Journal of Nepal Biotechnology Association <p>The Journal of Nepal Biotechnology Association (J. Nepal Biotech. Assoc.) is an annual scientific publication of the Nepal Biotechnology Association. It is a double-blind peer-reviewed journal that publishes articles on different fields of Biotechnology.</p> Nepal Biotechnology Association en-US Journal of Nepal Biotechnology Association 1728-1628 Screening of In vitro α-amylase Inhibitory Activity of Wild Orchids of Nepal <p>Orchids are the source of compounds like phenols, alkaloids, phenanthrenes widely used as a therapeutic agent. Inhibition of pancreatic <em>α</em>-amylase could be a better therapeutic approach in decreasing levels of post-prandial hyperglycemia. This study aimed to evaluate α-amylase inhibition of some orchid species to assess their inhibitory potential on PPA (porcine pancreatic <em>α</em>-amylase). Methanol extracts of the whole plant of <em>Gastrochilus distichus</em> (GDW), the pseudobulbs of <em>Otochilus albus</em> (OAP), the whole plant of <em>Papilionanthe uniflora</em> (PUW), pseudobulbs of <em>Eria graminifolia</em> (EGP), the leaves and pseudobulbs of <em>Pholidota articulata</em> (PAL and PAP) and stems of <em>Vanda cristata</em> (VCS) were screened for their phytoconstituents and role in <em>α</em>-amylase inhibition by modified 3,5-Dinitrosalicylic acid method. <em>V. cristata</em>, <em>E. graminifolia</em> and <em>G. distichus</em> extract showed moderate inhibition of <em>α</em>-amylase with IC<sub>50</sub> of 582.73 μg/ml, 710.89 μg/ml, 798.78 μg/ml respectively when compared to acarbose (26.85 μg/ml). Phytochemical analysis revealed the presence of alkaloids, tannins, glycosides, flavonoids, saponins and steroids with the major phytoconstituents. This study concluded that <em>V. cristata</em>, <em>E. graminifolia</em> and <em>G. distichus</em> exhibited moderate<em> α</em>-amylase inhibitory activity and they could be a potent source for antidiabetic phytochemicals.</p> Pusp Raj Joshi Siddharaj Joshi Mukti Ram Paudel Bijaya Pant Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2022-03-05 2022-03-05 4 1 1 7 10.3126/jnba.v4i1.53439 Growth Promoting Effect of Endophytic Bacteria Bacillus subtilis From Leaves of Vanda cristata and Its Potential Impact on In vitro Growth of Orchid <p>The orchids are well known for their microbial association. <em>Bacillus subtilis</em> has great diversity in <em>Vanda cristata</em> which is regarded as a potential growth enhancer for the development of plants. The isolation of its strain was confirmed as <em>Bacillus subtilis</em> by morphological and molecular sequencing of DNA. The isolate was tested for indole acetic acid (IAA) estimation, ammonia production and phosphate solubilization in qualitative and quantitative manners. Its growth-promoting activities were tested on in vitro-raised plantlets of <em>Cymbidium aloifolium</em>. <em>B. subtilis</em> synthesized the IAA with and without a precursor (tryptophan). A more amount of auxin was found by using more amount of tryptophan. During the qualitative test for ammonia and phosphate solubilization, both compounds were synthesized by <em>B. subtili</em>s, which was further confirmed by the quantification method. Further, <em>B. subtilis</em> was used as a biotic elicitor in the in-vitro mass propagation of <em>C. aloifolium</em>. The development of root and shoot was found significant in the tryptophan-treated elicitor than the other compared medium combination. This study concludes that <em>B. subtilis</em> can be used as a biological elicitor to promote the in vitro growth of <em>C. aloifolium</em> and puts the spectrum on microbial relationships with orchid species.</p> Krishna Chand Sujit Shah Bijaya Pant Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 8 16 10.3126/jnba.v4i1.53441 Growth Promoting Role of Phytase Producing Bacteria Isolated from Bambusa tulda Roxb. Rhizosphere in Maize Seedlings Under Pot Conditions <p>Phytase enzymes have focused on their role in agriculture to generate bioavailable phosphorus (P) requires for plant nutrition. Herein, the feasibility of phytase-producing microbes as biofertilizers was studied. Bacteria with greater potential for hydrolysing calcium phytate based on the halo-to-colony ratio from <em>Bambusa tulda</em> Roxb. rhizosphere was isolated. Phytase activity by incorporating wheat bran, phytase screening and Luria Bertani (LB) medium after acetone precipitation was measured. Bacterial genomes were screened for the presence of β-propeller phytase gene corresponding to the <em>Bacillus</em> spp. using polymerase chain reaction (PCR). Finally, the effect of the isolates on the growth of maize seedlings under pot conditions in P-deficient loamy soil was evaluated. Ten distinct bacterial isolates collected from <em>B. tulda</em> rhizosphere were capable of mineralizing phytate and the maximum effect was observed for designated SRBR-04. Most isolates solubilized Ca<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub> as a sole P source in Pikovskaya’s agar. Five isolates selected for the study synthesized auxin in the LB broth supplemented with 1 mg mL<sup>-1</sup> L-Tryptophan (1.63 to 4.5 μg mL<sup>-1</sup>). Phytase production was highest in wheat bran with isolate SRBR-04 producing a maximum of 0.34 U mg<sup>-1</sup>. Two isolates (SRBR-01, SRBR-04) screened positive for the presence of <em>Bacillus</em> phytase gene. Pot assay in P-deficient soil showed significant (p &lt; 0.05) biomass promotion for the isolate SRBR-07 in shoot height (57%), dry shoot weight (178%), dry root weight (104%) and leaf area (113%) over the untreated control. Amendment of P-deficient agricultural soils with phytase-producing bacteria would provide a sustainable approach for P nutrition management in <em>Zea mays</em>.</p> Dhurva Prasad Gauchan Shishir Pandey Bikash Pokhrel Nabin Bogati Puja Thapa Ashesh Acharya Bishnu Maya KC Janardan Lamichhane Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 17 26 10.3126/jnba.v4i1.53442 In vitro Propagation and Assessment of Genetic Homogeneity using RAPD and ISSR Markers in Tinospora cordifolia (Wild.) Hook. F. & Thoms, An Important Medicinal Plant of Nepal <p>The Menispermaceae family includes the widely glabrous, succulent, climbing shrub<em> Tinospora cordifolia</em> (Gurjo), which has been found to have a variety of pharmacological and ethnomedicinal characteristics. <em>T. cordifolia</em> is also one of the most commercially exploited plants in pharmaceuticals. The nodal segment explants were initially cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyl amino purine (BAP), and/or Kinetin (KIN) among which MS +BAP (2.0 mg/l) induced shoot initiation after 7 days of post-inoculation. The nodal segments were then excised and treated with various concentrations of BAP, BAP with KIN, and coconut water for the proliferation among which 5 mg/l induced significant nodal segment proliferation (9.0 nodal segments/per explant) and shoot length (8.0 cm). The efficacy of coconut water in increasing the nodal segment proliferation of <em>T. cordifolia</em> was tested and the shoot proliferation increased significantly at 5% and 10% of coconut water, however, the maximum response of shoot number (23.0), shoot length (12 cm) was in the MS medium supplementation with BAP (5mg/l) and 10% coconut water. The genetic fidelity of these plants was also confirmed by random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers in wild and in vitro cultures. This protocol is an efficient way for the in vitro mass propagation of true-to-type plantlets of <em>T. cordifolia</em> which provides a basis for germplasm conservation and sustainable utilization.</p> Sushma Pandey Lasta Maharjan Bijaya Pant Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 27 36 10.3126/jnba.v4i1.53443 Facile Synthesis of Zinc Carbonate and Zinc Oxide Nanoparticles and Their Antimicrobial Properties <p>ZnCO<sub>3</sub> nanoparticles (NPs) were prepared by the direct precipitation method using zinc nitrate hexahydrate (Zn(NO<sub>3</sub>)<sub>2</sub>.6H<sub>2</sub>O) and ammonium carbonate ((NH<sub>4</sub>)<sub>2</sub>CO<sub>3</sub>) as precursor materials and polyvinyl alcohol (PVA) as capping agent (R1) whereas ZnO NPs were prepared by thermal decarbonation of the prepared ZnCO<sub>3</sub> NPs in the absence (R2) and the presence (R3) of polyvinyl alcohol (PVA) as capping agent. Thus, prepared NPs were subjected to characterization by Fourier transform infrared (FTIR) spectroscopy and phase identification by X-ray diffraction (XRD) study. The effects of the prepared NPs towards different microbial species were also considered. The FTIR spectra of the synthesized ZnCO<sub>3</sub> nanoparticles showed characteristic peaks at about 480 cm<sup>-1</sup> and 1403 