Technical Sessions-abstracts-Poster Session 2-PS-21-PS-33

36th Ann. Meeting of MSI & Nat. Seminar on Fungal Biodiversity & Bioprospecting….. Goa, India Oct., 29-30, 2009


POSTER SESSION 2

PS – 21

Isolation, identification and purification of endophytic fungi from medicinal plants for the enumeration of extracellular enzymes

A. Amrita, J. Swetha, P. Sindhu, and K. P. Kannan*
Department of Biotechnology, Bannari Amman Institute of technology
Satyamangalam, Erode district 638 401
*E-mail: kp_kannan2001@yahoo.co.in

Endophytes are microorganisms, which inhabit in healthy living plant tissues for all or part of their life cycle without causing apparent harmful symptoms to the host. In resent years, much research has been focused on the bioactivities of endophytic fungi. This interest is due to the possibility of these microorganisms producing pharmacologically active substances with biotechnological potential such as antitumor agents (Taxol), antifungal agents (quercine), besides producing factors of plant growth, toxins and enzymes, including some microorganisms being used as biological controllers of many diseases and plagues. In the present investigation four medicinal plants viz., Adathoda vasica, Costus ingneus, Coleus aromaticus, and Lawsonia inerims were examined for the presence of endophytic fungi. Altogether 400 segments (approximately1 cm2) of 50 segments each from leaf and stem tissues of respective plants were screened for the enumeration of the endophytic fungi using the modified surface sterilization techniques. A total of 17 different species of endophytic fungi were recorded. Among them 9 belongs to Hyphomycetes, 7 belongs to Coelomycetes and one belongs to Xylariales. From the endophytic fungi recorded, 10 dominant endophytic fungi viz., Cladosporium cladosporioides, Curvularia brachyspora, C.verruciformis, Drechslera hawaiiensis, Colletotrichum carssipes, Colletotrichum falctum, Colletotrichum gleosporioides, Lasiodiplodia theobromae, Phyllosticta Sp. and Xylariales species were tested for their ability to produce extracellular enzymes i.e., Amylase, Cellulase, Laccase, Lipase, Protease and Tyrosinase activity by the qualitative assays, majority of the entophytic fungi showed the positive results. The results will be discussed in detail.

PS – 22

Screening of endophytic fungi from the medicinal plants for the production of Chitoson

A. Amrita, P. Sindhu, J. Swetha, and K. P. Kannan*
Department of Biotechnology, Bannari Amman Institute of Technology, Satyamangalam,
Erode district 638 401.
*E-mail: kp_kannan2001@yahoo.co.in

Endophytic fungi that live inside the tissues of living plants are under-explored group of microorganisms. Recently they have received considerable attention after they were found to protect their host against insect pests, pathogens and even domestic herbivores. Medicinal plants are reported to harbour endophytes which in turn provide protection to their host from infectious agents and also provide adaptability to survive in adverse environmental conditions. It is therefore important to determine the endophytic diversity of medicinal plants. In the present study four medicinal plants were screened for the presence of Endophytic fungi and were screened for the production of chitin and Chitoson. Industrially, Chitosan is derived from the chemical deacetylation of chitin, using strong alkali, a waste product of the crustacean exoskeletons obtained after industrial processing of seafood. This process is subject to seasonal, limited supply and it pollutes the environment, due to the large amount of waste of concentrated alkaline solution. An alternative to solve these problems is the fungal production of Chitosan, whose main advantage is that it is environmentally friendly. Therefore, physicochemical properties and yields of chitosan isolated directly from a fungus may be optimised by controlling fermentation and processing parameters. The development of applications for chitosan has expanded rapidly in recent years. Chitosan, being polycationic, nontoxic, biodegradable as well as antimicrobial, has many applications especially in the agriculture, food and pharmaceutical industries. It has been used in enzyme immobilization, wastewater treatment, as food additive and anticholesterolemic, for wound healing, and in pharmaceuticals in several drug delivery systems. The results will be discussed in detail.
PS – 23

Partial purification of exracellular enzyme cellulase from Trichoderma viride and T. reesei isolated from cotton seeds

N. Kavitha, C. Sivaranjani and K. P. Kannan*
Bannari Amman institute of Technology, Sathyamangalam Erode District Tamil Nadu.
E-mail: kp_kanann2001@yahoo.co.in

