Investigating the Production of Antimicrobial Nanoparticles by Chlorella vulgaris and the Link to Its Loss of Viability

Chlorella vulgaris from Al-Ahsa, KSA was proved to be an active silver and gold nanoparticle producer. Nanogold and nanosilver particles were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy, and scanning electronmicroscopy. Both nanoparticles were used in the antimicrobial bioassay. The two nanoparticles showed antibacterial activities, with the silver nanoparticles being the most effective. To investigate the argumentative nature of their biosynthesis (i.e., whether it is a biotic or abiotic process), we isolated total ribonucleic acid RNA as an indicator of vitality. RNA was completely absent in samples taken after one week of incubation with silver nitrate and even after one or two days. However, successful extraction was only achievable in samples taken after incubation for one and four hours with silver nitrate. Most importantly, the gel image showed recognizable shearing of the nucleic acid after 4 h as compared to the control. An assumption can be drawn that the synthesis of nanoparticles may start biotically by the action of enzyme(s) and abiotically by action of reducing entities. Nonetheless, with prolonged incubation, excessive nanoparticle accumulation can be deadly. Hence, their synthesis continues abiotically. From the RNA banding profile, we suggest that nanosilver production starts both biotically and abiotically in the first few hours of incubation and then continues abiotically. Nanosilver particles proved to have more of an antimicrobial impact than nanogold and hence are recommended for different applications as antibacterial agents.

Germplasm Conservation of Economically Important Medicinal Plant Nyctanthes Arbor-Tristis L. Through Encapsulation Technique and Maintenance Under Slow Growth Condition

An efficient encapsulation and germplasm conservation protocol were developed for Nyctanthes arbor-tristis L. In this study the gel matrix containing three percent sodium alginate (SA) and 100 mM calcium chloride (CaCl2. 2H2O) was found best for the formation of encapsulated seeds from node explant of this economically valuable species. The viability of encapsulated seeds and shoot sprouting potential was optimized. Encapsulated seeds stored at 4ºC and 24 ºC maintained its viability up to 90 days and showed sprouting potential 42.89±6.04 and 33.53±7.15 percent respectively. Node explant maintain under slow growth condition up to 180 days on one-eighth (1/8th) strength MS medium supplemented with 0.5 percent sucrose found suitable to maintain high span viability percent (40.28±2.04) with average number of shoots/ node (1.61±0.28) and shoots length (1.12±0.32 cm) respectively. One-eighth (1/8th) strength MS medium supplemented with 0.5 percent sucrose and enriched with 0.5 mg/l abscisic acid (ABA) prolonged the viability up to 40.36±1.01 percent of explant. The best rooting response was achieved on half (½) strength MS medium enriched with 4 mg/l indole-3-acetic acid (IAA). The rooted plant shows 65 percent survivability in open field condition. The true-to-type clonal fidelity assessment of tissue culture recovered acclimated plants with start codon targeted (SCoT) primer profile shows same banding mobility patterns as with source parent mother plant. The maximum banding profile is monomorphic and consistent. Hence on this basis it confirmed the true-to-type clonal stability among them. The protocols display the novel method for conservation of this species under in-vitro condition and facilitate easy exchange of plant germplasm.

Genotypic Variations of Mutans Streptococci Isolated from Dental Caries by REP-PCR

Mutans streptococci (MS) are a group of oral bacteria considered as the main cariogenic organisms. MS consists of several species of genus Streptococcus which are sharing similar phenotypes and genotypes. The aim of this study is to determine the genetic diversity of the core species of clinical strains of Streptococcus mutans, Streptococcus sobrinus and Streptococcus downei by using repitative extragenic palindromic (REP) primer. The DNA of the clinical strains of S. mutans (n=10), S. sobrinus (n=05) and S. downei (n=04) have been employed in the present study, which have been previously isolated from caries active subjects. The DNA of the clinical and reference strains was subjected to PCR amplification using REP primer. The phylogenetic dendrogram is constructed from the REP PCR banding profile by neighbour-joining method using PyElph 1.4 software. The size of the DNA amplicons generated by using REP primer were S. mutans (1500 bp to 250 bp), S. sobrinus (6000 bp to 250 bp) and S. downei (5000 bp to 400 bp). The results present common band at 480 bp in all the clinical strains of S. sobrinus. The current study is the first to demonstrate the genetic variety of S. sobrinus and S. downei by using REP primer. REP-PCR have been found to be a powerful method to study the molecular diversity of S. mutans, S. sobrinus and S. downei. Additionally, further studies are suggested to analyze the species specific bands and also to find the possibility to produce a new specific primer for S. sobrinus.

Insects’ RNA Profiling Reveals Absence of “Hidden Break” in 28S Ribosomal RNA Molecule of Onion Thrips,Thrips tabaci

With an exception of aphids, insects’ 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect’s RNA, the “hidden break” is absent in the 28S rRNA of onion thrips,Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies’ 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect’s 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.