cm<sup>-1</sup> correlated to the zinc oxide bond (Zn-O) and carbonate group respectively. Similarly, the FTIR spectra showing a peak at 400 cm<sup>-1</sup> suggested the formation of ZnO NPs. Likewise, the XRD pattern of the synthesized NPs confirmed the formation of single-phase crystalline ZnO NPs with FCC lattice structure. Using Scherrer’s equation, the crystallite diameter of the prepared nanoparticles was found to be 9, 21 and 17 nm for R1, R2 and R3 samples respectively. The antimicrobial actions against the different species of bacteria and fungi were also observed and it was found that ZnO NPs prepared by using a capping agent showed a higher zone of inhibition, than that without using the capping agent. Thus, in the present study, ZnCO<sub>3</sub> and ZnO NPs were prepared by simple, cost-effective precipitation followed by a decarbonation method and these NPs could be a potential antimicrobial agent.</p> Guna B. Karki Kshama Parajuli Subhav Adhikari Shankar P. Khatiwada Rameshwar Adhikari Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 37 43 10.3126/jnba.v4i1.53444 Wild and Cultivated Mushrooms of Nepal as a Source of Nutrients and Nutraceuticals <p>This study was carried out on nutrient and nutraceutical analysis of wild and cultivated edible mushrooms collected from different parts of Nepal. A total number of five species of mushroom were collected including two wild (<em>Morchella</em> sp. and <em>Ganoderma</em> sp.) and three cultivated species (<em>Pleurotus</em> sp., <em>Agaricus</em> sp. and <em>Lentinula</em> sp). The moisture, protein, crude fat, total ash, dietary fibre and carbohydrates were determined as nutrients whereas phenolic content, flavonoid content, antioxidant and mineral matter were determined as nutraceuticals. In this study, the highest amount of nutrients contained was protein (34%), fat (7%), fibres (42%), ash (11%), and carbohydrate (60%). The nutraceutical in these mushrooms is phenolic (0.79 μg of GAE/gm), flavonoid (0.585 μg of QE/gm) and antioxidant (0.261±0.036 mg/ml). The concentration of iron (26.19 mg/l) was found highest in all mushrooms, whereas lead and chromium were found in negligible amounts i.e., &gt;1. The comparative study revealed that the nutrients in cultivated mushrooms have highest than in wild mushrooms. Moreover, the nutraceutical is maximum in wild mushrooms (phenolic content of 0.79 μg of GAE/gm, flavonoid content of 0.58 μg of GAE/gm) than in cultivated mushrooms. Furthermore, wild edible mushrooms are a good source of antioxidants than cultivated ones. In conclusion, this study suggested that mushrooms are a good source of nutrients and nutraceuticals which are considered valuable good nutritional dietary supplements.</p> Roman Pandey Alisha Kunwar Rosa Ranjit Niranjan Koirala Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 44 51 10.3126/jnba.v4i1.53445 Production of Cellulase from the Municipal Waste Residue by a Novel Cellulolytic Fungi <p>Cellulases catalyze the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose and play a significant role in nature by recycling polysaccharide debris. This enzyme has enormous potential in industries such as textile wet processing, bio-stoning of denim fabric, biopolishing of textile fibres, softening of garments and removal of excess dye from the fabrics. Therefore, the research focused on obtaining new cellulose-producing microorganisms with higher specific activities and greater efficiency. By identifying a good strain, improving the production medium and using an alternative carbon source such as waste residue, this study aimed to lower the manufacturing cost of cellulase. This study was designed to assess the cellulase production by fungi isolated from water, soil, straw, dung, leaf and goat manure. In the present research, cellulase-producing fungal isolates obtained from waste samples were identified by morphological and microscopic features. On Congo red test, the largest zone of hydrolysis was found as 1.2 cm. From the morphological and microscopic test, the fungal strain was expected as <em>Aspergillus</em> sp. The assay of the enzyme cellulase was performed by measuring the release amount of reducing sugar. Optimization of process parameters was carried out for the isolate to maximize enzyme yield. On optimization, isolated fungal species showed maximum enzyme activity at a temperature of 30 °C and pH 6. Under optimized conditions of temperature and pH, agitation at 200 rpm with a 1 L/m air flow rate showed better cellulase activity. Cellulase production from <em>Aspergillus</em> sp. can be an advantage as the enzyme production rate is normally higher as compared to other fungi.