African Journal of Microbiology Research Volume 9 Number 32, 12 August, 2015 ISSN 1996-0808
ABOUT AJMR
The African Journal of Microbiology Research (AJMR) (ISSN 1996-0808) is published Weekly (one volume per year) by Academic Journals.
African Journal of Microbiology Research (AJMR) provides rapid publication (weekly) of articles in all areas of Microbiology such as: Environmental Microbiology, Clinical Microbiology, Immunology, Virology, Bacteriology, Phycology, Mycology and Parasitology, Protozoology, Microbial Ecology, Probiotics and Prebiotics, Molecular Microbiology, Biotechnology, Food Microbiology, Industrial Microbiology, Cell Physiology, Environmental Biotechnology, Genetics, Enzymology, Molecular and Cellular Biology, Plant Pathology, Entomology, Biomedical Sciences, Botany and Plant Sciences, Soil and Environmental Sciences, Zoology, Endocrinology, Toxicology. The Journal welcomes the submission of manuscripts that meet the general criteria of significance and scientific excellence. Papers will be published shortly after acceptance. All articles are peer-reviewed.
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Editors
Prof. Dr. Stefan Schmidt, Dr. Thaddeus Ezeji Applied and Environmental Microbiology Assistant Professor School of Biochemistry, Genetics and Microbiology Fermentation and Biotechnology Unit University of KwaZulu-Natal Department of Animal Sciences Private Bag X01 The Ohio State University Scottsville, Pietermaritzburg 3209 1680 Madison Avenue South Africa. USA.
Prof. Fukai Bao Department of Microbiology and Immunology Associate Editors Kunming Medical University Kunming 650031, Dr. Mamadou Gueye China MIRCEN/ Laboratoire commun de microbiologie IRD-ISRA-UCAD, BP 1386, Dr. Jianfeng Wu DAKAR, Senegal. Dept. of Environmental Health Sciences, School of Public Health, Dr. Caroline Mary Knox University of Michigan Department of Biochemistry, Microbiology and USA Biotechnology Rhodes University Dr. Ahmet Yilmaz Coban Grahamstown 6140 OMU Medical School, South Africa. Department of Medical Microbiology, Samsun, Dr. Hesham Elsayed Mostafa Turkey Genetic Engineering and Biotechnology Research Institute (GEBRI) Dr. Seyed Davar Siadat Mubarak City For Scientific Research, Pasteur Institute of Iran, Research Area, New Borg El-Arab City, Pasteur Square, Pasteur Avenue, Post Code 21934, Alexandria, Egypt. Tehran, Iran. Dr. Wael Abbas El-Naggar Head of Microbiology Department, Dr. J. Stefan Rokem Faculty of Pharmacy, The Hebrew University of Jerusalem Mansoura University, Department of Microbiology and Molecular Genetics, Mansoura 35516, Egypt. P.O.B. 12272, IL-91120 Jerusalem, Israel Dr. Abdel Nasser A. El-Moghazy Microbiology, Molecular Biology, Genetics Engineering Prof. Long-Liu Lin and Biotechnology National Chiayi University Dept of Microbiology and Immunology 300 Syuefu Road, Faculty of Pharmacy Chiayi, Al-Azhar University Taiwan Nasr city, Cairo, Egypt N. John Tonukari, Ph.D Department of Biochemistry Delta State University PMB 1 Abraka, Nigeria
Editorial Board
Dr. Barakat S.M. Mahmoud Dr. Haoyu Mao Food Safety/Microbiology Department of Molecular Genetics and Microbiology Experimental Seafood Processing Laboratory College of Medicine Costal Research and Extension Center University of Florida Mississippi State University Florida, Gainesville 3411 Frederic Street USA. Pascagoula, MS 39567 USA Dr. Rachna Chandra Environmental Impact Assessment Division Prof. Mohamed Mahrous Amer Environmental Sciences Poultry Disease (Viral Diseases of poultry) Sálim Ali Center for Ornithology and Natural History Faculty of Veterinary Medicine, (SACON), Department of Poultry Diseases Anaikatty (PO), Coimbatore-641108, India Cairo university Giza, Egypt Dr. Yongxu Sun Department of Medicinal Chemistry and Dr. Xiaohui Zhou Biomacromolecules Molecular Microbiology, Industrial Microbiology, Qiqihar Medical University, Qiqihar 161006 Environmental Microbiology, Pathogenesis, Antibiotic Heilongjiang Province resistance, Microbial Ecology P.R. China Washington State University Bustad Hall 402 Department of Veterinary Dr. Ramesh Chand Kasana Microbiology and Pathology, Pullman, Institute of Himalayan Bioresource Technology USA Palampur, Distt. Kangra (HP), India Dr. R. Balaji Raja Department of Biotechnology, Dr. S. Meena Kumari School of Bioengineering, Department of Biosciences SRM University, Faculty of Science Chennai University of Mauritius India Reduit
Dr. Aly E Abo-Amer Dr. T. Ramesh Division of Microbiology, Botany Department, Faculty Assistant Professor of Science, Sohag University. Marine Microbiology Egypt. CAS in Marine Biology Faculty of Marine Sciences Annamalai University Parangipettai - 608 502 Cuddalore Dist. Tamilnadu, India
Dr. Pagano Marcela Claudia Post doctoral fellowship at Department of Biology, Federal University of Ceará - UFC, Brazil.
Dr. EL-Sayed E. Habib Dr. Mohd Fuat ABD Razak Associate Professor, Institute for Medical Research Dept. of Microbiology, Malaysia Faculty of Pharmacy, Mansoura University, Dr. Davide Pacifico Egypt. Istituto di Virologia Vegetale – CNR Italy Dr. Pongsak Rattanachaikunsopon Department of Biological Science, Prof. Dr. Akrum Hamdy Faculty of Science, Faculty of Agriculture, Minia University, Egypt Ubon Ratchathani University, Egypt Warin Chamrap, Ubon Ratchathani 34190, Thailand Dr. Ntobeko A. B. Ntusi Cardiac Clinic, Department of Medicine, Dr. Gokul Shankar Sabesan University of Cape Town and Microbiology Unit, Faculty of Medicine, Department of Cardiovascular Medicine, AIMST University University of Oxford Jalan Bedong, Semeling 08100, South Africa and Kedah, United Kingdom Malaysia Prof. N. S. Alzoreky Dr. Kwang Young Song Food Science & Nutrition Department, Department of Biological Engineering, College of Agricultural Sciences & Food, School of Biological and Chemical Engineering, King Faisal University, Yanbian Universityof Science and Technology, Saudi Arabia Yanji, China. Dr. Chen Ding College of Material Science and Engineering, Dr. Kamel Belhamel Hunan University, Faculty of Technology, China University of Bejaia Algeria Dr Svetlana Nikolić Faculty of Technology and Metallurgy, Dr. Sladjana Jevremovic University of Belgrade, Institute for Biological Research Serbia Sinisa Stankovic, Belgrade, Dr. Sivakumar Swaminathan Serbia Department of Agronomy, College of Agriculture and Life Sciences, Dr. Tamer Edirne Iowa State University, Dept. of Family Medicine, Univ. of Pamukkale Ames, Iowa 50011 Turkey USA
Dr. R. Balaji Raja M.Tech (Ph.D) Dr. Alfredo J. Anceno Assistant Professor, School of Environment, Resources and Development Department of Biotechnology, (SERD), School of Bioengineering, Asian Institute of Technology, SRM University, Thailand Chennai. India Dr. Iqbal Ahmad Aligarh Muslim University, Dr. Minglei Wang Aligrah University of Illinois at Urbana-Champaign,USA India
Dr. Josephine Nketsia-Tabiri Prof. Dong Zhichun Ghana Atomic Energy Commission Professor, Department of Animal Sciences and Ghana Veterinary Medicine, Yunnan Agriculture University, Dr. Juliane Elisa Welke China UFRGS – Universidade Federal do Rio Grande do Sul Dr. Mehdi Azami Brazil Parasitology & Mycology Dept, Baghaeei Lab., Dr. Mohammad Nazrul Islam Shams Abadi St. NIMR; IPH-Bangalore & NIUM Isfahan Bangladesh Iran
Dr. Okonko, Iheanyi Omezuruike Dr. Anderson de Souza Sant’Ana Department of Virology, University of São Paulo. Faculty of Basic Medical Sciences, Brazil. College of Medicine, University of Ibadan, Dr. Juliane Elisa Welke University College Hospital, UFRGS – Universidade Federal do Rio Grande do Sul Ibadan, Brazil Nigeria Dr. Paul Shapshak Dr. Giuliana Noratto USF Health, Texas A&M University Depts. Medicine (Div. Infect. Disease & Internat Med) USA and Psychiatry & Beh Med. USA Dr. Phanikanth Venkata Turlapati Washington State University Dr. Jorge Reinheimer USA Universidad Nacional del Litoral (Santa Fe) Argentina Dr. Khaleel I. Z. Jawasreh National Centre for Agricultural Research and Dr. Qin Liu Extension, NCARE East China University of Science Jordan and Technology China Dr. Babak Mostafazadeh, MD Shaheed Beheshty University of Medical Sciences Dr. Xiao-Qing Hu Iran State Key Lab of Food Science and Technology Jiangnan University Dr. S. Meena Kumari P. R. China Department of Biosciences Faculty of Science Prof. Branislava Kocic University of Mauritius Specaialist of Microbiology and Parasitology Reduit University of Nis, School of Medicine Institute Mauritius for Public Health Nis, Bul. Z. Djindjica 50, 18000 Nis Serbia Dr. S. Anju Department of Biotechnology, Dr. Rafel Socias SRM University, Chennai-603203 CITA de Aragón, India Spain
Dr. Mustafa Maroufpor Iran
Prof. Kamal I. Mohamed Prof. Isidro A. T. Savillo State University of New York at Oswego ISCOF USA Philippines
Dr. Adriano Cruz Dr. How-Yee Lai Faculty of Food Engineering-FEA Taylor’s University College University of Campinas (UNICAMP) Malaysia Brazil Dr. Nidheesh Dadheech Dr. Mike Agenbag (Michael Hermanus Albertus) MS. University of Baroda, Vadodara, Gujarat, India. Manager Municipal Health Services, India Joe Gqabi District Municipality South Africa Dr. Omitoyin Siyanbola Bowen University, Dr. D. V. L. Sarada Iwo Department of Biotechnology, Nigeria SRM University, Chennai-603203 India. Dr. Franco Mutinelli Istituto Zooprofilattico Sperimentale delle Venezie Dr. Samuel K Ameyaw Italy Civista Medical Center United States of America Dr. Chanpen Chanchao Department of Biology, Prof. Huaizhi Wang Faculty of Science, Institute of Hepatopancreatobiliary Chulalongkorn University Surgery of PLA Southwest Hospital, Thailand Third Military Medical University Chongqing400038 Dr. Tsuyoshi Kasama P. R. China Division of Rheumatology, Showa University Prof. Bakhiet AO Japan College of Veterinary Medicine, Sudan University of Science and Technology Dr. Kuender D. Yang, MD. Sudan Chang Gung Memorial Hospital Taiwan Dr. Saba F. Hussain Community, Orthodontics and Peadiatric Dentistry Dr. Liane Raluca Stan Department University Politehnica of Bucharest, Faculty of Dentistry Department of Organic Chemistry “C.Nenitzescu” Universiti Teknologi MARA Romania 40450 Shah Alam, Selangor Malaysia Dr. Muhamed Osman Senior Lecturer of Pathology & Consultant Prof. Dr. Zohair I.F.Rahemo Immunopathologist State Key Lab of Food Science and Technology Department of Pathology, Jiangnan University Faculty of Medicine, P. R. China Universiti Teknologi MARA, 40450 Shah Alam, Selangor Dr. Afework Kassu Malaysia University of Gondar Ethiopia Dr. Mohammad Feizabadi Tehran University of medical Sciences Iran
