Journal of Basic and Applied Mycology Volume 12 (I) 2016

Patron Secretary Joint Secretary Prof. S.K. Hasija Prof. Akhilesh K. Pandey Dr. Surendra Sarsaiya Former Head, Chairman, Sri Satya Sai University of Dept. of Biological Sciences, M. P. Private University Technology and Medical Sciences R.D. University, Jabalpur, Regulatory Commission, Bhopal, Madhya Pradesh, India Madhya Pradesh, India Madhya Pradesh, India +91-9826838221

President Advisors Publication Cell Prof. C. Manoharachary Prof. R.C. Rajak Dr. Rakesh Pandey Department of Botany, Former Prof. M.C.N.U.J.C., Bhopal Osmania University, Dept. of Biological Sciences, Hyderabad, India R.D. University, Jabalpur, Dr. Monika Pandey Madhya Pradesh, India NRLM, Shahdol Vice President Dr. N.D. Sharma Dr. Mahendra Rai Business Manager JNKV, Jabalpur, Professor and Head Dr. Amit K. Pandey Madhya Pradesh, Sant Gadge Baba Amravati R.K.D.F. University India University Amravati, Madhya Pradesh, India Maharashtra, India Treasurer Mr. Saket Mishra Dr. Sneha Pandey Prof. T.N. Lakhanpal M.P. Pollution Control Board AISECT University Himachal Pradesh University, Madhya Pradesh, India Madhya Pradesh, India Shimla, India +91-8269247438

Editorial Board

Editor-in-chief Members Councilors Dr. Jamaluddin Prof. N.N. Tripathi, Gorakhpur Dr. R.K.S. Kushwaha, Kanpur Emeritus Scientist (UGC), Department of Biological Prof. Anil Prakash, Bhopal Dr. D.H. Tambekar, Amravati Sciences, R.D. University, Jabalpur, Madhya Pradesh, Prof. D.J. Bhat, Goa Dr. S.S. Bisht, Uttranchal India Prof. J.K. Mishra, Lucknow Dr. Rashmi Agarwal, Bilaspur

Prof. Deepak Vyas, Sagar Dr. G.K.N. Chhetry, Manipur Editor Prof. S.S. Sandhu Dr. S.K. Deshmukh, Mumbai Dr. Aswani Tapwal, Shimla Department of Biological Sciences,R.D. University, Dr. Tara Dubey, USA Dr. Rekha Shukla, USA Jabalpur, Madhya Pradesh, India Dr. M.K. Jaiswal, Gwalior Dr. Nikita Banerjee, Kolkata

Dr. Abhisekh K. Awasthi, China

Associate Editors Dr. Sanjai Saxena Dr. Deepak K. Rahi Dr. Rohit Sharma Thapar University, Punjab Punjab University, Punjab NCCS, Pune

Dr. S.R. Nawange Dr. Mukesh K. Awasthi F.D.D. Research Center, Jabalpur Northwest A&F University, China

© 2016 Society for Basic and Applied Mycology.

Disclaimer All rights reserved. No part of this journal may be reproduced in any form without permission in writing from SBAM. The information contained in this publication represents those of the respective authors and not those of the publisher, SBAM. JOURNAL OF BASIC AND APPLIED MYCOLOGY

AN INTERNATIONAL PEER REVIEWED JOURNAL OF MYCOLOGY

Journal of Basic and Applied Mycology is a peer-reviewed open access international journal that focuses on original and innovative basic and applied research studies, as well as learned reviews on all aspects of mycology.

Journal (ISSN: 0972-7167) is published bi-annually by the Society for Basic and Applied Mycology. The society is established in 2002, one of the oldest scientific society of the country and registered under the society registration act 1973 para 44 no. JJ 6039. Since its inception, it has contributed significantly towards dissemination of research and development of mycology in the country and abroad. The journal has a international impact factor 1.887.

The journal is abstracted/indexed in leading databases of the world. Its objective is to present the highest quality scientific research from throughout the world on such divergent topics as the fungal diversity, physiology, biochemistry, molecular characterization, application etc, excluding case reports. The articles are intended to provide a comprehensive information base on areas in the forefront of research and newly proposed revisions in reference concepts for use by mycologists, and microbiologists.

The official language of journal is english. Each manuscript submitted to the journal must be an original paper that not been submitted elsewhere. The manuscript can be submitted under following headings: Review Articles, Full length research paper, short communication, new report/record, and book review.

We would like to invite you to contribute papers for publication in the forthcoming issues of Journal of Basic and Applied Mycology.

Please submit your manuscript via email to:

Dr. Jamaluddin Prof. S.S. Sandhu Editor-in-chief Editor Journal of Basic and Applied Mycology Journal of Basic and Applied Mycology [email protected] [email protected]

For any enquiry please contact to: Email id: [email protected] Mob: +91-9826838221 www.sbamjournal.com

Printed and Published by

Society for Basic and Applied Mycology (SBAM) Dr. Surendra Sarsaiya C/o Madhya Pradesh Private University Regulatory Commission, Gyan Vatika, Walmi Road, Kaliasot Dam, Bhopal, Madhya Pradesh, India Pin - 462016 www.sbamjournal.com Email: [email protected]; Mob: +91-9826838221

Abstracted & Indexed In

and many more under processing..... Journal of Basic and Applied Mycology Volume 12 (I(I)) 2016

Contents Review Articles Page No.

Microbial xylanases : tool for biotechnology and industry 01 Monika Sharma and Madhu Smita

Bioremediation: An em erging technology for pesticide contaminated soil 12 detoxification Archana Mishra, A. K. Pandey and Jamaluddin

Full Length Research Papers

Diversity and utilization of wild macro fungi in Chirang district of Assam, 16 Northeast India Karuna Shrivastava, Karabi Devi and Jahnovi Brahma

Influence of seasonal variation on the concentration of fungal airspora over 24 Kachai lemon plantation in Ukhrul district, Manipur S. Nimyaola and N. Irabanta Singh

A new potential mycoherbicide Setospherica monoceras var agbioae for control 28 of Water hyacinth Ajay Kumar Singh and Praharaju Laxminarayana

Comparison of virulence and antifungal susceptibility profile of Candida 32 dubliniensis and Candida albicans isolated from Human Immunodeficiency Virus- infected patients with oropharyngeal candidiasis Sachin C. Deorukhkar and Santosh Saini

Phytochemical screening and antifungal activity of acetone extract of 39 Azardirachta indica, Ocimum sanctum and Catharnthus roseus leaves Shikha Gauri, Shikha Bansal and Pooja Tripathi

A new potential mycoherbicide Alternaria alternata f. sp. praharajae for control 43 of Parthenium hysterophorus Ajay Kumar Singh and Praharaju Laxminarayana

Short Communication

Taxonomical notes on Acaulospora species of Indian Thar Desert 47 Praveen Gehlot

New Record/Report

Identification of Basidiobolus haptosporus from a subcutaneous biospsy 50 specimen in Côte d’Ivoire Angora Kpongbo Etienne, Offianan Andre Toure, Vanga-Bosson Henriette, Ira- Bonouman Ama, Djohan Vincent, Sylla-Thanon Karidja, Angorantchi Assemian, Yavo William, Konate Abibatou, Kassi K. Fulgence, Kiki-Barro Pulcherie and Menan E.I. Herve

Journal of Basic and Applied Mycology Volume 12 (I) 2016

Prof . G. P. Agarawal: A tribute

Professor G.P. Agarawal, was born on 10 th July, 1926 at Karwi (Banda), U.P. and passed away on January 03, 2016 at New Delhi. His demise is matters of deep regret for most of us who have known him for many years. He obtained his masters M.Sc. in Botany and D.Phil in Mycology and Plant Pathology f rom the University of Allahabad. For his D.Phil degree , he worked under the internationally renowned and eminent Plant Pathology Professor R.N. Tondon. Professor Agrawal started his career as Lecturer in the College of Agriculture, Nagpur in 1955. A year l ater he moved to Jabalpur and joined Mahakosal Mahavidyalaya (Now Government Science College). In 1961 he became Asst - Professor and in 1967 he was conferred as a Professor of Botany. He had faced many anxious moments in his professional career, but due to his prudence and courage he succeeds in all his endeavors. He established a Centre of Botany at Jabalpur. In 1968 the teaching department of Botany at the University of Jabalpur was started and Professor Agrawal became its founder Head of the Department. In the year 1979 due to his continuous and committed efforts the present department of Botany was converted into the department of Biological Sciences, the post which he graced till his retirement in 1986. Professor Agrawal was a man of exceptional qualit ies. He was an excellent teacher, a dedicated researcher, balanced administrator, and with his intellectual superiority, courtesy and generosity he had won many friends and admirers throughout the country. He was a perfect gentleman who preferred to keep a way from controversies and dissensions. His lucidity in teaching and expression promoted many of his students to take up teaching as a profession. He went out of his way to help his student is gaining excellence in their fields. His spontaneous nature and sharp intellect had always left an indeclinable impression on all those who came in contact with him. Under his dynamic leadership in a short span of time, the department had excelled in teaching and research activities. The teaching of M.Sc. in Botany was started in 1973. It was again professor Agrawal‘s initiative and foresightedness that he had introduced the teaching of Microbiology, as an especial paper in M.Sc. Botany in 1973. M.Sc. Biological Sciences was started in 1980 and M.Sc. in Microbiology in 1983. Prof. Agrawal was known for his research contribution in Mycology Plant Pathology and Microbial Physiology. He had authored more than one hundred research papers and published them in leading national and international journals of repute. Professor A grawal pioneered the study of phytopathogenic fungi of Jabalpur and published a series of papers under the caption “Fungi cause plant diseases at Jabalpur". He described many new taxas and new records for the country e.g Pycnothera cordiae gen et sp. nov., Cercocladospora adinae gen et sp. nov., Beharia smilacis sp. nov Sporotrichum xylophila sp.nov. All fungi described upto 1971 have been compiled and published by him as “Fungi of Jabalpur”. A new genus Agarwalia terricola gen et sp. nov was established in recognition of his contribution in the field of mycology. Prof. Agarwal's thrust has also been on fungal physiology, physiology of parasitism, medical mycology and aerobiology. He and his co -workers have investigated various aspects of physiology and nut rition of phytopathogenic fungi to understand their virulence, survival and adaptability. Nutritional physiology of several pathogens has also been worked out with him. Studies on metabolic changes in host due to fungal infection were also investigated. He suggested that disease resistance in fruits largely depends on their phenolic level. In his presidential address to M.P. Vigyan Academy he emphasized the significance of pectic enzymes in pathogenesis and reported a wide variation in behavior of fungi in pectic enzyme production. In most instances the enzyme was produced inductively. He has concluded that the cultural conditions and length of incubation period of pathogen greatly influences the nature and quantity of enzyme. His work on the production of toxic metabolites by phytopathogen indicated that the thermos table toxic principle produced by the pathogen could cause permeability changes In the host tissues leading to cellular death. Prof. Agarwal also initiated the study of mycotic infections in human beings and has contributed substantially to this important area of study, studies on fungal diseases of the skin and nails of man, mycotic keratitis (fungal infection of the eye), Aspergillosis and Cryptococcosis in bronchopulmonary disorders have been his major fields of research. The work is being carried further by his students in the field of aerobiology. His emphasis has been “fungal aerospora”. The work mostly is pertained to identifying the air borne plant pathogens and to provide adequate information about disease forecasting, currently, his co-workers are busy with Studies on the insect pest control through entomogenous fungi (biological control) of Teactona grandis and Cicer arietinum . Prof. Agarwal was principal investigator of a number of projects. More than forty students have been awarded Ph.D degree and one DSc. degree under his supervision. He had also published a book entitled "Microorganisms in the laboratory" in collaboration with Dr. S.K. Hasija and had edited a symposium volume entitled “physiology of parasitism’’ in collaboration with Prof. K.S. Bilgrami. He had several honours to his credit. He was Founder, president of society of tropical forestry scientist, president of botany section of M.P Vigyan Academy, a fellow of the National Academy of India, fellow of Indian Phytopathological Society of India and fellow of India botanical society. In addition, Prof. Agarwal was also associated as a member of editorial boards of Indian Phytopathological and Biological Bulletin of India, as associated editor of Bulletin of Pure and Applied Sciences, as Councillor for the Society of Mycology and Plant Pathology and Society for Advanced Botany. Prof. Agrawal had also taken active part in extra-curricular activities. He was an outstanding sportsman and represented University of Allahabad as captain of the Volleyball team during his college days. He had served as Chairman of Sports Committee of Rani Durgawaati University, Jabalpur for several years. He also worked in the capacity of a hostel Warden, and also proctor and treasurer of the University 'students union and was connected with many social organizations of the town. His informal manner and cordial behavior towards students made him very popular and respectful leader. Besides being a good teacher and researcher, he was a superior man as well; indeed a quality rarely found today in people having high positions. For people of position, Binjamin Franklin once visualised" If you'd not be forgotten, As soon as you are dead or rotten, Either write things worth reading or write things worth writing". Prof. Agrawal had done both the things. Prof. Agrawal was a versatile figure having all attributes of an intellectual dynamic, social and magnetic personality. His pragmatic approach towards the society made him a popular figure. This undaunted inspiration was a motivation to many scholars. It is said that behind every great man there is a woman. Mrs. Madhuri Agrawal is the woman who actually supported Prof. Agrawal throughout. She is a lady with clear devotion, modesty, simplicity, and had warmth of hospitality. To sum up Dr. Agrawal as a man, I am reminded of Latin Phrase " managest veritas et praevalet " meaning great is the truth and prevails. A truthful man is always kind, helping and encouraging and that Prof. Agarawal was. His contribution in creating an environment for excellent quality research and teaching is not Proxime accessed .

Prof. Akhikesh Pandey Chairman M.P. Private University Regulatory Commission Gyan Vatika, WALMI Road, Bhopal, Madhya Pradesh

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Microbial Xylanases: tool for biotechnology and industry

Monika Sharma 1* and Madhu Smita 2

1Department of Biotechnology, Panjab University, Chandigarh, India 2Department of Microbial Biotechnology, Panjab University, Chandigarh, India

ARTICLE INFO ABSTRACT The hemicellulose xylan, is the second most abundant Article History: renewable resource in the world. It is the main component of Received 15 Jan 2014 the plant cell walls and has lot of commercial value. Xylans Accepted 01 Feb 2016 can be degraded by xylanolytic enzymes called xylanases (Endo-1, 4-α-xylanase) and produce xylooligosaccharides, xylobiose and xylose. Xylanases can be obtained from

microbes (fungal, bacterial) and plants. Microbial xylanases Keywords: have gained special attention as they have tremendous Xylan potential for various applications. They have been Endo -xylanases extensively studied for their application in paper and fibre Protein purification making industry, baking, brewing and juice making industry, Xylanase application feed and non-food (biofuels and soap making) industry. Recombinant DNA technology Extensive research on xylanases is further expanding its

applicability. The review highlights the structure, classification, purification and industrial applications of Article Type: different xylanases. It also discusses the methods of Full Length Review Article improvement of the enzyme production i.e. immobilization, protein engineering and recombinant DNA technology to make it more economical and to suit the commercial applications. ©2016 Society for Basic and Applied Mycology All Rights Reserved.

INTRODUCTION complex molecule is composed of 1,4 linked Plant cell walls have three major xylose chains with branches containing polymeric constituents: cellulose, arabinose and 4-O-methylgluconic acid [2]. hemicellulose and lignin. Hemicelluloses are This structure can be degraded by embedded in the cell walls of plants with xylanolytic enzymes. pectin. They contain D-pentose sugars 1,4- endoxylanases play a major role mainly and have xylose as its largest in degradation of xylan backbone by component [1]. Xylan, one of the catalyzing the random hydrolysis of hemicelluloses, is a polymer of xylose and xylosidic linkages. On the other hand, β- represents more than 20–40% of plant xylosidases release xylosyl residues by biomass, making it second most abundant endwise attack on xylooligosaccharides in polysaccharide in world. xylan [3]. Xylans are mainly found in The abundance of xylan clearly secondary walls of the plant cell wall. This indicates that xylanolytic enzymes can play *Corresponding author: [email protected] (Monika Sharma) J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 2 an important role in bioconversion. Due to cleave internal glycosidic bonds within the this ability, xylanases have potential main xylan backbone; xylan esterase (EC application in the pulp and paper industry 3.1.1.6) releases acetate group; [4], production of animal feed [5], xylitol Arabinofuranosidase (EC 3.2.1.55) (low calorie sugar) production, bread- hydrolyzes arabinose side-chains; α- making [6], brewing and juice clarification glucuronidase (EC 3.2.1.139) removes [7]. Constant technological advancements glucuronic acid side-chains from the xylosyl and innovations are helping to widen the units; β-xylosidases (EC 3.2.1.37) diversity of usages of enzymes especially in hydrolyzes xylobiose to xylose. developing countries. Potential usefulness of This structural heterogeneity has a this enzyme in industry has spurred great impact on enzymatic hydrolysis. The considerable research efforts towards main xylanase (endoxylanase) cannot cleave producing thermophilic, alkalophilic and glycosidic bonds between xylosyl units other extremophilic enzymes, depending on efficiently, if substituted. Therefore, side the type of need. This has been chains must be cleaved before the xylan accomplished by screening for naturally- backbone can be completely hydrolysed. occurring xylanases and their application in During enzymatic hydrolysis side chains get new fields [8]. The review deals with the released prior to degradation of backbone. microbial xylanases and their sources, As main backbone gets degraded by endo-1, distribution, structure and industrial 4-β-xylanase into monomers, it is considered application- in food, feed and non-food as the main xylanase [3]. technology. It also highlights the methods of improvement of xylanase production and Sources of xylanases activity. Xylanases were first reported in 1955 and were originally termed as pentosanases. Xylan and xylanolytic enzymes The International Union of Biochemistry Xylan derives its name from Xylose, and Molecular Biology (IUBMB) a sugar first isolated from wood, and named recognized them in 1961, when they were by Robert Koch in 1881. Xylose is an assigned the enzyme code EC 3.2.1.8. Their aldopentose type monosaccharide, official name is endo-1,4-β-xylanase, but containing five carbon atoms and includes a commonly used synonymous terms include formyl functional group. It is the precursor xylanase, endoxylanase, 1,4-β-D-xylan- to hemicellulose, one of the main xylanohydrolase, endo-1,4-β-D-xylanase, β- constituents of biomass and a part of neutral 1,4-xylanase and β-xylanase. detergent fibres which form the fibrous bulk Xylanases can be found in bacteria, of forage. Hemicelluloses have a random, marine algae, fungi, protozoans, crustaceans, amorphous structure with little strength. insects, gastropods, anthropods, snails and With its free carbonyl group, it is a reducing seeds of land plants [10]. Xylanases are sugar. There is a structural heterogeneity in produced mainly by microorganisms and xylans i.e they are present as take part in the breakdown of plant cell glucuronoxylans, glucuronoarabinoxylans, walls, along with other enzymes that arabinoxylans and arabinofuranoses. hydrolyse polysaccharides. It can also digest Glucuronoxylans and glucurono- xylan during the germination of some seeds arabinoxylans are located mainly in the (e.g. in the malting of barley grain). secondary wall and form covalent and non- covalent bonds with lignin, cellulose and Fungal xylanases other polymers essential to the integrity of Among microbial sources, the cell wall. The amount of each component filamentous fungi are especially desirable as varies from species to species and even from they secrete these enzymes into the medium tree to tree [9]. and their xylanase levels are much higher Xylans are degraded by a complex of than those found in yeasts and bacteria. xylanolytic enzymes like α-1,4- Interestingly, fungal xylanase has become endoxylanases (xylanases; EC 3.2.1.8), synonym with extracellular xylanase. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 3

Various fungal strains belonging to diverse activities are also important. The genus including Trichoderma sp .[11-12] , dinitrosalicylic acid (DNS) method by Aspergillus sp. i.e.( A.awanori, niger, Miller has been the most preferred assay fumigatius, japonicus, kavachii, oryzae, method. Arsenomolybdate method (ARS) of sulphurous, versicolor ), Aurobasidium, Nelson and Somogyi [20] can be used to Penicililum, Botrytinia sp., Trichomonas sp., measure the reducing sugar. HPLC-based Claviceps, Neocallimastix sp. have been IC method combined with pulsed reported for xylanase production. Among amperometric detection (PAD), is also a various strains of xylanase producing fungi, mean to determine enzyme kinetics. The Aspergillus, Trichoderma and Pencillium sp. IC/PAD analysis detects almost twice as are dominanting in xylanase production at much product as the DNS method [21]. industrial scale [9]. The xylanases obtained from fungi Xylanase classification and structure have been found to have high enzymatic Glycoside hydrolases are a activity over a range of pH and widespread group of enzymes which temperatures. Thermostable xylanases hydrolyse the glycosidic bond between two having activity maxima at higher or more carbohydrates or between a temperatures are highly desirable for carbohydrate and a non-carbohydrate industrial purposes. A number of fungal moiety. Glycoside hydrolases have been isolates have been reported for xylanase classified on the basis of structural and production (Table 1), and research on sequence similarities into 133 families. isolating newer and novel strains is under Among these 133 families, Xylanases have process. been classified as belonging to 5, 7, 8, 9, 10, Various bacterial strains belonging to 11, 12, 16, 26, 30, 43, 44, 51 and 62 families Paenibacillus sp. [13], Bacillus sp., of glycoside hydrolases [22]. Action Geobacillus, Thermobifida, Thermobacillus, mechanism of xylanase includes the Clostridium, Pseudomonas and hydrolysis of the glycosidic bond which is Enterobacter have been reported to produce catalyzed by two amino acid residues of the xylanases (Table 1). enzyme: a general acid (proton donor) and a The process of obtaining newer nucleophile/base. Depending on the spatial xylanases of microbial origin depends on position of these catalytic residues, action more reliable, robust, efficient and easy occurs via overall retention or overall screening methods. For xylanases, potato inversion of the anomeric configuration. dextrose agar with different xylan sources Xylanases are classified into two distinct (wheat bran, rice bran, oat meal, sugarcane families, family F/10 and G/11 of glycoside bagasse, birch wood etc.) has been used. M9 hydrolases. Family 11 xylanases media [14], basal salt media [15-16], ZoBell (E.C.3.2.1.8) regarded as “true family” have Marine agar [17] and nutrient agar media low molecular weight ( ∼20 KDa) and [18] with xylan source has also been used to hydrolyze xylan to form oligosaccharides of isolate the xylanase producing microbes. different lengths. Endo-1,4-β-xylanase (EC Most of the microorganisms have been 3.2.1.8); endo-1,3-β-xylanase (EC 3.2.1.32); isolated from soil contaminated with are the known activities of family G/11. industrial effluent [19]. Agro-industrial Their 3D- structure is β- jelly roll. 5 archea, waste, bagasse, composting waste material 425 bacteria and 310 eukaryotes are known are capable of producing a spectrum of cell to be belong to this family [22]. wall-degrading enzymes. They are generally In xylanases (3.2.1.8), the two active grown on finely powdered (having particular site glutamate residues, one acting as proton mesh size) agroresidues- wheat bran, rice donor (acid or base) and the other acting as bran, bagasse, corncobs, wheat straw and nucleophilic base, are found to be rice straw while working at laboratory scale responsible for enzyme hydrolysis. They are (Table 1). found to catalyse xylan by double High productivity is essential for displacement mechanism [23-24]. commercial value, so accurate measures of © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 4