The goal was to investigate the Cellulase production ability of fungal strains in batch culture using shake flasks. The cultures of Trichoderma viride and T.reesei were found to be the best producer of cellulases. Cellulose powder and agricultural by products such as wheat bran, sugarcane bagasse were evaluated as substrates by cultures for the production of enzymes.
Among the substrates used, natural substrates were found to be the ideal substrate providing all the nutrients for the synthesis of cellulases. Effect of concentration of cellulose powder and natural substrates, on enzyme production was studied using Folin Lowry’s method and Dinitro salicylic method. The enzyme activity was found to be maximum on 7th day for Trichoderma reesei and 8th day for Trichoderma viride. The partial purification of the cellulase enzyme on SDS PAGE revealed protein bands showing subunits of cellulase enzyme.
PS – 24

Growth responses of mycorrhizal and non-mycorrhizal bajra plants under salinity conditions

*Mahesh Borde, Mayura Dudhane and Paramjit Jite
Department of Botany, University of Pune, Pune 411007
E-mail:maheshmyco@gmail.com

Under salinity condition mycorrhizal bajra plants showed significant increase in shoot length, root length, plant fresh and dry weight. Mycorrhizal bajra plants were found to be dependent (Mycorrhizal dependency) on G. fasciculatum at second level of salinity condition. Mycorrhizal colonization was observed to be increased significantly in non saline and first level of salinity condition. After 90 days of AM inoculation mycorrhizal bajra plants showed increased photosynthetic activity upto second level of salinity. Shoot proline accumulation was higher in non mycorrhizal bajra plants and root proline accumulation was higher in mycorrhizal bajra plants. Root CAT activity was increased significantly in mycorrhizal bajra plants at second and third level of salinity, after 90 days of AM inoculation. After 60 days of AM inoculation, POD activity increased significantly in mycorrhizal bajra plants at third level of salinity (77.64%). After 90 days of AM inoculation, POD activity was found to increase significantly at all salinity levels in mycorrhizal bajra plants. Root SOD activity was found to be more in non-saline; first and second level of salinity after 60 days of AM inoculation. After 90 days, SOD activity was found to be more in second and third salinity levels in mycorrhizal bajra plants.
PS – 25

Effect of manganese concentration on growth responses of Gmelina arborea inoculated with Gigaspora margarita

Mayura Dudhane*, Mahesh Borde, and Paramjit Jite
Department of Botany, University of Pune, Pune 411 007.
*E-mail:mayuradudhane@gmail.com

Gmelina arborea Roxb. a medicinally and economically important tree species was selected for study. Mycorrhizal and nonmycorrhizal seedlings were treated with 50 mM, 100mM and 150mM concentration of Manganese. Gigaspora margarita treated Gmelina plants showed increase in shoot and root length, fresh and dry weight, greater percentage of mycorrhizal colonization, higher accumulation of proline and chlorophyll content with increasing levels of manganese. G. margarita colonization significantly increased acid and alkaline phosphatase activity, Proline content and also antioxidant enzymes like peroxidase and superoxide dismutase activity in mycorrhizal Gmelina plants as compared to non mycorrhizal plants.

PS – 26

Penicillium funiculosum rot of amla (Phyllanthus emblica L.) and its management with hot water dip treatment

Nisha Goswami* and Geeta Sumbali
Department of Botany, University of Jammu, Jammu 180 006.
*E-mail: nishagoswami23@yahoo.com

Fruits of Phyllanthus emblica L. are considered as potent gift of Mother Nature to mankind owing to their culinary usage and therapeutic potential. Approximately 15-20 % of the amla fruits are lost due to both pre- and post-harvest rot caused by Penicillium funiculosum. It is a unique pathogen requiring low pH (2.5) and low temperature (20-25oC) for its growth and the acidic fruits of amla serve as an excellent host.
Penicillium funiculosum grows aggressively, softening the fruit completely within 3-4 days, sporulating heavily on its surface and causing 100 percent loss. Healthy amla fruits get infected even by a brief contact with P. funiculosum infected fruits, suggesting thereby, the enzymatic potential of the pathogen.
In view of the extensive loss of amla fruits that occurs every year, an attempt was undertaken for the management of this disease by giving hot water dip treatment. Both pre-infection and post-infection dip treatments were given to amla fruits at 45oC, 50oC, 55oC, 60oC, 65oC, 70oC for 5 minutes. Results showed that hot water dip at 55 oC for 5 mins. gave maximum control (98.2%) without changing the texture and colour of the fruit. This method is considerably advantageous due to low cost, simplicity of application and good disease management.