Prof. Ahmed H Mitwalli Dr. Adibe Maxwell Ogochukwu State Key Lab of Food Science and Technology Department of Clinical Pharmacy and Pharmacy Jiangnan University Management, P. R. China University of Nigeria, Nsukka. Dr. Mazyar Yazdani Nigeria Department of Biology, University of Oslo, Dr. William M. Shafer Blindern, Emory University School of Medicine Oslo, USA Norway Dr. Michelle Bull Dr. Ms. Jemimah Gesare Onsare CSIRO Food and Nutritional Sciences Ministry of Higher, Education Australia Science and Technology Kenya Prof. Dr. Márcio Garcia Ribeiro (DVM, PhD) School of Veterinary Medicine and Animal Science- Dr. Babak Khalili Hadad UNESP, Department of Biological Sciences, Dept. Veterinary Hygiene and Public Health, Roudehen Branch, State of Sao Paulo Islamic Azad University, Brazil Roudehen Iran Prof. Dr. Sheila Nathan National University of Malaysia (UKM) Dr. Ehsan Sari Malaysia Department of Plan Pathology, Iranian Research Institute of Plant Protection, Prof. Ebiamadon Andi Brisibe Tehran, University of Calabar, Iran. Calabar, Nigeria Dr. Snjezana Zidovec Lepej University Hospital for Infectious Diseases Dr. Julie Wang Zagreb, Burnet Institute Croatia Australia
Dr. Dilshad Ahmad Dr. Jean-Marc Chobert King Saud University INRA- BIA, FIPL Saudi Arabia France
Dr. Adriano Gomes da Cruz Dr. Zhilong Yang, PhD University of Campinas (UNICAMP) Laboratory of Viral Diseases Brazil National Institute of Allergy and Infectious Diseases, National Institutes of Health Dr. Hsin-Mei Ku Agronomy Dept. NCHU 250 Kuo Dr. Dele Raheem Kuang Rd, Taichung, University of Helsinki Taiwan Finland
Dr. Fereshteh Naderi Dr. Li Sun Physical chemist, PLA Centre for the treatment of infectious diseases, Islamic Azad University, Tangdu Hospital, Shahre Ghods Branch Fourth Military Medical University Iran China
Dr. Biljana Miljkovic-Selimovic Dr. Pradeep Parihar School of Medicine, Lovely Professional University, Phagwara, Punjab. University in Nis, India Serbia; Referent laboratory for Campylobacter and Helicobacter, Dr. William H Roldán Center for Microbiology, Department of Medical Microbiology, Institute for Public Health, Nis Faculty of Medicine, Serbia Peru
Dr. Xinan Jiao Dr. Kanzaki, L I B Yangzhou University Laboratory of Bioprospection. University of Brasilia China Brazil
Dr. Endang Sri Lestari, MD. Prof. Philippe Dorchies Department of Clinical Microbiology, Laboratory of Bioprospection. University of Brasilia Medical Faculty, Brazil Diponegoro University/Dr. Kariadi Teaching Hospital, Semarang Dr. C. Ganesh Kumar Indonesia Indian Institute of Chemical Technology, Hyderabad Dr. Hojin Shin India Pusan National University Hospital South Korea Dr. Farid Che Ghazali Universiti Sains Malaysia (USM) Dr. Yi Wang Malaysia Center for Vector Biology, 180 Jones Avenue Rutgers University, New Brunswick, NJ 08901-8536 Dr. Samira Bouhdid USA Abdelmalek Essaadi University, Tetouan, Dr. Heping Zhang Morocco The Key Laboratory of Dairy Biotechnology and Engineering, Dr. Zainab Z. Ismail Ministry of Education, Department of Environmental Engineering, University Inner Mongolia Agricultural University. of Baghdad. China Iraq
Prof. Natasha Potgieter Dr. Ary Fernandes Junior University of Venda Universidade Estadual Paulista (UNESP) South Africa Brasil
Dr. Alemzadeh Dr. Papaevangelou Vassiliki Sharif University Athens University Medical School Iran Greece
Dr. Sonia Arriaga Dr. Fangyou Yu Instituto Potosino de Investigación Científicay The first Affiliated Hospital of Wenzhou Medical Tecnológica/División de Ciencias Ambientales College Mexico China
Dr. Armando Gonzalez-Sanchez Dr. Galba Maria de Campos Takaki Universidad Autonoma Metropolitana Cuajimalpa Catholic University of Pernambuco Mexico Brazil
Dr. Kwabena Ofori-Kwakye Dr. Hans-Jürg Monstein Department of Pharmaceutics, Clinical Microbiology, Molecular Biology Laboratory, Kwame Nkrumah University of Science & Technology, University Hospital, Faculty of Health Sciences, S-581 KUMASI 85 Linköping Ghana Sweden
Prof. Dr. Liesel Brenda Gende Dr. Ajith, T. A Arthropods Laboratory, School of Natural and Exact Associate Professor Biochemistry, Amala Institute of Sciences, National University of Mar del Plata Medical Sciences, Amala Nagar, Thrissur, Kerala-680 Buenos Aires, 555 Argentina. India
Dr. Adeshina Gbonjubola Dr. Feng-Chia Hsieh Ahmadu Bello University, Biopesticides Division, Taiwan Agricultural Chemicals Zaria. and Toxic Substances Research Institute, Council of Nigeria Agriculture Taiwan Prof. Dr. Stylianos Chatzipanagiotou University of Athens – Medical School Prof. Dra. Suzan Pantaroto de Vasconcellos Greec Universidade Federal de São Paulo Rua Prof. Artur Riedel, 275 Jd. Eldorado, Diadema, SP Dr. Dongqing BAI CEP 09972-270 Department of Fishery Science, Brasil Tianjin Agricultural College, Tianjin 300384 Dr. Maria Leonor Ribeiro Casimiro Lopes Assad P. R. China Universidade Federal de São Carlos - Centro de Ciências Agrárias - CCA/UFSCar Dr. Dingqiang Lu Departamento de Recursos Naturais e Proteção Nanjing University of Technology Ambiental P.R. China Rodovia Anhanguera, km 174 - SP-330 Araras - São Paulo Dr. L. B. Sukla Brasil Scientist –G & Head, Biominerals Department, IMMT, Bhubaneswar Dr. Pierangeli G. Vital India Institute of Biology, College of Science, University of the Philippines Dr. Hakan Parlakpinar Philippines MD. Inonu University, Medical Faculty, Department of Pharmacology, Malatya Prof. Roland Ndip Turkey University of Fort Hare, Alice South Africa Dr Pak-Lam Yu Massey University Dr. Shawn Carraher New Zealand University of Fort Hare, Alice South Africa Dr Percy Chimwamurombe University of Namibia Dr. José Eduardo Marques Pessanha Namibia Observatório de Saúde Urbana de Belo Horizonte/Faculdade de Medicina da Universidade Dr. Euclésio Simionatto Federal de Minas Gerais State University of Mato Grosso do Sul-UEMS Brasil Brazil
Dr. Yuanshu Qian Dr. N Saleem Basha Department of Pharmacology, Shantou University M. Pharm (Pharmaceutical Biotechnology) Medical College Eritrea (North East Africa) China Prof. Dr. João Lúcio de Azevedo Dr. Helen Treichel Dept. Genetics-University of São Paulo-Faculty of URI-Campus de Erechim Agriculture- Piracicaba, 13400-970 Brazil Brasil
Dr. Xiao-Qing Hu Dr. Julia Inés Fariña State Key Lab of Food Science and Technology PROIMI-CONICET Jiangnan University Argentina P. R. China Dr. Yutaka Ito Dr. Olli H. Tuovinen Kyoto University Ohio State University, Columbus, Ohio Japan USA Dr. Cheruiyot K. Ronald Prof. Stoyan Groudev Biomedical Laboratory Technologist University of Mining and Geology “Saint Ivan Rilski” Kenya Sofia Bulgaria Prof. Dr. Ata Akcil S. D. University Dr. G. Thirumurugan Turkey Research lab, GIET School of Pharmacy, NH-5, Chaitanya nagar, Rajahmundry-533294. Dr. Adhar Manna India The University of South Dakota USA Dr. Charu Gomber Thapar University Dr. Cícero Flávio Soares Aragão India Federal University of Rio Grande do Norte Brazil Dr. Jan Kuever Bremen Institute for Materials Testing, Dr. Gunnar Dahlen Department of Microbiology, Institute of odontology, Sahlgrenska Academy at Paul-Feller-Str. 1, 28199 Bremen University of Gothenburg Germany Sweden
Dr. Nicola S. Flanagan Dr. Pankaj Kumar Mishra Universidad Javeriana, Cali Vivekananda Institute of Hill Agriculture, (I.C.A.R.), Colombia ALMORA-263601, Uttarakhand India Dr. André Luiz C. M. de A. Santiago Universidade Federal Rural de Pernambuco Dr. Benjamas W. Thanomsub Brazil Srinakharinwirot University Thailand Dr. Dhruva Kumar Jha Microbial Ecology Laboratory, Dr. Maria José Borrego Department of Botany, National Institute of Health – Department of Infectious Gauhati University, Diseases Guwahati 781 014, Assam Portugal India
Dr. Catherine Carrillo Dr. Tang Ming Health Canada, Bureau of Microbial Hazards College of Forestry, Northwest A&F University, Canada Yangling China Dr. Marcotty Tanguy Institute of Tropical Medicine Dr. Olga Gortzi Belgium Department of Food Technology, T.E.I. of Larissa Greece Dr. Han-Bo Zhang Laboratory of Conservation and Utilization for Bio- Dr. Mark Tarnopolsky resources Mcmaster University Key Laboratory for Microbial Resources of the Canada Ministry of Education, Yunnan University, Kunming 650091. Dr. Sami A. Zabin School of Life Science, Al Baha University Yunnan University, Kunming, Saudi Arabia Yunnan Province 650091. China Dr. Julia W. Pridgeon Aquatic Animal Health Research Unit, USDA, ARS Dr. Ali Mohammed Somily USA King Saud University Saudi Arabia Dr. Lim Yau Yan Monash University Sunway Campus Dr. Nicole Wolter Malaysia National Institute for Communicable Diseases and University of the Witwatersrand, Prof. Rosemeire C. L. R. Pietro Johannesburg Faculdade de Ciências Farmacêuticas de Araraquara, South Africa Univ Estadual Paulista, UNESP Brazil Dr. Marco Antonio Nogueira Universidade Estadual de Londrina Dr. Nazime Mercan Dogan CCB/Depto. De microbiologia PAU Faculty of Arts and Science, Denizli Laboratório de Microbiologia Ambiental Turkey Caixa Postal 6001 86051-980 Londrina. Dr Ian Edwin Cock Brazil Biomolecular and Physical Sciences Griffith University Dr. Bruno Pavoni Australia Department of Environmental Sciences University of Venice Prof. N K Dubey Italy Banaras Hindu University India Dr. Shih-Chieh Lee Da-Yeh University Dr. S. Hemalatha Taiwan Department of Pharmaceutics, Institute of Technology, Dr. Satoru Shimizu Banaras Hindu University, Varanasi. 221005 Horonobe Research Institute for the Subsurface India Environment, Northern Advancement Center for Science & Dr. J. Santos Garcia A. Technology Universidad A. de Nuevo Leon Japan Mexico India