A disulphide bond and more than ten products of molecular weight 135 kDa and salt bridges are there in the structure. In 145 were observed which showed similar particular, Arg124-Glu232, bridges the β- hydrolytic, immunological and physiological strands β4 and β7 and glutamate residues properties [28]. may play an important role in the stability Synergistic and cooperative effects and activity at elevated temperature. Proline among the xylan-degrading enzymes resides in the middle of the α-helix α6 which enhance the susceptibility of the may be contributing to better packing. The heteropolymeric xylan to be attacked by members of family 11 xylanases form a endoxylanases. Complex substrates such as structure that resembles a “partly closed wheat bran, which contains quite large right hand” [25]. amounts of arabinoxylan, get easily The known activities of family F/10 degraded by adding α-arabinofuranosidase are endo-1,4-β-xylanase (EC 3.2.1.8); endo- along with endoxylanases, thus enhancing 1,3-β-xylanase (EC 3.2.1.32) and the saccharification of arabinoxylan [9]. tomatinase. Their 3D- structure includes Tim Therefore, for biotechnological barrel shaped structure containing eight purposes, the ideal microorganism would be strands of α/β and glutamate as catalytic the one that produces an adequate amount of nucleophilic base and proton donor. 14 each of the enzymes of the xylanolytic archea, 938 bacteria and 295 eukaryotes are complex. “Xylanosome” thus refers to known to be belonging to this family. structures observed as protein aggregates of many subunits with xylanolytic activity and Multiple xylanases have a very high MW (500–600 kDa). In Xylan is known to exhibit some microorganisms, there may be polydiversity and polymolecularity that has association of xylanosome with cellulosome resulted in the occurrence of multiple (multienzyme cellulase complex) forming xylanases. These multiple xylanases have large complexes responsible for the been reported in many microorganisms. hydrolysis of both cellulose and xylan [29]. Aspergillus kawachii shows three endo- xylanases [26] and one of the Streptomyces Enzyme purification sp . also produces three endo-xylanases [27] Purification of target enzymes from a but each of different molecular weight. pool of proteins requires tedious purification During enzymatic action, all xyloside steps thereby increasing their costs [30]. It linkages are not accessible to a single owns a lot to the cost of the enzyme at enzyme and accessibility gets changed industrial scale. Due to these steps, industry during course of hydrolysis. So, a group of is aptly penchant towards extracellular enzymes each with a specific function brings enzymes. Column chromatographic efficient hydrolysis. Different types of techniques, mainly ion exchange, size xylanase differ in their pattern of cleavage of exclusion, hydrophobic interaction column xylosidic bonds. These include Arabinose chromatography are the generally utilised liberating endo-xylanases; Non-arabinose for xylanase purification. liberating endo-xylanases, exo-β-1,4-D- There are many applications of xylanase, β-Xylosidase, acetylxylan xylanases, where it is either required in pure esterase, arabinase, ferulic acid esterase and form or free from impurities like cellulose or p-coumaric acid esterase. protease. 31 Ammonium sulphate fractionation Most of the times, a single xylanase (40–70% saturation) of crude xylanase was gene encodes for multiple xylanases but reported to yield 84.7% of the enzyme with posttranslational modifications such as 4.98-fold purification [32]. As this enzyme differential glycosylation or proteolysis or was extracellular, it was used for many both are responsible for multiple forms of applications like whole wheat bread, xylanases. Redundancy in gene expression probiotics after this partial purification step. also leads to xylanase multiplicity in plant The separation of proteins can be done by pathogens. When xylanase from Aeromonas conducting bioseparation in aqueous two sp. was expressed in E . coli , two gene phase systems where two mutually © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 5 incompatible polymers or polymer and salt hemp, sisal, abaca, and jute [40]. Xylanase are mixed at certain concentration. Xylanase mixtures have been used for enzyme retting from Trichoderma viride was separated by process, i.e. for flax fibre separation from precipitation with an ionic polymer Eudragit core cells [41] leading to separation of fibre S 100 with high yield [33]. Purification of bundles from non-fibre parts. In keeping xylanase was carried out by batch treatment with the requirements of the pulping with DEAE cellulose and the lyophilized operations in paper and fibre industry, the enzyme sample was then applied to search for cellulase-free xylanases from superdex75 FPLC column [34]. Xylanase diverse microbial strains that are active was purified to near homogeneity using under highly alkaline pHconditions with packed and expanded beds [35]. Another different temperature stabilities have gained technique employed for the purification of momentum [42]. xylanase was by eluting the adsorbed extracellular enzyme from Amberlite IRC- 2. In baking, brewing and juice industry 50, thus providing a yield as high as 82% Xylanases are becoming cynosure in [36]. baking and brewing industry, related to cereal technology where they are used to Application of xylanases reduce processing time and increase Progress in the understanding the separation and product quality. In baking structure and function of xylanase, has industry, xylanases have been instrumental paved the way towards its broader in improving the quality, color and volume applicability. Paper and pulp bleaching by of the bread. They help to improve the xylanase has remained the foremost usage crumb structure, reduce the stickiness of the till date but nowadays, xylanases have been dough and increase the shelf life of the used for other applications such as bread. They have been reported to modify production of xylitol, biofuels, feed and food the rheological properties and consistency of industry, fibres making, baking and brewing the spaghetti dough [43]. (Table 2). Xylanases have been utilized to increase the wheat flour separation into 1. Paper and fibre industry starch and gluten. The potential of During the Kraft process in the paper Aspergillus foetidus MTCC 4898 strain as a industry there is lot of lignin deposition that bread improver was tested in whole wheat confers a dark colour to the pulp. It requires bread by Shah [32]. There was an a bleaching step with lots of chlorine to be improvement in cohesiveness and decline in used. The use of chlorine results in the springiness and gumminess of the bread. production of highly toxic and mutagenic Xylanases have been incorporated in beer organochlorides from the degradation making process to assist in modifying β- products of lignin, making treatment of the glucan/xylan haze and increase the filtration effluents from the paper-making plant to be performance of beer wort and improve necessary. It also increases the cost, but lautering as an aftermath. The Ceremix about 10% of the bleaching chemical can be Plus™ and Ultraflo Max™enzyme replaced by xylanases [37]. Use of xylanase preparations containing xylanase have been in paper and textile is its major application used in the mashing process [44]. and the requirement of more and more Beside bread and baking related environment friendly processes has made processes, xylanase mixtures are used for these enzymatic processes still more clarifying fruit juices. Bacillus pumilus SV- desirable. The relative low MW of GH-10 85S xylanase has been used for enzymatic and GH-11 of xylanases help in penetrating clarification of apple, pineapple, and into the inner part of the cell wall [38], tomatojuices [42]. Likewise, a cocktail of which results in the better deletion of lignin xylanases from Sclerotiniasclerotiorum polymers from the wood pulp [39]. helps in juice clarification by releasing Xylanases can also be used in oligo- and mono-saccharides, and synthesis of long plant fibres from flax, concomitantly decreasing the level of © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 6 insoluble materials up to 27% [45]. The positive effect on growth performance and treatment with xylanases lead to an digestion. It also affects immune parameters increased juice yield and transmittance. It and gut microflora [51]. helped in bringing down the turbidity and viscosity of the product without any 5. Biofuels significant effect on acid neutrality. Among the second generation biofuels ethanol consumption is increasing 3. Xylanases as probiotic day by day. Lignocellulosic biomass Xylanases have also been reported provides a more economical and greener for their probiotic potential. Xylanase from approach for bioethanol production than Aspergillus foetidus MTCC 4898 has been other sources. Xylanase break down xylan in used for production of xylo- the lignocellulosic material into a mixture of oligosaccharides(XOS) from corncobs [46]. pentoses (xylose and arabinose from Partially purified β-xylosidase free enzyme hemicelluloses) and other short was found efficient in releasing only short oligosaccharides, which are subsequently chain XOS (xylobiose, xylotriose, fermented into ethanol by different xylotetraose and xylopentaose). Probiotic mechanisms [9]. effect of XOS was evaluated by in vitro As compared to chemical processes, fermentation of XOS using known probiotic enzymes are critical components by strains viz. Bifidobacterium adolescentis, facilitating more sugars for fermentation and Bifidobacterium bifidum, Lactobacillus giving higher yield [52]. fermentum, Lactobacillus acidophilus [46] . Sun [53] studied the effect of dilute Other released XOs havealsobeen reported sulphuric acid pre-treatment on rye straw to have antioxidant property, low density and Bermuda grass for ethanol production. lipid (LDL) peroxidation and aid in The results revealed astonishing progress prevention and treatment of anaemia and with increase in sugar yield of Bermuda arteriosclerosis [47]. grass from 46% to 81% depending upon dilute sulfuric acid concentration and 4. Feed technology retreatment time. Thus, physical Application in the feed technology is characteristics including enzyme size and other arena of synergistic xylanases. Xylan the interwoven cell wall network in the or arabinose are among the major non-starch lignocellulosic biomass are considered as polysaccharides present in the feed and relevant factors for determining the xylanase increase the viscosity of the feed. The performance [38]. molecular weight, the structure of xylan and the arabinose to xylose (A/X) residue ratio 6. Soap technology of the feed alter the solubility [48]. Hence Xylanases can be used in substitution hydrolysis of feed is essential to make it of conventional chemical soap treatment for more digestible. BioSorb – ACDX is a wool and specialty hair fibres [54]. Itis xylanase enzyme to break down xylan / advantageous to incorporate alkaline arabinose in the animal feed. Facchini [49] xylanases in detergent formulations to studied and characterized fibrolytic enzymes solubilise stubborn stains of plant origin by Aspergillus japonicas C03 with their [55]. They can clean fibres efficiently by stability in the presence of goat ruminal synergistic action and do not cause any environment, while prying to use it in physical damage. In textiles they can be used ruminant feed applications. Similarly for bleaching action. Other worker checked xylanase was immobilized on glutaraldehyde the compatibility of the alkaline xylanase activated aluminum oxide pellets for with commercially available detergents and increasing digestibility of poultry feed [50]. proteases [55]. In the presence of detergents, However, for those productions, enzyme the enzyme retained its full activity (40°C processing involves generally more than one for 1 h). Significant activity (80% for 1h) in Glycoside hydrolases. In poultry production, the presence of the proteases Alcalase and xylanase supplementation in diet have Conidiobolus protease was also retained. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 7

Supplementation of the enzyme enhanced The polymer was first activated with the cleaning ability of the detergents. glutaraldehyde and xylanase bound polymer was characterized using FTIR. Immobilized 7. Xylanase-based production of XOS for enzyme exhibited better thermal stability, plant defense reusability and storage stability along with Ethylene-inducing xylanase is a lower Km values. potent elicitor of plant defense responses in Pal and Khanum [64], compared a some species of tobacco ( Nicotiana few kinetic properties of covalently tabacum ) and tomato ( Solanum immobilized xylanase on the surface of lycopersicum ) [56]. Its products have been glutaraldehyde activated alginate beads with reported to induce ethylene biosynthesis, free xylanase. The immobilized enzyme electrolyte leakage, media alkalinisation. displayed lower kcat/Km value. But, Xylanase has also been reported to induce provided ‘low temperature stabilization’ expression of pathogenesis-related (PR) effect to xylanase which makes it more proteins and the hypersensitive response thermostable at temperatures lower than the (HR) in specific plant species and/or Tisokin (Isokinetic temperature). varieties by many researchers [57-58]. Partially purified enzyme was Ethylene-inducing xylanase (EIX) is immobilized using Barium alginate gel using a 22-kD fungal protein ( β-1-4-endoxylanase) entrapment method for its use in continuous and has been reported to be isolated from fluidized bed column reactor by Pal [65] . xylan-induced Trichoderma viride [59-60] . Xylanase from fungus, T. lanuginosus Xylanases have also been identified in SSBP, was immobilized on the polymers xylan-induced filtrates of plant pathogenic alginate, chitosan, and Eudragit S-100. The fungi. The proposed mechanism of action is activity of the precipitated enzyme was that the oligosaccharides come from cell highest with Eudragit S-100 immobilization. wall components and activate plant defence The xylanase retained 62% of its activity systems by mimicking pathogen aggression after six precipitation cycles on the polymer in the plant [61]. matrix [66]. Nagar [50] reported covalent Methods of enzyme improvement immobilization of xylanase by response Although many wild-type xylanases surface methodology on glutaraldehyde- contain certain desired characteristics, such activated aluminum oxide pellets. The bound as thermostability, pH stability or high enzyme gave an immobilization yield of activity, an individual xylanase is not 83.65% and displayed increase in optimum capable of meeting all of the requirements of temperature, pH, half-lives and Vmax.. the feed and food industries. Moreover, as Immobilized xylanase could be reused for 10 industrial applications require cheaper consecutive cycles retaining 60% of its enzymes, the elevation of expression levels initial activity. and the efficient secretion of xylanases are Polymethyl methacrylate (PMMA) crucial to ensure the viability of the process; nanofiber membrane (NFM) was therefore, recombinant DNA technology and synthesized by an electrospinning technique genetic engineering have a significant role in [67]. The pH and temperature stability of the large-scale expression of xylanases. In xylanase were enhanced upon addition, techniques like enzyme immobilization. The immobilized enzyme immobilization and protein engineering have was active on repeated use and retained contributed to applicability. ∼80% of its initial activity even after 11 reaction cycles. 1. Immobilization Immobilization of enzymes helps in 2. Protein Engineering their recovery and reuse for various A new directed evolution method applications [62]. Chemically synthesized was used to enhance the thermostability of polyaniline (PANI) was used as polymeric the wild-type GH11 xylanase from support for xylanase immobilization [63]. Hypocrea jecorina . Both Look-Through © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 8

Mutagenesis (LTMTM), and Combinatorial indicating that glycosylation is not essential Beneficial Mutagenesis (CBMTM), were for maintaining the activity of this enzyme. employed to enhance the stability of an Endo-xylanase expression levels are enzyme. When these mutations were relatively higher in Lactobacillus species combined into a single construct, the and Bacillus subtilis than in E. coli as these purified protein was active even after gram positive bacteria can perform N- heating at 100 oC for 20 min [68]. glycosylation [73]. A combinatorial strategy to engineer the KRICT PX1 gene (GB: FJ380951) of GH11 xylanase from Thermobacillus 996 bp and mol wt. of 38.1 kDa from xylanilyticus (Tx-Xyn) was implemented Paenibacillus sp. strain HPL-001 [69] to improve the hydrolytic performance (KCTC11365BP) has been cloned and for its use in biorefining. expressed in E. coli [74]. Similarly, The enzymatic activity of xylanases xylanase gene, xyn40, from marine B. varies considerably under various physico- subtilis cho 40 was cloned, and expressed in chemical conditions such as temperature, E. coli [75]. pH, high salt and presence of proteases. A Moreover, Lactobacillus possesses better understanding of the structural GRAS status (generally regarded as safe), implications help in protein enginnering. So, providing it an easier acceptability for the in Silico study of Family 10 or glycosyl recombinant proteins expressed in these hydrolase 10 (GH10) xylanases having Tim- bacterial cells to be accepted for use in food barrel fold structure was done. It revealed and dairy industries. that N- and C-terminal might contact to provide protein stability and help proteins to 3.2. Yeast expression system retain function under extreme conditions To secrete proteins into fermentation [70]. media and to grow to very high cell densities has projected Saccharomyces cerevisiae and 3. Recombinant DNA (rDNA)technology Pichia pastoris as attractive hosts for Recombinant DNA technology has expressing heterologous and homologous turned the DNA into a play station, to play proteins. Studies on bacterial β-xylanase are with its genes, for large scale expression of limited to the genera Bacillus , Streptomyces these enzymes in both homologous and [76] and Clostridium [77]. S. cerevisiae has heterologous protein expression hosts. It GRAS status and causes minimal makes the enzyme production more contamination of recombinant proteins by economical and applicable for industry. secreting very few native proteins in the medium [78]. Several studies have been 3.1. Bacterial expression system conducted on the secretion of heterologous Different bacterial expression xylanases like T. reesei xylanase gene systems have been reported to express endo- (XYN2) [79], Aspergillus kwachii [78] by S. xylanases, these includes E. coli, cerevisiae . Lactobacillus, Bacillus subtilis and Oleaginous yeast Yarrowia Clostridium . E. coli is considered to be an lipolytica has emerged as excellent protein ideal system for expressing recombinant expression host. A comparison of expression proteins. Whereas, the repetitive appearance systems in the S. cerevisiae, Hansenula of rare codons, absence of N-glycosylation polymorpha, Klyveromyces lactis, [71], requirement for specific post Schizosaccharomyces pombe and Y. translational modifications, usually makes it lipolytica was done by Muller [80]. Y. difficult for many heterologous proteins lipolytica was found as a highly attractive such as xylanases to be functionally alternative to S. cerevisiae as a host expressed in E. coli. organism for expression cloning. To A glycosylated β-xylosidase from a facilitate the direct conversion of xylan into thermophilic fungi P. thermophila was ethanol, construction of xylan-fermenting functionally expressed extracellularly in E. yeast strain through co-display of coli with a titer up to 98.0 U/ml [72], xylanolytic enzymes was done on the © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 9 surface of xylose-utilizing Saccharomyces technology have played an important role in cerevisiae cells [81]. the large-scale expression of xylanases in Similarly, highly efficient production homologous or heterologous protein- of a thermostable β-1,4-xylanase from expression hosts. We believe that in near Thermomyces lanuginosus was achieved future tailor made xylanases will be in P. pastoris under the control of the AOX1 exploited to revolutionize their industrial promoter making this expression system application. attractive for industrial application [82]. Heterologous expression of a gene for REFERENCES thermostable xylanase from Chaetomium 1. Ota, M., H. Sakuragi, H. Morisaka, K. thermophilum in P. pastoris GS115 was also Kuroda, H. Miyake, Y. Tamaru and M. performed to qualify it for the potential use Ueda (2013). Biotechnol. Progr . 29(2): in paper, pulp and animal feed applications 346-351. [83]. 2. Emilie, A., E.A. Rennie and H.V. Scheller (2014). Curr. Opin. Biotechnol . 3.3. Fungal expression system 26 :100-107. Fungal expression system is another 3. Sharma, M. and A. Kumar (2013). important alternative for production of both British Biotechnol. J. 3(1): 1-28. homologous or heterologous proteins by 4. Sandrim, V.C., A.C.S. Rizzatti, Terenzi, fermentation. They are more applicable over H. F., J.A. Jorge, A.M.F. Milagres and developed bacterial and yeast systems for M.L.T.M. Polizeli (2005). Process recombinant protein expression, for Biochem . 40:1823-1828. expressing foreign xylanases from other 5. Panwar, D., P.K. Srivastava and M. sources. Fungal systems handling offer Kapoor (2013). Biocatal. Agric. easier cultivation and cheap solid state Biotechnol . http://dx.doi.org/ 10.1016/ fermentation. Bacterial xylanase gene, j.bbr. 2011.03.031. Nonomuraea flexuosa xyn11A , was 6. Shah, A.R., R.K. Shah and M. expressed in the filamentous fungus Madamwar (2006). Bioresource Technol . Trichoderma reesei [84]. The xylanase gene 97 : 2047-2053. from P. griseofulvum has been expressed in 7. Kumar, L., S. Nagar, D. Kumar, N. A. oryzae [85]. However, constitutive Garg and V.K. Gupta (2013). Int J. Cell expression is more preferable in some cases Science Biotechnol . 2: 1-10. than inducible expression. The constitutive 8. Guha, S., S. Bhutty, S.M.P. Khurana promoters of fungus T.reesei were screened and U.K. Kohli (2013). Int J. Res. Pure and successfully used for high level Appl. Microbiol . 3(4): 116-120. homologous expression of xylanase II [86]. 9. Polizeli, M.L.T.M., A.C.S. Rizzatti, R. Monti, H.F. Terenzi, J.A. Jorge and CONCLUSION D.S. Amorim (2005). Appl. Microbiol. Xylanase, a complex enzyme system Biotechnol. 67 : 577–591. of microbial origin, plays an important role 10. Sunna, A. and G. Antranikian (1997). in the degradation of xylan, the most Crit. Rev. Biotechnol . 17(1): 39-67. abundant polysaccharide in nature. Endo- 11. Voronovsky, A.Y., O.V. Rohulya, C.A. 1,4-β-xylanase, cleaves the glycosidic bonds Abbas and A.A. Sibirny (2009). Metab. between xylosides,generating short Eng. 11 : 234–242. xylooligosaccharides. The majority of 12. Mohan, G., P. Guhankumar and V. xylanases have been classified into the Balakrishnan (2011). Research Plant GH10 or GH11 families. These enzymes Biology . 1(3): 15-20. have a wide range of industrial applications. 13. Hwang, I.T., H.K. Lim, H.Y. Song, S.J. Improved screening and purification Cho, J.S. Chang and N.J. Park (2010). methods have been developed for easier and Biotechnol. Adv . 28 : 594-601. cheaper production of these enzymes to 14. Chandra, S., R. Sharma, N.D. Jasuja, fulfill the demands of various industries. N. Saxena and S. Rana (2012). Ind. J. Protein engineering and recombinant DNA Fund. Appl. Life Sci . 2(4): 7-12. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 10

