The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Marine Yeasts from the Slope Sediments of Arabian Sea and Bay of Bengal

The present study provides an account of the occurrence and diversity of marine yeasts in the slope sediments of Arabian Sea and Bay of Bengal. It also gives a clear idea about the role of yeasts in the benthic realm of marine ecosystem. The lipolytic potential of the organisms indicate the presence of rich lipid moieties in the study area. The isolates, Candida sp. SD 302 and Pichia guilliermondii SD 337 were proved to have potential oil degrading property and can be employed as bioremediators of oil spill after further characterization. The black yeasts isolated during the study area were found to have high commercial value by virtue of the by-products obtained from them. The melanin and the melanin degrading enzyme extracted from these organisms are potential bioactive materials for application in cosmetology.

Marine yeasts — a review

Yeasts are ubiquitous in their distribution and populations mainly depend on the type and concentration of organic materials. The distribution of species, as well as their numbers and metabolic characteristics were found to be governed by existing environmental conditions. Marine yeasts were first discovered from the Atlantic Ocean and following this discovery, yeasts were isolated from different sources, viz. seawater, marine deposits, seaweeds, fish, marine mammals and sea birds. Nearshore environments are usually inhabited by tens to thousands of cells per litre of water, whereas low organic surface to deep-sea oceanic regions contain 10 or fewer cells/litre. Aerobic forms are found more in clean waters and fermentative forms in polluted waters. Yeasts are more abundant in silty muds than in sandy sediments. The isolation frequency of yeasts fell as the depth of the sampling site is increased. Major genera isolated in this study were Candida, Cryptococcus, Debaryomyces and Rhodotorula. For biomass estimation ergosterol method was used. Classification and identification of yeasts were performed using different criteria, i.e. morphology, sexual reproduction and physiological/biochemical characteristics. Fatty acid profiling or molecular sequencing of the IGS and ITS regions and 28S gene rDNA ensured accurate identification.

Marine yeasts as source of single cell protein and immunostimulant for application in penaeid prawn culture systems

The study revealed the potential of marine yeasts as a source of single cell protein and immunostimulant for prawns. Prawns fed with the selected marine yeasts were showing more growth compared to the control feed and commercial feed. Yeasts being rich with proteins, vitamins and carbohydrates serve as a growth promoter for prawns as being evidenced in this study. The better performance of marine yeasts, D. hansenii S8 and S100 and C. tropicalis S186 compared to S. cerevisiae S36 as a feed supplement is worth investigating. Besides being a rich nutritional source, yeasts act as immunostimulants by virtue of its high carbohydrate (Beta, 1-3 glucan) and RNA content. Beta, 1-3 glucan, a cell wall component of yeasts /fungi is the most commonly used immunostimulant in aquaculture. The present study shows that even the whole cell yeast could serve as a good immunostimulant when supplied through diet. Extraction of Beta-1,3 glucan results in the removal of nutrients like proteins, vitamins etc. from the cell biomass.Utilization of the yeast biomass as such in the diet would help perform a dual role as nutritional component and immunostimulant for aquaculture applications.

Studies on fungal flora with special reference to Yeasts in the Cochin backwaters

Man's concern with environmental deterioration is one of the major reasons for the increased interest in marine and estuarine microbes. Microbes form an important link in the biogeochemical cycling and their cyclinq activites often determine to a large measure the potential productivity of an ecosystem In the recycling of the nutrients in the estuary, bacteria and fungi therefore play a particularly significant role.The allochthonous plant materials contain biopolymers such as cellulose, lignin, humus etc., that are difficult to degrade into simpler substances. The fungi have the ability to degrade _substances, thereby making them available for cycling within the system. The present study is devoted to find the composition and the activity of myco populations of Cochin backwater. For convenience the thesis is divided into eight chapters. The opening chapter briefly reviews the literature and projects the importance of work and the main objectives. Second chapter discusses the materials and methods. In the third chapter the systematic and taxonomy of estuarine yeasts are examined in detail since this information is scarcely available for our waters. The general ecological aspects of the yeasts and filamentous fungi in the area of study are examined in the fourth chapter using appropriate statistical techniques. A special reference to the fungi in a small mangrove ecosystem is attempted in the fifth chapter. The biochemical studies are discussed in the sixth chapter and the penultimate chapter provides an overall discussion. In the last chapter the summary of the work is presented.

Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.

Black yeasts from the slope sediments of Bay of Bengal: phylogenetic and functional characterization

Occurrence of black yeasts in the slope sediments of Bay of Bengal was investigated during FORV Sagar Sampada cruises 236 and 245. The black yeast population was found to be very scanty in the area and the isolates could be obtained from 200m to 1000m depth regions in the slope sediments. The isolates were identified as Hortaea werneckii by Internal Transcribed Spacer (ITS) sequencing. The biodegradation potential of these strains was found to be very high with all the strains exhibiting protease, lipase and amylase production. The optimum growth conditions were pH 8, salinity 30 ppt and temperature 30oC. The pigment melanin, in these organisms was identified to be of dihydroxynaphthalene type by NMR. The melanin was found to exhibit inhibitory activity against different human and fish pathogens. Melanin degrading enzyme could also be extracted from these organisms

Selection of Marine Yeasts for the Generation of Single Cell Protein from Prawn-shell Waste

Marine yeasts (33 strains) were isolated from the coastal and offshore waters off Cochin. The isolates were identified and then characterized for the utilization of starch, gelatin, lipid, cellulose, urea, pectin, lignin, chitin and prawn-shell waste. Most of the isolates were Candida species. Based on the biochemical characterization, four potential strains were selected and their optimum pH and NaCI concentration for growth were determined. These strains were then inoculated into prawn-shell waste and SCP (single cell protein) generation was noted in terms of the increase in protein content of the final product.

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Isolation of culturable yeasts from market wines and evaluation of the 5 center dot 8S-ITS rDNA sequence analysis for identification purposes

Aims: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. Methods and Results: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. Conclusions: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. Significance and Impact of the Study: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.