Dr. Somboon Tanasupawat Dr. Mick Bosilevac Department of Biochemistry and Microbiology, US Meat Animal Research Center Faculty of Pharmaceutical Sciences, USA Chulalongkorn University, Bangkok 10330 Dr. Nora Lía Padola Thailand Imunoquímica y Biotecnología- Fac Cs Vet-UNCPBA Argentina Dr. Vivekananda Mandal Post Graduate Department of Botany, Dr. Maria Madalena Vieira-Pinto Darjeeling Government College, Universidade de Trás-os-Montes e Alto Douro Darjeeling – 734101. Portugal India Dr. Stefano Morandi Dr. Shihua Wang CNR-Istituto di Scienze delle Produzioni Alimentari College of Life Sciences, (ISPA), Sez. Milano Fujian Agriculture and Forestry University Italy China Dr Line Thorsen Dr. Victor Manuel Fernandes Galhano Copenhagen University, Faculty of Life Sciences CITAB-Centre for Research and Technology of Agro- Denmark Environment and Biological Sciences, Integrative Biology and Quality Research Group, Dr. Ana Lucia Falavigna-Guilherme University of Trás-os-Montes and Alto Douro, Universidade Estadual de Maringá Apartado 1013, 5001-801 Vila Real Brazil Portugal Dr. Baoqiang Liao Dr. Maria Cristina Maldonado Dept. of Chem. Eng., Lakehead University, 955 Oliver Instituto de Biotecnologia. Universidad Nacional de Road, Thunder Bay, Ontario Tucuman Canada Argentina Dr. Ouyang Jinping Dr. Alex Soltermann Patho-Physiology department, Institute for Surgical Pathology, Faculty of Medicine of Wuhan University University Hospital Zürich China Switzerland Dr. John Sorensen Dr. Dagmara Sirova University of Manitoba Department of Ecosystem Biology, Faculty Of Science, Canada University of South Bohemia, Branisovska 37, Ceske Budejovice, 37001 Dr. Andrew Williams Czech Republic University of Oxford United Kingdom Dr. E. O Igbinosa Department of Microbiology, Dr. Chi-Chiang Yang Ambrose Alli University, Chung Shan Medical University Ekpoma, Edo State, Taiwan, R.O.C. Nigeria. Dr. Quanming Zou Dr. Hodaka Suzuki Department of Clinical Microbiology and Immunology, National Institute of Health Sciences College of Medical Laboratory, Japan Third Military Medical University China
Prof. Ashok Kumar Dr. Guanghua Wang School of Biotechnology, Northeast Institute of Geography and Agroecology, Banaras Hindu University, Varanasi Chinese Academy of Sciences India China
Dr. Chung-Ming Chen Dr. Renata Vadkertiova Department of Pediatrics, Taipei Medical University Institute of Chemistry, Slovak Academy of Science Hospital, Taipei Slovakia Taiwan Dr. Charles Hocart Dr. Jennifer Furin The Australian National University Harvard Medical School Australia USA Dr. Guoqiang Zhu Dr. Julia W. Pridgeon University of Yangzhou College of Veterinary Medicine Aquatic Animal Health Research Unit, USDA, ARS China USA Dr. Guilherme Augusto Marietto Gonçalves Dr Alireza Seidavi São Paulo State University Islamic Azad University, Rasht Branch Brazil Iran Dr. Mohammad Ali Faramarzi Dr. Thore Rohwerder Tehran University of Medical Sciences Helmholtz Centre for Environmental Research UFZ Iran Germany Dr. Suppasil Maneerat Dr. Daniela Billi Department of Industrial Biotechnology, Faculty of University of Rome Tor Vergat Agro-Industry, Prince of Songkla University, Hat Yai Italy 90112 Thailand Dr. Ivana Karabegovic Faculty of Technology, Leskovac, University of Nis Dr. Francisco Javier Las heras Vazquez Serbia Almeria University Spain Dr. Flaviana Andrade Faria IBILCE/UNESP Dr. Cheng-Hsun Chiu Brazil Chang Gung memorial Hospital, Chang Gung University Prof. Margareth Linde Athayde Taiwan Federal University of Santa Maria Brazil Dr. Ajay Singh DDU Gorakhpur University, Gorakhpur-273009 (U.P.) Dr. Guadalupe Virginia Nevarez Moorillon India Universidad Autonoma de Chihuahua Mexico Dr. Karabo Shale Central University of Technology, Free State Dr. Tatiana de Sousa Fiuza South Africa Federal University of Goias Brazil Dr. Lourdes Zélia Zanoni Department of Pediatrics, School of Medicine, Federal Dr. Indrani B. Das Sarma University of Mato Grosso do Sul, Campo Grande, Jhulelal Institute of Technology, Nagpur Mato Grosso do Sul India Brazil
Dr. Tulin Askun Dr. Hongxiong Guo Balikesir University STD and HIV/AIDS Control and Prevention, Turkey Jiangsu provincial CDC, China Dr. Marija Stankovic Institute of Molecular Genetics and Genetic Dr. Konstantina Tsaousi Engineering Life and Health Sciences, Republic of Serbia School of Biomedical Sciences, University of Ulster Dr. Scott Weese University of Guelph Dr. Bhavnaben Gowan Gordhan Dept of Pathobiology, Ontario Veterinary College, DST/NRF Centre of Excellence for Biomedical TB University of Guelph, Research Guelph, Ontario, N1G2W1, University of the Witwatersrand and National Health Canada Laboratory Service P.O. Box 1038, Johannesburg 2000, Dr. Sabiha Essack South Africa School of Health Sciences South African Committee of Health Sciences Dr. Ernest Kuchar University of KwaZulu-Natal Pediatric Infectious Diseases, Private Bag X54001 Wroclaw Medical University, Durban 4000 Wroclaw Teaching Hospital, South Africa Poland
Dr. Hare Krishna Dr. Hongxiong Guo Central Institute for Arid Horticulture, STD and HIV/AIDS Control and Prevention, Beechwal, Bikaner-334 006, Rajasthan, Jiangsu provincial CDC, India China
Dr. Anna Mensuali Dr. Mar Rodriguez Jovita Dept. of Life Science, Food Hygiene and Safety, Faculty of Veterinary Scuola Superiore Science. Sant’Anna University of Extremadura, Spain Dr. Ghada Sameh Hafez Hassan Pharmaceutical Chemistry Department, Dr. Jes Gitz Holler Faculty of Pharmacy, Mansoura University, Hospital Pharmacy, Egypt Aalesund. Central Norway Pharmaceutical Trust Professor Brochs gt. 6. 7030 Trondheim, Dr. Kátia Flávia Fernandes Norway Biochemistry and Molecular Biology Universidade Federal de Goiás Prof. Chengxiang FANG Brasil College of Life Sciences, Wuhan University Dr. Abdel-Hady El-Gilany Wuhan 430072, P.R.China Public Health & Community Medicine Faculty of Medicine, Dr. Anchalee Tungtrongchitr Mansoura University Siriraj Dust Mite Center for Services and Research Egypt Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University 2 Prannok Road, Bangkok Noi, Bangkok, 10700, Thailand
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Electronic submission of manuscripts is strongly Regular articles encouraged, provided that the text, tables, and figures are included in a single Microsoft Word file (preferably in Arial All portions of the manuscript must be typed double- font). spaced and all pages numbered starting from the title page. The cover letter should include the corresponding author's full address and telephone/fax numbers and should be in The Title should be a brief phrase describing the contents an e-mail message sent to the Editor, with the file, whose of the paper. The Title Page should include the authors' name should begin with the first author's surname, as an full names and affiliations, the name of the corresponding attachment. author along with phone, fax and E-mail information. Present addresses of authors should appear as a footnote. Article Types Three types of manuscripts may be submitted: The Abstract should be informative and completely self- explanatory, briefly present the topic, state the scope of Regular articles: These should describe new and carefully the experiments, indicate significant data, and point out confirmed findings, and experimental procedures should major findings and conclusions. The Abstract should be be given in sufficient detail for others to verify the work. 100 to 200 words in length.. Complete sentences, active The length of a full paper should be the minimum required verbs, and the third person should be used, and the to describe and interpret the work clearly. abstract should be written in the past tense. Standard Short Communications: A Short Communication is suitable nomenclature should be used and abbreviations should for recording the results of complete small investigations be avoided. No literature should be cited. or giving details of new models or hypotheses, innovative Following the abstract, about 3 to 10 key words that will methods, techniques or apparatus. The style of main provide indexing references should be listed. sections need not conform to that of full-length papers. Short communications are 2 to 4 printed pages (about 6 to A list of non-standard Abbreviations should be added. In 12 manuscript pages) in length. general, non-standard abbreviations should be used only when the full term is very long and used often. Each Reviews: Submissions of reviews and perspectives covering abbreviation should be spelled out and introduced in topics of current interest are welcome and encouraged. parentheses the first time it is used in the text. Only Reviews should be concise and no longer than 4-6 printed recommended SI units should be used. Authors should pages (about 12 to 18 manuscript pages). Reviews are also use the solidus presentation (mg/ml). Standard peer-reviewed. abbreviations (such as ATP and DNA) need not be defined.