PS – 27

Studies on the antagonistic effect of Bacillus sp. and Pseudomonas sp. against soil borne plant pathogenic fungi.

P. Rajapriya*, M.Pandi** and J. Muthumary**
Department of Biotechnology, Srinivasan College of Arts and Science, Perambalur 621 212.
**Centre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai 600 025.
*E-mail: pandypriya@rediffmail.com

The suppression of the growth of soil-borne plant pathogens by the rhizosphere microorganisms is termed as biocontrol. It is the reduction of the amount of inoculums or disease producing activity of pathogen accomplish by or through one or more organism. Biocontrol of plant disease with bacterial antagonist is a potential alternative of chemical control because chemical control is expensive and result in accumulation of hazardous compounds being toxic to the soil biota. However, recently some of beneficial bacterial strains applied in agriculture as biological control agents to suppress plant disease. Hence, the present study is deals with the antagonistic effect of bacillus sp. and pseudomonas sp. active against the pathogenic fungi i.e. aspergillus sp. and fusarium sp. The results will be discussed.

PS – 28

Biochemical changes in Sweet Potato infected with Charcoal Rot

V. B. Patil, A. R. Apte and S. S. Kamble*
Department of Botany, Shivaji University, Kolhapur 416 004.
*E-mail: s20sk@yahoo.co.in

Infection of sweet potato roots by M. phaseolina decreased the reducing sugars, DNA, RNA, calcium, manganese and zinc. Polyphenols were increased due to the infection by M. phaseolina isolates (sensitive and resistant). The increase was more in sweet potato roots infected by resistant isolate of M. phaseolina. Starch content was also increased in the sweet potato roots infected by sensitive and resistant isolates of M. phaseolina. There was also increase in iron content of sweet potato root due to infection by sensitive and resistant isolate of M. phaseolina.

PS – 29

Production of chitinase enzyme by Aspergillus species

Sandhya Ravishankar, C. V. Sushma, J. Sandhya, Pushpa Agrawal and Ashwani Sharma Department of Biotechnology, R.V. College of Engineering, R.V. Post, Mysore road,
Bangalore-59, Karnataka.

Fungi are used extensively to produce industrial enzymes such as lipases, amylases, cellulases, chitinases, invertases, proteases and xylanases. Among different species of fungi, Aspergillus species produces many enzymes, and the present study was aimed at production of chitinase enzyme from three different strains of Aspergillus. In vitro production of chitinases was examined under various cultural conditions. Three strains of Aspergillus were cultured in liquid synthetic medium containing different concentrations of colloidal chitin and at different incubation period. The highest specific activity of 0.257 U/mg of NAG was obtained with A.nigerS1 at 2ml concentration of colloidal chitin for 96hours incubation period. Antagonistic effect of the chitinase enzyme produced was tested by zone inhibition method for important phytopathogenic fungi (Alternaria, Fusarium, Colletotrichum and Sclerotium). The growth of these pathogens was inhibited by the action of crude extract of chitinase enzyme.


PS – 30

Ergosterol in Mucoraceous fungi

R. Rajeshwari, M. Rajeshwari, Preeti Garai, S. K. Sukrutha and J. Savitha*
Department of Microbiology & Biotechnology, Bangalore University,
Jnanabharathi Campus, Bangalore 560 056.
*E-mail: drsvtj@yahoo.co.in

Microorganisms have received broad attention as sources of novel lipids. Ergosterol [I (22E, 24R)- 24 methyl-cholesta-5,7,22, trienol] and its metabolic precursors are major lipid components of industrial and commercial interest. Ergosterol is considered as predominant sterol in fungi and yeast and is responsible for vital membrane functions such as fluidity and permeability similar to the way of cholesterol in mammalian cells. Ergosterol is the major precursor of vitamin D and is an important pharmaceutical intermediate that can be converted into cortisone and flavone hormones. Our present work involves screening of fungi belonging to the order Mucorales isolated from different natural sources for ergosterol content. The amount of ergosterol was determined using colorimetric estimation. Out of 24 isolates, the isolate 12 (Mucor sp.) showed highest ergosterol content i.e. 64mg/gm dry weight of mycelium whereas in other isolates, the ergosterol content ranged from 10mg-40mg/gm. Ergosterol in these isolates were further confirmed by HPLC analysis.
PS – 31

Exploring hidden dimension of soil fungal biodiversity: A simple technique to detect soil fungi resistant to antifungal compounds