15. Khandeparkar, R.D.S. and N.B. Bhosle 35. Savitha, S., S. Sadhasivam and K. (2006). Enzyme Microbial. Technol. Swaminathan (2009). Bioresour. 39(4): 732-742. Technol. 100 : 883–889. 16. Sharma, A., R. Pujari and P. Patel 36. Santos, E.S.D., R. Guirardello and T.T. (2009). Inter. J. Biotechnol. 8: 110-114. Franco (2002). J. Chromat. A. 944 : 217- 17. Khandeparker, R., P. Verma, R.M. 24. Meena and D.D. Deobagkar (2011). 37. Breccia, J.D., R.H. Kaul, G.R. Castro Shelf Sci . 95 : 359-366. and F. Sineriz (1999). Bioseparation . 8: 18. Mahilrajan, S., S. Balakumar and V. 273-79. Arasaratnam (2012). Advances Appl. Sci. 38. Kulkarni, N., A. Shendye and M. Rao Research . 3(1): 242-250. (1999). FEMS Microbiol Rev. 23 : 411- 19. Kaur, A., R. Mahajan, A. Singh, G. 456. Garg and J. Sharma (2010). Bioresour. 39. Beaugrand, J., G. Paes, D. Reis, M. Technol. 101(23): 9150-9155 Takahashi, P. Debeire, M.J. Odonohue 20. Miller, G. L. (1959). Anal. Chem. 131 : and B. Chabbert (2005). Planta . 222 : 426-8. 246-257. 21. Somogyi, M. (1952). J. Biol 40. Beg, Q.K., M. Kapoor, L. Mahajan and Chem . 195(1): 19–23. G.S. Hoondal (2001). Appl. Microbiol. 22. Jeffries, T.W., V.W. Yang and M.W. Biotechnol . 56 : 326-338. Davis (1998). Appl. Biochem. Biotechnol . 41. Ibarra, D., V. Kopcke, P.T. Larsson, 72 : 257-265. A.S. Jaaskelainen and M. Ek (2010). 23. Carbohydrate - active enzyme database Bioresour. Technol. 101 : 7416–23. (1998). Cazy database. Available at: 42. Mussig J. (2010). Wiley Series in (http://www.cazy.org). Renewable Resources. New York: John 24. Koshland, Jr.D.E. (1953). Biological Wiley & Sons, Inc. Reviews . 28(4): 416-436. 43. Nagar, S., A. Mittal and V.K. Gupta 25. Henrissat, B. and G.J. Davies (1997). (2012). Asian J. Biolog. Sci . 5(8): 384- Curr. Opin. Struct. Biol . 7(5): 637-644. 394. 26. Torronen, A. and J. Rouvinen (1995). 44. Ingelbrecht, J.A., K. Moers, J. Biochemistry. 34(3): 847–856. Abecassis, X. Rouau and J.A. Delcour 27. Ito, K., K. Iwashita and K. Iwano (2001). Cereal Chem. 78 : 721–9. (1992). Biosci. Biotechnol. Biochem. 56 : 45. Aastrup, S. and H.S. Olsen (2008). 1338–1340. Biozoom . 2:1-10. 28. Georis, J., E.F. de Lemos, J. Lamotte- 46. Olfa, E., M. Mondher, I. Smaali, I. Brasseur, V. Bougnet, B. Devreese, F. Ferid and M.M. Nejib (2007). J. Food Giannotta, B. Granier and J.M. Frere Biochem . 31(1): 96-107. (2000). Protein Sci . 9: 466–475. 47. Chapla, D., P. Pandit and A. Shah 29. Wong, K.K., L.U. Tan and J.N. Saddler (2011). Bioresource Technol. 115 (2012) : (1988). Microbiol. Rev . 52 : 305–317. 215-221. 30. Sun, J., F. Wei, T. Si, J.H. Xu and H. 48. Moure, A., P. Gullon, H. Dominguez Zhao (2012). Appl. Environ. and J.C. Parajo (2006). Process Microbiol. 78(11): 3837-3845. Biochem. 41 : 1913-1923. 31. Kormelink, F.J.M., L.M.J.F. Searle- 49. Broekaert, W.F., C.M. Courtin, K. Van, T.M. Wood and A.G.J. Voragen Verbeke, T. van de Wiele, W. (1993). J. Biotechnol. 27: 249-265. Verstraete and J.A. Delcour (2011). 32. Raj, A., S. Kumar, S.K. Singh and M. Crit. Rev. Food Sci. Nutr. 51 : 178-194. Kumar (2013). Scientific World J. 50. Facchini, F.D.A., A.C. Vici, V.R.A. Reis, doi: 10.1155/2013/386769. J.A. Jorge, H.F. Terenzi, R.A. Reis and 33. Shah, A.R. and D. Madamwar (2005). M.L.T.M. Polizeli (2011). Bioprocess Process Biochemistry . 40 :1763–1771. Biosyst. Eng. 34 : 347–355. 34. Gupta, M.N., D. Guoqiang, R. Kaul and 51. Nagar, S., A. Mittal, D. Kumar, L. B. Mattiasson (1994). Biotechnol. Tech . Kumar and V.K. Gupta (2012). Process 8:117-22. Biochemistry . 47(9): 1402-1410. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 01-11, 2016 11

52. Gao, F., Y. Jiang, G.H. Zhou and Z.K. 71. Bhardwaj, A., P. Mahanta, S. Han (2007). Br. Poult. Sci . 48 : 480-488. Ramakumar, A. Ghosh, S. Leelavathi 53. Margeot, A., B. Hahn-Hagerdal, M. and V. S. Reddy (2012). Comput Struct Edlund, R. Slade and F. Monot (2009). Biotech J. 2(3): doi.org/10.5936/csbj. Curr. Opin. Biotechnol . 20 : 372–80. 201209014. 54. Sun, Y. and J.J. Cheng (2005). 72. Messner, P. (2009). Chembiochem. Bioresource Technol. 96 : 1599-1606. 10 :2151–4. 55. Das, T. and G.N. Ramaswamy (2006). 73. Teng, C., H. Jia, Q. Yan, P. Zhou and Z. Text Res. J. 76 : 126–33. Jiang (2011). Bioresour. Technol. 56. Kumar, B.K., H. Balakrishnan and 102 :1822–30. M.V. Rele (2004). J. Ind. Microbiol. 74. Upreti, R.K., M. Kumar and V. Biotechnol. 31 : 83–87. Shankar (2003). WILEY-VCH Verlag. 57. Bar, M., M. Sharfman, M. Ron and A. 363–79. Avni (2010). The Plant J. 63 : 791–800. 75. Hwang, I.T., H.K. Lim, H.Y. Song, S.J. 58. Elbaz, M., A. Avni and M. Weil (2002). Cho, J.S. Chang and N.J. Park (2010). Cell Death Differ . 9: 726–733. Biotechnol. Adv . 28: 594-601. 59. Bargmann, B.O., A.M. Laxalt, B.T. 76. Khandeparker, R., P. Verma, R.M. Riet, E. Schouten, W. van Leeuwen, Meena and D.D. Deobagkar (2011). H.L. Dekker, C.G. de Koster, M.A. Shelf Sci. 95 : 359-366. Haring and T. Munnik (2006). Plant J . 77. Dekker, R.F.H. (1985). New York: 45 : 358–368. Academic Press. pp. 505-533. 60. Fuchs, Y., A. Saxena, H.R. Gamble and 78. Lee, S.F., C.W. Forsberg and J.B. J.D. Anderson (1989). Plant Physiol . 89 : Rattray (1987). Appl. Environ. Microbiol . 138–143. 53 : 644-650. 61. Dean, J.F.D., K.C. Gross and J.D. 79. Crorus, J.M., I.S. Pretorius and W.H. Anderson (1991). Plant Physiol . 96 : 571– van Zyl (1995). Current Genetics. 28(5): 576. 467-473. 62. Lagaert, S., T. Belien and G. Volckaert 80. La Grange, D.C., I.S. Pretorius and (2006). Semin Cell Dev Biol . 20 : 1064- W.H. van Zyl (1996). Appl. Environ. 1073. Microbiol . 62(3): 1036-1044. 63. Cao, L. (2005). Curr. Opin. Chem. 81. Muller, S., T. Sandal, P. Kamp-Hansen Biol . 3:217-226. and H. Dalboge (1998). Yeast. 14 : 1267- 64. Madakbas, S., O. Danıs, S. Demir and 1283. M.V. Kahraman (2013). Starch . 65: 82. Katahira, S., Y. Fujita, A. Mizuike, H. 146–150. Fukuda and A. Kondo (2004). Appl 65. Pal, A. and F. Khanum (2012). J Environ. Microbiol. 70(9): 5407-5414. Biochem. Tech . 3(4): 409-413. 83. Damaso, M.C.T., M.S. Almeida, E. 66. Pal, A., Ray, L. and Chattopadhyay, P. Kurtenbach, O.B. Martins, N. Pereira, (2006). J Biotechnol. 5:163-168. C.M.M.C. Andrade and R.M. Albano 67. Edward, V.A., V.L. Pillay, P. Swart and (2003). Appl. Environ. Microbiol . 69(10): S. Singh (2002). South Afr. J. Sci. 98 : 553- 6064-6072. 554. 84. Ghaffar, A., S.A. Khan, Z. Mukhtar, 68. Kumar, L., S. Nagar, D. Kumar, N. M.I. Rajoka and F. Latif (2011). Garg and V.K. Gupta (2013). Int. J. Cell Molecular Biol. Reports . 38(5): 3227- Science Biotechnol . 2: 1-10. 3233. 69. Hokanson, C.A., G. Cappuccilli, T. 85. Paloheimo, M., A. Mantyla, J. Kallio Odineca, M. Bozic, C.A. Behnke, M. and P. Suominen (2003). Appl. Environ. Mendez and R. Crea (2011). Protein Microbiol. 69(12): 7073-7082. Eng. Des. Sel. 24: 597-605. 86. Nevalainen, K.M.H., V.S.J. Te'o and 70. Song, L., B. Siguier, C. Dumon, S. P.L. Bergquist (2005). Trends Bozonnet and M.J. ODonohue (2012). Biotechnol. 23 : 468–74. Biotechnol. For Fuels . 5: 3.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Bioremediation: An emerging technology for pesticide contaminated soil detoxification

Archana Mishra 1, A. K. Pandey 2 and Jamaluddin 1

1Department of Biological Sciences, Rani Durgawati University, Jabalpur, Madhya Pradesh, India 2Chairman, Madhya Pradesh Pvt. University Regulatory Commission, Bhopal, Madhya Pradesh, India

ARTICLE INFO ABSTRACT Pesticides are widely used worldwide in the agriculture to Article History: increase the annual crop yield to meet out the requirements Received 17 July 2015 of fast growing population. They are being used by the Accepted 31 March 2016 farmers to protect the crops from the damages caused, due to pests. These pesticides are very hazardous in nature and during their applications in the field these leach through

the ground water and become dangerous to human, animals Keywords: and the environment. Though they are biodegradable in Pesticides nature, some are acutely toxic and their residues are found in Bioremediation the environment. Thus, it becomes extremely essential to Microbial Degradation detoxify their levels in the environment. Various traditional Phytoremediation physico-chemical techniques are used to detoxify these

chemicals such as incineration, land filling excavation etc. But, because of their tedious procedure and high expenditure Article Type: cost, they are not used frequently. Microbial degradation is Full Length Review Article considered to be a major factor determining the fate of pesticides in the environment in a cost effective way .Thus the present paper reviews the efficacy and usefulness of bioremediation technique for the detoxification of pesticide in soil. ©2016 Society for Basic and Applied Mycology All Rights Reserved.

INTRODUCTION such myriad contaminant which is used Environmental pollution has become extensively in the agricultural sector is a major universal concern due to rapid pesticides. It is a substance/mixture of growth of industrialization, urbanization and substances used to kill pest [2]. It may be an modern agricultural expansion. The main antimicrobial disinfectant or device used sources of pollution include: chemical against any pest, a chemical substance, or a plants, coal-fired power plants, oil refineries biological agent (such as virus or bacteria), [1], petrochemical plants, nuclear waste Pests comprise insects, mammals, plant disposal activity, incinerators, large line pathogens, fish, nematodes (roundworms) stock farms, PVC factories, metal weeds, molluscs, birds, and microbes that production factories, plastics factories, many compete with humans for food, devastate industries and spraying of pesticides. One the property, spread or are a vector for

*Corresponding author: [email protected] (Archana Mishra) J. Basic Appl. Mycol. Vol. 12 (I), pp. 12-15, 2016 13 diseases or cause a nuisance. The common Animals may be poisoned by formulations types of pesticides are liquids, pesticide residues that remain on food after wettable powder, emulsifiable concentrates spraying for eg. when wild animals enter in and dusts. There are used for diverse the sprayed fields or nearby areas shortly purposes viz., to sanitize drinking and after spraying. These chemicals can travel recreational water, to disinfect indoor areas up to the food chain eg. birds can be harmed (eg. kitchens, operating rooms, nursing when they eat insects and worms that have homes) as well as dental and surgical consumed pesticides [9-10]. The uses of instruments. The major routes through which such chemicals have thus proven to be pesticides contaminate the water resources detrimental to the ecosystem. Therefore, it are as follows: becomes extremely important to detoxify (i) They may drift outside of the their levels by use of techniques that are safe intended area when sprayed, and at the same time cost effective. The (ii) They may percolate or leach conventional techniques which were used through the soil, for the detoxification previously have met (iii) They may be carried to the water as serious opposition from the environmental runoff or by spill for eg. accidentally groups because of their operational or through ignorance. difficulties and high expenditure cost. (iv) They may be also carried to water by eroding soil [3]. Bioremediation-an emerging tool for the environmental clean–up Factors that affect a pesticide’s Bioremediation overcomes the ability to contaminate water include its water limitations of traditional methods of solubility, the distance from an application hazardous chemical disposal by bringing site to a body of water, weather, soil type, about the actual destruction of many organic presence of a growing crop and the method contaminants at the reduced cost. It serves as used to apply the chemical [4]. In addition to a boon in solving environmental related air and water, migrating birds and oceanic problems since it safe, least disruptive and currents are also responsible for the pesticide cost effective [11-12]. This technology uses migration from thousands of miles away microorganisms to reduce, eradicate or alter from the area, where it was applied initially. the contaminants present in soils, sediments or water. It depends on the presence of The harmful effects of pesticides usage particular microorganisms in the correct The widespread use of pesticides amounts, in exact combination and at the causes fatal effects on wildlife populations appropriate favorable conditions. and humans [5]. According to an estimate Bioremediation process involves made by the WHO, each year 3,000,000 biotransformation and biodegradation. It cases of pesticide poisoning including works by transforming contaminants to non- 2,20,000 deaths are reported across the globe dangerous or less dangerous forms. [6]. Humans get in touch with these chemicals, in the field, during pesticide Different methods of bioremediation application, weeding, pruning, harvesting, 1. Degradation with the help of microbes re-entry to collect fire wood or vegetables, When microorganisms are used for or in their homes, to kill mosquitoes, the remediation of myriad chemicals, the cockroaches, fleas and flies. Previous studies process is called as microbial degradation revealed that the children’s of farm workers [13]. Many amazing characteristics of were highly exposed to different types of microbes such as their small size, ubiquitous pesticides and have leukemia, cancer of distribution, high specificity, surface area, brain, and kidney [7]. It was observed that potentially rapid growth rate and unrivaled the loss of fatal miscarriage is common in enzymatic and nutritional adaptability make spouses of pesticide applicators than the them, as one of the most important recycling spouses of non- agricultural workers and is agents of pesticides in nature. The attacked more common during the spray season [8]. substances are used by the microbes, as a © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 12-15, 2016 14 source of carbon, energy, nitrogen or other Table 1. Microorganisms responsible for nutrients or as final electron acceptor. the pesticide degradation. Bioremediation is the collection of number Name of the Microorganisms Reference of processes like bioaccumulation, Pesticide used biodegradation, biostimulation and Carbamates Pseudomonas [15], melophthora, [16],[17] bioaugmentation, all these techniques, Pseudomonas involve livings organisms but using different aeruginosa, strategies for using them [14].The technique A consortium that involves the stimulation of Pyrethroid Alcaligenes YF11 [18] microorganisms by adding the nutrients and Organochlorines Trichoderma viridae [19] Organophosphates Flavobacterium sp. [20], [21], other chemicals required for the metabolism Pseudomonas sp.A3 [22], [23], of microorganisms that promote the Aspergillus sp. [24] degradation of contaminants of native Burkholderia cepacia microorganisms at the contaminated site, is Pseudomonas biostimulation. This is one of the most aeruginosa Nitro aromatic Arthrobacter [25], favored and cheapest techniques to compounds (p- protophormiae [26],[27] , decontaminate the environment. If the nitro phenol) Achromobacter [28] contaminated site has no native xylosoxidans Ns microorganisms, then another way is to Burkholderia sp. SJ98 introduce some microorganisms that have Pseudomonas putida the ability to degrade these contaminants .This process is known as bioaugmentation. CONCLUSION The following table gives a glimpse, of some In agriculture sector, the use of of the microorganisms which are used in the pesticides has become indispensable since, pesticide’s degradation. their usage results in the enhancement in agricultural production, soil productivity and 2. Use of earthworms in pesticide products quality which is reflected in bioremediation economic benefits .The usage of chemical Due to their biological, chemical and fertilizers and pesticides ,no doubt increase physical actions, earthworms can be directly the annual crop yield but at the same time it employed within bioremediation strategies exerts, toxic effects on man and the to promote biodegradation of organic environment . Some pesticides can also bio- contaminants [29]. Their application within accumulate or build up to toxic levels in the waste management involves the digestion of bodies of organisms that consume them over a wide range of organic wastes [30]. the time. A number of food items have shown alarming level of pesticides .A large 3. Phytoremediation number of pesticides manifests their toxicity It is the use of plants to clean up through functional and biochemical action in polluted soil and water resources. Although central and peripheral nervous system. Thus plants have the inherent capability to it becomes extremely important to reduce detoxify xenobiotics, they lack the catabolic the toxicity of such chemical. pathway for the complete degradation of Bioremediation serves an answer in solving, these compounds compared to the problem of pesticide discharge in the microorganisms [31]. This technique can be atmosphere ,in a cost effective way. Thus used for the removal of myriad compounds , the present paper provides an insight on the since plants are robust in growth , are a usefulness of bioremediation technology. renewable resource and can be used for in situ remediation[32-33]. The technology of ACKNOWLEDGEMENT using plants in remediation has proven to be The authors are thankful to the efficient for the treatment of organic Director and Dr. Juwarkar (Chief Scientist), pollutants including chlorinated solvents, National Environmental Engineering polyaromatic hydrocarbons and explosives Research Institute, Nagpur-(MS), India, for [34]. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 12-15, 2016 15 providing all the laboratory and 18. Yu, Y.L., H. Chen, D.F. Fan, G.Y. characterization facilities. Sheng and J.M. Fu (1998). Environmental Sciences . 19: 66-69. REFERENCES 19. Matsumura, F., G.M. Boush and A. Tai 1. Beychook, M.R. (1967). Aqueous waste (1968). Nature. 219(5157) : 965-967. from petroleum and petrochemicals plants. 20. Sethunathan, N. and T.A. Yoshida 1st edition. John Wiley and sons LCCN (1973). Canadian J. Microbiol. 19(7) : 67019834. 873-875. 2. Doxtader, K.G. and R.L. Croissant 21. Ramanathan and Lalitakumari (1997). (1992). Fate of pesticides in soil. Colarado Appl. Biochem. and Biotech . 80 : 1-12. state University, cooperative extension. 22. Liu, Z., Q. Hong, J.H. Xu, J. Wu, X.Z. The National Ag. Safety. Zhang, X.H. Zhang, A.Z. Ma, J. Zhu 3. Papendick, R.D., D. Young and H. and S.P. Li (2003). Acta Genetica Sinica . Krauss (1986). Soil Erosion and Crop 30(11) : 1671-4083. Productivity. 1(1) : 306-319. 23. Keprasertsup, C., E.S. Upatham, N. 4. Bingham, I.J., J. Blake, M.J. Foulkes Sukhapanth and P. Prempree (2001). and Spink, J. (2007). Field Crop Science Asia . 27 : 261-270. Research . 101 : 212-220. 24. Balamurugan, K., M. Ramakrishna and 5. Younes, M. and H. Galal (2000). Food R. Senthil kumar, S. Ignacimuthu. Chem. Toxicol. 38 : 87-90. (2010). Asian Journal of Science and 6. Down to Earth (DTE) (2001). 9(19) : 28- Technology. 6: 123-126. 35. 25. Labana S., O.V. Singh and R.K. Jain 7. Daniels, J.L., A.F. Olshan and D. A. (2005). Appl. Microbiol. Biotech. 68 : 417- Savitz (1997). Environ. Health Perspect. 424. 105 : 1068-1077. 26. Wan, N., Ji. Gu And Yan (2007). 8. Garry, V.F.M., A. Harkins, L. International Biodeterioration and Lyubimov, Erricson and Long, L. Biodegradation. 59: 90-96. (2002). J. Toxicol. Environ. Health. 65 : 27. Vikram, S., J. Pandey, N. Bhalla, G. 769-786. Pandey, A. Ghosh, F. Khan, R.K. Jain 9. Bai, Y.L. and W. Zhou (2006). Food and G.P.S. Raghava (2012). A Springer Chem. 98 : 240-242. Open Journal . 2(30): 1-7. 10. Rekha, S.N., R. Naik and Prasad (2006). 28. Guleria, A. (2014). Indian Journal of Chem. Health and Safety. 12-19. Research . 3(5): 127-130. 11. Alexander, M. (1999). Biodegradation 29. Hickman, A. and B.J. Reid (2008). and Bioremediation. 2 nd edition, Environmental International. 34: 1072- Academic Press, New York. 1081. 12. Frazar, C. (2000). The bioremediation and 30. Edwards, C.A. and C.L. Bohlen (1996). phytoremediation of pesticide Biology and ecology of earthworms 3 rd contaminated sites. National Network of edition. Chapman & hall, London. Environmental studies. 31. Abilash, P.C. and N. Singh (2009). 13. Eapen, S., Singh and D’Souza, S.F. Environ. Sci. Pollut. Res. Doi.10.1007/ (2007). Biotechnol. Adv. 25: 442-451. s11356-009-0133-5. 14. Walter, M.V. and R.L. Crawford 32. Suresh, B., P.D. Sherkhane, S. Kale, S. (1997). Biotransformation and Eapen, and G. Ravishankar (2004). Cri. Biodegradation . 707-708. Rev. Biotechnol . 24: 97-124. 15. Matsumura, F. and G.M. Boush (1967). 33. Parameswaran, A., B. Leitenmaier, M. Science. 156 :956. Yang, P.M. Kroneck, W. Welte, G. 16. Chapalamadugu, S. and G.R. Lutz, A. Popayan and H. Kupper Choudhary (1992). Crit. Rev. (2007). Biochem. Biophys. Res. Commun. Biotechnology. 12 :357-389. 36: 51-56. 17. Naqvi, T.A., N.A. Kanhar, A.H. Shar, 34. Pilon- Smits, E. (2005). Annu. Rev. Plant M. Hussain and S. Ahmed (2011). Pak. Biol. 56: 15-39. J. Bot. 43(2) :1079-1084. © 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Diversity and utilization of wild macro fungi in Chirang district of Assam, Northeast India

Karuna Shrivastava 1* , Karabi Devi 2 and Jahnovi Brahma 3

1,2Laboratory of Plant-Microbe Interactions and Biotechnology, Department of Forestry, North Eastern Regional Institute of Science and Technology, Nirjuli, Arunachal Pradesh, India 3Department of Botany, Science College, Kokrajhar, BTAD, Assam, India

ARTICLE INFO ABSTRACT The present study aimed to document diversity, ecological Article History: relationship and utilization of wild macro fungi as an Received 03 March 2016 important source of food by the Bodo tribe of Chirang Accepted 31 March 2016 district of Assam, Northeast India. Owing to rich biodiversity, a variety of wild macro fungi were collected from forest fringe areas of Chirang reserve forest during the

rainy period in 2012 - 2013. A total of 14 macrofungal Keywords: species representing 12 genera, 10 families and 4 orders Wild edible fungi were collected. Most species belonged of order Agaricales Bo do tribe (64.3%) followed by Polyporales (21.4%) and Biodiversity Auriculariales and Phallales (7.2% each). The maximum Chirang district (10) species were saprophytic in nature, while parasitic and Assam symbiotic (2 each) were also recorded. Overall four edible

species including a medicinal were recorded for the first time from Chirang district of Assam, North-East India. Among Article Type: all, Termitomyces sp. showed maximum frequency (83.3%) Full Length Research Paper and Bovista plumbea recorded with maximum density (33.71). ©2016 Society for Basic and Applied Mycology All Rights Reserved.

