bacillus altitudinis, Bacillus anthracis, Bacillus cereus, Bacillus ginsengisoli, Bacillus pumilus, Bacillus safensis, Bacillus stratosphericus, Bacillus subtilis, Bacillus tequilensis, Bacillus thermocopriae, Bacillus thuringiensis, Brevibacillus agri strain, Lysinibacillus boronitolerans, Lysinibacillus pakistanensis and Lysinibacillus sphaericus.

Aquatic bacterial diversity: Magnitude, dynamics, and controlling factors.

The primary aspiration in the microbial observatory is to advance the understanding of freshwater bacterioplankton, whose diversity and population dynamics are currently the least understood off all freshwater planktonic organisms. Through identification and characterization of bacterial populations in a suite of fresh water bodies, we are able to gain significant new insight into the ecological niches of bacteria in diverse freshwater ecosystems. Given the facts that lakes and other inland freshwaters play a more critical role in the global carbon budget and that lakes have been described as early indicators of both regional and global environmental change, the role of microbes in these processes is of renewed interest. In this review, general overview will be given highlighting the characteristic features of bacterial species thriving in different water bodies. In the following sections, different cultural approaches vis a vis the controlling factors of bacterial diversity have been elaborated. In the concluding sections, the prospects of aquatic microbial diversity are well mentioned.

Antimicrobial activity of crude fractions and morel compounds from wild edible mushrooms of North western Himalaya.

The antimicrobial properties of morel compounds from wild edible mushrooms (Morchella esculenta and Verpa bohemica) from Kashmir valley was investigated against different clinical pathogens. The butanol crude fraction of most popular or true morel M. esculenta showed highest 19 mm IZD against E.coli while as same fraction of Verpa bohemica exhibited 15 mm IZD against same strain. The ethyl acetate and butanol crude fractions of both morels also exhibited good antifungal activity with highest IZD shown against A. fumigates. The three morel compounds showed quite impressive anti bacterial and fungal activities. The Cpd 3 showed highest inhibitory activity almost equivalent to the synthetic antibiotics used as control. The MIC/MBC values revealed the efficiency of isolated compounds against the pathogenic strains. In the current study significant inhibitory activity of morel compounds have been obtained paying the way for their local use from ancient times.

Breaking the resistance of Escherichia coli: Antimicrobial activity of Berberis lycium Royle.

The antimicrobial activity of root bark of Berberis lycium and its principal component berberine was tested against a panel of microbial strains using agar well diffusion test and further analyzed using micro-broth dilution method. Preliminary analysis, on the basis of zone of Inhibition (ZOI) showed that the methanolic extract of B. lycium was highly effective against Escherichia coli (ZOI 41 ± 1 mm). Among the bacterial strains E. coli was found to be most susceptible and among fungi Candida albicans was the most susceptible for berberine as well as the crude methanolic extract of the plant. Methanolic extract of the plant was more effective for E. coli (MIC 1.7 ± 1.18; MBC 2.4 ± 1.18) than berberine (MIC 3.5 ± 0.57) (p < 0.05), whereas berberine was more effective than crude extracts for C. albicans. In addition, E. coli showed the development of resistant colonies after 72 h when tested with berberine but the development of such colonies was not observed with the methanolic extract of the plant. This could be due to the presence of resistance breaking molecules in the crude methanolic extract of B. lycium. Also the MIC index of crude methanolic extract was 1.39 for E. coli, which showed the mode of action to be bactericidal. HPLC analysis revealed the presence of berberine at highest concentration in methanolic extract of the plant, followed by aqueous extract. Potentiation of this berberine by resistance breaking molecules in the crude extract could be a possible explanation for its strong effectiveness.

Opportunistic fungi in lake water and fungal infections in associated human population in Dal Lake, Kashmir.