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African Journal of Microbiology Research International Journal of Medicine and Medical Sciences
Table of Content: Volume 9 Number 32, 12 August, 2015
ARTICLES
Isolation and characterization of Lactobacillus plantarum BLS29 as a potential probiotic starter culture for pork sausage production Luciana Senter, Jamile Queiroz Pereira, Manoela Alano Vieira, Eliane Maria Zandonai Michielin and Eduardo Cesar Tondo
Response of single and co-inoculation of plant growth promoting rhizobacteria on growth, flowering and nutrient content of chrysanthemum Anop Kumari, R. K. Goyal, Mahesh Choudhary and S. S. Sindhu
Isolation and genetic characterization of endophytic and rhizospheric microorganisms from Butia purpurascens Glassman Cintia Faria da Silva, Jaqueline Alves Senabio, Liliane Cira Pinheiro, Marcos Antonio Soares and Edson Luiz Souchie
Isolation and identification of Sparicotyle chrysophrii (Monogenea: Microcotylidae) from gilthead sea bream (Sparus aurata L.) in the Mediterranean Sea, Greece Jin Woo Jun
Vol. 9(32), pp. 1887-1895, 12 August, 2015 DOI: 10.5897/AJMR2015.7606 Article Number: D8FE13D55003 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR
Full Length Research Paper
Isolation and characterization of Lactobacillus plantarum BLS29 as a potential probiotic starter culture for pork sausage production
Luciana Senter1,4,, Jamile Queiroz Pereira2, Manoela Alano Vieira1, Eliane Maria Zandonai Michielin1 and Eduardo Cesar Tondo3,4
1Laboratory of Food Microbiology, Federal Institute of Education, Science and Technology of Santa Catarina, Campus Xanxerê, 1603, Euclid Hack Street, Venice Neighborhood, Xanxerê, SC, Brazil. 2Laboratory of Applied Biochemistry and Microbiology, Department of Food Science and Technology, ICTA, Federal University of Rio Grande do Sul - UFRGS, 9500, Bento Gonçalves Avenue, building 43212, Campus do Vale, Agronomy, Porto Alegre, RS, Brazil. 3Laboratory of Food Microbiology and Food Control, Institute of Food Science and Technology, Federal University of Rio Grande do Sul - ICTA /UFRGS, 9500, Bento Gonçalves Avenue, building 43212, Campus do Vale, Agronomy, Porto Alegre, RS, Brazil. 4Pos-Graduation Program in Agricultural and Environmental Microbiology (PPGMAA/UFRGS). Federal University of Rio Grande do Sul, 500, Sarmento Leite street, center. Porto Alegre, RS, Brazil.
Received 2 June, 2015; Accepted 3 August, 2015
The aim of this study was to isolate and characterize lactic acid bacteria (LAB) from naturally fermented pork sausages and test them for use as a probiotic starter culture in the production of fermented sausages. LAB (n=26) were isolated from natural fermented pork sausages. Isolates were identified using 16S rDNA or Internal Transcribed Spacer (ITS) region sequencing. After that, strains were characterized based on technological, functional and safety proprieties. A LAB strain was chosen and its survival was tested in simulated gastric juice and intestinal juice. Results indicate that Lactobacillus plantarum (n=15) was the predominant species in fermented sausage, followed by Enterococcus faecium (n=8), Lactobacillus brevis (n=1), Enterococcus durans (n=1) and Enterococcus hirae (n=1). L. plantarum BLS29 strain was selected because it was not able to produce CO2 and was able to multiply at temperatures ranging from 15 to 37°C, usually practiced during the sausage cure process. BLS29 showed better growth capacity than other isolated LABs when exposed to curing salts, and also demonstrated antimicrobial activity against foodborne pathogens. According to our findings, BLS29 can be a promising strain to be used as a probiotic starter culture for the production of fermented pork sausage.
Key words: Probiotics, Lactobacillus plantarum, fermented pork sausage.
INTRODUCTION
The demand for new food products has greatly influenced though these products have been considered unhealthy the development of new fermented meat products, even because of the high saturated fat, additives and spice 1888 Afr. J. Microbiol. Res.
contents (Arihara, 2006; De Vuyst et al., 2008; Macedo et ensure the safety of the product is of great interest. al., 2012). According to Fiorentini et al. (2010) and Generally, probiotic bacteria of the genus Lactobacillus Raigorodsky (2011), the evolution of fermented sausages are capable to produce bacteriocins, inhibiting the growth is relatively old, having emerged from the need of using of Gram-positive bacteria like Staphylococcus aureus and all body parts of animals as food. The production of Listeria monocytogenes (Incze, 1998; Guetouache and fermented sausages was introduced in Brazil by Italian Guessas, 2015). Some species of LAB are used as immigrants and, more recently, gave rise to small starter cultures, contributing to the sensory quality and factories, transforming the economy, especially in with the microbiological safety of industrial fermented Southern Brazil. Salamis and sausages are raw products (Leroy et al., 2006). One way to obtain both embedded, cured, fermented, matured and dry, and may characteristics is to use the new generation of ‘functional or may not be smoked (Terra et al., 2004; Ordoñez starter cultures' (De Vuyst, 2000). Wild strains are Pereda, 2005). In Brazil, these products are classified naturally dominant in natural fermentation and show a into eight different classes, according to the composition high capacity for metabolic formation of flavor. Natural of meat and fat, type of raw material used and seasoning selected strains are interesting because frequently they (Brazil, 2000). Among these classes are the salamis that produce antimicrobial compounds, enhancing the are fermented meat products with moisture content below competitiveness against pathogens (Ayad et al., 2000; 40%. Maldonado et al., 2002). Functional foods are defined as: ‘foods that contain In recent years, the study of functional cultures has some health-promoting component(s) beyond traditional aroused great attention (Erkkilä et al., 2001; Arihara, nutrients’. One way in which foods can be modified to 2006; De Vuyst et al., 2008), either using the addition of become functional is by the addition of probiotics (Soccol free probiotics (Pennachia et al., 2006; Leroy et al., 2006) et al., 2010). Probiotics are defined as live micro- or using encapsulated forms (Muthukurasami and Holley, organisms which, when consumed in adequate amounts 2006). The market for probiotics has been very as part of the diet, may play an important role in promising, and are rare products containing meat respiratory, immune, and gastrointestinal functions and probiotics (De Vuyst, 2000; Pennacchia et al., 2004; have a significant effect on the clearance of infectious Pennacchia et al., 2006; Leroy et al., 2006; De Vuyst et diseases in children and lactose intolerance (FAO/WHO, al., 2008). This market has been dominated mainly by 2001). dairy products, but these cannot be consumed by people Foods containing probiotic are considered functional with lactose intolerance. In this sense, meat products foods and present great commercial interest because its containing probiotics may have competitive advantage to consumption has increased expressively. Although dairy products (Erkkila et al., 2001; Arihara, 2006; Metchnikoff (1907) has described the benefits of Macedo et al., 2012). Most of the probiotic bacteria have microorganisms to consumers’ health, it was only in the been isolated from human or animals intestinal tracts, last 30 years that the study of probiotics has been milk or vegetables, however the use of bacteria isolated intensified, especially in the selection and characteriza- from meat may contribute to a better sensory quality of tion of probiotic species (FAO/WHO, 2001). Numerous the meat fermented product produced (De Almeida Junior studies claim that probiotic species contribute to the et al., 2015). Considering the need to find new strains reduction of cholesterol levels in the blood, prevention with probiotic characteristics that can be used as and treatment of food-related allergies, assist in the functional starter cultures, while preserving the treatment of infections caused by Helicobacter pylori, technological and sensory characteristics of the prevention of cancer and modulation of intestinal flora sausages, the present study aimed to isolate LAB from (Arihara, 2006; El-Ghaish et al., 2011; Yoon et al., 2013). fermented pork sausages and to characterize potential Probiotics have been extensively used in dairy product probiotic starter cultures for the use in sausage production, but few studies have demonstrated its usage production. in fermented meat products (Erkkilä et al., 2001; Arihara, 2006; De Vuyst et al., 2008). In 1998, Germany was the first country to produce salami containing three probiotic MATERIALS AND METHODS strains of intestinal lactic acid bacteria (Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium Isolation of LABs spp.). This study highlights the promise usage and Microorganisms were isolated from fermented pork sausages development of new probiotic meat products (Hammes produced without the addition of starter cultures from three different and Hertel, 1998). The development of sausage con- batches, produced in a commercial manufacturing plant in the State taining probiotics able to provide health benefits and that of Santa Catarina, Southern Brazil. Samples of 25 g of pork