INTRODUCTION value. They therefore contribute towards Macro fungi or mushrooms are diet, human health and income. Many fleshy fruit bodies of higher fungi. They species play a vital ecological role through mostly grow in forests in association with symbiotic relationships known as woody parts of trees either as parasite, mycorrhizas that they establish with trees. saprophyte or as symbionts. They may also Numerous varieties of edible mushrooms grow on animal’s dung, termite’s hive, leaf exist in nature with more than 1100 species litter etc. Wild macro fungi may be edible or recorded so far [1], however; less than 25% deadly poisonous in nature. They are natural are widely accepted as food and only a few resource and often considered amongst most have attained the level of an item of valuable non-wood forest products collected commerce [2]. Substantial quantities are for food due to their rich nutritional and eaten through personal collections from wild occasionally for their supposed medicinal that go unrecorded. Wild edible macro fungi *Corresponding author: [email protected] (Karuna Shrivastava) J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 17 are good source of proteins and [3,4] of Assam, Northeast India. The paper contributing substantially to the diets of rural discusses some traditional and contemporary and poor people. They are also increasing uses of these fungi as food or in medicine. source of income for small-scale enterprises in developing as well as developed countries MATERIALS AND METHOD [5,6]. During our ecological and botanical Wild edible fungi are being survey, field tours were conducted at Ripu- consumed by the people since ancient times Chirang reserve forest of Assam, India ( 26° as reported in Greek and Roman literature 0.06´ 56.05½ N to 26° 0.54´ 21.95½ N [7]. They are utilised in more than 80 latitude and 90° 0.12´ 03.76½ E to 90°0 29´ countries. A huge range of wild fungi are 07.02½ E longitude). Chirang district is collected from forests and fields and also amongst one of the four districts of marketed widely [1]. India is known for its Bodoland Territorial Area District (BTAD) variety of cultures, languages, tribes etc. as under the Government of Assam, India. being inhabited by more than 2000 ethnic Inventorization of macrofungi was done tribes who eat and exploit wild macro fungi during rainy season for a period of three since time immemorial. Tribal people months for two consecutive years i.e., June possess tremendous knowledge about the to August, 2012 & 2013. Based on utilization of various forest products, possess information from local residents, two village adequate knowledge about wild macro fungi areas within Chirang forests namely and able to distinguish the edible fungi from Kumarsali and Subhaijhar were selected the poisonous ones. The macro fungi (Figure 1) for sample collection at weekly consumed by the people are seasonally intervals. Information regarding their harvested from forest areas every year but no edibility and mode of preparation were efforts have been made to cultivate them. collected by semi-structured open ended The local people collect the macro fungi questionnaire and continued discussions with directly from forests for their consumption the local village people. because of their taste and health benefits. However, the ancient tradition of gathering and consuming mushrooms from the wild is gradually on the decline. This may be due to their non-availability all the time, fondness of people leading to immediate harvesting from wild and their host specific nature. Uses of wild edible fungi have been documented from different parts of India but despite of favorable climate for fungal Figure 1. Map showing study sites. growth, only a few reports belong to north eastern region of the country [8-11] . The Ethno mycological information was Indian state of Assam, which lies in the recorded from reliable sources such as northeast of Himalaya, is a rich repository of Ojhas, elderly persons and local inhabitants the unexplored macro fungal wealth due to who were considered to have good its varied climatic and topographic knowledge of the wild resources of this conditions, thus providing congenial region. They also accompanied us to the environment for the lavish growth of this forests as guide cum informants. The heterogeneous group of fungi. Chirang sporocarps encountered were collected and district is dominated by Bodo tribe and one identified on the basis of their macro and of the four districts of Bodoland Territorial microscopic characteristics, spore prints and Area District (BTAD) under the Government comparison with standard manuals [12-15] of Assam. The present study aims at as well as online mycokeys documenting and identifying the most (www.wildfooduk.com/mushroom-guides; widely collected and consumed macro fungi www.mycokey.com; www.mushroomexpert. by the ethnic Bodo people of Chirang district com) to facilitate proper identification. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 18

Photography was done using digital camera uses. All samples have been deposited in the (Sony 12 Megapixel). herbarium of Forestry Department, NERIST, The substrate of each specimen was and Arunachal Pradesh. The frequency and also recorded and labeled with number, date density of each species were determined [16] of collection, locality and ethnomycological using the following formulas:

No. of site in which the species is present Frequency of fungal species (%) = ------x 100 Total no. of sites

Total no. of individuals of a particular species in all quadrats Relative Density = ------x 100 Total no. of individuals of all species in all the quadrats

Table 1. Wild macro fungi of Chirang Reserve Forest, Assam, Northeast India. Sl. Name of the Order Family Ecological Host/Sub stratum Uses by Frequ- Relativ No. species Relationship Bodo ency of e people occur- Density rence (%) (%) 1. Agrocybe Agaricales Strophariaceae Saprophytic In open area Edible 33.3 7.58 pedides (Fr.) associated with Fayod grasses, especially with Cynodon sp. 2. Auricularia Auriculari- Auriculariaceae Saprophytic Both on dead Non 50 2.53 auricula -judae ales wood edible (Bull. ex St. Amans) Wettst. 3. Bjerakandera Polyporales Meruliaceae Parasitic On tree trunk and Non 33.3 9.27 spp. branches of edible hardwoods 4. Bovista plumbea Agaricales Agaricaceae Saprophytic Grows on old Edible/m 33.3 33.71 Pers. pastures, on ground edicinal as solitary 5. Coprinus Agaricales Coprinaceae Saprophytic Growing on lawns Non 33.3 7.58 comatus (Fr) or at the edges edible Pers. often in large groups 6. Coprinus Agaricales Coprinaceae Saprophytic Growing on lawns Non 33.3 3.93 plicatilis (Fr.) Fr. and other grassy edible places 7. Coprinus sp. Agaricales Coprinaceae Saprophytic Growing on ground Non 33.3 2.25 at grassy places Edible 8. Dictyophora sp. Phallales Phallaceae Saprophytic On ground amongst Non 16.6 1.69 leaf litter in damp Edible wood land 9. Entoloma sp. Agaricales Entolomataceae Symbiotic Growing on ground Non 33.3 2.53 near tree roots Edible 10. Ganoderma Polyporales Ganodermataceae Parasitic Grows on trunk of Non 16.6 0.28 applanatum standing tree Edible Patouillard 11. Lepiota sp. Agaricales Agaricaceae Saprophytic On ground with Non 33.3 4.21 rich humus Edible 12. Macrolepiota Agaricales Agaricaceae Saprophytic On old pastures, Edible 66.6 12.92 procera (Scop. mixed forest and on ex Fr. ) Singer woods 13. Podoscypha sp. Polyporales Meruliaceae Saprophytic On the ground in Non - 33.3 5.90 deciduous woods edible 14. Termitomyces Agaricales Lyophyllaceae Obligate On compost heap Edible 83.3 5.62 heimii Natarajan Symbiotic of termite nest in evergreen forest The Wild edible fungi of Chirang district of Assam is highlighted by bold.

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 19

Table 2. Importance of four wild edible macro fungi collected from Chirang, Assam.

Sl. Name of the Local Edible preparations Medicinal Other uses no edible specimen name by the Bodo people uses by Bodo people 1. Agrocybe pedides Mwikhun Eaten by preparing Not used as a Show antimicrobial and (Fr.) Fayod curry. Sometimes medicine by antioxidant properties cooked with fish. them. [17] 2. Bovista plumbea Mwikhun Fresh fruiting bodies Used in the Shows anti oxidant daudwi are used by the Bodos treatment of property. [18] for making vegetable. sores , ulcers They first boil them, and skin decant the water and infection by then fry them in oil. powder of the fruit bodies 3. Macrolepiota Mwikhun Eaten by frying the Not used as a Demonstrates anti tumor procera fruit bodies in oil. medicine by activity and exhibit an Sometimes cooked them. antibiotic activity against with meat. Gram-negative bacteria. As food, it is known to sustain physiological homeostasis of the body [19] 4. Termitomyces Uri They are highly prized Not used as a Contains anti heimii mwikhun mushrooms and are medicine by hypertensive property. eaten by making curry. them. Used as food. It has higher consumer preferences due to their unique flavor and texture. [20]

RESULTS AND DISCUSSION procera (12.92) then by Bjerkandera sp. The wild edible fungi are locally (9.27) while the least density was recorded called as ‘Mwikhun’ by Bodos. Daudwi of Ganoderma applanatum (0.28). (Table: 1) Mwikhun or Uri Mwikhun names indicate From the Chirang district four wild specific edible species. From the present macro fungal species viz. Agrocybe study, fourteen wild macro fungal species pediades, Bovista plumbea, Macrolepiota representing twelve genera, nine families procera and Termitomyces heimii. were and four orders were recorded from Ripu- recorded as edible (Figure 2). These fungi Chirang forest reserve of Chirang district of were much relished and eaten by the ethnic Assam. Of them, three each belonged to Bodo people of this region. Out of them, Agaricaceae and Coprinaceae, two to Bovista plumbea was reported to be used Meruliaceae and one each to topically as medicine to cure skin ailments Ganodermataceae, Strophariaceae, (Table 2). Auriculariaceae, Phallaceae, Entolomataceae The ecological preference of the and Lyophyllaceae families. The order macrofungi revealed that maximum species Agaricales contributed maximum (64.3%) were saprophytic. Ten out of fourteen followed by Polyporales (21.4%) and species were saprophytic, two were parasitic Auriculariales and Phallales (7.2% each). and one was mycorrhizal in nature. The The maximum frequency was recorded for parasitic fungi were Bjerkandera sp . and Termitomyces sp. (83.3%) followed by Ganoderma applanatum whereas Entoloma Macrolepiota procera (66.6%) and sp. was mycorrhizal (Figure 3). The Auricularia judae (50%). The frequency of Termitomyces sp., an obligate symbiont, the rest of the species ranged between showed coexistence with termites and vice- 16.6%-33.3%. Almost in the similar way versa for their sustenance appeared with maximum density was recorded for Bovista maximum frequency of occurrence (83.3%). plumbea (33.7`) followed by Macrolepiota

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 20

Description of the wild edible macrofungi preserve and eaten later. Bovista plumbea is 1. Agrocybe pediades used by Bodos by mixing it in other This mushroom was found inhabited vegetables after boiling in water. in grassy forest area, mostly saprobic, It also medicinally used by them in growing scattered alone or gregariously in the powdered form in the treatment of sores, grass. Cap plano convex, 1-3.0 cm broad, skin infections and ulcers. broadly convex or nearly flat, yellow brown or paler, margin at first slightly incurved, then decurved, nearly plane at maturity; margin surface smooth, sticky when moist. Gills adnate to adnexed, moderately broad, attached to the stem; pale grayish brown to rusty or cinnamon brown at maturity, covered by an ephemeral white partial veil when young, slender and equal. stipe 3-4 cm long, 1-2mm broad, stem 2-6cm long, 1.5- 2mm thick; more or less equal; smooth; concolorous with the cap and sometimes twisted grooved. Flesh usually pale and thin, found scattered in grasses and fruit in summer. Spores are elliptical with cellular cuticle smooth with an apical pore. Spore print is brown. Spores are 10-13x8-9µm. They are eaten by the Bodo people soon after collection as fresh when gleba is white and are eaten by preparing a good fry curries. Sometimes it is cooked with fish.

2. Bovista plumbea It is commonly called as the rolling puffball, habitat amongst short grasses and pasture, fruit body is sub-spherical, smooth 2-5cm across and stemless, the outer scale or the exoperidium is white at first which gradually turns grayish laterally splitting revealing a grayish inner peridium. The inner peridium forms a roughly circular apical hole on bursting, with white gleba that turns olive brown as the spore mass matures Figure 2. Four wild edible macro fungi of reveals brownish spores. Capillitium thread Chirang district of Assam. was 2mm thick with reddish brown thick wall, dichotomously branched that branches 3. Macrolepiota procera terminate in sharp tapered tips without septa It is a basidiomycete fungus with or pores. Spores sub-globose to broadly large prominent fruiting body resembling ellipsoidal with a very warted surface, 4.5- aparasol. Its cap is 5-8 cm and more oval in 7.2 x 5-5.8 µm tapering freely creating a young stage which later becomes convex to tadpole like appearance, thick-walled and broadly convex with age with a dark central nearly smooth. Sprouting period May to bump, initially spherical and brown with a October and are edible when young. Both dark brown area at the apex, coarsely scaly, young and old sporocarps of Bovista are but crown smooth, not sharply scaly. When eaten but young ones are preferred more. matured, it expands into a flattened structure They are also sometimes sundried to © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 21 with an umbo. Cap flesh is white. Ring 4. Termitomyces heimii membranous, on drying moving freely It is a rare and highly sought edible spores usually over 7 µm usually with mushroom that grows seasonally and in clamps. Gills are white and free from the symbiosis with other termites. Pileus 7cm in stems (stipe) which becomes movable. A diameter, surface smooth, convex, when ring persists on the stipe. Stipe is smooth young prominently sub-umbonate, margin and white with brown scales. Stipe is 14- un-curved, white with the umbo region when 20cm in length or 0.5-1.5cm broad, with young, grey and browinish grey when stems long, slender with an enlarged base, mature, context fleshy white, lamellae free, pale above the ring but below the ring, it white becoming pink, up to 8mm broad, consists of small brown scales that break up margin serrate, lamellulae present, stipe up as the mushroom matures creating zones or to 19cm long and up to 1.5cm wide, white, sometimes disappearing with a double smooth, cylindrical, stuffed with a thick edgedring that moves freely up and down annulus. Stipe is 15cm long and up to 1.5cm the stem. Spores are ellipsoid and smooth wide, smooth and cylindrical. Pseudorhiza with a germ pore. It is 12-17x9-12 µm. present up to 12 cm below the ground level. The fruiting bodies of this macro fungus Its spores are 7.0-8.3 x 4.3x5.4 m, ellipsoid, are eaten as fries and also along with meat. smooth and hyaline . The fruiting bodies of this species are eaten as fries by Bodo people. They also sale this species in local market to fetch good price as it is sold at a high price. This is the first report of diversity of wild macro fungi and use of four wild edible species from Chirang district of Assam. As a result of survey 14 species were obtained whose frequency and relative density were calculated. (Table 1)The diversity of macro fungi is determined by the type of habitat in which it occurs. Many factors like geographic location, elevation, humidity, light, temperature as well as the flora of the surrounding area greatly influence the growth and development of macrofungi. Earlier, the uses of twenty nine wild macro fungi from Kachugaon, Haltugaon, Parbatjhora that also includes parts of Chirang reserve forest of western Assam were recorded [10] but this does not involves Kumarsali and Subhaijhar area under Ripu-Chirang forest reserve of Assam, India. Studies on diversity of macrofungi were done in many parts of India [21-23] while much less work was undertaken in north east including Assam. [10, 24]. The edible species of these villages were documented as a result of personal interview. Four species were found edible which are listed in Table 2. The traditional Figure 3. Some other wild macro fungi knowledge and ethnic relevance of wild collected from the study area. edible macrofungi by Khasi tribe of Meghalaya was also documented [9].

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 22

Ethnomycological knowledge of tribes of 3. Adewusi, S.R.A., F.V. Alofe, O. Mokokchung, Nagaland, and North East Odeyami, O.A. Afolabi and O.L. Oke India on wild mushrooms was done recently (1993). Plant Food Human Nutrition . [25, 26]. However, the wild edible fungi 43: 115-121. from other parts of India were documented 4. Aletor, V.A. (1995). Food Chem . 54: extensively [22, 27-29]. 256-268. The Bodo people are fond of wild 5. Hosford, D., D. Pilz, R. Molina and M. mushrooms but use a limited species as food Amaranthus (1997). Ecology and item (Table 1, Figure 2). They are also management of commercially harvested aware about their medicinal values. They American Matsutake mushroom, General use the fruit bodies of Bovista plumbea in Technical Report PNW-GTR-412, the treatment of sores, ulcers and skin USDA Forest Service Pacific Northwest infection as powder. B. plumbea is also Research Station, 68 pp. known for the treatment of head affections, 6. Wong, J.L.G., K. Thornber and N. diabetes, ovarian cysts, acne etc. [30]. Baker (2001). Non Wood Forest Though not used by Bodos, Auricularia Products (13, FAO, Rome). 68(3): 221- judae is used medicinally by other tribes in 26. Assam (Table 2) [10]. Dictyophora and 7. Buller, A.H.R (1914). Transactions of Coprinus comatus were also recorded as the British Mycological Society (5): 21- edible in Gujrat [31]. Potential antioxidant 66. activity of Cantharellus cibarius, Bovista 8. Tapwal, A., R. Kumar and S. Pandey plumbea, Coprinus comatus and C. (2013). Biodiversitas . 14(2): 73-78. atramentarius growing in Kashmir valley 9. Khaund, P. and S.R. Joshi (2013). was determined [18]. The Ganoderma Indian Journal of Natural Products & applanatum is used in Nigeria as medicinal Resources . 4(2): 197-204. due to its antioxidant, hypoglycemic and 10. Sarma, T.C., I. Sarma and B.N. Patiri antihypertension activities (Table 2) [32]. (2010). Intl. J. Life Sci. 3: 613-625. 11. Singh, M.N. and G.K.N. Chhetry CONCLUSION (2010). Indian Phytopathology . 63(1): The use of wild edible fungi play a 79-82. very important role and enriches the socio- 12. Lamaison, J.L. and J.M. Polese (2005). economic life of the tribal people. The The Great Encyclopedia of Mushrooms. present investigation focuses upon a great Germany: Konemann. 138 pp. need for thorough, careful and 13. Konemann, H.F.U. (2005). The Pocket comprehensive macro fungal investigation in Guide to Mushrooms, Konemann, pp. locations of Chirang district of Assam, India. 381. This ethno-mycological study is the first 14. Keizer, G.J. (1998). The Complete documentation about the use of four edible Encyclopedia of Mushrooms. Rebo macro fungi by Bodo tribes of Assam. Productions, The Netherland. Seeing increasing importance of the wild 15. Zoberi, M.H. (1973). Niger. Field . 38: edible mushrooms to fulfill dietary 81-90. requirement of rural populace, we 16. Ambasht, R.S. and N.K. Ambasht recommend regular and seasonal surveys to (2008). A Textbook of Plant Ecology be done over an extended period in order to (15 th edition), CBS Publishers & assess the patterns of abundance in this Distributors Pvt.ltd.; pp. 444. unexplored area. 17. Al-Fatimi, M., G. Schroder, H. Kreisel and U. Lindequist. (2013). Pharmazie . REFERENCES 68(3): 221-6. 1. Boa, E. (2004b). Non Wood Forest 18. Wani, A.H. R.H. Boda, Taskeen-un- Product . (17) (FAO, Rome). pp. 147. Nisa and A. Latif (2010). Mycopath. 2. Smith, J. (1972). Process Biochemistry . 8(2): 71-75. 7: 24-26. 19. Sung, M.S., H. Yun, K.R.L. Oh, H.K. Seong, H.I. Kyung, W.K. Jung, L. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 16-23, 2016 23

