Research Journal of Biotechnology

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Vol. 14(6) June 2019

Characterization and molecular phylogeny of Magnaporthe oryzae causing rice blast disease in eastern India

Yadav Manoj Kumar, Aravindan S., Prabhukarthikeyan S.R., Keerthana U., Raghu S., Bal Archana, Samal Pankajini, Behera Motilal, Kar Meera Kumari, Rath P.C. and Devanna Pramesh

Page No: 1-7

Abstract: Rice blast, caused by the hemibiotrophic fungus Magnaporthe oryzae, is one of the most potent threats to rice production and causing tremendous yield loss. In the present study, seventy-one M. oryzae isolates were collected from three states of eastern India. Based on colony color, the blast isolates were grouped in five; grayish (11), grayish black (9), grayish white (19), blackish (20) and white (12). Most of the isolates were smooth (60) and few were rough (11) in colony appearance. The blast isolates produced characteristic spindle shaped symptoms on susceptible plant.

Five isolates were recorded as highly virulent, eight as moderately virulent isolates and only two isolates were observed as mild isolates. The sequence similarities among fifteen M. oryzae isolates varied from 78 to 98.8%. The phylogenetic analysis showed uniform distribution of 71 blast isolates into two clusters and indicated the existence of high genetic variation among blast isolates originated from the same location and existence of genetic similarity among blast isolates originated from different geographical origins. The outcome of the present work would help to formulate strategies for improved disease management against rice blast through resistance breeding, genetic studies and host-pathogen interaction.

Prediction of putative functional role of hsa-miR-1277 and hsa-miR-5095 microRNAs in MCF7 by computational approach

Bhardwaj Vaishali and Mandal Abul Kalam Azad

Page No: 8-20

Abstract: MicroRNAs (miRNAs) are small, non-coding regulatory RNA molecules in both plant and animal. Its function has been associated with several cancer-related genes that regulate biological and metabolic processes such as carcinogenesis and drug resistance. The computational homology-based search of expressed sequence tags (ESTs) is an important approach to identify miRNAs. In the present study, we have used EST sequences of a breast cancer cell line MCF7 and identified several potential miRNAs. Among these, three miRNAs (hsa-miR-548d-5p, hsa-miR-1277 and hsa-miR-5095) were further used for target prediction, gene ontology and pathway analysis.

TargetScan has identified multiple target genes namely LRP6, Wnt, APC, GSK3β and FZD for these miRNAs. GO and KEGG pathway analysis indicated a significant involvement of these miRNAs in PI3K-AKT and WNT pathways. Wide network regulations of these miRNAs have opened insights to study their enormous and diverse role.

Hematite Nanoparticles: synthesis, characterization and aquatic ecotoxicity effects

Suman Thodhal Yoganandham, Radhika Rajasree Santha Ravindranath, Gayathri Sathyamoorthy, Remya Rajan Renuka and Aranganathan Lakshminarayanan

Page No: 21-30

Abstract: Iron oxide nanoparticles have been investigated recently for their useful applications in numerous biomedical areas, in environmental remediation and in different industrial applications. In any case, additional risks have been identified with the release of nanoparticles into the environment. In the present study the toxicity of hematite nanoparticles to marine algae, Chlorella vulgaris was studied with focus on oxidative stress and cytotoxicity analysis. The synthesized hematite nanoparticles are in the range of 26-50 nm. Result showed that Chlorella vulgaris growth reduced with increasing concentrations. The nanoparticles induced oxidative stress was the main toxic mechanism.

The nanoparticles and Chlorella vulgaris cell physical interaction also contributed to the nanotoxicity. Field Emission Scanning Electron Microscope shows the morphological changes and cell damage. In 24h, treatment mortality was 20 – 70 % and LC50 value for 24h was 393.60 mg/L. Toxicity study on copepod showed mortality increased from 20- 100% for 48hr and LC50 value for 44h was 221.34 mg/L.

Boosting lipid assimilation of a novel microalgae in mixotrophic cultivation and incorporating silver nanoparticles for improved cell recovery towards biodiesel application

Vasistha Shrasti, Khanra Anwesha and Rai Prakash Monika

Page No: 31-40

Abstract: In present study, Chlorosarcinopsis sp. (VKR02) was isolated from natural water body of New Delhi and screened for its growth in sugarcane vinasse a waste from distillery. Cultivation of microalgae was optimized in a mixotrophic mode by varying media concentration using response surface method (RSM). Microalgae was presented with the higher biomass productivity of 0.244 g/L/d and lipid productivity of 0.058 g/L/d in presence of 15% v/v sugarcane vinasse. Physicochemical properties of sugarcane vinasse were also estimated including chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP) and total carbon (TC) before and after microalgae cultivation for its reuse. Chlorosarcinopsis sp. has the potential of nitrogen and phosphorous removal by 20 % and 6.13 % respectively. Post-cultivation harvesting of microalgae biomass was proposed by using silver nanoparticle (Ag NPs) mediated flocculation technique.