*Corresponding author. E-mail: [email protected]. Tel: +55-49-3441-7900.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Senter et al. 1889
sausage were diluted in 225 ml of 0.1% peptone water and were Growth at different temperatures homogenized inside sterile plastic bags with a stomacher (Easy Mix, AES Chemunex, Rennes, France). One aliquot of 1000 µl of The capacity of one probiotic growing at different temperatures is a each dilution was inoculated on MRS agar (Man Rogosa and very important property for the production of sausages. The Sharpe, Acumedia, Baltimore EUA), using pour-plate method, and temperatures of growing were investigated using MRS broth plates were incubated at 37°C for 48 h using anaerobic jars incubated for 72 h at 15, 37 and 45°C. The growth at 4°C for seven (Permution, Curitiba, Brazil). After incubation, different colonies days was tested as well (Dalla Santa, 2008) and checked with were randomly selected and checked for its purity, being analyzed MacFarland scale. after Gram staining. All selected isolates were stored at -20°C in 30% (v / v) glycerol and propagated into tubes containing 5 ml of MRS broth, before next experiments. Determination of antagonistic activity
The antagonistic activity of each LAB isolate was determined by the Molecular Identification of Isolates presence of a growth inhibition zone of a specific foodborne indicator strain around the spot of LAB. For assessment of Genomic DNA of isolates was extracted and the 16S rDNA genes antagonistic activity, 5 µl of overnight LAB cultures were spotted on were amplified by PCR using the following universal primers: 27F MRS agar plates and incubated at 37°C, for 24 h, in microaerophilic (5’-AGAGTTTGATCMTGGCTCAG-3') and 1525R (5’- conditions. After this period, the plates were treated with AAGGAGGTGWTCCARCC-3') (Lane, 1991). For those samples chloroform, during 30 min, and residual chloroform was evaporated, whose identification by 16S rDNA was not possible or inaccurate, with the lids open for another 30 min. Then, 108 colony forming units the 16S-23S rDNA intergenic spacer region (ITS) was amplified (CFU)/ml of the pathogen in BHI soft-agar (0.75% w/v) (Himedia, using bacterium specific universal primers 13BF (5'- Mumbai, India) were inoculated using the overlay method. LAB GTGAATACGTTCCCGGGCCT-3') and 6R (5'- isolates were tested against Salmonella enterica serovar Enteritidis GGGTTYCCCCRTTCRGAAAT-3') (where Y=C or T, R=A or G) SE86 (one of most important foodborne pathogen in the State of (Gurtler and Stanisich, 1996). The sequencing reactions were Rio Grande do Sul, Southern Brazil) (Tondo and Ritter, 2012), carried out using the same primers previously used in both PCR Salmonella Typhimurium (ATCC 14078), Staphylococcus aureus reactions (Ludwig Biotec, Porto Alegre, Brazil). The 16S and ITS (ATCC 14458), Escherichia coli O157:H7 (ATCC 43888), Bacillus sequences obtained were submitted to the BLAST search program cereus (ATCC 33010), and Listeria monocytogenes (ATCC 6477). of the National Center for Biotechnology Information website (NCBI, After incubation under anaerobic conditions, the diameters of the http://www.ncbi.nlm.nih.gov) in order to search for homologous inhibition zones were determined with a caliper (Martins et al., sequences. BioEdit (Hall, 1999) was used to edit sequences and for 2010). the construction of 16S and ITS contigs.
Tolerance to simulated gastric juice and intestinal juice CO2 and exopolysaccharides production The BLS29 strain was tested for its tolerance to the upper The production of carbon dioxide gas (CO2) was determined using gastrointestinal tract by using simulated gastric and intestinal juices, MRS broth inside glass tubes with inverted Durham tubes. according to Meira et al. (2012) with modifications. The simulated Ammonium citrate was replaced by ammonium sulfate in order to gastric juice solution was daily prepared with 3 mg/ml of pepsin induce glucose use for fermentation. The tubes were incubated (Sigma Aldrich, St. Louis, USA), 0.5% NaCl and acidified with HCl during 48 h at 37°C and gas production was verified from the until pH 4.0, 3.0, 2.5, and 2.0. The simulated intestinal juice glucose fermentation. Exopolysaccharides production was consisted of 1 mg/ml of pancreatin (Sigma Aldrich, St. Louis, USA), determined checking the growth of the strains on MRS agar, 0.5% NaCl, with or without 5 g/l of porcine bile, and pH 8.0 (Sigma substituting glucose by 5% of sucrose. The plates were incubated Aldrich, St. Louis, USA). The solutions were sterilized using 0.22 for 7 days at 25°C and checked for exopolysaccharides production µm membrane filter. After two propagations, L. plantarum BLS29 by negative staining (Hugas et al., 1993). cultures incubated 24 h in MRS broth were concentrated by centrifugation (12,000 g for 5 min), washed twice in phosphate buffer pH 7.0 and suspended in 0.5% NaCl solution. Two hundred Acidification capacity microliters (200 μl) of the cell suspension were incubated at 37°C in a bath (De Leo, Porto Alegre, Brazil) in the presence of 1.0 ml of The acidification capability of isolates was determined using tubes simulated gastric juice. Viable cell counts were carried out at initial containing 10 ml of MRS broth (pH 6.6) without KH2PO4, but time (0h), 3 and 4 h of exposure to simulated gastric juice, in order containing ammonium citrate, 0.3% of sodium citrate (Dinamica, to simulate intestinal transit tolerance. The BLS29 cells that São Paulo, Brazil) (Hugas et al., 1993). The pH level was evaluated survived to pH 2.0 (acid-exposed) were inoculated in simulated after 48 h of incubation at 37°C (Tec-5 electrode Mettler Toledo, intestinal juice as well as a control consisting in a suspension of Tecnal, Piracicaba, Brazil). The mean value of three pH non-acid-exposed BLS29 cells. measurements was calculated.
Hemolysis Tolerance to sodium chloride and sodium nitrite Lactobacilli strains were cultured on blood agar plates and Isolates were checked according to their tolerance for 5 and 8% incubated for 48 hours at 30 ºC (Maragkoudakis et al., 2006; Meira NaCl (Vetec, Duque de Caxias, Brazil), to the tolerance of 3% NaCl et al., 2012). Strains that did not change the medium (γ-hemolysis) associated with NaNO2 (Vetec, Duque de Caxias, Brazil) and of 200 or that produced a clear green halo around the colony (α- ppm for check the ability of growing in MRS broth after 72 h, at hemolysis) were classified as non-hemolytic, while strains that 37°C incubation (Hugas et al., 1993). Growth was determined by showed a lytic zone around the colony were classified as hemolytic the McFarland scale. (β-hemolysis). 1890 Afr. J. Microbiol. Res.
Table 1. 16S rDNA gene and 16S-23S ITS sequence identification of lactic acid bacteria isolated from starter-free pork sausage produced in Southern Brazil.
Corresponding GenBank Isolate Sequence identity Sequence length % similarity strain accession number BLS02 Lactobacillus plantarum2 438 100 AB083123 BLS03 Lactobacillus plantarum2 441 100 AB362387 BLS04 Lactobacillus plantarum1 720 100 KF472174 BLS05 Enterococcus durans1 847 100 KF317897 BLS06 Enterococcus hirae1 1148 100 AB841310 BLS07 Enterococcus faecium1 658 100 KF012874 BLS08 Enterococcus faecium1 1145 100 JN792505 BLS09 Lactobacillus plantarum1 1161 100 KF472174 BLS10 Lactobacillus plantarum2 471 100 AB362387 BLS11 Enterococcus faecium1 1148 100 EU717954 BLS12 Enterococcus faecium1 858 99 HQ293078 BLS13 Enterococcus faecium1 1166 100 KF318400 BLS14 Enterococcus faecium1 1162 100 KF149621 BLS15 Enterococcus faecium1 988 99 KF318400 BLS16 Enterococcus faecium1 1149 99 KF149621 BLS17 Lactobacillus plantarum2 467 100 AB102857 BLS19 Lactobacillus plantarum2 481 100 AB083123 BLS20 Lactobacillus plantarum2 464 100 AB102857 BLS21 Lactobacillus plantarum1 1153 100 KF472174 BLS23 Lactobacillus plantarum2 760 100 JN587506 BLS26 Lactobacillus plantarum2 817 100 HE646365 BLS27 Lactobacillus brevis1 1126 100 KF307784 BLS29 Lactobacillus plantarum1 1363 100 KF472174 BLS30 Lactobacillus plantarum2 466 100 AB102857 BLS31 Lactobacillus plantarum2 481 100 AB083119 BLS32 Lactobacillus plantarum2 468 100 AB102857
1Identification based on the 16S rDNA sequence data. 2 Identification based on the ITS sequence data.
RESULTS and only BLS27 L. brevis producing CO2.