Uyoun, O.S. Jae, J.S. Mi, W.L. Min, Life Sciences . 1 (3): 118-123. S.L. Hyun and S.L. Tae (2005). 27. Vishwakarma, M.P., R.P. Bhatt and S. Mycobiology . 33(1): 15–18. Joshi (2012). Indian Journal of Science 20. Hasni, M.F.N., C.F. Misnan, N. and Technology . 5(1): 1928-32. Aminudin and N. Abdullah (2013). 28. Chandrawati, P. Singh, N. Kumar and Journal of Health and Translational N.N. Tripathi (2014). American Medicine . 16: 67. International Journal of Research in 21. Swapana, S., A. Syed and M. Formal, Applied and Natural Sciences . Krishnappa (2008). J. Mycol. Pl. 5(1) : 71-75. Pathol. 38(1): 21-26. 29. Chauhan, J., A.K. Negi, Rajasekaran, 22. Mani, S. and V. Kumaresan (2009). A. Nazir and A. Pala. (2014). Indian Journal of Threatened Taxa. 1(1): 54-57. Journal of Medicinal Plants Studies . 2 23. Anand, N., A. Mathur and P.N. (1): 40-44. Chowdhary (2014). 3(12): World J. 30. Jain, J.P. (1997). Highlights of Pharmacy and Pharmaceutical Sciences. Homoeopathic Materia. B Jain Pub. Pvt. 3(2): 1385-1402. Ltd., 1 edition (June 29, 1997). 24. Kumari B. and N.S. Atri (2012). 31. Parihar S., E.A. Pithawala, S.K. Mycosphere. 3(6): 949–955. Lahiri, M.D. Shukla, N.K. Jain and 25. Kumar, R., A. Tapwal, S. Pandey, H.A. Modi (2015). Indian Journal of R.K. Borah, D. Borah and J. Fundamental and Applied Life Sciences. Borgohain (2013). Nusantara Biosci . 5 5 (4): 43-51. (1): 1-7. 32. Oyetayo, O.V. (2011). Afr. J. Tradit. 26. Kumar, R., S. Pandey, K. Giri, G. Compl. Altern. Med. 8(3): 267-274. Mishra and R.R. Rishi (2015). Current

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167

Available online at www.sbamjournal.com

Influence of seasonal variation on the concentration of fungal airspora over Kachai lemon plantation in Ukhrul district, Manipur

S. Nimyaola and N. Irabanta Singh

Aerobiology Laboratory, Centre of Advanced Study in Life Sciences, Manipur University, Manipur

ARTICLE INFO ABSTRACT The present investigation deals with the study on the Article History: seasonal variation of fungal airspora over lemon orchard at Received 19 March 2016 Kachai in Ukhrul District, Manipur. The incidence of fungal Accepted 06 April 2016 spores in the air was studied by employing Rotorod air sampler supplemented by Petriplate exposure technique from May, 2013-April, 2015. Emphasis has been given to the

occurrence of airborne fungal spores and the effect of Keywords: meteorological parameters on their dissemination. A total Fungal airspora count of 33 fungal spore types was identified. Occurrence of Lemon orchard fungal spores in air was in correlation with changes in Meteorological parameters weather parameters. These studies will be of great help in Disease forecasting providing information to the farmers towards disease

forecasting. ©2016 Society for Basic and Applied Mycology All Rights Reserved. Article Type: Full Length Research Paper

INTRODUCTION processes influenced by environmental There has been an increasing interest parameters such as climatic condition. Many in the study of airspora because of its plant pathogens are dispersed by the wind applications in medicine (inhalant allergy), infecting crops and causing significant agriculture (epidemiology of airborne plant economic losses in agriculture [5]. Airborne diseases), biodeterioration and spoilage of micro organisms especially bacteria and food stuffs and other useful materials fungi attack the biotic flora, deteriorate food including air pollution [1-3]. The knowledge and damage crops. The fungal spores are of air-spora not only contributes to the disseminated by fruiting bodies of fungi and understanding of their abundance and blown by air facilitating attack on plants. In seasonal variations, but is also helpful in this concern, the study on airborne forecasting the epidemics of crop plants [4]. biological materials mainly fungal spores The increasing concern about the effects of and their impact on biological species is an the airborne fungi on plants, animals and important aspect. Although the fungal spore humans creates new incentives for further content of the atmosphere has attracted advancement in this field. The diversity and significant interest during recent decades, it the concentration of the fungal spores in the still remains underexplored concerning the atmosphere are the result of dynamic mycoflora presence in the lemon orchard at *Corresponding author: [email protected] (S. Nimyaola) J. Basic Appl. Mycol. Vol. 12 (I), pp. 24-27, 2016 25

Kachai. Therefore, the main objective of this identification of colonies was based on their study was to quantify and identify the fungal colour, size, shape and their morphological airspora present in this environment as well features [8-9]. The temperature, relative as to investigate the climatic conditions that humidity and rainfall of the air during the could influence microbial counts. experiment were also recorded.

MATERIALS AND METHODS RESULTS AND DISCUSSION Sampling site A total of 33 fungal spore types were The Kachai village is located at identified. Out of 33 fungal spore types, 20 extreme west in Ukhrul District, boundary spores belonging to Deuteromycotina, 9 to with Senapati district which is about 140 km Ascomycotina, 2 to Phycomycotina and 2 to away from Imphal and 48 km from Ukhrul Basidiomycotina. Unidentified spore types, District Headquarters lying between 25 ̊ 14 ̍ pollen grains, hyphal fragments and insect N -21 ̊ 25 ̍ N latitude and 94.16 ̊ E - 94.32 ̊ E parts were classified as other types. The longitude with an altitude ranging from average percentage contribution of each 1280-1387m (MSL). The climate of Kachai spore group were as follows: area is Sub-tropical characterized by Deuteromycotina (64.42%, 61.23%) coolness and extreme humidity. The village followed by Ascomycotina (13.33%, is inhabited by 360 households with 15.81%) other types (11.6%, 13.31%), population of 2107 (Census 2011). It is Basidiomycotina (3.34%,5.82%), known as one of the highest lemon producer Phycomycotina (3.12%, 3.745). Among the village in Manipur state. The fruit is spores, Alternaria has maximum endemic to Kachai village and hence known concentration (8.72%) to the total airspora, as Kachai Lemon. followed by Aspergillus (8.29%), Torula (4.92%), Fusarium (4.41%), Penicillium Air Sampling and Slide Preparation (4.31%), Helminthosporium ( 4.30%), etc. Rotorod Air Sampler with sampling The most dominant species isolated were rate of 100 Lmin -1 was used to analyze the Aspergillus niger, A.flavus, A.fumigatus, total spore counts. Air samplings were Cladosporium herbarum, Penicillium conducted for 2 years (May 2013 to April chrysogenum, P.italicum, Fusarium 2015) by monitoring Rotorod air sampler. oxysporum, F.moniliforme and Trichoderma The air sampler was installed on the lemon viride. The majority of these fungal species tree which is about 2 metres above ground were present throughout the year (Table 1). level for 1 hour twice a month i.e. at 15 days Monthly and seasonal fluctuations intervals. The exposed cellophane tapes were recorded in the spore concentrations. were mounted with glycerine jelly on a slide Quantitative and qualitative analysis of air and examined under a microscope. The samples over the plantation field showed trapped spores were identified with the help contamination by some major fungal spore of reference slides as well as available types such as Alternaria, Aspergillus, standard literatures [6-7]. For the qualitative Fusarium, Penicillium and analysis of fungi, two different media viz Helminthosporium. Besides the factors like Czapek’s Dox Agar (CZA) and Potato time of the day, weather condition, condition Dextrose Agar (PDA) were used. The of the surrounding areas, local source of sterilized petriplates (3 replicates) containing spores, etc., the number and type of fungi of each medium with streptomycin were vary with the season also [10-11]. The carried to the study sites and exposed to the calendar of Kachai area was divided into air for 5 minutes being kept at the height of four season viz. spring (February-April), 1metre above the ground level to receive the summer (May-June), Monsoon/Rainy (July- sedimentation of airborne fungal spores. October), winter (November-January). After exposure, the petriplates were brought During investigation period, the maximum to the laboratory and incubated at 25±1 ̊ C incidence of fungal spores was recorded in for 5-7 days. Fungal colonies developed in rainy season, moderate in summer season plates were counted and identified. The and the least was recorded during spring. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 24-27, 2016 26

Table 1. Total concentration and percentage contribution of each airborne component over Lemon plantation field from May 2013 to April 2015. May 2013 to April 2014 May2014 to April 2015 Fungal types Concentration Percentage (%) Concentration Percentage (%) (spores/m 3) Contribution (Spores/m 3) contribution Deuteromycotina Alternaria 195 8.71 210 8.75 Pithomyces 105 4.69 90 3.75 Torula 90 4.02 140 5.84 Drecshlera 35 1.56 40 1.67 Tetraploa 25 1.12 10 0.42 Curvularia 35 1.56 15 0.63 Diplodia 35 1.56 55 2.29 Cercospora 40 1.79 30 1.25 Helminthosporium 95 4.24 105 4.38 Bispora 25 1.12 30 1.25 Heterosporium 20 0.89 10 0.42 Aspergillus 185 8.26 200 8.33 Fusarium 95 4.24 110 4.58 Penecillium 105 4.69 95 3.96 Trichoderma 55 2.46 40 1.67 Cordana 50 2.23 55 2.29 Cladosporium 50 2.23 65 2.71 Periconia 65 2.90 60 2.5 Sporothrix 45 2.01 30 1.25 Nigrospora 95 4.24 80 3.34 Ascomycotina Sporormia 60 2.68 55 2.29 Epicocum 5 0.22 25 1.04 Spegazzinia 10 0.45 40 1.67 Lacanidion 25 1.12 20 0.84 Didymosphaeria 45 2.01 50 2.08 Passeriniella 45 2.01 65 2.71 Chaetomium 25 1.12 45 1.88 Massarina 40 1.79 25 1.04 Sordaria 45 2.01 55 2.29 Phycomycotina Circinella 50 2.23 65 2.71 Cunninghamella 20 0.89 25 1.04 Basidiomycotina Smut 50 2.23 95 3.96 Uredospores 25 1.12 45 1.88 Other types Hyphal fragments 90 4.02 85 3.54 Insect parts 80 3.57 95 3.96 Pollen grains 75 3.35 105 4.38 Unidentified 10 0.45 35 1.46 Total 2240 100 2400 100

The incidence of airborne fungal fall and their germination in hot and wet spores of study fields reveals that the conditions prevailing in and around the concentration of spores fluctuate irrespective lemon plantation field. The abundance of of crop growth indicating the concentration organic matter in rainy season provides of spores is not governed by crop growth but opportunity to the growth of fungi for both that meteorological parameters have the groups (saprophytic and plant influence of their occurrence (Fig. 1). pathogenic) of fungi. The present Frequent precipitation in rainy investigation also revealed that irrespective season is an important factor for the spores of the years, there is uniformity in the © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 24-27, 2016 27 growth curve of different fungal species in detection of phytopathogenic airborne spores the form of both degree of percentage and leading to prevention, avoidance and month of occurrence (Table 1). treatment of lemon disease in time.

ACKNOWLEDGEMENT 30 Percent contribution of aero mycoflora The first author is grateful to Head,

25 Department of Life Sciences, Manipur University, Canchipur for providing 20 laboratory facilities. Thanks are also due to 15 UGC, New Delhi for awarding a fellowship

10 to the first author. %Contribution 5 REFERENCES

0 1. Gregory, P.H. (1973). The Aug Sep Oct Nov Dec Jan Feb Mar April May Jun Jul nd Months Microbiology of the Atmosphere. 2 Ed. Leonard Hill, London. Figure 1. Mean monthwise percent 2. Edmonds, R.L. (ed) 1979. Aerobiology: contribution of aeromycoflora over lemon The Ecological Systems Approach, plantation in Kachai from Aug 2013 to Dowden, Hutchinson and Rose, Inc. July 2015. Strondsburg/ Pennsylvania. 3. Shivpuri, D.N. (1982). Asp. Allergy Appl. Immunol . 15: 19-30. 5% 4. Waggoner, P.E. (1960) . Plant 10% Pathology. An Advanced Treatise 12% Spring (Ed.Horsfall and Dimmod). Academic summer Press, New York. 5. Leyronas, C. and P.C. Nicot (2013). Rainy Aerobiologia. 29(2): 291-299. winter 6. Tilak, S.T. and R.L. Kulkarni (1970). 73% Experiential . 26: 443-444. 7. Lacey, M.E. and S.W. Jonathan. (2006). The Air Spora: A manual for Figure 2. Seasonal contribution of catching and identifying airborne Aeromycoflora over lemon plantation in biological particles. Springer US, pp. 14. Kachai, Ukhrul district, Manipur. 8. Barnett, H.L. (1969). Illustrated Genera of Imperfect Fungi. Burgess Pub. Co. The number of fungal colony Minneapolis Minnesola. forming unit (CFUs) showed significant 9. Ellis, M.B. (1971). Dematiaceous positive correlation (r=0.841) with the prime Hypomycetes. Commonwealth meteorological parameters like temperature, Mycological Institute, London. rainfall and relative humidity. The present 10. Ianovici, N. and D. Tudorica (2009). findings agree with the previous studies in Not. Sci. Boil . 1(1): 21-28. respect of meteorological parameters which 11. Pepeljnjak, S. and M. Klaric Segvic affect air fungi both qualitatively and (2003). Aerobiologia . 19: 11-19. quantitatively [12-13]. This study also would 12. Gravesen, S (1972). Acta Allergol. 27: help the lemon cultivators to understand the 337-354. various components of air and their 13. Banerjee, U.C., P. Weber, J. Ruffin occurrence in the environment. The and S. Banerjee (1987). Grana . 26: 103- continuous air monitoring would also serve 108. as a useful data to forecast the probable 14. Premila, A. (2013). International J. period of infection thereby avoiding losses Pharmaceutical Science Invention. 2(2): due to fungal plant diseases by early 1-4.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

A new potential mycoherbicide Setospherica monoceras var agbioae for control of Water hyacinth

Ajay Kumar Singh 1* and Praharaju Laxminarayana 2 1Principal Scientist & Head R&D, AG BioSystems Private Limited, Hyderabad, India 2Managing Director, AG BioSystems Private Limited, Hyderabad, India

ARTICLE INFO ABSTRACT Water hyacinth, Eichhornia crassipes (Mart.) Solms.- Article History: Laubach, is considered one of the world’s worst weeds, Received 22 Dec 2015 invading lakes, ponds, canals, and rivers. During survey for Accepted 31 March 2016 fungal pathogens of water hyacinth infested areas, an isolate AGWH#11 of the fungus Setospherica monoceras was collected from infect leave of water hyacinth. The 16s rRNA

sequence analysis provided enough evidences to show that Keywords: isolate AGWH#11 belonged to the Setospherica genus and 18S rRNA was distinct variation from Setospherica monoceras . Based Setospherica m onoceras on these evidences, the new fungal isolate was named as Eichhornia crassipes “Setospherica monoceras var agbioae ”. Mycoherbicide ©2016 Society for Basic and Applied Mycology All Rights Reserved. Non viable substance

Article Type: Full Length Research Paper

INTRODUCTION of aquatic fish production [4]. Bio-control Water hyacinth ( Eichhornia has been considered as the most adequate crassipes ) has spread worldwide causing control strategy against water hyacinth [5]. widespread problems to millions of users of Among possibilities offered by the bio- water bodies and water resources. It is one control management, fungal pathogens could of the most troublesome aquatic weed all be an efficient control tool against this over the world. It is a free floating aquatic aquatic weed. Application of fungi is easy weed with beautiful liliac violet flowers and and inexpensive to produce and therefore continues to be most dangerous aquatic have potential for development as bio weed [1]. Most of the problems associated herbicides. During research on fungal with this weed are due to its rapid growth pathogens of water hyacinth and the rate, its ability to successfully compete with isolation and identification of fungi we have other aquatic plants and its ease of found isolate AGWH#11 as a potential strain propagation [2,3]. Its rapid multiplication for water hyacinth. The IMTECH and NCIM and mat like proliferation can lower Collection identified isolate AGWH#11 as dissolved oxygen level leading to reduction Setospherica monoceras with reference *Corresponding author: [email protected] (Ajay Kumar Singh) J. Basic Appl. Mycol. Vol. 12 (I), pp. 28-31, 2016 29

NCIM and MTCC. However, 16s rRNA nuclear ribosomal DNA [rDNA] characters displayed by this isolate including transcriptional unit were performed between the pattern and ITS region of this isolate isolate AGWH#11 and the closest species or hardly match with those currently used to isolates for which sequences were available. describe known species in the genus. It was Comparison of the ITS rDNA gene showed therefore assumed that this isolate could that AGWH#11 isolate was 99% identical to belong to a novel species of Setospherica 2 other isolates (Table 1).Concerning the monoceras. The present study was based on study, isolate AGWH#11 showed 96% molecular evidence and host test we identity with Setosphaeria monoceras . conclude that ‘‘ Setospherica monoceras f. Similarly to the ITS rDNA gene, the 18S agbioae ’’ is distinct from Setospherica rDNA gene sequence of isolate AGWH#11 monoceras and the taxon Setospherica showed than a 91% identity with 4 different monoceras nov is described. Alternaria sp. Similarly to the its rDNA gene, the 18S rDNA gene sequence of MATERIALS AND METHODS isolate AGWH#11 showed than a 93% Isolation of AGWH#11 identity with Cochliobolus lunatus and one The indigenous fungal strain other Cochliobolus sp showing 94% Setospherica monoceras f. agbioae similarity. Similarly it is showing 95% AGWH#11 was isolated and purified from identity with Bipolaris elecusines . the diseased leaves of water hyacinth. GGTGGTGGTGCATGGCCGTTCTTAGTTCGTGGGGTGAC 18s rRNA identification TTGTCTGCTTAATTGCGATAACGAGCGAGACCTTCCTCT To confirm the species of the fungi, GCTAAATAGCCAGGCTAGCTTTGGCTGGTCGCCGGCTT the identification of culture using 18S–ITS CTTAGAGAGACTATCAGCTCAAGCTGATGGAAGTTGGA GGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGG sequence of rRNA based molecular CCGCACGCGCGCTACACTGACAGAGCCAACGAGTTCTT technique was done. CACCTTGGCCGAAAGGTCTGGGTAATCTTGTTAAACTCT GTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCA Culture deposit and characterization ACGAGGAATACCTAGTAAGCGCGTGTCATCAGCATGCG The isolates also deposited in the TTGATTACGTCCCTGACCTTTGTACACACCGCCCGTCGC NCIM, Pune, India and given an accession TACTACCGATTGAATGGCTCAGTGAGGCGTTCGGACTG number NCIM 1370. This strain has also GCTCGGGGAGGTTGGCAACGACCACCCCAAGCCGGAA been deposited at IMTECH, Chandigarh, AGTTCGTCAAACTCGGTCATTTAGAGGAAGTAAAAGTC GTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCA India under the Budapest treaty under TTACACAACAAAAATATGAGGGTGTGGTTTGCTGGCAA accession number. MTCC 5974. The strain CAGCGTCCGCCCCAAGTATTTTTCACCCATGTCTTTTGC has identified as Setospherica monoceras . GCACTTTTTGTTTCCTGGGCGAGTTCGCTCGCCACCAGG ACCCAACCATAAACCTTTTTTTATGCAGTTGCAATCAGC RESULTS AND DISCUSSION GTCAGTATAATAATTCAATTTATTAAAACTTTCAACAAC 18s rRNA identification GGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGA Setospherica monoceras AATGCGATACGTAGTGTGAATTGCAGAATTCAGTGAAT (AGWH#11) were characterized based on CATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTC CAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAA partial DNA sequence of the internal GCTTTGCTTGGTGTTGGGCGTCTTTTTGTCTCTCCCCTTG transcribed spacer regions of the nuclear TTGGGGGAGACTCGCCTTAAAACGATTGGCAGCCGACC ribosomal RNA gene. TACTGGTTTTCGGAGCGCAGCACAAATTTGCGCCTTCCA Consensus sequence of 18S region ATCCACGGGGCGGCATCCAGCAAGCCT was generated (Fig. 1) from forward and Figure 1. Consensus sequence reverse sequence data. It was then used to of Setospherica monoceras (AGWH#11). carry out BLAST with the database gene bank database and based on maximum The ITS sequence was obtained identity score, 11 sequences were selected using the deposited database of ITS. The (Table 1) for preparing the phylogenetic tree isolates ITS sequence obtained were not (Fig. 2) 100% identical to any ITS sequence Internal transcribed spacers 1 and 2 deposited in Gene Bank. This unique ITS [ITS-1 and 2] and 5.8 S regions of the sequence of the isolates provides strong © 2016 Society for Basic and Applied Mycology . Printed in the India.

Figure 2. Phylogenetic tree constructed from 9 closely related sequences, showing similarities between Setospherica monoceras (AGWH#11). evidence to consider these pathogens as new which the name Setospherica monoceras var from specialis. Based on these evidences, the agbioae was provided. Results of analysis new fungal isolate was named as were confirmed by the phylogenetic data “Setospherica monoceras var agbioae . obtained in this study. Because ITS sequence of AGWH#11 was then 96% Table 2. BLAST report - Nine sequences identical to those of same isolates (Table 1). with maximum identity score from Based on these evidences, the new fungal BLAST report. isolate was called “ Setospherica monoceras Organism GenBank % identity var agbioae ”. Accession(s) (bp) This strain is under investigation for Setosphaeria DQ337380 96 more host specificity, its biocontrol efficacy monoceras Bipolariseleusines DQ337382 95 in field and formulation studies to be Pleosporaceae sp EF060604 99 developed as a mycoherbicide from India in Pleosporaceae sp EF060407 99 the near future. Cochliobolus lunatus DQ337381 93 The research reported in this paper Pleosporaceae sp EF060433 99 explores the possibility of evaluating a new Cochliobolus sp. FJ235087 94 Alternaria AM237287 91 strain of Setospherica monoceras var multirostrata agbioae ” an Indigenous fungal pathogen/non Alternaria cichorii AM237286 91 viable substance to control water hyacinth Alternaria jesenskae AM237084 91 by applying the principle of bio-control of Alternaria AM237288 91 weeds with fungal pathogen/biorational so passiflorae as to reduce the use of chemical pesticides. Further experiments and green house field This study records the discovery of trials will determine the potentiality of this an unknown subspecies in the Setospherica pathogen to control water hyacinth. genus. Comparisons of 18s rRNA sequences of isolate AGWH#11 and all other fungal ACKNOWLEDGEMENT sequences existing in GenBank, suggest that We would like to express our this fungus represents a new taxon, for grateful appreciation to all staff from AG *Corresponding author: [email protected] (Ajay Kumar Singh) J. Basic Appl. Mycol. Vol. 12 (I), pp. 28-31, 2016 31

BioSystems for assistance in this work. We M.H. Jijakli (2012). Biotechnol. Agron. would like to especially thank to Mr. V.N. Soc. Environ. 16(3): 360-368. Anjanleu for his assistance. 4. Dagno, K., M. Diourté, R. Lahlali, M.H. Jijakli (2011b). Afr. J. Microbiol. REFERENCES Res. 5: 924-929. 1. Babu, M.R., A. Sajeena, K. 5. Shabana, Y.M., R. Charudattan and Seetharaman, P. Vidhyasekaran, P. M.A. Elwakil (1995). Plant Dis. 279- Rangasamy, P.M. Som, R.A. Senthil 319. and K.R. Biji (2002). Crop Prot. 22: 6. Charudattan, R. (2005). Biol. Cont. 35: 1005-1013. 183-196. 2. Aneja, K.R. and K. Singh (1989). Trop. 7. Nag Raj, T.R. and K.M. Ponnappa Pest Manage. 35: 354-356. (1970). Trans. Br. Mycol. Soc. 55: 123- 3. Dagno, K, M. Diourté, R. Lahlali, 130.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Comparison of virulence and antifungal susceptibility profile of Candida dubliniensis and Candida albicans isolated from human immunodeficiency virus - infected patients with oropharyngeal candidiasis

Sachin C. Deorukhkar * and Santosh Saini Department of Microbiology, Rural Medical College, Pravara Institute of Medical sciences (Deemed University), Loni, Maharashtra. India ARTICLE INFO ABSTRACT Oropharyngeal candidiasis (OPC) is one of the most Article History: common opportunistic mycotic infections among persons Received 05 Nov 2015 living with HIV/AIDS. Among non-albicans Candida Accepted 30 March 2016 species a high prevalence of C. dubliniensis in the oral cavities of HIV-infected individuals has been reported. The present was conducted with an aim to investigate virulence

factors and antifungal susceptibility profile of Candida Keywords: dubliniensis isolated from HIV infected patients with OPC Antifungal Resistance and compared it with those of C. albicans . A total of 50 HIV Infection isolates of C. dubliniensis and C. albicans isolated from HIV Oropharyngeal Candidiasis infected patients with OPC were included in the study. The Virulence Factors isolates were screened for the production of virulence

factors, and antifungal susceptibility profile was determined. C. dubliniensis showed high adherence property and Article Type: proteinase production. Biofilm formation and phospholipase Full Length Research Paper expression was low in C. dubliniensis . Amphotericin B resistance was high in C. albicans whereas, resistance to azole group of antifungal drug was higher in C. dubliniensis . C. dubliniensis not only shares phenotypic properties with C. albicans but is also capable of producing virulence factors once attributed to C. albicans . The availability of antifungal susceptibility profile can guide physicians in choosing appropriate antifungal agents. ©2016 Society for Basic and Applied Mycology All Rights Reserved.