Natural habitats of opportunistic fungal pathogens are outside of the host; therefore, it is critically important to understand their ecology and routes of transmission. In this study, we investigated the presence of human pathogenic opportunistic fungi in lake water and incidence of fungal infections in associated population in Kashmir, India. Six hundred forty water samples were taken on seasonal basis from a wide network of sampling stations of the lake for an extended period of two years for screening their occurrence. The samples were inoculated onto rose bengal agar, malt extract agar, potato dextrose agar and other specified culture media supplemented with Chloramphenicol and Streptomycin followed by incubation at 37 °C. All the samples were positive for fungi, which were later identified by sequencing the rDNA internal transcribed spacer region aided by classical morphological culture techniques and physiological profiling. The whole process led to the isolation of sixteen species of opportunistic fungal pathogens belonging to genus Aspergillus, Candida, Penicillium, Cryptococcus, Fusarium, Rhizopus and Mucor in decreasing order of prevalence. Furthermore, 20% population (n = 384) of Dal inhabitants was examined for possible fungal infections and it was observed that only 8.07% individuals were positive for fungal infections with 4.68% skin infection cases, 2.34% onychomycosis cases and 1.04% candidiasis cases. Scrapings from onychomycosis and candidiasis patients showed the presence of Aversicolor and Calbicans respectively, resembling exactly the strains isolated from the lake water. However, the skin infection was because of a dermatophyte not isolated for the lake water. Higher prevalence of infection (6.77%) was seen in people using lake water followed by a positive prevalence of 1.30% using tap water. The results of present study suggest that the lake inhabitants are at a greater risk of getting life threatening fungal diseases which may lead to various morbidities.

Detection and characterization of potentially pathogenic Aeromonas sobria isolated from fish Hypophthalmichthys molitrix (Cypriniformes: Cyprinidae).

The current study focuses on the detection and characterization of potentially pathogenic Aeromonas sobria from fish silver carp (Hypophthalmichthys molitrix). Assessment of clinical, microbiological, pathological and biochemical characteristics of A. sobria were taken into account in order to understand the epidemiology, frequency and occurrence of this infection. Clinically the infected fish (H. molitrix) was observed for various types of symptoms. A total of 33 colonies of A. sobria strain were isolated from 20 cultured H. molitrix, collected from controlled fish pond. Microscopic examination revealed that the strains were rod-shaped, Gram negative bacteria. The revealed percent probability identification of A. sobria from the biochemical characterization in VITEK system was 93% with gram negative (GN) card. The histopathology of Gills caused by this bacterium, A. sobria indicate haemorrhagic gill epithelia and epithelial hyperplasia. Lamelar epithelial hypertrophy and hyperplasia with degenerative changes of the epithelium and hypertrophic epitheliocystis infected cells on gills of H. molitrix were observed during the present study.

Synergistic approach for treatment of chicken coccidiosis using berberine--A plant natural product.

Despite the advent of anticoccidial drugs and vaccines, coccidiosis continues to result in substantial economic losses to the poultry industry. Berberine, a natural alkaloid is well known in studies involving synergistic approaches, thereby reducing the dosage of principal drugs. Therefore, a study was designed to see whether a synergistic anticoccidial effect could be obtained between amprolium and berberine, in vivo using broiler chicken. Anticoccidial activity was measured in comparison to the reference drug amprolium on the basis of oocyst output reduction, mean weight gain and feed conversion ratio. Oocyst output was measured using Mc-Masters counting technique. Different combinations of berberine and amprolium were tested and out of which 1:1 ratio was the most effective for controlling these parasites. Oral gavaging of 100(50 + 50) mg/kg body weight of 1:1 ratio of amprolium and berberine caused the equivalent reduction in number of oocysts (38.85 ± 9.61) one day prior to that of standard drug amprolium (49.95 ± 16.65) as well as pure berberine (44.4 ± 9.61) used in the study. Weight gain of birds was also highest in the synergistic group (1547.43 ± 12.86) among all the infected groups. Besides feed conversion ratio in the synergistic group was also better (1.387 ± 0.026). The results of this study proved the effectiveness of both amprolium and berberine and revealed synergism between amprolium and berberine against coccidian oocysts, confirmed by significant reduction in the number of coccidian oocysts shed in the feces, leading to better weight gain and improved feed conversion ratio. The study deep-rooted the synergistic potential of berberine, a natural bioactive compound for controlling a protozoan parasite and the results of this study corroborate with its use for treatment of severe diarrhoea, amoebiasis and intestinal infections.

Enhanced production of α-amylase by Penicillium chrysogenum in liquid culture by modifying the process parameters.

In this paper, we have assessed the role of changing physicochemical parameters and substrate types on the production of α-amylase enzyme from Penicillium chrysogenum, with a view to determining the optimal conditions required for its maximum production. The findings of this research revealed that, at pH 6 using linseed oil cake as substratum, α-amylase enzyme production was maximum (550.0 U/mL), when the fungi was incubated for 6 days at 30 °C in 0.1 M acetate buffer. Further, reasonably good production of the α-amylase enzyme was also observed at pH 9 with all the experimented carbon sources as substrates. Moreover, statistical analysis, using analysis of variance (ANOVA) carried out to study the impact of different studied parameters on the α-amylase enzyme production revealed that incubation period of 6-18 days is highly significant (p = 0.01) factor in amylotic activity of the P. chrysogenum. Under the researched out optimal conditions, P. chrysogenum is an economically viable option for the industrial and biotechnological production of α-amylase enzyme.

Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: A review.

Endophytic fungi are those that live internally in apparently healthy and asymptomatic hosts. Endophytic fungi appear to be ubiquitous; indeed, no study has yet shown the existence of a plant species without endophytes. High species diversity is another characteristic of endophytic mycobiota which is depicted by the fact that it is quite common for endophyte surveys to find assemblages consisting of more than 30 fungal species per host plant species. Medicinal plants had been used to isolate and characterize directly the bioactive metabolites. However, the discovery of fungal endophytes inside these plants with capacity to produce the same compounds shifted the focus of new drug sources from plants to fungi. Bioactive natural products from endophytic fungi, isolated from different plant species, are attracting considerable attention from natural product chemists and biologists alike which is clearly depicted by the steady increase of publications devoted to this topic during the recent years. This review will highlight the chemical potential of endophytic fungi with focus on the detection of pharmaceutically valuable plant constituents as products of fungal biosynthesis. In addition, it will cover newly discovered endophytic fungi and also new bioactive metabolites reported in recent years from fungal endophytes. It summarizes the up-to-date and comprehensive information on bioactive compounds from endophytic fungi by having done a thorough survey of literature.

Municipal solid waste generation rates and its management at Yusmarg forest ecosystem, a tourist resort in Kashmir.

The present study was carried out at Yusmarg, a forest ecosystem and tourist resort, in the Kashmir valley during 2012 with the objectives of determining the municipal solid waste (MSW) generation rates per capita and on a daily basis, and assessing the existing MSW system. It was estimated that daily generation of MSW at Yusmarg by tourists, as well as residents, was 107.74 kg; on average, the MSW generated at each site was about 36.48 kg/day. The per capita generation of MSW was highest (0.97 kg/person/day) at site 1 followed by 0.288 kg/person/day at site 2 and 0.201 kg/person/day at site 3, with an average per capita MSW generation rate of 0.484 kg/person/day. Manual segregation of the collected wastes showed that it comprised some recyclable, combustible, compostable and inert materials. Among the different waste categories, 56% of waste was recyclable materials, 29% was compostable wastes, 9% was combustible wastes and 6% was inert materials. The present study infers that MSW management in Yusmarg was inappropriate, and infrastructure, skilled manpower and a proper scientific disposal mechanism is lacking in the area. In order to conserve the forest wealth of the area there is a great need to focus on the solid waste problem of the tourist resort.

Antimicrobial and antioxidant activities of alcoholic extracts of Rumex dentatus L.

In-vitro antimicrobial and antioxidant activities of various concentrations ranging from 150 to 500 µg/ml of alcoholic (methanol and ethanol) extracts of Rumex dentatus were analyzed on different clinical bacterial strains (Shigella flexneri, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus) and fungal strains (Aspergillus versicolor, Aspergillus flavus, Acremonium spp., Penicillium dimorphosporum, Candida albicans, Candida kruesie, Candida parapsilosis) using agar disk diffusion method and broth dilution method (MIC and MBC determination) for antimicrobial activity and DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, Riboflavin photo-oxidation assay, deoxyribose assay, lipid peroxidation assay for antioxidant activity. The extracts showed maximum inhibitory effect against K. pneumonia and P. aeruginosa with no activity against S. typhimurium from among the bacterial strains while as in case of the fungal strains the maximum effect was observed against C. albicans by both the extracts. MIC and MBC values determined for active fractions of the extracts against some bacterial strains (S. flexneri, K. pneumonia and E. coli) revealed that the test organisms were inhibited by all the extracts with methanol showing lower values of both MIC and MBC indicating it as a better antimicrobial agent. The antioxidant activity showed that the extracts exhibited scavenging effect in concentration-dependent manner on superoxide anion radicals and hydroxyl radicals leading to the conclusion that the plant has got a broad spectrum antimicrobial and antioxidant activity and could be a potential alternative for treating various diseases.

Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant.

SR4 genotype of rice is high altitude Himalayan rice prone to various abiotic stresses such as cold stress and therefore gives a poor yield. An efficient protocol for callusing and regeneration via direct and indirect means was established using mature seeds as an explant which can be utilized for molecular studies for genetic advancement of Himalayan rice genotype SR4 through transformation. Highest frequency (96.6%) of callus induction was obtained on MS media 3.0 mg/L 2, 4-D. While maximum regeneration frequency (100%), number of shoots with maximum length 9.14 ± 0.204 (cm) from callus was recovered from MS media amended with 5.0 mg/L BAP in combination with 0.5 mg/L NAA with highest number of shoots having an average shoot length 9.14 ± 0.204 (cm) after four weeks of culture. Direct multiple shoot regeneration from seed explants was obtained using various concentrations of TDZ and BAP with highest regeneration frequency was observed on MS media fortified with 6 mg/L of TDZ with maximum number of shoots. The shoots developed roots on MS media supplemented with 0.6 mg/L IBA.