Isolation and characterization of microorganisms Acidification capacity, salt tolerance and growth at Twenty-six (26) isolates of LAB were isolated from pork different temperatures sausage starter-free manufactured in a meat processing unit in the western part of the State of Santa Catarina, All the isolated strains were tested for their ability of Southern Brazil. On the basis of 16S rRNA gene and ITS acidification, tolerance to curing salts and capacity of region sequencing data, isolates were identified as growing at different temperatures (Table 2). This was belonging to the genus Lactobacillus and Enterococcus done in order to select a fermentative strain with the (Table 1). L. plantarum was the most frequently isolated potential to be used in the preparation of sausage with microorganisms. The accession numbers were identified functional culture. and corresponded to the sequences of Genbank, where Among the L. plantarum strains, BLS29 was chosen the highest identified scores after submitting the and tested for its probiotic potential, based on the high sequence query to the BLAST tool were obtained. acidification of MRS broth; BLS29 grow well in the presence of curing salts and grow at temperature of 15°C, what is usually practiced during sausages ripening. Exopolysaccharide, CO2 and hemolysis production
All strains produced exopolysaccharides on MRS plates Antimicrobial activity supplemented with 5% of sucrose. None of the Lactobacillus strains were positive for hemolytic reaction All lactobacilli isolated from pork sausages were tested Senter et al. 1891
Table 2. Ability of acidification, tolerance to curing salts and growth at different temperatures of lactic bacteria isolated from fermented pork sausage produced in Southern Brazil.
Salts tolerance Growth at different temperatures Strain NaCl 3% + 5% 8% pH 4ºC 15ºC 37ºC 45ºC NaNO2 200 ppm NaCl NaCl BLS02 + + + - + + - 3.89±0.03a BLS03 + + + - + + - 3.87±0.00a BLS04 + ++ + - + + - 3.92±0.02a BLS05 + + - - + + - 4.54±0.05b BLS06 + + - - + + - 4.67±0.01b BLS07 + ++ - - + + + 4.56±0.00b BLS08 + ++ - - + + + 4.53±0.01b BLS09 + ++ + - + + - 3.90±0.00a BLS10 + ++ + - + + - 3.96±0.00a BLS11 + ++ - - + + + 4.58±0.00b BLS12 + + - - + + + 4.58±0.00b BLS13 + + - - + + + 4.58±0.00b BLS14 + + - - + + + 4.59±0.01b BLS15 + ++ - - + + + 4.59±0.01b BLS16 + ++ - - + + + 4.61±0.01b BLS17 + ++ + - + + - 4.00±0.01c BLS19 ++ + ++ - + + - 4.00±0.01c BLS20 + ++ + - + + - 3.90±0.01a BLS21 + ++ + - + + - 3.83±0.06a BLS23 + + + - + + - 3.81±0.00a BLS26 + + + - + + - 3.90±0.01a BLS27 + + + - + + - 4.61±0.01b BLS29 ++ ++ ++ - + + - 3.89±0.01a BLS30 + ++ + - + + - 3.91±0.07a BLS31 + + + - + + - 3.90±0.01a BLS32 + + + - + + - 3.82±0.01a
-, No growth; +, Growth; ++ Strong growth. a,b,c Different letters indicate significant difference (P<0,05) between pH values by Tukey test.
for inhibitory potential against Salmonella enterica resistance to simulated gastric juice and intestinal juices serovar Enteritidis SE86, Salmonella Typhimurium (ATCC (Tables 4 and 5). 14078), S. aureus (ATCC 14458), E. coli O157:H7 (ATCC BLS29 was incubated at 37°C, and then subjected to 43888), B. cereus (ATCC 33010) and L. monocytogenes simulate gastric conditions for up to four hours. An initial (ATCC 6477) (Table 3), and demonstrated to be able to reduction of 3 log was observed, however about 5 log of inhibit all pathogens. BLS29 remained viable. Both preparations of L. plantarum BL29 exposed to the gastric juice at pH ranging from 4.0 to 2 as the control non-acid-exposed Tolerance to simulated gastric juice and intestinal were able to survive and could also tolerate the exposure juice to the bile salts for four hours, demonstrating tolerance to the passage through the intestinal tract, emphasizing that The BLS29 strain showed good potential to be used as a there were no significant difference in the viable cells probiotic for the production of pork sausages, because counts, demonstrating its potential as a probiotic demonstrated ability to produce exopolysaccharides, but microorganism. not CO2, was tolerant to cure salts, was able to acidify MRS broth, was able to grow at different temperatures and demonstrated the ability for inhibiting food DISCUSSION pathogens. Based on these characteristics, BLS29 was chosen for further experiments and was tested for the In spontaneous fermentation, it is common to find several 1892 Afr. J. Microbiol. Res.
Table 3. Antimicrobial activity of Lactobacillus bacteria isolated from pork sausages in Southern Brazil against foodborne pathogens.
E. coli S. Enteritidis S. Typhimurium S. aureus B. cereus L. monocytogenes Strains O157:H7 SE86 ATCC 14028 ATCC 14458 ATCC 33010 ATCC 6477 ATCC 43888 L. plantarum BLS2 54.95aA 51.20abABC 48.45bA 55.80aACE 32.55cACD 54.60aACE L. plantarum BLS3 53.00aA 49.25Babc 59.05cC 53.00cC 44.05dB 58.75cAC L. plantarum BLS4 44.50aB 47.4aABC 39.00abBD 45.15aBCD 33.35bACD 45.80aBDE L. plantarum BLS9 56.65aA 43.70abAB 49.15abA 46.05abABCD 36.30bACD 55.95aAC L. plantarum BLS10 51.30aAB 49.35aAB 52.10aA 50.10aABC 42.50aBC 42.75aBD L. plantarum BLS17 44.40aB 42.25aABC 51.75bA 43.65aBCD 34.00cABCD 49.20bABE L. plantarum BLS19 43.05aB 38.00bABC 40.05abA 48.40cABCD 34.65bACD 44.55acBDE L. plantarum BLS20 42.00aBC 39.60abAB 38.05bdBDE 55.30cACE 35.15dACD 51.55cABCE L. plantarum BLS21 51.70aAB 37.85bB 37.05bdBDE 52.90aABC 35.80bACD 48.60aBDE L. plantarum BLS23 48.30acAB 44.95acB 33.70bBDE 43.65acBCD 34.05bACD 52.40adBDE L. plantarum BLS26 43.50aBC 37.75bB 43.45aAB 39.15abBD 34.85bcACD 40.55abBD L. brevis BLS27 45.85abBC 44.65abB 41.85abAB 56.40cBD 37.10bACD 55.60cBD L. plantarum BLS29 57.10acA 57.55acAC 60.80acC 64.00aAE 37.05bABCD 53.20cACE L. plantarum BLS30 44.85aB 41.00aAB 37.70bDE 47.90aABCD 34.80abACD 45.30aBDE L. plantarum BLS31 49.50aAB 42.15abAB 39.70bBD 47.00abABCD 30.50cACD 45.40abBDE L. plantarum BLS32 51.50aAB 48.15aABC 35.10bBDE 47.60aABCD 38.70bABC 49.55aABE
aADifferent letters indicate significant difference (P < 0,05) between halos diameter of growth inhibition measured in mm on the agar surface.
Table 4. Lactobacillus plantarum BLS29 isolated from a pork sausage survival at different pH. simulating gastric conditions.
pH Strain Time (h) 2 2.5 3 4 0 8.23 ± 0.17aA 8.48 ± 0.08aA 8.23 ± 0.17aA 8.23 ± 0.17aA Lactobacillus plantarum BLS29 3 5.44 ± 0.13bB 6.28 ± 0.25bB 8.71 ± 0.13aA 8.31 ± 0.0aA
aADifferent letters in the same column or same line differ significantly (P < 0.05) by Tukey test between the different pH values.