INTRODUCTION clinical significance are now one of the The emergence of HIV/AIDS has important causes of opportunistic infections changed the scenario of infectious diseases. [1]. Organisms previously considered non Oropharyngeal candidiasis (OPC) is one of virulent or less virulent have emerged as the most common opportunistic mycotic potent pathogens. Fungi once considered as infections in persons living with HIV/AIDS “microbiological curiosities” with little or no (PLHA) [2]. Although OPC is rarely fatal, it *Corresponding author: [email protected] (Sachin C. Deorukhkar) J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 33 contributes significantly to morbidity and Laboratories Pvt. Ltd., Mumbai, India) and reduces the quality of life of PLHA [3]. growth at 45 0C [6]. Candida albicans is widely The virulence factors studied include recognized as the most important cause of adherence to human buccal epithelial cell superficial and disseminated infections, (HBEC), biofilm formation, production of though in recent years infections due to non extracellular hydrolytic enzymes albicans Candida (NAC) species are being (phospholipase and proteinase) and increasingly reported [2]. Among NAC spp, haemolytic activity. a high prevalence of C. dubliniensis in oral cavities of PLHA has been reported [4]. (1) HBEC adherence assay Although C. dubliniensis shares The adherence assay method many phenotypic characteristics with C. described by Al Abied et al [7] was used albicans , it differs with respect to virulence with minor modifications. HBECs were characteristics and the ability to develop obtained from healthy human volunteers (no fluconazole resistance in vitro [5]. As most signs or symptoms of OPC or other oral of the research on virulence factors in lesions and not receiving any antibiotics at Candida spp. is directed towards C. albicans the time of study) after obtaining consent. and other common NAC spp. like C. As fresh HBECs were used for adherence tropicalis and C. glabrata , there is a paucity assay, they were collected in the morning on of information about virulence attributes of the day of assay by gentle rubbing of the C. dubliniensis . Therefore in the present inner side of the cheeks with a sterile cotton study we have investigated virulence factors swab. and antifungal susceptibility profile of C. Candida isolates were inoculated in dubliniensis isolated from HIV infected 0.67% (w/v) yeast nitrogen base (YNB) patients with OPC and compared it with broth supplemented with 2.5% (w/v) glucose those of C. albicans . and incubated at 37 0 C for 24 h. 2 ml of this overnight culture was inoculated in a flask MATERIALS AND METHODS containing 50 ml of the same medium and This descriptive study was conducted grown for 24 h in a shaking water bath at for a period of 3 years from January 2011 to 37 0C. The cells were harvested by December 2013 and is a part of a PhD thesis centrifugation (1200 g for 10 min) and conducted in the Department of washed thrice with phosphate buffered Microbiology of rural tertiary care teaching saline (PBS). The yeast cells were hospital of Maharashtra .The protocol of the suspended in PBS and concentration was study was approved by the Institutional adjusted to 1x10 7 cells/ ml using a Ethics Committee. A total of 50 isolates of turbidometer. C. dubliniensis and C. albicans isolated Two ml of yeast suspension was from HIV infected patients with OPC were mixed with 2 ml of HBECs suspension. The included in the study. The type of OPC and mixture was incubated at 37 0 C for 2 h with most recent CD4 cell count were obtained gentle shaking. After incubation, the mixture from the clinical records. The isolates were was passed through a 20 m filter to remove screened for the production of virulence unattached yeast cells. The HBECs on the factors, and antifungal susceptibility profile filter were washed with PBS and finally was determined. The virulence factors and suspended in 5 ml of PBS. A smear was antifungal susceptibility profile of C. prepared from a drop of this suspension and dubliniensis were compared to those of C. stained by Gram staining. The adherence albicans . was determined microscopically by counting Speciation of C. albicans and C. the mean number of yeast cells adhering to dubliniensis was done on the basis of germ 100 HBECs. Each assay was repeated three tube test, carbohydrate assimilation, colony times and average of three readings was colour on Hichrom Candida agar, taken. C. albicans ATCC 90028 was used as Hi Candida identification kit (Himedia the control strain.

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 34

(2) Biofilm formation occasions. The mean of the nine readings Biofilm formation was determined was taken as the Pz value. using the tube method described by Yigit et al [8] with minor modifications. Candida Proteinase production colonies form Sabouraud dextrose agar The proteinase activity of Candida (SDA) were inoculated in saline and isolates was measured in terms of bovine incubated for 24 h at 35 0 C. One ml of this serum albumin (BSA) degradation by the saline suspension was transferred to screw method described by Staib [10]. capped conical polystyrene tubes containing Approximately 10 l of standard inoculum 5 ml of Sabouraud dextrose broth containing 10 6cells/ml was aseptically supplemented with glucose (final inoculated onto 1% BSA agar plate. The concentration of 8%). The tubes were plate was incubated at 37 0C for 5 days. After incubated at 37 0 C for 24 h without agitation. incubation, further proteinase activity was After incubation, the broth from the tube inhibited by adding 20% tricholoroacetic was gently aspirated using a Pasteur pipette. acid and the plate was stained with 1.25% The tube was washed thrice with PBS (pH amidoblack. A zone of proteolysis 7.2) and stained with 1% saffranin. The stain surrounding the colony that could not be was decanted after 10 min and the tube was stained indicated proteinase activity. rinsed with PBS to remove excess stain. The proteinase index (Prz) was Presence of visible adherent film on measured in terms of the ratio of the the wall and the bottom of the tube indicated diameter of the colony to the diameter of biofilm formation by the isolate. Ring unstained zone. A Prz value of 1 indicated formation at the liquid interface was not no proteinase activity; Prz<1 denoted considered as an indication of biofilm proteinase expression by Candida isolate. production. C. albicans ATCC 90028 and The lower the Prz value, the higher the C. albicans ATCC 10231 were used as activity. C. albicans ATCC 10231 was used control strains. as positive control. For each isolate the assay was conducted thrice on three different (3) Extracellular enzymes activity occasions. The mean of the nine readings The Candida isolates were screened was taken as the Prz value. for the production of extracellular hydrolytic enzymes like phospholipase and proteinase. (4) Haemolytic activity Phospholipase activity: The Candida isolates The haemolytic activity of Candida were screened for phospholipase production isolates was determined on sheep blood by the method described by Samaranayake SDA by the method described by Luo et al et al [9]. Approximately 5 l of standard [11]. Candida isolates were inoculated onto inoculum of test strain containing 10 8 sheep blood SDA plate and incubated at 37 0 cells/ml was aseptically inoculated onto egg C in 5% CO 2 for 48 h. The presence of a yolk agar. After inoculation, the plates were distinct halo around the inoculum site, dried at room temperature and then viewed with transmitted light, indicated incubated at 35 0 C for 3days. The plates haemolysin production. Haemolytic activity were examined for the presence of zone of (Hz) was determined by calculating the ratio precipitate around the colony (phospholipase of the diameter of the colony to that of the production). translucent zone of haemolysis. The phospholipase index (Pz) was C. albicans ATCC 90028 and C. defined as the ratio of the diameter of the parapsilosis ATCC 22019 were used as colony to the total diameter of the colony positive and negative controls, respectively. plus the precipitation zone. A Pz value of 1 One strain each Streptococcus pyogenes denoted no phospholipase activity whereas (Lancefield group A) and Streptococcus Pz<1 indicate phospholipase expression by sanguis , were used as positive controls for the isolate. C. albicans ATCC 10231 was beta and alpha haemolysis, respectively. used as positive control. For each isolate, the For each isolate, the assay was assay was conducted thrice on three different conducted thrice on three different © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 35 occasions. The mean of the nine readings RESULTS AND DISCUSSION was taken as the Hz value. A total of 36 (72%) of C. albicans were isolated from pseudomembranous Table 1. Production of various virulence candidiasis and 14 (28%) from erythematous factors by Candida albicans and Candida candidiasis. In case of C. dubliniensis, dubliniensis . 41(82%) isolates were obtained from Virulence factor C. albicans C. dubliniensis erythematous variant of OPC and 9 (18%) (%) (%) from pseudomembranous type. Of the 50 C. HBEC adherence 30(60) 39(78) albicans isolates, 36(72%) were isolated Biofilm formation 26(52) 09(18) -3 Haemolytic activity 36(72) 32(64) from patients with CD4 ≤200 cells mm and Phospholipase 47(94) 11(22) 14(28%) were isolated from patients with -3 production CD4>200 cells mm whereas, 48(96%) C. Proteinase 34(68) 39(78) dubliniensis were isolated from patients with production CD4>200 cells mm -3 and only 2(4%) were isolated from patients with CD4 ≤200 cells Antifungal susceptibility testing mm -3. The in vitro activities of As shown in table 1, biofilm amphotericin B (range 0.002-32 g/ml), formation, haemolysin production and fluconazole (range 0.016-256 g/ml), phospholipase production were major itraconazole (range 0.002-32 g/ml), virulence factors produced by C. albicans . ketoconazole (range 0.002-32 g/ml) and As compared to C. albicans , C. dubliniensis voriconazole (range 0.002-32 g/ml) were showed high HBEC adherence and assessed. The minimum inhibitory proteinase production. Haemolytic activity concentration (MIC) was determined using TM was shown by 32(64%) C. dubliniensis Ezy MIC strips (Himedia Laboratories isolates. Biofilm formation and Pvt. Ltd., Mumbai, India). phospholipase expression was low in C. The inoculum was prepared by dubliniensis . inoculating 3-4 colonies of the Candida Table 2 shows the antifungal isolate in saline. The turbidity of suspension susceptibility profile of C. albicans and C. was matched to 0.5 McFarland standard. The dubliniensis . Amphotericin B resistance was yeast suspension was inoculated on an agar high in C. albicans (34%) as compared to C. plate containing RPMI 1640 supplemented dubliniensis (22%). Resistance to azole with 2% glucose, by lawn culture method group of antifungal drugs was higher in C. using tipped cotton swab. The dubliniensis as compared to C. albicans . manufacturer’s instructions were adhered to Candida spp. is capable of initiating throughout the test. The antifungal strips infection in immunocompetent as well as were aseptically placed on the media with immunocompromised individuals, but the the help of forceps and the plates were 0 incidence of Candida infections is more in incubated at 35 C 24-48 h. C. albicans immunocompromised hosts. The continuing ATCC 90028 and C. parapsilosis ATCC AIDS epidemic, malignancies, and 22019 were used for quality control. chemotherapeutic interventions have The results of antifungal increased the population of susceptibility test were interpreted as immunocompromised hosts highly sensitive (S), dose-dependent sensitive vulnerable to mycotic infections in general, (DDS) and resistant (R). Interpretative and candidiasis in particular [17]. criteria for azoles were those recommended OPC is one of the most commonly by the Clinical and Laboratory Standard diagnosed mycotic infection in HIV infected Institute (CLSI) [12, 13]. Due to the lack of individuals and individuals with AIDS [18]. defined breakpoints for amphotericin B, It is often the first visible sign of HIV arbitrary values based on the studies of other infection [19].Therefore, it can be rightly researchers were used [14-16]. said ‘Candida is a better physician since it can discover abnormalities in a person’s immune system much sooner, than we with © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 36 our diagnostic tests’. A number of studies Candida spp. can produce several postulate that the widespread and increased exoenzymes like proteinases, phospholipase use of antifungal drugs to suppress OPC in and lysophospholipases. These extracellular HIV patients has favoured the selection of hydrolytic enzymes modify the host NAC spp [20]. membrane components, causing a In the present study C. dubliniensis malfunction [2]. In our study, C. was isolated more from the erythematous dubliniensis isolates showed less variant of OPC whereas C. albicans was phospholipase activity as compared to C. isolated more frequently from the albicans . The phospholipases enzymes pseudomembranous type. In case of C. hydrolyze one or more ester linkages in dubliniensis , oral infections are more glycerolipids of host cells. It facilitates the erythematous rather than growth of Candida and remodels the host pseudomembranous [21]. Erythematous cell membrane. Phospholipases are also candidiasis is a poorly understood condition implicated in the mechanism of host tissue associated with corticosteroids and topical or invasion and dissemination of infection [16]. systemic broad spectrum antibiotics or Limited phospholipase activity may be one HIV/AIDS. It is the most common of the reasons for limited ability of C. manifestation of candidiasis encountered in dubliniensis to cause systemic infections like HIV infected individuals and AIDS patients candidemia. C. dubliniensis isolates [21, 22]. exhibited higher levels of proteinase activity In our study, majority of C. as compared to C. albicans . A similar dubliniensis isolates were obtained from observation was reported by Moran et al HIV infected individuals with a CD4 count [25]. Proteinases degrade the host epithelial >200 cells mm -3 whereas, C. albicans were and mucosal barrier proteins such as isolated mostly from individuals with a CD4 collagen, keratin and mucin [16]. count ≤200 cells mm -3. This observation Ability of Candida to form biofilm highlights the possibility that OPC due to C. plays an important role in pathogenesis. dubliniensis occur more frequently in PLHA Candida spp . are capable of forming biofilm with low CD4 counts. Sullivan et al. [5] on most, if not all, medical devices [26]. expressed the possibility of acquisition of C. Medical device related infections are dubliniensis from exogenous sources potentially life threatening and their following sufficient depletion of an management is difficult and costly [2]. In the individual’s T cell count as HIV infection present study C. dubliniensis demonstrated proceeds. less biofilm forming capacity as compared to The establishment of infection by C. albicans . This observation can explain the Candida spp. involves a very complex fact that although many recent studies interaction between a wide range of host reports the increase incidence of NAC spp. predisposing factors and virulence in various clinical manifestations, C. determinants of infecting species. One of the albicans remains the major cause of health most important virulence factors of Candida care associated infections. spp. is its ability to adhere to certain human Haemolysin produced by a cells. Adherence of Candida spp. to host cell microorganism destroy host erythrocytes. is the initial step in the establishment of Iron is a vital inorganic element for the colonization or infection. Our results establishment of an infectious process by demonstrated that C. dubliniensis isolates Candida [2]. A review of the available showed greater adherence ability to HBEC literature has revealed a dearth of compared to C. albicans. This finding is in information regarding haemolysin accordance with studies by Gilfillan et al production in C. dubliniensis . In our study [23] and McCullough et al [24]. Adherence 64% of C. dubliniensis and 72% of C. to the host tissue helps in penetration of albicans showed haemolytic activity. Candida spp. and also helps to resist Antifungal resistance once rarely phagocytosis. documented in Candida spp . has emerged as a significant clinical problem worldwide. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 37

Table 2. Antifungal susceptibility profile of Candida albicans and Candida dubliniensis . Antifungal agent Sensitive Dose-dependent sensitive Resistant C. albicans C. dubliniensis C. albicans C. dubliniensis C. albicans C. dubliniensis Amphotericin B 32(64%) 38(76%) 01(02%) 01(02%) 17(34%) 11(22%) Fluconazole 21(42%) 17(34%) 04(08%) 01(02%) 25(50%) 32(68%) Itraconazole 18(36%) 30(60%) 03(06%) 02(04%) 29(58%) 18(36%) Ketoconazole 23(46%) 15(30%) - - 27(54%) 35(70%) Voriconazole 32(64%) 35(70%) 01(02%) - 17(34%) 15(30%)

The use of oral fluconazole, often at sub- will help in prevention of emergence of drug therapeutic concentration, for treatment of resistance. OPC in PLHA not only led to the widespread emergence of fluconazole- ACKNOWLEDGEMENT resistant C. albicans but also selected This study was conducted under the innately resistant NAC spp [20]. Many aegis of Laboratory, Department of Candida spp . belonging to NAC group are Microbiology, Rural Medical College. We either intrinsically resistant or acquire are grateful to the management of Rural resistance during course of therapy, or both, Medical College and Rural Hospital of to commonly used antifungal drugs [16]. Pravara Institute of Medical Sciences, The emergence of antifungal Deemed University, Loni, Maharashtra, resistance and gradual increase in the India for their encouragement and support number of new and broad spectrum throughout the study. We also thank the antifungal drugs has complicated the choice technical staff of Department of of antifungal drug for treatment of Microbiology for their assistance in the candidiasis. Several drugs are available for study. the treatment of OPC, including azole drugs such as ketoconazole and fluconazole, and REFERENCES the polyenes nystatin and amphotericin B 1. Chakrabarti, A., R. Kaur and S. Das [27]. (2000). Indian J. Med. Microbiol . In our study, azole resistance was 18: 146-52. more common in C. dubliniensis isolates as 2. Sardi, J.C.O., L. Scorzoni, T. compared to C. albicans . Amphotericin B Bernardi, A.M. Fusco-Almeida and resistance was more common in C. M.J.S Mendes Giannini (2013). J. Med dubliniensis isolates as compared to C. Microbiol . 62: 10-24. albicans . Resistance to fluconazole in C. 3. Junqueria, J.C., B.B. Fuchs, M. dubliniensis isolates has been reported in Muhammed, J.J. Coleman, J.M.A.H. studies by other researchers, and the Suleiman and S.F.G Vilela (2011). frequency of resistance is more in isolates BMC Microbiology . 11: 247-55. from HIV infected individuals [5]. 4. Hamza, O.J.M., M.I.N. Matee, M.J. Resistance to fluconazole is of concern Moshi, E.N.M. Simon, F. Mugusi, because its water solubility, oral F.H.M Mikx et al (2008). BMC bioavailability and good safety profile, make Microbiology . 8: 135-43. it the drug of choice in management of OPC 5. Sullivan, D.J., G.P. Moran, E. Pinjon, in PLHA [27]. A. Al-Mosaid, C. Stokes, C. Vaughan, C. dubliniensis is reported to be et al (2004). FEMS Yeast Research . 4: innately susceptible to azole, polyene and 369-76. echinocandin antifungal agents but can 6. Deorukhkar, S., R. Katiyar and S. rapidly acquire resistance [5]. Therefore the Saini (2012). National J. Integrated availability of antifungal susceptibility Research in Medicine . 3: 86-90. profiles can guide physicians in choosing 7. Al-Abied, H.M., K.H. Abu-Elteen, appropriate antifungal agents rather than an A.Z. Elkarni and M.A. Hamad (2004). empirical prescription of drugs. This will Jpn. J. Infect Dis . 57: 279-84. not only limit the use of toxic drugs but also 8. Yigit, N., E. Aktas, S. Dagistan and A. Ayyildiz (2011) . EAJM . 43: 27–32. © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 32-38, 2016 38

9. Samaranayake, L.P., J.M. Raeside Fothergill, D.I. MCCarthy et al and T.W. MacFarlane (1984). (1998) . J. Clin. Microbiol . 36: 3007- Sabouraudia . 22: 201–07. 3012. 10. Staib, F. (1965) . Sabouraudia . 4: 187– 19. Jeddy, N., K. Ranganathan, U. Devi 93. and E. Joshua (2011) . J. Oral 11. Luo, G., L.P. Samaranayake and J.Y. Maxillofac Pathol . 15: 182-6. Yau (2001) . J. Clin. Microbiol . 39: 20. Coleman, D.C., D.J. Sullivan, D.E. 2971-74. Bennet, G.P. Moran, H.J. Barry and 12. Clinical and Laboratory Standards D.B. Shanley (1997). AIDS . 11: 557-67. Institute (CLSI) (2002) . Reference 21. Sullivan, D. and D. Coleman (1998) . J. Method For Broth Dilution Antifungal Clin. Microbiol . 36: 329-34. Susceptibility Testing of Yeasts, 22. Samaranayake, L.P and R.G. Nair Approved standard M27-A2, Clinical (1995) . Indian J. Dent. Res . 6: 69-82. Laboratory Standard Institute, Wayne, 23. Gilfillan, G.D., D.J. Sullivan, K. Ind. USA, 2nd edition. Haynes, T. Parkison, D.C. Coleman 13. Pfaller, M.A. and D.J. Diekema and N.A. Coleman (1998) . Microbiol . (2012) . J. Clin. Microbiol . 5: 2846–56. 144: 829-38. 14. Mane, A., S. Panchvalli, S. Bembalkar 24. McCullough, M., B. Ross and P. and A. Risbud (2010) . Indian J. Med. Reade (1995). J. Clin. Microbiol . 33: Res . 31: 836–38 . 696-700. 15. Deorukhkar, S. and S. Saini (2014) . 25. Moran, G.P., D.C. Coleman and D.J. BMRJ . 4: 35–45 . Sullivan (2012) . Int. J. Microbiol . 16. Deorukhkar, S.C., S. Saini and S. Article ID 205921:7 pages, Mathew (2014) . Int. J. Microbiol . doi:10.1155/2012/205921. Article ID 456878: 6 pages, doi: 26. Seneviratne, C.J., L. Jin and L.P 10.1155/2014/456878. Samaranayake (2008) . Oral Dis . 14: 17. Jabra-Rizk, M.A., W.A. Falker, W.G. 582–590. Merz, A.A. Baui, J.I. Kelley and T.F. 27. Samaranayake, L.P., P.L. Fidel, J.R. Meiller (2000) . J. Clin. Microbiol . 38: Naglik, S.P. Sweet, R. Teanpaisan, M. 2423-2426. Coogan et al (2002) . Oral Dis . 8 (suppl. 18. Kirkpatrick, W.R., S.G. Revankar, 2): 151-160. R.K. Mcatee, J.L. Lopez-Ribot, A.W.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Phytochemical screening and antifungal activity of acetone extract of Azardirachta indica, Ocimum sanctum and Catharnthus roseus leaves

