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Background: Endophytic fungi are largely underexplored in the discovery of natural bioactive products though being rich sources of novel compounds with promising pharmaceutical potential. In this study, Taxus wallichiana, which has huge medicinal value, was investigated for its endophytic diversity and capability to produce bioactive secondary metabolites by analyzing antioxidant, antimicrobial and cytotoxic properties. Methods: The endophytes were identified by ITS-PCR using genomic DNA samples. The secondary metabolites were extracted by solvent extraction method using ethyl acetate. The antioxidant activity was analyzed by Thin Layer Chromatography, Total Phenol Content (TPC), Total Flavonoid Content (TFC) and DPPH assay, and the antimicrobial activity was analyzed by agar-well diffusion method. Brine shrimp lethality assay was used to analyze the cytotoxicity of the fungal extracts. Results: Out of 16 different Taxus trees sampled from different locations of Dhorpatan, 13 distinctive endophytic fungi were isolated and grouped into 9 different genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. The ethyl acetate extracts isolated from three endophytic fungi: Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae showed significant TPC values of 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively, and TFC values of 177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. Furthermore, these three extracts showed a dose dependent radical scavenging activity with IC50 concentration of 22.85, 22.15 and 23.001 µg/ml, respectively. The extracts of C. cladosporioides and A. brassicae also showed promising antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis with a minimum inhibitory concentration of 250μg/ml for all bacteria. Both the samples showed cytotoxic property against shrimp nauplii with LC50 of 104.2 and 125.9µg/ml, respectively. Conclusions: The crude fungal extracts obtained from endophytes: A. alternata, C. cladosporioides and A. brassicae upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents.

Background: Endophytic fungi are largely underexplored in the discovery of natural bioactive products though being rich sources of novel compounds with promising pharmaceutical potential. In this study, Taxus wallichiana, which has huge medicinal value, was investigated for its endophytic diversity and capability to produce bioactive secondary metabolites by analyzing antioxidant, antimicrobial and cytotoxic properties. Methods: The endophytes were identified by ITS-PCR using genomic DNA samples. The secondary metabolites were extracted by solvent extraction method using ethyl acetate. The antioxidant activity was analyzed by Thin Layer Chromatography, Total Phenol Content (TPC), Total Flavonoid Content (TFC) and DPPH assay, and the antimicrobial activity was analyzed by agar-well diffusion method. Brine shrimp lethality assay was used to analyze the cytotoxicity of the fungal extracts. Results: Out of 16 different Taxus trees sampled from different locations of Dhorpatan, 13 distinctive endophytic fungi were isolated and grouped into 9 different genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. The ethyl acetate extracts isolated from three endophytic fungi: Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae showed significant TPC values of 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively, and TFC values of 177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. Furthermore, these three extracts showed a dose dependent radical scavenging activity with IC50 concentration of 22.85, 22.15 and 23.001 µg/ml, respectively. The extracts of C. cladosporioides and A. brassicae also showed promising antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis with a minimum inhibitory concentration of 250μg/ml for all bacteria. Both the samples showed cytotoxic property against shrimp nauplii with LC50 of 104.2 and 125.9µg/ml, respectively. Conclusions: The crude fungal extracts obtained from endophytes: A. alternata, C. cladosporioides and A. brassicae upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents.

24Endophytes are microbial colonizers that reside in plants by symbiotic association produces 25 several biological classes of natural products. The current study focuses on the isolation and 26 characterization of bioactive compounds produced by endophytic fungi isolated from the 27 Himalayan yew (Taxus wallichiana) collected from the Mustang district of Nepal. The plant 28 materials were collected from the Lower-Mustang region in the month of October 2016 and the 29 endophytic fungi were isolated on artificial media from inner tissues of bark and needles. 30 Antimicrobial and antioxidant activity, along with total phenolic-and flavonoid-content assays, 31 were used in the evaluation of bioactivity of the fermented crude extracts along with the in vitro 32 ability of the endophytes to produce the anticancer compound Taxol was analyzed. A total of 16 33 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. 34 wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed 35 antibacterial and antifungal activity against all test-pathogens used, with significant inhibition 36 against Pseudomonas aeruginosa ATCC 27853 (MIC: 250 µg/ml) and the pathogenic yeast, 37 Candida albicans (MIC: 125 µg/ml). Antioxidant activity was also evaluated using 2,2-diphenyl-38 1-picrylhydrazyl (DPPH). At a concentration of 100 µg/ml, the % radical scavenging activity 39 was 83.15±0.40, 81.62±0.11, and 62.36±0.29, for ascorbic acid, butylated hydroxytoluene (BHT) 40 and the EA fraction of MUS1, respectively. The DPPH-IC50 value for the EA fraction was 81.52 41 µg/ml, compared to BHT (62.87 µg/ml) and ascorbic acid (56.15 µg/ml). The total phenolic and 42 flavonoid content in the EA fraction were 16.90±0.075 µg gallic acid equivalent (GAE) and 43 11.59±0.148 µg rutin equivalent (RE), per mg of dry crude extract, respectively. Isolate MUS1, 44 identified as an Annulohypoxylon sp. by ITS sequencing, also produced Taxol (282.05 µg/L) as 45 shown by TLC and HPLC analysis. Having the ability to produce antimicrobial and antioxidant 46 3 compounds, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain 47 MUS1, a promising candidate for further study given that naturally occurring bioactive 48 compounds are of great interest to the pharmacological, food and cosmetic industries.49

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 Bambusa tulda 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 Bacillus 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 B. tulda rhizosphere were capable of mineralizing phytate and the maximum effect was observed for designated SRBR-04. Most isolates solubilized Ca3(PO4)2 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-1 L-Tryptophan (1.63 to 4.5 μg mL-1). Phytase production was highest in wheat bran with isolate SRBR-04 producing a maximum of 0.34 U mg-1. Two isolates (SRBR-01, SRBR-04) screened positive for the presence of Bacillus phytase gene. Pot assay in P-deficient soil showed significant (p < 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 Zea mays.