Silicon supplementation improves early blight resistance in Lycopersicon esculentum Mill. by modulating the expression of defense-related genes and antioxidant enzymes.

Early blight is the most devastating disease in tomato which causes huge yield losses across the globe. Hence, development of specific, efficient and ecofriendly tools are required to increase the disease resistance in tomato plants. Here, we systematically investigate the defensive role and priming effect of silicon (Si) in tomato plants under control and infected conditions. Based on the results, Si-treated tomato plants showed improved resistance to Alternaria solani as there was delay in symptoms and reduced disease severity than non-Si-treated plants. To further examine the Si-mediated molecular priming in tomato plants, expression profiling of defense-related genes like PR1, PR2, WRKYII, PR3, LOXD and JERF3 was studied in control, Si-supplemented, A. solani-inoculated and Si + A. solani-inoculated plants. Interestingly, Si significantly increased the expression of jasmonic acid (JA) marker genes (PR3, LOXD and JERF3) than salicylic acid (SA) marker genes (PR1, PR2 and WRKYII). However, Si + A. solani-inoculated plants showed higher expression levels of defence genes except WRKYII than A. solani-inoculated or Si-treated plants. Furthermore, pre-supplementation of Si to A. solani-infected tomato plants showed increased activity of antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase (POD) than control, Si-treated and A. solani-inoculated plants. Altogether, present study highlights the defensive role of Si in tomato plants in response to A. solani by increasing not only the transcript levels of defense signature genes, but also the activity of antioxidant enzymes.

Phytochemical screening of Prunella vulgaris l. - an important medicinal plant of Kashmir.

Kashmiri medicinal plant (Prunella vulgaris) was analyzed for its chemical composition and amount of bioactive constituents. The results showed that the herb contains on an average alkaloid (1120 mg %), saponins (350 mg %), phenolics (55.785 mg %) and tannins (52.25 mg %). The medicinal plant contained carbohydrates (375 mg %), proteins (441.6 mg %) and lipids (2403.8 mg %). Role of these bioactive principles are discussed according to their folkloric use in Kashmir valley. Besides the herb is of great importance as far as its other clinical application are concerned. This quantitative estimation can be used for comparative evaluation of bioactive constituents with other populations of Prunella vulgaris present in different parts of the world and can be used for selection of superior quality of this herb to use in pharmaceutical industries.

Ecology, distribution mapping and conservation implications of four critically endangered endemic plants of Kashmir Himalaya.

During the last few decades, human-driven activities have led to indiscriminate habitat destruction and exploitation of many plant species in Kashmir Himalaya. As a result, many species have become threatened and are struggling for survival. Of particular concern are the endemic and critically endangered species which have the highest risk of the extinction, hence warranting immediate conservation actions. Therefore the current study was carried out to understand the distribution, ecology and conservation implications of the four critically endangered endemic plants of Kashmir Himalaya. Habitat distribution modelling showed that the suitable potential areas for the species occurred from subalpine to alpine meadowlands with an elevational range of 1500-4600 m asl. The output of the MaxEnt model and field surveys have revealed that their highest potential distribution is in Panchari, Khrew, Ramnagar, Pahalgam, Gurez, Sonamarg, Gulmarg and Kishtwar forest ranges. Based on the field explorations and herbarium records, Saussurea costus (Falc.) Lipsch have 27 distribution areas, Gentiana kurroo Royle 18, Lilium polyphyllum D. Don 12 and Aconitum chasmanthum Stapf have 15. Precipitation of the driest month and annual mean temperature played an important role in the distribution of the studied species. The species started their lifecycle with the onset of the spring season, flowered in summer, fruit in autumn and senesce in the winter season. Under natural conditions, the number of days required for germination ranged from 180 to 210 where cold stratification played a pivotal role. Since last few decades, the populations of these species have been shrinking in their natural habitats due to over-exploitation for medicinal purposes and habitat destruction through amplified humanoid interferences like the expansion of agricultural land, road building, grazing and urbanization. Thus there is an urgent need to come up with positive strategies to save whatever is left and plan long term rescue measures not only to protect these species from extinction but also to reintroduce them along with framing the plans to supply sustained raw materials for medicine.