Sonashia Velho-Pereira and Nandkumar Kamat*
Department of Botany, Goa University, Goa 403206.
*E-mail: nkamat@unigoa.ac.in

Soils are known to be ultimate and complex reservoirs of microbial diversity. The complex dimensions of bacterial and fungal diversity in tropical soils and microbial community dynamics are underexplored. Isolation techniques aimed at Actinomycetes generally employ highly selective media, powerful antibiotics and antifungal compounds to suppress undesirable bacteria and fungi. However some soil fungi may show their resistance towards these antifungal compounds. During our work to explore soil actinomycetes diversity, slides coated with Arginine Vitamin agar (AVA) incorporating a cocktail of antibiotics and antifungal compounds such as Nystatin, Cycloheximide, Terbinafin, Griseofulvin, and Flucanazole were exposed to soil environment and were retrieved at intervals of 4, 7, 15 and 28 days for detail microscopic studies of surface colonies. Alongwith actinomycetes the presence of unidentified aseptate and septate fungi was revealed indicating their resistance to combination and concentration of antifungals. Heat treatment of the soil was found to cause considerable decrease in fungal contamination probably due to elimination of heat labile fungi. Our results have led us to develop a simple procedure to sample the interesting and industrially useful strains of soil fungi resistant to common antifungal compounds. Some fungal strains are reported resistant to certain antifungals with resulting therapeutic failures as use of these antifungals inevitably selects resistant fungi, thereby pressing the urge for continuing and cyclical need of new antifungals. This technique could prove useful to detect novel antifungal resistant strains with potential to emerge as novel human pathogens. It has not escaped our notice that the probability of such finding could also help to verify whether these fungi could utilize such antifungal compounds through use of hitherto undiscovered metabolic pathways and novel enzymes leading to identification of genes responsible for antifungal resistance.

PS – 32

Biochemical investigations of fungal laccase from Pleurotus sp.

Sunil S. More* and Renuka Sudarshan
Department of Biochemistry, Center for Post Graduate studies, Jain University, 18/3,
9th Main Jayanagar, 3rd Block Bangalore 560 011.
*E-mail:sunilacr@yahoo.co.in

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are polyphenol oxidases that require O2 to oxidize phenols, polyphenols, aromatic amines and different non-phenolic substrates by one electron transfer resulting in the formation of reactive radicals.Laccasea are widely distributed in plants and fungi. In fungi they involve in morphogenesis and virulence.
In the present investigation A positive strain was isolated and characterized as non-spore forming Basidiomycetes Pleurotus sp. Laccase activity was determined using ABTS as substrate at 420 nm.Maximum laccase activity (30.8 U) was observed on 20th day in the medium with pH 6.5 laccase was purified by conventional ion exchenge chromatography. The purified laccase was a monomer showed a molecular mass of 39 ±1 kDa as estimated by SDS- PAGE with 72 fold purification with a 22 % yield. The Km and Vmax values are 250 (mM) and 0.33 (µmol/min) respectively for ABTS as substrate where as Km values for phenolic compound 2,6-DMP was 38.46 mM and Vmax 20 (µmol/min) which shows that phenolic compounds are better substrates for Pluerotus laccase than commonly used ABTS. Metal ions like CuSO4, BaCl2, MgCl2, FeCl2, ZnCl2 has no effect where as HgCl2, MnCl2 moderately decreases enzyme activity. SDS,completely inhibited enzyme where as sodium azide inhibited enzyme activity with Ki of 0.03mM and EDTA inhibited laccase to a lesser extent with a Ki of 3.2 .Urea, PCMB, DTT and Mercaptoethanol has no effect on enzyme activity.

PS – 33

Studies on Coelomycetous fungi with special reference to taxol production

S. Yogeswari*, M. Pandi, S. Kamalraj and J. Muthumary
Centre for Advanced Studies in Botany,
University of Madras, Guindy campus, Chennai 600 025.
E-mail: yogi_sagi2009@yahoo.co.in, mm_j@rediffmail.com

This paper gives an account of isolation of Coelomycetes from different places in Tamilnadu. Fifteen Coelomycetous fungi were isolated and examined. 12 were selected for screening for the production of taxol. Botryodiplodia theobromae Pat., Phomopsis sp. and Pestalotiopsis sp. were selected for taxol producing potential. Intracellular taxol was extracted from each fungus and
studied using TLC, Ultraviolet (UV) Absorption, Infrared (IR) Spectrophotometer and High Performance Liquid Chromatography (HPLC) analysis to detect Taxol. The results are presented.