Flocculation efficiency of 94.28% was observed with 15 mg/L of Ag nanoparticle. Fluorescence microscopy revealed the presence of neutral lipid in the cell that was converted to fatty acid methyl ester (FAME) by transesterification. The GC-MS result of microalgal FAME showed appropriate fatty acid composition that could be explored for biodiesel application. This study highlights the recycle of industrial waste material sugarcane vinasse as a nutrient source for microalgae growth and lipid production for its potential use.

Toxicity of commonly used food preservative sodium nitrate using Allium cepa L. as a test plant

Pandey Himadri and Kumar Sanjay

Page No: 41-49

Abstract: The possible cytotoxicity of sodium nitrate (SN) on root tip cells of Allium cepa L. using mean root length, EC50 value, apoptotic activity, mitochondrial activity, protein content, antioxidant activity and anatomical changes was investigated. Treatments were divided into 5 groups as one control group and four treatments (1000, 1500, 2000 and 2500 ppm) groups for 16 h. The parameter for mean root length was analyzed after the treatment for 5, 10 and 15 days.

As a result, significant inhibition of root growth, physiological changes, biochemical changes, anatomical changes, apoptotic and metabolic activity was observed.

Puriﬁcation and characterization of fructosyltransferase: A low molecular weight enzyme from Aspergillus niger NFCCI2736

Belorkar Seema A. and Gupta A.K.

Page No: 50-57

Abstract:A novel transfructosylating enzyme derived from A. niger NFCCI2736 was puriﬁed 7.99-fold by ammonium sulphate precipitation (30–80%) followed by DEAE-cellulose ion exchange chromatography. The minimum molecular mass of the puriﬁed enzyme was 45 kDa by SDS-PAGE. The Km for fructosyltransferase was 333 mM of sucrose and Vmax was 1.25 x103 mM/ mg/ min. The optimum enzyme activity of the purified enzyme was at 5.5 and the maximum stability of the enzyme was at pH 5.00.

The optimum temperature for enzyme activity and enzyme stability coincided at 55 °C. The ammonium ions enhanced the activity of the purified enzyme whereas sodium and manganese ions decreased the activity of purified Ftase.

Statistical optimization of medium for enhancing siderophore production by Bacillus tequilensis DB1

- Patel Dhara and Saraf Meenu

Page No: 58-66

Abstract:The evaluation of media component for enhanced siderophore production by Bacillus tequilensis DB1 was studied by applying statistical method i.e. Plackett-Burman and response surface methodology (RSM) using central composite design (CCD).The Plackett-Burman factorial design experiment revealed that pH, incubation time and succinic acid were the three factors significantly influencing siderophore production.

The optimum concentrations of these significant parameters were determined employing second stage of optimization i.e. response surface central composite design. The optimal medium was found to contain pH (6.0), incubation time (30 hrs) and succinic acid (0.56g/100ml) that yielded maximum siderophore production (71.42%). Validation of these experiments showed 8.65% increase in yield of siderophore.

Simple molecular assay for validation of interspecific hybridity in annona species

Vinay G.M., Sakthivel T., Honnabyraiah M.K., Lakshmana Reddy D.C. and Priyanka H.L.

Page No: 67-73

Abstract: Inter-simple sequence repeats (ISSR) molecular markers were used to confirm the hybridity of annona hybrids. Genomic DNA was extracted from young leaves of hybrids and their respective parents to access the hybridity. The results revealed that among six primers pair used, the primer UBC 827 clearly showed polymorphism with differentiating ability of all four interspecific hybrids viz. Arka Sahan, 19/26, 16/10 and 16/14. The primer UBC 834 confirms the hybridity only in Arka Sahan and UBC 890 confirms the hybridity of two hybrids viz. 16/14 and 16/10 respectively.

Compared to other dominant markers like RAPD, the ISSR distinguishes the hybridity very clearly. The interspecific hybridization in annona was successful mainly due to the genetic compatibility between the species. One molecular marker was enough to validate the hybridity in hybrids demonstrating to be trustworthy, fast and efficient methodology. The validated ISSR can be used in hybridity confirmation of offspring’s.

Seed Traits as factors influencing Pre-Germination Anaerobic Stress Tolerance in Soybean

Ambika Rajendran, Lal S.K., Jain S.K. and Dhandapani Raju

Page No: 74-79

Abstract: Waterlogging is one of the causes of crop loss in soybean during early stages of production. Pre-germination anaerobic stress tolerance in seeds can overcome adverse waterlogging during germination phase of crop development in field. Controlled experiment was done to understand the influence of seed test weight (large, medium, small) and seed colour (yellow, black) of soybean for improving pre-germination tolerance in water logging. Tolerance is characterized by higher germination and seedling performance in waterlogging treatment.