Table 5. L. plantarum BLS29 strain tolerance nonacid-exposed however in the present study we reported the prevalence and acid- exposed (BLS29ae) in simulated intestinal juice. NC of L. plantarum and E. faecium in a pork sausage. L. (negative control), PC (intestinal juice containing pancreatin), plantarum are homofermentative and facultatively PC+SB (intestinal juice containing pancreatin and bile salts). heterofermentative microorganisms widely distributed in -1 nature (fermented vegetables, meats, dairy products and Log10 (CFU/ml ) Strain Time (h) intestines), and possesses an expressive metabolic NC PC PC+SB a a b versatility, what is an important characteristic for potential BLS29 0 8.78± 0.09 7.99 ± 0.14 8.21 ± 0.05 probiotics intended to be used in foods. Depending on b a b BLS29ae 0 8.03 ± 0.25 8.18± 0.07 8.07± 0.01 the glycolytic pathway, L. plantarum can produce different b a a BLS29 4 7.92 ± 0.11 8.17 ± 0.10 8.5 ± 0.08 amounts of lactic acid, being that in the homo- BLS29ae 4 8.02 ± 0.06b 8.27± 0.11a 8.49± 0.03a fermentative route, more than 90% of substrate is
aDifferent letters in the same column differ significantly (P < 0.05) converted in lactic acid under anaerobic conditions. On by Tukey test. the other hand, the heterofermentative pathway produces, in addition of lactic acid, acetic acid and carbon dioxide, both undesirable by-products in sausage. Nevertheless, it was demonstrated that the salt species of lactobacilli like L. sakei and L. curvatus (Hugas concentrations (6-8% NaCl) found in dry sausages may et al., 1993), staphylococci, micrococci, and enterococci, alter the pattern of fermentation, leading to the production Senter et al. 1893
of less acetic acid and more acid latic under aerobic and also L. monocytogenes, and S. aureus. The authors conditions, showing the plasticity of using this kind of indicated that this bacterium has potential to be used as a microorganism for meat fermentation (Bobillo and biopreservative in the food industry. Gong et al. (2010) Marshall, 1991). have reported the action of plantaricin MG against L. The flavor of sausages can be attributed to monocytogenes and S. Typhimurium. Lee et al. (2014) Lactobacillus, enterococci, and other LAB that have reported that L. plantarum HY7712 demonstrated good several metabolic pathways, such as proteolytic, lipolytic results against the impairment of NK-Cells activity caused and esterolytic (Sarantinopoulos et al., 2001). As by y-irradiation in mice. These studies showed the benefit examples of this, the flavor both of the Mediterranean action of L. plantarum in functional food, highlighting the cheeses and the fermented sausages have been possible use of this functional starter. attributed to LAB (Hugas et al., 1993). Enterococci are In order to provide beneficial effect to the hosts, generally associated with the fermentation of meat microorganisms used as probiotics must reach and products, being detected in low numbers as 102 – 105 colonize the intestine, and for that they must survive the CFU g-1 at the final products (Samelis et al., 1998; Franz gastric acidic fluid and bile salts. These abilities were et al., 2003). These bacteria are especially found in demonstrated by BLS29. It is remarkable that BLS29 handmade products of Southern Europe, and can demonstrated a population increase in the presence of contaminate meat at the time of slaughter. pancreatin with or without bile salts and similar results In our study, only bacteria belonging to the genus were obtained by Pennacchia et al. (2004) studying Lactobacillus were tested, since, in spite of being Lactobacillus strains from fermented sausages, in Italy. characterized as probiotics, enterococci may possess Some factors may negatively impact on the virulence factors that precludes its use as a food starter development of functional cultures in meat environment, (Franz et al., 2003). Although some Enterococci strains as the concentration of curing salts, low pH and water have been characterized as functional microorganisms, activity (De Vuyst et al., 2008), so it is appropriate to use some of these bacteria may have plasmids, including strains adapted to these conditions (Hugas et al., 1993; some that may be related to resistance factors to Pennacchia et al., 2006). Therefore, the BLS29 strain antibiotic and pathogenicity. Therefore, is not recommen- was chosen for follow-up study, since the strain survived ded to use this type of microorganism in food as starter well to this kind of conditions, i.e. growing at (Franz et al., 2003). In the production of sausages, it is temperatures prevailing during maturation of fermented important that LAB be homofermentative to prevent the meat products and developed well in the presence of production of voids caused by the production of carbon curing salts. dioxide, which would result in a displeasing appearance Besides the Good Manufacturing Practices (GMP) that (Terra et al., 2004;Ordoñez Pereda, 2005). Although should be adopted in the production of meat products in BLS29 to be facultatively homofermentative, the bacteria order to prevent contamination, a quick acidification did not produced gas during tests, because it is isolated carried out by LAB and the production of bacteriocins from sausage with sensory characteristics acceptable to may contributes to the inhibition of food pathogens such the population. Heterolactic bacteria can produce acetic as Salmonella, E. coli, S. aureus and L. monocytogenes. acid, which in high quantities can produce astringent The flavor of fermented foods depends on several taste, as is the case L. brevis BLS27. factors, mainly by the quantity and quality of the source, Production of exopolysaccharides by the isolates amount and type of ingredients (meat, salt, and spices), investigated in this study were considered a positive temperature, processing time, smoking, and type of characteristic for the production of sausages, because it starter culture used (Leroy et al., 2006). The involvement can contribute to the organoleptic properties, such as of proteolytic enzymes also have their contribution to the flavor and texture of the final fermented food, since the liberation of peptides that together with free fatty acids, exopolysaccharides can improve thickening and resulting in the action of lipases, lead to volatile emulsifying properties of the product (Ballús et al., 2010). compounds, giving the characteristic odor and flavor Furthermore, production of exopolysaccharides may characterized of each fermented sausage (Leroy et al., contribute to the adhesion of probiotics to the intestine, 2006). These enzymes may originate in parts by the raw contributing to the colonization of this surface (Sanders material itself, and most often by the starter culture and Klaenhammer, 2001). fermentation. With the exception of the post- mortem Although bacteriocins produced by LAB are generally glycolysis, the catabolism of carbohydrates is carried inefficient to inhibit Gram-negative bacteria, our results mostly by LAB which produces compounds such as lactic demonstrate that S. Enteritidis and S. Typhimiurium were acid, acetic acid, ethanol, acetoin, and other complex inhibited by the LAB isolated. Similar results were compounds which contribute for the flavor of sausages demonstrated by Hu et al. (2013), who described a (Viallon et al., 1996; Leroy et al., 2006). The fast acid bacteriocin called plantaricin 163, produced by L. production observed in BLS29 is an important factor that plantarum that showed broad inhibitory activity against contributes not only to the development of pleasant foodborne pathogens including S. Typhimurium, E. coli, sensory characteristics, but also to the microbiological 1894 Afr. J. Microbiol. Res.
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Vol. 9(32), pp. 1896-1906, 12 August, 2015 DOI: 10.5897/AJMR2015.7610 Article Number: 405B37C55005 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR
Full Length Research Paper
Response of single and co-inoculation of plant growth promoting rhizobacteria on growth, flowering and nutrient content of chrysanthemum
Anop Kumari, R. K. Goyal1*, Mahesh Choudhary1 and S. S. Sindhu2
1Department of Horticulture, College of Agriculture, CCS Haryana Agricultural University, Hisar, 125 004, Haryana, India. 2Department of Microbiology, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar, 125 004, Haryana, India.
Received 6 June, 2015; Accepted 3 August, 2015
A pot experiment was conducted in the screen-house of Department of Horticulture, College of Agriculture, CCS Haryana Agricultural University, Hisar during the two successive seasons of 2011-12 and 2012-13 to investigate the potential effect of different strains of Bacillus (BS1- SYB101, BS2- SB155 and BS3- SB127), Pseudomonas (PS1- WPS73, PS2- CPA152 and PS3-P20) and their combination on growth, flowering and nutrient content of chrysanthemum. Strains of Bacillus and Pseudomonas significantly influenced the observed parameters of chrysanthemum. Maximum plant height and number of branches per plant were recorded in plants inoculated with PS2 strain of Pseudomonas (CPA152) and BS3 strain of Bacillus (SB127) in both the years. The minimum number of days taken to bud initiation, days for first flowering from bud initiation and days taken for 50% flowering were recorded in plants inoculated with PS2 strain of Pseudomonas (CPA152) and BS3 strain of Bacillus (SB127). The maximum flower size was noticed with PS3 strain of Pseudomonas (P20) in the first year whereas, in second year, the response of Pseudomonas strains was found non-significant. Among Bacillus strains, the plants inoculated with BS3 (SB127) recorded maximum flower size. Maximum flower yield/plant was recorded in plants inoculated with PS2 strains of Pseudomonas (CPA152) and BS3 strains of Bacillus (SB127). The N content of plant was recorded maximum with PS2 (CPA152) and PS3 (P20) strains of Pseudomonas and BS3 (SB127) strains of Bacillus, whereas, P content of plant was noticed maximum with PS3 strains of Pseudomonas (P20) in the first year, while it was found non-significant in second. The response of Pseudomonas strains to K content of plant was found non-significant in both years, while K content of plant was influenced significantly by Bacillus strains.
Key words: Chrysanthemum, PGPR, rhizosphere bacteria, Bacillus, Pseudomonas, nutrient content.
INTRODUCTION
Chrysanthemum (Chrysanthemum morifolium Ramat.) flowers and perfection in the form of plants are the which occupies a prominent place in ornamental important objectives to be reckoned in commercial flower horticulture is one of the commercially exploited flower production. Though the quality of flowers is primarily a crops. It is mainly grown for cut and loose flowers for varietal trait, it is greatly influenced by climatic, geogra- garland making, general decoration, hair adornments and phical and nutritional factors among which nutrition play religious function. Increased flower production, quality of major role (Laishram et al., 2013). At present, these Kumari et al. 1897
nutrients are supplied through chemical fertilizers. The Department of Horticulture, College of Agriculture, CCS Haryana use of chemical fertilizers has resulted not only in the Agricultural University, Hisar, India during the year 2011-2012 and deterioration of soil health but also has led to some major 2012-2013. Hisar is situated at 29° 10 East longitude with an elevation of 215.2 m above mean sea level. environmental problems, such as soil and water pollution The tract falls in the semi-arid subtropical region having the and other health related problems, besides increasing the characteristic extremes of weather conditions with hot dry winds input cost for crop production especially on the marginal during summers and severe cold in winters. For experimental farmers. purpose, soil was collected from pure sand dune near to Hisar and Plant growth-promoting rhizobacteria (PGPR) are mixed thoroughly. Each pot was lined with polythene sheet and naturally occurring soil bacteria that aggressively colonize filled with 5 kg of soil. The experimental soil was sandy in texture having 0.19% organic carbon, 47.5 ppm available nitrogen, 5 ppm plant roots and benefit plants by providing growth available phosphorus and 51 ppm available potassium. One month promotion (Saharan and Nehra, 2011). They are a old rooted cuttings of Chrysanthemum morifolium Ramat. cv. ‘Dolly heterogeneous group of bacteria that can be found in the Orange’ having almost equal size (5-7 cm) and vigour were rhizosphere, at root surfaces and in association with transplanted in the centre of pot in the month of September. Soil roots, enhancing the growth of the plant either directly was firmly pressed around the plant and light watering was done immediately. The biofertilizers used in the experiment were and/or indirectly (Glick, 1995). Inoculation of crop plants procured from the Department of Microbiology, Chaudhary Charan with certain strains of PGPR at an early stage of Singh Haryana Agricultural University, Hisar, India during both the development improves biomass production through direct years of investigation. Three strains of Bacillus (BS1- SYB101, BS2- effects on root and shoots growth. They not only promote SB155 and BS3- SB127), three strains of Pseudomonas (PS1- plant growth but also help in sustainable agricultural WPS73, PS2- CPA152 and PS3-P20) and their combination were development and protecting the environment (Das et al., applied in three replications having CRD experimental design. In addition to the above treatments recommended dose of fertilizers 2013). It is well established that only 1 to 2% of bacteria that is NPK 30, 20, 20 g/m2 or 150, 100, 100 ppm were also promote plant growth in the rhizosphere (Antoun and applied. Nitrogen, phosphorus and potassium were applied through Kloepper, 2001). The mechanisms by which PGPR urea (46% N), single super phosphate (16% P2O5) and murate of promote plant growth are not fully understood, but it is potash (60% K2O), respectively. Half dose of nitrogen and full dose believed that the plant growth promoting rhizobacteria of phosphorous and potash were applied as a basal dose just enhance plant growth and yield either by direct or indirect before planting of rooted cuttings, while the remaining half of the nitrogen was applied after 30 days of planting by top dressing mechanisms (Glick, 1995). The direct promotion of plant method. The culture of Bacillus and Pseudomonas strains were growth by PGPR entails either providing the plant with a grown in LB broth media for three days. About 20 ml suspension compound that is synthesized by the bacterium, for was applied to rhizosphere of the plant after 6 days of plantation as example plant growth regulators, or facilitating the uptake per treatments. Data on various growth, yield and quality of certain nutrients from the environment (Glick, 1995). parameters viz., plant height, number of branches per plant, number of days taken for bud initiation, number of days taken for The indirect promotion of plant growth occurs when first flowering from bud initiation, number of days taken for 50% PGPR lessen or prevent the deleterious effects of one or flowering, size of flower (cm), flower (capitulum) yield per plant (g), more phytopathogenic organisms. This can happen by and nutrient content in chrysanthemum plant (%) were recorded producing antagonistic substances or by inducing and average data were analyzed statistically as per method resistance to pathogens (Glick, 1995). A particular PGPR suggested by Panse and Sukhatme (1978). Estimation of nitrogen may affect plant growth and development by using any was done by Nessler’s reagent method as per standard procedure (Jackson, 1967). Phosphorus content in plant sample was one, or more, of these mechanisms. PGPR, as biocontrol determined by ‘Vanado-Molybdate method’ (Jackson, 1967). The agents, can act through various mechanisms, regardless intensity of yellow colour was read at 430 nm in Elico spectrometer of their role in direct growth promotion, such as by known model CL-24. Potassium content of plant tissue was determined by production of auxin phytohormone (Patten and Glick, flame photometer method. Readings were taken in Elico flame 2002), decrease of plant ethylene levels (Glick et al., photometer; model C-140 after digesting the samples with triacid 2007) or nitrogen fixing associated with roots mixture (Jackson, 1967).