Shikha Gauri, Shikha Bansal and Pooja Tripathi Department of Botany and Microbiology, St. Aloysius College (Auto) Jabalpur, Madhya Pradesh, India

ARTICLE INFO ABSTRACT Screening of leaves of different plants viz. Azardirachta Article History: indica, Ocimum sanctum and Catharnthus roseus shows Received 06 Jan 2016 antimicrobial effects against large number of Accepted 06 April 2016 microorganisms. The antimicrobial efficacy of acetone extract of leaves of Azardirachta indica, Ocimum sanctum and Catharnthus roseus was compared against Alternaria,

Fusarium, Aspergillus niger, Aspergillus flavus, Penicillium Keywords: and Rhizopus by agar well diffusion . Phytochemical analysis Antimicrobial activity gave positive results for protein, glycoside, gum mucilage Azardirachta indica and steroids. All the leaf extracts exhibited significant Ocimum sanctum inhibition. The extract was then further optimized from the Catharnthus roseus results of anti-microbial studies. Acetone e xtract of Acetone extract Catharnthus roseus showed the best antifungal activity

against Alternaria with inhibition zone of 11.2 cm. ©2016 Society for Basic and Applied Mycology All Rights Reserved. Article Type: Full Length Research Paper

INTRODUCTION secondary metabolites in plant. The present Plants are used medicinally in study deals with the antimicrobial efficacy different countries and are source of many of acetone extracts of leaves, of [Neem potent and powerful drugs [7]. The presence (Azardirachta indica ), Tulsi ( Ocimum of antifungal compound is an important sanctum ), and Sadasuhagan ( Catharnthus factor for disease control in higher plants. roseus )] against A lternaria , Fusarium sp., These compounds are biodegradable and Aspergillus niger, Aspergillus flavus, toxic to a considerable value for suppressing Penicillium, Rhizopus. Azadirachta indica some plant disease. The preharvested losses (Neem), is one of the mostly concerned tree due to fungal disease in world crop by the scientist because it contains many protection may reach up to 12% or even natural substances in its different parts , higher in developing countries. Modern leaves , seeds , bark , and has many agrochemical research research influence the biological activities against disease causing application of plant derived fungicides and it organisms, and it contains about 140 has enormous potential against microbial chemical compounds [4]. Ocimum sanctum pathogens attack due to presence of is a popular home remedy for many ailments *Corresponding author: [email protected] (Shikha Bansal) J. Basic Appl. Mycol. Vol. 12 (I), pp. 39-42, 2016 40 such as wound, bronchitis, liver diseases, Evans Harborne, Baker and Thormsberg catarrhal fever, lumbago, hiccough, [4,6,3,2]. ophthalmia, gastric disorders, genitourinary disorders, skin diseases, various forms of Determination of antimicrobial activity poisoning and psychosomatic stress The acetone extracts of leaf of Neem disorders [1,15]. Catharanthus roseus is an (Azardirachta indica ), Tulsi ( Ocimum important medicinal plant and is cultivated sanctum ), and Sadasuhagan ( Catharnthus mainly for its alkaloids, which are having roseus ) were screened for antifungal anticancer activities [8]. activity by agar well diffusion method. For the detection of antifungal activity 1ml spore MATERIALS AND METHODS suspension of test fungus was spread over Collection and preparation of plant the PDA plate. Then equally spaced well leaves were made in agar with the help of a The leaves of Azardirachta indica, sterilized core borer having a diameter of 5.0 Ocimum sanctum, Catharnthus Roseus and mm size. Extract was prepared and 500 µl of Allium sativum were collected in three each plant extract was added to the wells and polythene bags from botanical garden of St. then kept for incubation for 3 – 5 days at Aloysius’ college (Auto) Jabalpur. Freshly 27 0C. After incubation the plates were collected leave s of Azardirachta indica, observed and the zones of inhibition were Ocimum sanctum, Catharnthus Roseus and measured. Allium sativum were cleaned and dried under the shade at normal room temperature. After Effect of temperature on the activity of drying, leaves were ground using pestle and extract mortar into smaller particles and then To study the effect of temperature on blended to powder using an electric blender. the activity of leaf extract of different 100 g of the powdered sample was then experimental plants,extract of each plant was stored in airtight containers and kept under taken in four test tube each of the test tube normal room temperature until containing 10ml extract and were incubated required.Leaves of all the three plants in a water bath for 30 min at different species were collected and treated separately temperature of 60 C,30 0 C,4 C,90C after that throughout the procedure. 1ml extract from each test tube was introduced into wells bored on PDA plates Isolation and identification and incubated at 28 C for 48 hrs . The zone The tested microorganisms were of inhibition was measured. isolated from the soybean seeds collected from Seed pathology department , JNKVV Effect of pH on the activity of extracts Jabalpur (M.P) by blotter method and was A set of five test tube was taken each tentatively identfied on the basis of its tube containing 10 ml of plant extract was microscopic and morphological adjusted to pH 2,4,7,10 respectively by characteristics. adding 1N HCL or 1N NaOH as per requirement and the fifth tube was served as Preparation of acetone extract control and the antifungal activity The first Leaves were placed into the extractor four tubes were adjusted to pH 2,4,7,10 by of a Soxhelet. The extraction was carried out adding 1N HCl or 1N NaOH dropwise as by using acetone (solvent). required. The fifth tube was served as control, and their antifungal activity was Phytochemical analysis of plant extracts determined using agar well diffusion The extracts were subjected to method. phytochemical tests for secondary metabolites viz. gum and mucilage, tannins, RESULT AND DISCUSSION saponins, steroid, alkaloids, carbohydrate, Experiments showed that the acetone protein, flavonoids and glycosides in is efficient solvent. accordance with the methods of Trease, © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 39-42, 2016 41

Isolation and Identification Sadasuhagan (Catharnthus Roseus ) (Table The isolated fungal culture from 2). soybean seeds were tentatively identified as Aspergillus niger, Alternaria, Fusarium, Effect of different pH Rhizopus, Aspergillus flavus on the bases of In Tulsi ( Ocimum sanctum ), microscopic and macroscopic charaterstics. Sadsuhagan ( Catharnthus roseus ) and Neem ( Azardirachta indica ) maximum zone of inhibition of 23.21,15.76 and 25.86 cm respectively was observed against Fusarium, Alternaria at pH 4.

Effect of different temperature In tulsi ( Ocimum sanctum ), Figure 1. Alternaria. Figure 2. Rhizopus. sadsuhagan ( Catharnthus roseus ) and neem (Azardirachta indica ) maximum zone of inhibition of 21.68 cm, 18.68 cm and 26.86 cm respectively was observed against Aspergillus flavus at temperature 6 0C. In phytochemical test carbohydrate, glycoside, steroids was found in acetone extract of Neem ( Azardirachta indica ), Tulsi (Ocimum sanctum ) and Sadasuhagan

Figure 3. Aspergillus niger. (Catharnthus Roseus ). Kumara & Gupta [9] found alkaloids, steroids, tannin, phytosterol

Phytochemical screening with leaves extracts of Catharnthus Roseus . The phytochemical analysis Susmitha [13] found alkaloid, steroid, performed on acetone extract of Neem saponins, tannin, and flavanoid with acetone (Azardirachta indica ), Tulsi ( Ocimum and methanol extract of Azardirachta indica. sanctum ) and Sadasuhagan ( Catharnthus Choudhury [5] reported alkaloid, glucoside, Roseus ) leaves show the presence of gum & mucilage, protein, amino acid, steroids, protein and gum & mucilage in tannin, phenolic compound, steroid, acetone extracts (Table 4). saponins, flavanoids in acetone & methanol extract of Ocimum sanctum. Quinlan [11] worked on steroidal extracts from some medicinal plants which exhibited antibacterial activities on some bacterial isolates. Neumann [10] also confirmed the antiviral property of steroids. Similarly flavonoids were also reported to exhibit antimicrobial, anti-inflammatory, anti- angionic, analgesic, anti-allergic, cytostatic and antioxidant properties [7]. Fig. 1, 2 & 3 shows the varying Figure 4. Antimicrobial activity of acetone degree of antifungal activity of acetone extract of Sadasuhagan against Alternaria . extract of leaves of Neem ( Azardirachta indica ), Tulsi ( Ocimum sanctum ) and Antimicrobial activity Sadasuhagan ( Catharnthus Roseus ).Highest Maximum zone of inhibition of 11.2 antifungal activity of 3.48 cm (Neem), 11.2 cm was observed against Alternaria in cm (Sadasuhagan) and 4.42 cm (Tulsi) was extract of sadasuhagan and no inhibition observed for Alternaria . No zone of zone was observed against Rhizopus in the inhibition was observed for Rhizopus in extract of Tulsi (Ocimum sanctum) and sadasuhagan and Tulsi extract. According to Kumari & Gupta (2013) reported methanolic © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 39-42, 2016 42 leaf extract of Catharnthus roseus was 2. AOAC (1990). Official methods of analysis, maximum against Aspergillus fumigates. Association of Official Analytical Chemists, The experiments based on antifungal Washington, D.C. USA. 15th Edition activity of acetone extract of all the three pp.807- 928. plant leaves showed that alkalinity had slight 3. Baker C. and C. Thormsberg (1983). J. diminishing effect on the antifungal activity Chin. Microbial. 17: 140-145. of the extract. For example at pH 2 acetone 4. Biswas, K., C. Ishita, K.B. Ranajit and B. extract (Sadasuhagan) exhibited Uday (2002). Current Science . 82(11): 1336-1345. antimicrobial activity with a diameter of 5. Choudhury, G.B., M.A. Behera, J.P. zone of inhibition of 4.42 cm, 16.6 cm and Kumar and S.K. Tripathy (2011). 23.01cm for Aspergillus niger, Aspergillus Indian J. Med. Res. 2(2): 605-610. flavus and Fusarium respectively although at 6. Harborne, J.B. (1973). Phytochemical pH 10 antifungal activity decreased to methods: A guide to modern Techniques of 1.56cm, 12.16cm and 19.6 cm for plant Analysis. Chapman and Hall Ltd, Aspergillus niger, Aspergillus flavus and London. p.279. Fusarium . 7. Hodek, P., P. Trefil and M. Stiborova During the present investigation (2002). Chemico-Biol. Intern. 139(1): 1- when different temperature were given then 21. the acetone leaf extract (sadsuhagan) at 4 ℃ 8. Jaleel, C.A., R. Gopi and R. inhibit the growth of Aspergillus flavus, Paneerselvam (2009). Plant Omics J. 2: Alternaria and Fusarium by producing zone 30-40. size of 16.5cm, 9.5 cm, 11.6 cm whereas at 9. Kumara, K. and S. Gupta (2013). International Journal of Research in 90 ℃ 14.9 cm, 11.84 cm and 16.9 cm zone Pure and Applied Microbiology. 3(3): size is observed for Aspergillus flavus, 77-82. Alternaria and Fusarium. 10. Neumann, U.P., T. Berg, M. Baha, G.

CONCLUSION Puhl, O. Guckelbeger, J.M. Langreh and P. Neuhaus (2004). The present research work suggest Transplantation . 77(2): 226-231 that the compound extracted from 11. Quinlan, M.B., R.J. Quinlan and J.M Sadasuhagan have great antimicrobial Nolan (2000). J. Ethanopharmacol. 80: activity and can be used in the treatment of 75-83. infectious disease caused by resistant 12. Srivastava, J., J. Lambert and N. microbes. Vietmeyer (1996). World Bank

ACKNOWLEDGEMENT Technical Paper. No. 320. 7: 579-586.

Authors are thankful to the Principal, 13. Susmitha, S., K.K. Vidyamol, P. St Aloysius'College (Auto), Jabalpur for Ranganayaki and R. Vijayaragavan (2013). Global Journal of Pharmacology encouragement and providing necessary 7(3): 316-320. research facilities. 14. Trease, G.E. and W.C. Evans (1989).

REFERENCES Pharmacognosy 13th Edition, Bailere Traiadal, London p.69. 1. Ashoka, S., C. Shastry and G. Sridevi 15. Udupa, S.L., S. Shetty, A.L. Udupa (2009). J. Pharm. Res. 2(7): 1218-1220. and S.N. Somayaji (2006). Indian J. Exp. Biol. 44: 49-54.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

A new potential mycoherbicide Alternaria alternata f. sp. praharajae for control of Parthenium hysterophorus

Ajay Kumar Singh 1* and Praharaju Laxminarayana 2 1Principal Scientist & Head R&D, AG BioSystems Private Limited, Hyderabad, India 2Managing Director, AG BioSystems Private Limited, Hyderabad, India

ARTICLE INFO ABSTRACT Parthenium hysterophorus L. commonly called as congress grass is among the top ten worst weeds of the world. It is Article History: noxious because it is highly adaptable to almost all type of Received 22 Dec 2015 environmental conditions, can invade all types of land, and also causes high losses in the yield of field crops and direct Accepted 30 March 2016 contact with plant or plant parts for long time causes dermatitis sometimes it may lead to death of person. A foliar pathogen Alternaria alternata f sp praharajae was isolated from infected leave of Parthenium hysterophorus . Based on Keywords: 16s rRNA study of this fungal pathogen demonstrated in 18S rDNA studies provides strong evidence to propose these pathogens of Parthenium as ‘‘ A. alternata f. sp. praharajae ’’. A. alternata ©2016 Society for Basic and Applied Mycology All Rights Reserved. Parthenium

Mycoherbicide

Non viable substance

Article Type:

Full Length Research Paper

INTRODUCTION agricultural or horticultural system but is Parthenium hysterophorus L. also a potential hazard to livestock and (Asteraceae ), is regarded as one of the worst humans [1-3]. The plant is responsible for weeds because of its invasiveness, potential allergenic eczematous contact dermatitis, for spread, and economic and environmental allergenic rhinitis, nasobrochial allergies, impacts. It is not only a major threat to *Corresponding author: [email protected] (Ajay Kumar Singh) J. Basic Appl. Mycol. Vol. 12 (I), pp. 43-46, 2016 44 and seasonal pollenosios eventually leading Table 1. BLAST report - Nine sequences with to death in humans [4-5]. maximum identity score from BLAST report. It inhabits many parts of the world, Organism GenBank % identity in addition to its native range in North and Accession(s) (bp) 6 Alternaria alternata AF21879 95 South America and the West Indies . AF21879.1 According to Holm et al. [7] this noxious Alternaria cichorii AM237286.1 95 invasive species is considered to be one of Alternaria jesenskae AM237084 95 the worst weeds currently known. Alternaria multirostrata AM237487 95 Conventional methods of management rely Alternaria passiflorae AM237288 94 Alternaria sp AY154699 98 mainly on use of synthetic herbicide. Public Alternaria sp AY154698 98 concern over the safety due to indiscriminate Alternaria tenuissima AY154712 98 use of synthetic herbicides has generated Alternaria tenuissima AY154711 98 significant pressures on weed managers to search an alternative of these chemicals. RESULTS AND DISCUSSION Exploitation of fungi as herbicide has 18s rRNA analysis generated significant interest worldwide [8- A. alternata f. sp. praharajae 10]. AGPH#04 are potential, highly hosts The fungus, Alternaria alternata f. specific mycoherbicide for control of the sp. praharajae AGPH#04 a phyllosphere Parthenium weed (Table 1 & Fig 3). The pathogen was found to cause extensive fungal isolates were characterized based on damage to Parthenium plants. In the present partial DNA sequence of the internal communication we assess the Host range transcribed spacer regions of the nuclear studies using tomato, chilly, brinjal, and ribosomal RNA gene. The ITS sequence was similar family indicated that only obtained using the deposited database of Parthenium hysterophorus was susceptible. ITS. Both isolates ITS sequence obtained 16s rRNA study of this fungal pathogen. was not 100% identical to any ITS sequence Based on molecular evidence and host test deposited in Gene Bank. This unique ITS we conclude that ‘‘ A. alternata f. sp. sequence of the two isolates provides strong praharajae ’’ is distinct from A. evidence to consider these pathogens as new alternata and the taxon A. alternata nov is from speicalis that will facilitate and described. encourage the introduction and acceptance by regulatory authorities for practical field MATERIALS AND METHODS application. Recovery of strain Consensus sequence of 18S region A. alternata f. sp. praharajae was generated (Fig. 1) from forward and (AGPH#04) was obtained from AGBIO Lab reverse sequence data. It was then used to collection, Hyderabad. The indigenous carry out BLAST with the database gene fungal strain was isolated earlier from bank database and based on maximum diseased leaves of Parthenium . identity score, 9 sequences were selected The isolates also deposited in the (Table 1) for preparing the phylogenetic tree NCIM, Pune, India and given an accession (Fig. 2). number NCIM 1371. This strain has also been deposited at IMTECH, Chandigarh, 5’GCTCTTTCTTGATTTTTCAGGTGGTGGTGCATGG India under the Budapest treaty under CCGTTCTTAGTTCGTGGGGTGACTTGTCTGCTTAAT TGCGATAACGAGCGAGACCTTACTCTGCTAAATAGC accession number. MTCC 5973. The strain CAGGCTAACTTTGGTTGGTCGCCGGCTTCTTAGA has identified as Alternaria alternata . GAGACTATCAACTCAAGTTGATGGAAGTTTGAGGCA ATAACAGGTCTGTGATGCCCTTAGACCGCACGCG 18s rRNA identification CGCTACACTGACAGAGCCAACGAGTTCTTTTCCTTG TTCGAAAGAATTGGGTAATCTTGTTAAACTCTGT To confirm the species of the fungi CGTGCTGGGGATAAAGCATTGCAATTATTGCTTTTC and the identification of culture using 18S– AACGAGGAATGCCTAGTAAGCGCGTGTCATCAGC ITS sequence of rRNA based molecular ATGCGTTGATTACGTCCCTGACCTTTGTACACACCG technique was done. CCCGTCGCTACTACCGATTGAATGGCTCAGTGAG

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 43-46, 2016 45

GCCTTCGGACTGGCTCGAGCAGGTTGGCCACGACCA CCTCAAGCCGGAAAGTTCGTCAAACTCGGTCATT TAGAGAAAGTGAAAGTCGTAACACGGTCTCCGTAGG TGAACCTGCGGAGGGATCATTACACAAATATGAA GGCGGGCTGGAATCTCTCGGGGTTACAGCCTTGCTG ATTATTCACCCTTGTCTTTGCGTACTTCTGTTTC CTTGGTGGGTCGCCCACCACTGGAACAAACATAAAC CTTTGTAATTGCATCGCGTCTGTCACAATTAATA ATTACAACTTTCAACAACGGATCTCTTGGTTCTGGC ATCGATGAAGAACGCAGCGAAATGCGATAAGTAG TGTGAATTGCAGAATTCAGTGAATCATCGAATCTTT GAACGCACATTGCGCCCTTTGGTATTCCAAAGGG CATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTT TGCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGC TGGAGACTCGCCTTAAAGTAATTGGCAGCCGGCCTA CTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCT ATCAGCAAAGGTCTAGCATCCATTAAGCCTTTTTTC AACT’3 Figure 1. Consensus sequence of Alternaria alternata (AGPH#04).

Results of analysis were confirmed by the phylogenetic data obtained in this study. Because ITS sequence of AGPH#04 was then distinctly variable to those of 9

Figure 2. Phylogenetic tree constructed from 9 closely related sequences, showing similarities between Alternaria alternata (AGPH04).