Seed test weight had a relative impact on pre-germination tolerance to water logging and appeared to be seed colour dependent. Smaller black seeds germinated well with better root development and higher dry weight than yellow seeds in water logged condition. Seed test weight and seed colour can be reasonable indicators of pre-germination tolerance to water logging in soybean seeds.

Effect of different tissues and growth hormones on the in vitro propagation from Curculigo orchioides Gaertn., an endangered medicinal Herb in Vietnam

Thanh Pham, Thi Thiet Hoang and Minh Duc Tran

Page No: 80-87

Abstract: Curculigo orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Using MS medium supplemented with TDZ (0.03, 0.05, 0.07, 1 mg/l) and BAP (0.5, 1, 1.5 mg/l) alone or in combination with 0.05 and 0.1 mg/l NAA, in vitro shoot regenerations were tested from three different tissues (rhizome apical tip, rhizome, leaf) of C. orchioides. Result analysis found that shoot regeneration in MS medium supplemented with TDZ (0.07 mg/l) combination with NAA (0.1 mg/l) from rhizome apical tip of C. orchioides was significantly highest compared among treatment.

In this treatment, 100 % cultures responded with an average number of 8.67 shoots per explant. Additionally, rooting per shoot regeneration in the half MS medium supplemented with 1.5 mg/l IBA was significantly highest. The rooted plants were successfully transplanted to soil with 100% success. Based on our results, we suggest that the best effect in vitro regeneration of C. orchioides may be to use MS medium supplemented with TDZ (0.07 mg/l) combination with NAA (0.1 mg/l) from rhizome apical tip tissue.

Antimicrobial and phytochemical analysis of some indigenous plants

Dixit Himisha, Kumar Prakash and Kaushik B.D.

Page No: 88-95

Abstract:The antimicrobial activity of the extracts of the Calotropis procera, Datura metel, Cuscuta reflexa, Pongamia pinnata and Nerium oleander has been studied against the pathogenic bacteria Staphylococcus aureus, Escherichia coli, Pseudomonas aureuginosa, Klebsiella pneumoiniae, Paenibacillus and Bacilus subtilis. Significant antibacterial activity was observed with extract of ethanol, methanol and chloroform. The ethanol extract of D. metel showed maximum antibacterial activity against the all tested bacteria except the P. aureuginosa.

Aqueous extract showed no measurable antibacterial activity. Inhibition was seen as concentration dependent phenomenon. Phytochemical screening revealed the presence of alkaloids, flavanoids, glycosides, steroids, tannins, phenol and saponin.

Cloning and expression of Apis mellifera melittin cDNA in E. coli

Diego Jáuregui, Miquel Blasco and Santiago Mafla

Page No: 96-99

Abstract:Honey bee venom, known as apitoxin, is composed of several peptides, the most important of which is melittin. This peptide is a current focus of research since it can improve the immune system and act against cancer due to its bactericidal, anti-mutagenic, anti-inflammatory and even contraceptive effects. This makes it very desirable to obtain melittin producing bacteria and for this reason, this study has aimed at the cloning of E. coli with the melittin gene from western bee. In order to do this, the total RNA of the western honey bee (Apis mellifera) has been extracted and a reverse transcription polymerase chain reaction (RT-PCR) has been carried out, at different annealing temperatures (68.0, 68.2, 68.4, 68.6, 68.8 and 69.0 ° C) to amplify the melittin cDNA.

The annealing temperature of 68.4 ºC has allowed the highest production. Subsequently, this cDNA has been cloned into the pGEM-T vector, which has transformed E. coli JM109‎. This transformation has been corroborated by the blue/white test mediated by X-gal.

Isolation of Saccharomyces cerevisiae yeast strains from Macedonian “Tikveš” wine-growing region and their impact on the organoleptic characteristics of Vranec and Cabernet Sauvignon wines

Ilieva F., Kostadinović Veličkovska S., Dimovska V., Mirhosseini H. and Spasov H.

Page No: 100-110

Abstract:Isolation of autochthonous yeast strains from 15 microregions from Tikveš winegrowing region of Macedonia was first object of this study. Furthermore, morphological and physiological characterization of yeast strains were performed in order to reveal small species diversity belonging to Saccharomyces cerevisiae genera. Moreover, this study estimated of impact of the isolated yeasts on the organoleptic profile of wines produced from Vranec and Cabernet Sauvignon grape variety.

Our study confirmed improved organoleptic characteristics of wines produced from Vranec and Cabernet Sauvignon grape variety fermented by autochthonous yeast strains from second stage of selection in comparison to commercial yeast strain SiHa. Oeno-chemical parameters obtained from semi-industrial production of Vranec wine fermented by third-stage selected yeast strain can be used as general recommendation for specific regional wines production.