</p> Puja Bhatt Garima Bista Mukesh Yadav Sujeeta Maharjan Pravesh Paudel Usha Lamsal Sanoj Katharia Jarina Joshi Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 52 57 10.3126/jnba.v4i1.53446 Detection of Respiratory Syncytial Virus in Suspected Asthmatic Children by Conventional RT-PCR Assays in Kathmandu <p>Respiratory Syncytial Virus (RSV) is the most common causative agent for lower respiratory tract infections in children particularly in infants. At least 50% of children are infected with RSV by the age of 2 to 3 years of age. However, the diagnosis of the RSV infection and its association with risk factors for asthma in asthmatic children is not studied in Nepal. The objectives of this study were to determine the prevalence of RSV in asthmatic children. This work was a hospital based cross-sectional prospective study and conducted from September 2017 to February 2018. Ethical approval was obtained from the Institutional Review Committee (IRC) of Kanti Children’s Hospital, Maharajgunj. Suspected asthmatic children visiting the Special Asthma OPD of Kanti Children’s Hospital, Maharajgunj were selected with the help of asthma specialist using a set of inclusion criteria for asthma. A total of 32 nasal swab samples were obtained from suspected asthmatic children. Initially, samples were processed for RNA extraction. The extracted RNA was then used for cDNA synthesis followed by PCR using primers for the Nucleocapsid (N) gene of RSV. Out of total 32 samples, 9 (28%) samples were positive for the N gene of RSV. There was no significant association of different variables including age (P=0.187), sex (P=0.264), family history of asthma (P=0.115), passive smoking (P=0.88), birth weight (P=0.954), seasonality (P=0.298) and history of pneumonia (P=0.457) with the RSV infection in this study.</p> Stuti Khadka Anil Kumar Sah Jeena Amatya Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 58 63 10.3126/jnba.v4i1.53447 Lipids: A Key Regulatory Hub in Plant Stress Adaptation <p>The plants exert rapid responses to environmental stresses to adapt with the stress. Plants respond in many ways to different abiotic and biotic stresses. The first cellular component to sense stress signals is cell membrane. Cell membrane is a biological membrane, and its main constituents are Lipids that can sense conditions outside the cell. Remodeling of cellular membrane lipids is one of the critical mechanisms in the plant cells to withstand stresses. Lipids play a critical role in triggering and regulating cellular hormonal signaling cascades. There is a close correlation between ABA-dependent signaling and lipid metabolic pathways to maintain the integrity and the viability of cellular membranes. Phosphatidic acid, lysophospholipid, inositol phosphate, fatty acid, oxylipins, diacylglycerol, and sphingolipid are considered as the signaling molecules. Lipids also act as a stress reliever to lower the negative effects of stress. This review highlights the key regulatory roles of lipids in plant adaptation against the environmental stresses.</p> Deyala M. Naguib Kishor Regmi Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 64 71 10.3126/jnba.v4i1.53448 Fungal Diseases of Tomato in Kathmandu Valley <p>The infected parts of the tomato plant were collected from Jitpurphedi of Kathmandu, Nepal. The isolated fungi from the infected parts were <em>Septoria lycopersici</em>, <em>Cladosporium oxysporum</em> responsible for leaf spot, <em>Phytophthora infestans</em> and <em>Rhizoctonia solani</em> responsible for leaf blight, <em>Cladosporium cladosporioides</em> responsible for fruit rot, <em>Leveilulla taurica</em> responsible for powdery mildew and <em>Plasmopara viticola</em> responsible for Downey mildew disease. In the survey period, the highest incidence was found at leaf blight (30.08%) and the lowest at stem rot (4.64%). In the case of severity, the maximum severity was found at Downey mildew (77%) and the minimum was recorded at fruit rot (5.25%).</p> Sanjay Kumar Jha Sita Lamichhane Copyright (c) 2023 Nepal Biotechnology Association, Kathmandu, Nepal 2023-03-22 2023-03-22 4 1 72 74 10.3126/jnba.v4i1.53449