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 43-46, 2016 46 other isolates, making it not pertinent to 1. Basak, S.L. (1984). Indian build a molecular phylogeny. Agriculturist. 28: 137-143. Genetic results confirmed that the 2. Valliappan K. and G.H.N. Towers fungal isolate AGPH#04 belonged to the (1989). Indian J. Weed Sci. 20: 18–22. genus Alternaria and was distinct from any 3. Swaminathan, C., R.R.S. Vinaya and Alternaria alternata and isolates which had K.K. Sureshi (1990). Inter. Tree Crops been previously characterized. Based on J. 6: 143-150. these evidences, the new fungal isolate was 4. Towers, G.H.N. and P.V. Subbha Rao called “ A. alternata f. sp. praharajae ”. (1992). In: Richardson RG (ed) This strain is under investigation for Proceedings of the first international more host specificity, its bio-control efficacy weed control congress, Melbourne, in field and formulation studies to be Australia, Weed science society of developed as a mycoherbicide from India in Victoria, pp 134–138. the near future. 5. McFadyen, R.E. (1995). Australian The research reported in this paper Family Physician. 24: 1455-1459. explores the possibility of evaluating a new 6. Picman, J. and A.K. Picman. (1984). strain of A. alternata f. sp. praharajae an In: Biochemical Systematics and Indigenous fungal pathogen/non viable Ecology . 12(3): 287–292. substance to control Parthenium by applying 7. Holm, L., J. Doll, E. Holm, J. V. the principle of bio-control of weeds with Pancho and J.P. Herberger (1997). fungal pathogen/biorational so as to reduce World Weeds: Natural Histories and the use of chemical pesticides. Further Distribution , John Wiley & Sons, New experiments and green house field trials will York, NY, USA. determine the potentiality of this pathogen to 8. Pandey, A.K., R.C. Rajak and S.K. control Parthenium . Hasija (1997). In: Achievements and prospects in Mycology and Plant ACKNOWLEDGEMENT Pathology (S S Chahal, I B Parashar, H We would like to express our S Randhawa and S. Arya eds). grateful appreciation to all staff from AG International Book Distributions, Dehra BioSystems for assistance in this work. We Dun: pp 59-85. would like to especially thank to Mr. V.N. 9. Pandey, A.K., Jaya Singh and A.M. Anjanleu for his assistance. Varghese (1999). PCIN . 31: 1-6. 10. Pandey, A.K., S. Lal and S. Joseph REFERENCES (2000). PCIN . 32: 11-23.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Taxonomical notes on Acaulospora species of Indian Thar Desert

Praveen Gehlot

Mycology and Microbiology Laboratory, Department of Botany, JNV University, Jodhpur, India

ARTICLE INFO ABSTRACT The present investigation deals with to explore the Article History: different species of Acaulospora inhabiting in Indian Received 16 Oct 2015 Thar desert and studied out through colour Accepted 07 March 2016 microphotographs. The results showed that rhizosphere soils of xerophytic plants of Indian Thar desert,

Rajasthan, were used and three species of Acaulospora Keywords: Acaulospora isolated. ©2016 Society for Basic and Applied Mycology All Rights Reserved. Species Diversity Taxonomy Indian Thar Desert

Article Type: Short Communication

Taxonomy of Arbuscular usually membranous, hyaline may be Mycorrhizae fungi has been the subject of laminated and ornamented. At maturity the much interest and debate [1]. In AM Fungi, spore does not show a any subtending Acaulospora Gerd. & Trappe emend. Berch. hypha. is one of the important genus. The Greek A survey of literature on the name Acaulospora mean (a (without) + occurrence of AM fungi in India revealed caulos (stem) + spora (spore) i.e. sessile that species of Acaulospora have been spore. The spores are borne laterally from reported only camera lucida drawn the neck of a pre-differentiated sporiferous illustration [2, 3, 4]. Therefore, the present saccule (Fig.1). Spores are globose or aimed to explore the different species of ellipsoid, range from 100 to 400 µm in Acaulospora inhabiting in Indian Thar desert diameter, hyaline, yellow or reddish brown. and studied out through colour The surface of the spore wall may be microphotographs. ornamented with pits, projections of various During the survey of rhizosphere shapes, folds, spines or reticulations. The soils of xerophytic plants of Indian Thar wall is up to 12 µm thick, of two distinct, desert, Rajasthan, three species of separable wall groups, part of the outer wall Acaulospora were isolated by using of continuous, may be pigmented, laminated or sieving and decanting method [5]. composed of distinct walls. Inner wall Identification and taxonomic composed of one or more layers that are characterization were done by using of *Corresponding author: [email protected] (Praveen Gehlot) J. Basic Appl. Mycol. Vol. 12 (I ), pp. 47-49, 2016 48 manual [6]. The description of all collected Acaulospora laevis Gerd. & Trappe species given with taxonomical features. Azygospore formed single in soil. Spores globose, sessile, smooth, size 200- 400 µm in diameter, brown to light reddish brown when young, becoming honey colour at maturity. Spore wall consisting of two layers. Outer wall thick, smooth redish brown with homogenous matrix. Inner wall thin, membranous. Associate with rhizosphere soil of Tecomella undulata (Fig. 2). Figure 1. Camera lucida drawing of Spore formation of Aaulospora. Acaulospora mellea Spain & Schenck Sporocarp without a peridium. Spores borne laterally on hyphae tapering to a globose to subglobose. Hyphal terminus contents white, emptying during spore formation, resulting in a transparent to subhyaline receptacle attached to the azygospore. Old spores in soil usually devoid of a hyphal terminus. Spores coloured to yellow brown, globose to subglobose, 100 – 125 µm. two layered. Outer spore wall thick, yellowish Figure 2. Acaulospora laevis Gerdemann brown, 12-15 µm. Inner wall thin, hyaline & Trappe. and membranous. Spore contents yellow to

brown granules. Associated with rhizosphere soil of Moringa oleifera (Fig 3).

Acaulospora bireticulata Rothwell &Trappe Spores globose or subglobose, range from 200-300 µm in diameter, brown to reddish brown. Spore wall not much distinct. The surface of the spore wall ornamented with a polygonal reticulum. Figure 3. Acaulospora mellea Spain & Mature spore doesn’t show Schenck. subtending hyphae, the vesicular structure that gives rise to it loses cytoplasm and collapses during development of the spore. Associate with rhizosphere soil of Prosopis cineraria (Fig. 4). Acaulospora species are an important component of the soil in all the types of environments and are beneficial for plant growth and development by increasing the nutrient uptake. These species are an also important to contribute substantially to the Figure 4. Acaulospora bireticulate establishment, productivity and longevity of Rothwell and Trappe. natural and manmade ecosystem.

© 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I ), pp. 47-49, 2016 49

ACKNOWLEDGEMENT Rizvi (1991). The Fungi of India . IIed. Authors are thankful to J. Panwar, R. List and References. Today and Negi, and S. Kausal for manifold help Tomorrows Printers and Publishers, New during diversity assessment of AM fungi. Delhi, pp. 798. 4. Jamaluddin, M.G. Goswami and B.M. REFERENCES Ojha (2004). Fungi of India (1989- 1. Schüßler, A., D. Schwarzott and C. 2001). Scientific Publ. India, pp.326 Walker (2001). Mycol. Res. 105 : 1413- 5. Gerdemann, J.W. and T.H. Nicolson 1421. (1963). Trans. Br. Mycol. Soc. 46 : 235- 2. Bilgrami, K.S., Jamaluddin and M.A. 244. Rizvi (1979). The Fungi of India. Part I. 6. Schenck, N.C. and Y. Perez (1990). List and references. Today and Manual for the identification of Tomorrows Printers and Publishers, New Vesicular arbuscular Mycorrhizal Delhi, pp. 467. Fungi. Synergistic Publication. 3. Bilgrami, K.S., Jamaluddin and M.A. Gainsville, Florida, USA. pp. 283.

© 2016 Society for Basic and Applied Mycology . Printed in the India.

Journal of Basic and Applied Mycology Volume 12 | Issue I | 2016 ISSN: 0972-7167 (P) ISSN: 2455-3875 (O) Available online at www.sbamjournal.com

Identification of Basidiobolus haptosporus from a subcutaneous biospsy specimen in Côte d’Ivoire

Angora Kpongbo Etienne 1,2, Offianan Andre Toure 2, Vanga-Bosson Henriette 1,2, Ira- Bonouman Ama 2, Djohan Vincent 1, Sylla-Thanon Karidja 2, Angorantchi Assemian 3, Yavo William 1, Konate Abibatou 1, Kassi K. Fulgence 1, Kiki-Barro Pulcherie 1 and Menan E.I. Herve 1 1Laboratoire de parasitologie-mycologie, UFR des Sciences Pharmaceutiques et Biologiques, Abidjan (Côte d’Ivoire) 2Unité de Mycologie, Institut Pasteur (Côte d’Ivoire) 3Institut de Dermatologie-Vénérologie, CHU de Treichville (Côte d’Ivoire)

ARTICLE INFO ABSTRACT Basidiobolomycose is phycomycosis subcutaneous endemic Article History: that preferentially affects young people in rural tropical Received 04 Sep 2015 areas. The case of Basidiobolus haptosporus described in the Accepted 06 Feb 2016 present report has been identified from tender indurated subcutaneous swelling occurred in a 13-year-old, Ivorian male from a rural area of the N'zi-comoé, eastern area of

Côte d'Ivoire. The biological specimen was sent by Keywords: dermatology department of University Hospital (CHU) of Basidiobolus haptosporus Treichville in Abidjan for mycology diagnososis of a tender Children indurated subcutaneous swelling on the right thigh over the Ketoconazole anterolateral and medial aspect. Microbiological Côte d’ivoire examination identified the fungus as Basidiobolus Treatment haptosporus. Treatment with keteconazole was

successfulled. ©2016 Society for Basic and Applied Mycology All Rights Reserved. Article Type: New Report

INTRODUCTION class of Zygomycetes and is mainly Basidiobolomycosis is a rare disease associated with subcutaneous fat tissue that, unlike other fungal infections, affects infection involving the limbs, trunk, or immunocompetent individuals. It is caused buttocks. It is presumed that infection is by an environmental saprophyte fungus acquired after minor trauma to skin or insect named Basidiobolus haptosporus . This bites. Most cases of basidiobolomycosis disease usually appears as a subcutaneous have been reported from tropical and infection. subtropical regions of Africa, South B. haptosporus is a fungus belonging America, and Asia [1-3]. to family of Entomophthoraceae and the

*Corresponding author: [email protected] (Offianan Andre TOURE) J. Basic Appl. Mycol. Vol. 12 (I ), pp. 50 -53, 2016 51

There is a rare fungus which to the family of Basidiobolaceae and the preferentially affects children, often genus of Basidiobolus [7]. Entomo- adolescents and rarely a dults [1, 4]. It is due phthoromycosis has been known as a to B. haptosporus that grow produce hyphae saprophytic fungus present in soil, decaying and zygospores [5]. The zygospores are fruit, an d vegetable matter as well as in the presented in culture sub-bodies of spherical gut of amphibians and reptiles [8]. shape measuring about 30 mm in diameter with a conjugating spout [6]. Diagnosis is based on the demonstration of the fungus in a skin fragment pat hological examination and mycological examination whose culture is on a medium Sabouraud-Chloramphenicol without Actidione. In our study, we report a case of Basidiobolus haptosporus identified in Mycology Laboratory of Institute Pasteur of Côte d'Ivoire.

Basidiobolus haptosporus identification A 13-year-old, Ivorian male from a rural area of the N'zi-comoé region in Figure 1 . Basidiobolus haptosporus eastern Ivory Coast was admitted to the colonies on Sabouraud -chloramphenicol dermatology department of the teaching medium without Actidione (Mycology hospital of Treichville in Abidjan in October Laboratory of Institut Pasteur of Côte 2012 for tender indurated subcutaneous d’Ivoire). swelling on the right thigh over the anterolateral and medial aspect. Culture cutaneous biopsy on Sabouraud-chloramphenicol medium without Actidione after four days of incubation at 28°c showed flat creamy brown, furrowed colonies with pale reverse (Fig. 1). After about a week, satellite colonies Figure 2. Furrowed colonies of were developed and the colonies hav e turned Basidiobolus haptosporus on Sabouraud- brown in colour (Fig. 2). chloramphenicol medium without Microscopic examination Actidione. Picture A: recto. Picture B demonstrated aspect hyphae and smooth reverse (Mycology Laboratory of Institut walled zygospores with characteristic Pasteur of Côte d’Ivoire). conjugation beaks, which confirmed the fungus to be B. haptosporus (Fig. 3). Following the confirmation of the diagnosis as basidiobolomycosis by culture and microsco py, the patient was put on ketoconazole at 7mg/kg/day during one month.

DISCUSSION Figure 3. Zygospores with characteristic Zygomycosis is considered as a large conjugation beaks of Basidiobolus group of infections that are caused by the haptosporus (Mycology Laboratory of fungi of the zygomycota class and Institut Pasteur of Côte d’Ivoire). Entomophthorales order [2]. Basidiobolus , a filamentous fungus, is one the three species The route of transmission is still of the Entomophthorales order and belongs unclear [2] but presumed to be through the © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 50-53, 2016 52 insect bite or fungus exposure via minor treat Basidiobolus infection include trauma to the skin [1, 9, 10]. This disease potassium iodide, trimethoprim- may also be transmitted from soil and sulfamethoxazole, amphotericin B, oral vegetation that were contaminated with azoles, and potassium iodide combined with animal feces. Iatrogenic infection has also oral azoles.17, 18, 19, 20]. been reported. Following the confirmation of the The disease begins with a small diagnosis as basidiobolomycosis by culture papule on the site of a scratch or skin and microscopy, the patient was treat with puncture. The most common initial ketoconazole at 7mg/kg/day during one presentation is an indolent infection of the month. Therefore, antifungal imidazole subcutaneous tissues with a firm, non- derivatives represent the best alternative in ulcerated lesion [11]. the treatment of phycomycosis. Similar The clinical diagnosis of results have been reported by several authors basidiobolomycosis is usually suspected in a with ketoconazole [12, 16, 22]. Moreover, in pseudo-tumor infiltration non inflammatory our case a good tolerance was observed with firm consistency to clean edge and ketoconazole as reported previously in Côte mobilized over deep planes. This d’Ivoire by Kanga et al. [15]. phycomycosis mainly affects rural youth Other derivatives imidazole issues in the tropics as in our case [8, 10, antifungals such as itraconazole, 12]. fluconazole, miconazole and terbinafine In a study of ten cases of have also been used successfully in the entomophthoromycosis by Krishnan et al. treatment of basidiobolomycosis [17, 18, 19, [13], eight were caused by Basidiobolus 21]. haptosporu s predominantly in children It seems that it has been undiagnosed below ten years of age, and thigh was the or probably unnoticed in other parts of the most commonly involved site, as in our case country. [13]. The case of subcutaneous ACKNOWLEDGEMENT basidiobolomycosis described in the present We would like to thank the patient report occurred in a 13-year-old, Ivorian and their parents. We are grateful to male from a rural area of the N'zi-comoé, physicians of the department of dermatology eastern area of Côte d'Ivoire. The biological of the teaching hospital of Treichville specimen was sent by the department of (Abidjan) for their contribution to this work. dermatology of the teaching hospital of Treichville in Abidjan for mycology REFERENCES diagnosis of a tender indurated subcutaneous 1. Ribes, J.,A., C.L. Vanover-Sams and swelling on the right thigh over the D.J. Baker (2000). Clin. Microbiol. Rev . anterolateral and medial aspect. 13(2): 236-301. Unfortunately, picture of subcutaneous was 2. Gugnani, H.C. (1999). Eur. J. not available as the patient was discharge Epidemiol. 15(10): 923-929. from the clinic at the time of diagnosis. 3. Bittencourt, A.L., S.M. Aruda, J.A. de Fungal culture and microbiological Andrade and E.M. Carvalho (1991). examination identified the fungus as B. Pediatr. Dermatol . 8(4): 325-328. haptosporus as responsible of the 4. Saka, B., K. Kombaté, A. Mouhari- subcutaneous in the patient who was an Toure, S. Akakpo, B. Tchangaï, K. immunocompetent patient with a negative Amégbor, P. Pitché and K. Tchangaï- retroviral serology. Walla (2010). Bull. Soc. Path - Exot . Few cases of suspected subcutaneous 103(5): 293-295. basidiobolomycosis have been already 5. Hussein, M.R., A.O. Musalam, M.H. observed in Côte d’Ivoire since 1963 [14, Assiry, R.A. Eid, A.M. El Motawa and 15, 16]. A.M. Gamel (2007). Mycol. Res. 111: The most commonly pharmaceutical 926-930. agents that have been used to successfully © 2016 Society for Basic and Applied Mycology . Printed in the India. J. Basic Appl. Mycol. Vol. 12 (I), pp. 50-53, 2016 53

6. Khan, Z.U., B. Prakash, M.M. Exot . 56: 112-114. Kapoor, J.P. Madda and R. Chandy 14. Kanga, J.M., D. Djeha, P. Yoboue, R. (1998). Clin. Infect. Dis . 26: 521-523. Roux, I. Dao and P. Heroin (1983). 7. Radjou, A.N. and N.G. Rajesh (2011). Ann. Université Abidjan. Med . 17: 161- Indian J. Med. Microbiol. 29(2): 186– 166. 188. 15. Sangare, A., P. Yoboue, Bamba, B. 8. Sujatha, S., C. Sheeladevi, A.B. Aka, I. Gbery, D. Djeha, S. Sanni and Khyriem, S.C. Parija and D.M. J.M. Kanga (2000). Médecine d'Afrique Thappa (2003). Indian J. Medical Noire . 47(8/9): 390-393. Microbiol. 21(3): 205–206. 16. Guido, E.L., N. Arnold, J. Ruud, L. 9. Hussein, M.R., A.O. Musalam, M.H. Jeannouel, A.B. Willem, M.P. Jan Assiry, R.A. Eid, El A.M. Motawa and (2006). BMC Infect. Dis . 6: 140. A.M. Gamel (2007). Mycol. Res . 111: 17. Khan, Z.U., M. Khoursheed, R. 926-930. Makar, S. Al-Waheeb, I. Al-Bader, Al- 10. Chandrasekhar, H.R., P. Shashikala, Muzaini and R. Chandy (2001). J. R. Haravi and R.S. Kadam (1998). Clin. Microbiol . 39(6): 2360–2363. Indian J. dermatol. Venerol. Leprol . 18. Marshall Lyon, G., J.D. Smilack, K.K. 64(2): 89-90. Komatsu, T.M. Pasha, J.A. Leighton, 11. Saka, B., K. Kombaté, A. Mouhari- J. Guarner, T.V. Colby and M.D. Toure, S. Akakpo, B. Tchangaï, K. Lindsley (2001). Clin. Infect. Dis . 32: Amégbor, P. Pitché and K. Tchangaï- 1448-1455. Walla (2010). Bull. Soc. Path-Exot . 103 19. Roy, A.K., J.N. Sorkar and P.K. Maiti (5): 293-295. (2000). India J. Dermatol . 45(1): 22-23. 12. Krishnan, S.G.S., G. Sentamilselvi, A. 20. Foss, N.T., M.R. Rocha and V.T. Lima Kamalam, K.A. Das and C. Janaki (1996). Dermatology . 193(3): 258-260. (1998). Mycoses. 41(1-2): 55–58. 21. Pitche, P., G. Napo-Koura and K. 13. Angate, Y., H. Ouedraogo, S. Diarra Tchangai- Walla (1998). Nouv. and R. Camin (1963). Bull. Soc. Path- Dermatol . 17(6): 376-377.

© 2016 Society for Basic and Applied Mycology . Printed in the India. Journal of Basic and Applied Mycology Volume 12 (I(I)) 2016

MEMBERSHIP FORM Member Information Membership No.: Name:......

Designation:...... Photograph Affiliation:...... DOB:...... Sex:...... Specialization:...... Address for correspondence:...... Pin...... Tel:...... Mob:...... Email:...... Mode of Payment (DD/cheque/cash):...... no:...... dated...... Membership Type (Student/Annual/Life):...... Fee Amount:...... I wish to become a Student/Annual/Life membership of society for basic and applied mycology. Date: Name and Signature

SUBSCRIPTION FORM Subscription No.: Name:...... Designation...... Membership (Student/Annual/Life):...... Membership No:...... Year:...... Affiliation:...... DOB:...... Sex:...... Specialization:...... Subscription Type (Annual):...... Address for correspondence:...... Pin...... Tel:...... Mob:...... Email:...... Mode of Payment (DD/cheque/cash):...... no:...... dated...... Date: Name and Signature

Membership Fee Journal Subscription Fee Student Member: Rs. 200/- ($ 50) Annual Subscription Annual Member: Rs. 300/- ($ 50) Member: Rs. 300/- ($ 50) Life Member: Rs. 1500/- ($ 100) Non Member: Rs. 500/- ($ 100) Patron Member: Rs. 5000/- ($ 300) Institutional: Rs. 1500/- ($ 300)

Admission fee for all the categories are Rs. 100.00 (US$2) Send DD in favour of Treasurer, SBAM payable at Jabalpur, Rs. 100/- for out station cheque. Send your application form along with DD to: Dr. Surendra Sarsaiya C/o Madhya Pradesh Private University Regulatory Commission, Gyan Vatika, Walmi Road, Kaliasot Dam, Bhopal, Madhya Pradesh, India Pin - 462016 www.sbamjournal.com; Email: [email protected]; Mob: +91-9826838221 Journal of Basic and Applied Mycology Volume 12 (I(I)) 2016

Society for Basic and Applied Mycology

No matter which aspect we look at fungi, they are highly diverse and versatile organisms adapted to all kind of environments. In their biochemical activities fungi show variation and plasticity of extreme types. They are of direct concern to man from many points of view. On one hand they are extreme harmful, causing serious disease in plants, in human and in animal while on the other they are highly beneficial organisms. There are no limits to the variety of useful products that fungi can produce in nature as well as in laboratories. Some of the fungi are of direct use as food such as protein rich food and mushroom which are already with big business concerns all over the world. India population being largely vegetation, fungus based protein has a great future to lesser protein hunger particularly in the weaker sections of the society.

In fact fungal exploitation for human society has already made for reaching advances in some well advanced countries. However, their useful role in developing countries can hardly be overemphasized. Even today in the era of highly defined information system, people are still unaware about the application of these novel organisms. This may be because of the luck of any origination, which makes these organisms popular among not only the scientific communities but also the common people for their betterment.

To quote Subramanian (1986) "The relevance of mycology is global as it touches every facet of man's life and progress on this planet" . There are scientific organizations dealing with plant pathology, microbiology and bacteriology, molecular biology figures prominently. A challenge for the whole community of the mycologist at present is to raise the level of recognition of the importance of fungi. These required the briefing of peers in other scientific discipline and further those formulating and implanting policies. Establishment of a good society of organization, which help rather acts as bridge between the traditional research and global demand of innovations is urgently needed.

Therefore, the need to form a Society for Basic and Applied Mycology (SBAM) was felt for long time by the active mycologist working in various regions of country.

"To create awareness and develop scientific temperament among students and young researchers, and society"

The Society for Basic and Applied Mycology (SBAM) is established in 2002, one of the oldest scientific society of the country. Since its inception, it has contributed significantly towards dissemination of research and development of mycology in the country. The society published a bi- annual journal, “Journal of Basic and Applied Mycology” since 2002 and organized a National level programs like "MICROTECH: YOUNG SCIENTIST AWARD" and " WINTER SUMMER TRAINING PROGRAMME" in the country.

The Society has introduced the following awards:

Microtech - Junior Scientist Awards ;
Late Shri. Y.P. Sharma Memorial Senior Scientist Award ;
Shayama Kamal Young Scientist Award ;
Prof. G.P. Agarwal Young Scientist Award ; and
Prof. S.M. Paul Khurana Young Scientist .

It is not an exaggeration if I say that this has been possible only by the support of mycologist like you and blessing of our senior mycologist. This is our initial effort to bring all the active workers at one platform and future growth of the society will depend upon your active and sincere corporation. I request you to encourage your students and colleagues to do so for the benefits of the subject.

With best wishes, Prof. A.K. Pandey Secretary, SBAM