Isolation and Characterization of Stress Inducible Protein (TaSti/Hop) from Heat-Tolerant Wheat Cultivar C306

Vishwakarma Harinder, Sharma Jyoti, Solanke Amolkumar, Singh G.P. and Padaria Jasdeep

Page No: 111-121

Abstract:Heat stress adversely affects wheat crop plants leading to immense yield losses. To cope up with the stress, plants respond by overexpressing stress-associated transcription factors and genes. Here, we have identified and cloned a putative heat stress-responsive gene TaHSti/Hop (Accession no. MF383198) from heat tolerant Indian bread wheat cv. C306. Based on in silico analysis, we report here 3D protein structure and digital expression analysis for TaSti/Hop. Computational studies on TaSti/Hop confirmed its role in wheat heat stress tolerance. This gene can be prospective resource for development of abiotic stress tolerant transgenic crops.

Study of Enterococcus faecium strain LF3(1) as potential probiotics for Cyprinus carpio to prevent Pseudomonas aeruginosa infection

Chauhan Arun and Singh Rahul

Page No: 122-129

Abstract:The antibiotic resistance among the pathogens by extensive use of antibiotics requires the search for an alternative to control the disease in aquaculture. Probiotics, renowned as valuable microbes, are recommended as an efficient and environment friendly approach to reduce the use of antibiotics. The present study aims at the isolation of putative probiotic bacteria. In the study, 80 different bacteria isolated from 20 different fish of Malwa region, Punjab (India), have been characterized for their probiotic potential and their application against Pseudomonas aeruginosa infection in common carp (Cyprinus carpio). The isolated bacterial strains were found to fulfill all the criteria for them to be classified as a potent probiotic. The in-vitro evaluation showed a strong antagonistic response of Enterococcus faecium strain LF3(1) (identified by 16s rRNA sequencing) towards potential fish pathogen Pseudomonas aeruginosa (MTCC 4673).

This isolate was further screened in response to bile tolerance (0.3% ox-gall), pH tolerance, adhesion and drug susceptibility to different antibiotic discs. Also, the in-vivo results indicated improved growth and survival against the infection (Pseudomonas aeruginosa) after oral administration of the probiotics. The present study indicates that the putative bacteria isolated from the gut of teleost serves as a potential candidate to improve immunity and enhances the production in aquaculture.

Genetic relationship and diversity of Taif-roses plant by using three different types of molecular markers

Alotaibi Saqer S., Hassan Mohamed M., Gaber Ahmed and Aljuaid Bandar S.

Page No: 130-138

Abstract:Taif roses (Rosa x damascena) are important ornamental crops that are widely cultivated in the Taif region. Taif roses are well known in the Arabian world for their deep and intense fragrance and they have a favourable economic value. In this study, the genetic variation between 30 individual plant samples of Taif roses was evaluated. This was done by using three different molecular markers, 10 primers each for ISSR, SCoT and CDDP markers. The results obtained from ISSR-PCR analysis of the 30 local rose samples revealed approximately 106 different banding patterns; 98 of which are considered monomorphic bands (92.5%) and 8 fragments of which are considered polymorphic bands (7.5%). The SCoT marker revealed approximately 100 different banding patterns; 71 of which are considered monomorphic bands (71%) and 29 fragments of which are considered polymorphic bands (about 29%). Finally, the CDDP marker revealed approximately 103 different banding patterns; 82 of which are considered monomorphic bands (79.7%) and 21 fragments of which are considered polymorphic bands (20.3%).

Of the three molecular markers, the SCoT marker is a useful tool for the detection of genetic diversity among Taif roses. Even though the cluster analysis for the three markers separated the 30 individuals into different groups, the dendrogram obtained from the SCoT molecular marker presented the best clustering pattern. Thus, the ISSR, SCoT and CDDP markers provide a useful technique for plant genotyping and fingerprinting.

Proline and betaine suppressed cell death of tobacco cultured cells under saline stress

Jahan Md. Sarwar

Page No: 139-141

Abstract:We measured salinity-induced cell death in the presence of proline and betaine to justify if proline and betaine suppressed salinity effect on tobacco bright yellow-2 cultured cells (BY2 cells). Evans blue dye was used in BY2 cells in the presence of sodium chloride (NaCl) and proline and betaine. Different NaCl solutions increased cell death differently compared to a control condition. Proline decreased NaCl-induced death of tobacco BY2 cells compared to the proline-untreated BY2 cells.

In addition, betaine treatment showed similar results to the proline treatment. These results suggest that proline and betaine might decrease saline effects on Tobacco BY2 cells and therefore decreased cell death in saline condition.