Molecular characterization of four morphological mutants of Neurospora crassa

Four morphological mutants (albino, ropy, conidial band and buff) of Neurospora crassa were characterized based on morphological features and molecular markers. The mycelial color of mutants buff, ropy and conidial band were more or less orange like wild parents. Albino mutant showed colorless mycelium. The germination time of conidia of wild and other mutants ranged from 5 to 7 days while in albino it was 12 days. Esterase and acid phosphatase isozymes analysis of the N. crassa mutants clearly indicated that mutation altered the carotenoid biosynthesis pathway creating the albino mutant due to the effect UV light. Most of the mutants viz; albino, conidial band and buff showed characteristic RAPD banding profile. However, no band was found in wild EmA, Ema and mutant ropy. Highest number of RAPD bands were found in albino. The mutant albino showed very different morphological and molecular features from the rest specimens. DOI: http://dx.doi.org/10.3329/dujbs.v23i1.19831 Dhaka Univ. J. Biol. Sci. 23(1): 85-91, 2014

Cucumber Mosaic Virus on Asiatic Hybrid Lilies in India

Asiatic hybrid lilies are popular cut flowers with a range of bright colors. Of the several viruses reported from lily (2,3), cucumber mosaic virus (CMV) reduces flower quality and yield (1). Classical symptoms of CMV were observed in recently introduced plants of Asiatic hybrid lilies in Kangra Valley, Himachal Pradesh. The symptoms were mild leaf mosaic, ring spot, transient vein yellowing, occasionally with growth deformation, and flower breaking. Leaf samples from cvs. MonteNegro, Yellow Present, Apeldoorn, Toscana, Connecticut King, and Adelina were collected randomly on the basis of symptom expression. Viral-associated double-stranded RNA (dsRNA) analysis was used to analyze tissue from symptomatic and asymptomatic plants for evidence of a possible cucumovirus. dsRNA analysis resulted in a banding profile typical of that seen with cucumoviruses. There was no evidence of dsRNA in the asymptomatic tissue. Presence of CMV in the symptomatic plants was also confirmed by enzyme-linked immunosorbent assay with antiserum from Agdia (Elkhart, IN). Virus from symptomatic tissues was purified and 30 nm polyhedral, viruslike particles were observed that were subsequently tested for CMV with counter immunoelectrophoresis with antibodies of CMV-C and CMV-D (antibodies obtained from H. A. Scott, University of Arkansas) and ATCC CMV antisera PVAS 242-A. Our isolate differs from other prevalent CMV isolates of Kangra Valley, having a narrow host range and not being readily sap transmissible. However, this isolate is normally transmitted to progeny bulblets. Lack of fallow periods, continuous cropping of other CMV-susceptible bulbous crops, and occasional sprouting of uncollected lily bulblets enhance inoculum build-up. Planting of CMV-tested lilies is recommended to avoid disease losses and to reduce viral inoculum floriculture fields. This is the first report of CMV in Asiatic hybrid lilies in India. References: (1) M. P. Benettii and L. Tomassoli. Acta Hortic. 234:465, 1988. (2) P. Brierley. Phytopathology 30:250, 1940. (3) L. Tomassoli and M. P. Benettii. Adv. Hortic. Sci. 2:117, 1988.

First Report of Cucumber Mosaic Virus in Eryngium amethystinum, Canna spp., and Aquilegia hybrids in Ohio

Eryngium amethystinum (amethyst sea holly) is a herbaceous plant commonly grown as an ornamental perennial in U.S.D.A. hardiness zones 3 to 8. The plant thrives in dry areas with infertile soils and the flowers are often used in dried floral arrangements. Canna spp. (Canna), soft perennials (U.S.D.A. zone 9 and above), are becoming popular flowering plants because of their bright flowers and spectacular foliage. There are a variety of species that fall under the heading Canna spp., of which the most popular are C. glauca, C. indica, C. edulis, and C. iridiflora. Hybrids of Aquilegia (garden columbine), a hardy perennial (U.S.D.A. zones 3 to 9), flower in late spring through early summer. The genus is made up of a wide variety of cultivars. E. amethystinum exhibiting severe mosaic, yellowing, and stunting, along with Canna plants exhibiting severe stunting, chlorotic and distorted foliage, and mosaic, and garden columbine plants exhibiting stunting, leaf curl, chlorosis, and mosaic, collected from commercial plantings throughout the central Ohio area, were analyzed for the presence of virus infection with viral-associated, double-stranded RNA (dsRNA) analysis. dsRNA analysis resulted in a banding profile typical of that seen with members of the cucumovirus family of plant viruses. Plants positive for cucumovurus-like dsRNA were tested with a direct antibody sandwich enzyme-linked immunosorbent assay (ELISA). ELISA results confirmed the presence of cucumber mosaic virus (CMV) in all symptomatic plants tested. No evidence of dsRNA or CMV was found in any of the asymptomatic plants tested. Because all of these hosts are common in the perennial garden, they could serve as a reservoir host of CMV for other plants in the garden. This is the first report of CMV in E. amethystinum, Canna spp., and Aquilegia hybrids in Ohio.