(Dobereiner, 1992). PGPR and their interactions with plants are exploited commercially and hold great promise RESULTS AND DISCUSSION for sustainable agriculture. Thus the aim of this study was to determine the effect of plant growth promoting Plant height (cm) rhizobacteria on growth, flowering and nutrient content of chrysanthemum. The data recorded on the response of different strains of biofertilizers and their interactions on plant height are
MATERIALS AND METHODS presented in Table 1. It is apparent from the data that plant height was significantly influenced by different The present experiment was carried out in the screen-house of the strains of Pseudomonas (Figure 1) and the maximum plant
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1898 Afr. J. Microbiol. Res.
Table 1. Response of single and co-inoculation of PGPR on plant height (cm) in chrysanthemum.
2011-12 2012-13 Biofertilizer strains Biofertilizer strains Treatment PS3- PS3- PS0-Control PS1-WPS73 PS2-CPA152 Mean PS0-Control PS1-WPS73 PS2-CPA152 Mean P20 P20
BS0-Control 21.13 22.50 23.97 26.50 23.53 19.21 24.60 27.47 25.20 24.12
BS1-SYB101 22.63 23.43 28.40 25.53 25.00 21.63 23.63 26.50 25.63 24.35
BS2-SB155 24.13 24.93 30.10 24.20 25.84 24.43 25.83 29.73 27.50 26.88
BS3-SB127 25.13 24.70 29.13 31.80 27.69 27.80 24.07 27.97 28.10 26.99 Mean 23.26 23.89 27.90 27.01 23.27 24.53 27.92 26.61 C.D. at 5% Pseudomonas 1.01 1.61 Bacillus 1.01 1.61 Pseudomonas x 2.01 3.22 Bacillus
Figure 1. Effect of different Pseudomonas strains on chrysanthemum.
height (27.90 and 27.92 cm) was recorded with inoculation 2012, whereas, in the year 2012-2013, the tallest plant of PS2 treatment, which remained at par with PS3 (27.01 (29.73 cm) was recorded with application of BS2PS2, and 26.61 cm), whereas, it was recorded minimum (23.26 which was at par with BS3PS3 (28.10 cm), BS3PS2 (27.97 and 23.27 cm) in control during the year 2011-2012 and cm), BS3PS0 (27.80 cm), BS2PS3 (27.50 cm) and BS0PS2 2012-2013, respectively. It is inferred from the data that (27.47 cm) treatments. The smallest plant (21.13 and the effect of Bacillus strains was also found to be 19.21 cm) was observed in control during both the years, significant for plant height (Figure 2). The maximum plant respectively. Indole acetic acid (IAA) is one of the height (27.69 and 26.99 cm) was observed with the naturally occurring auxins with broad physiological effects application of BS3 treatment in the year 2011-2012 and of plants. Many rhizosphere bacteria including Bacillus, 2012-2013, respectively, but in the year 2012-2013, it Pseudomonas, Azotobacter, Azospirillum, etc. were was at par with BS2 application with the value of 26.88 found to have the ability to produce IAA or related auxins cm. The minimum plant height (23.53 and 24.12 cm) was (Salma et al., 2013). Auxins have been implicated in recorded in control during both the years, respectively. initiation of lateral and adventitious roots, in stimulation of Among the interactions of Bacillus and Pseudomonas cell division and elongation of stems and roots (Malamy strains, the maximum plant height (31.80 cm) was and Benefy, 1997). Increased of plant height with the recorded with the inoculation of BS3PS3, which was at par application of Bacillus (SYB101) and Pseudomonas with inoculation of BS2PS2 (30.10 cm) in the year 2011- (CPS63) strain in gladiolus was also reported by Singh Kumari et al. 1899
Figure 2. Effect of different Bacillus strains on chrysanthemum.
Table 2. Response of single and co-inoculation of PGPR on number of branches per plant in chrysanthemum.
2011-12 2012-13 Biofertilizer strains Biofertilizer strains Treatment PS - PS3- PS3- 0 PS -WPS73 PS -CPA152 Mean PS -Control PS -WPS73 PS -CPA152 Mean Control 1 2 P20 0 1 2 P20
BS0-Control 5.33 5.67 7.33 5.67 6.00 5.67 6.00 6.67 6.00 6.08
BS1-SYB101 5.67 6.33 6.67 7.67 6.58 5.33 5.67 6.00 7.67 6.17
BS2-SB155 5.33 5.67 7.33 6.00 6.08 6.00 7.00 7.67 7.33 7.00
BS3-SB127 6.67 7.00 7.33 7.67 7.17 6.67 7.33 7.67 7.67 7.33 Mean 5.75 6.17 7.17 6.75 5.92 6.50 7.00 7.17 C.D. at 5% Pseudomonas 0.45 0.50 Bacillus 0.45 0.50 Pseudomonas x 0.90 NS Bacillus
(2009). Dua and Sindhu (2012) reported that in wheat which remained at par with PS2 (7.00). The minimum single inoculation of Pseudomonas strain WPS3 or number of branches per plant (5.75 and 5.92 cm) was WPS90 resulted in increased plant growth as compared observed with control during both the years, respectively. to control. Such a positive effect was in line with the It is inferred from the data that among the Bacillus findings of Prasad et al. (2012) in chrysanthemum and strains, the number of branches per plant was noted Jayamma et al. (2008) in jasmine. maximum with application of BS3 strain (7.17 and 7.33) in both the years, respectively, which was at par with the treatment of BS2 (7.00) in the year 2012-13. The Number of branches per plant minimum number of branches per plant (6.00 and 6.08) was found with control during both the years, The data regarding number of branches per plant are respectively. given in Table 2, which reveal that the number of The interaction effects of different strains of Bacillus branches per plant was influenced significantly by and Pseudomonas on number of branches per plant was different strains of biofertilizers. In the year 2011-2012, found significant in first year, whereas, it was non- the maximum number of branches per plant (7.17) was significant in second year. During the year 2011-2012, observed with the inoculation of PS2 treatment, which BS3PS3 and BS1PS3 treatment combination recorded the remained at par with PS3 (6.75), whereas, in the year maximum number of branches per plant (7.67), which 2012-2013, it was observed maximum with PS3 (7.17), was at par with BS2PS2 and BS3PS2 (7.33) and BS3PS1 1900 Afr. J. Microbiol. Res.
Table 3. Response of single and co-inoculation of PGPR on number of days taken for bud initiation in chrysanthemum.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS3- PS3- PS -Control 1 PS -CPA152 Mean PS -Control PS -WPS73 PS -CPA152 Mean 0 WPS73 2 P20 0 1 2 P20
BS0-Control 76.00 74.00 63.33 67.00 76.00 74.33 72.33 64.67 69.00 70.08
BS1-SYB101 71.00 74.33 69.67 63.33 71.00 73.33 70.33 71.00 61.67 69.08
BS2-SB155 72.00 69.33 64.67 68.67 72.00 71.00 68.67 63.33 67.33 67.58
BS3-SB127 69.67 64.00 62.00 63.33 69.67 69.00 66.33 62.00 64.00 65.33 Mean 72.17 70.42 64.92 65.58 71.92 69.11 65.25 65.50 C.D. at 5% Pseudomonas 2.39 1.70 Bacillus 2.39 1.70 Pseudomonas 4.78 3.40 x Bacillus
(7.00) treatment combinations, whereas number of The interaction between Bacillus and Pseudomonas branches per plant was recorded minimum (5.33) in strains was found to be significant during both the years. control. The increase in number of branches per plant In first year, the minimum number of days taken for bud could be attributed to increased uptake of nutrients and initiation (62.00 days) was recorded with inoculation of increased activity of hormones like auxins, cytokinins, BS3PS2, which was at par with treatment of BS0PS2, etc. in biofertilizers-inoculated plants. They also help in BS1PS3, and BS3PS3 (63.33 days), BS3PS1 (64.00 days) the absorption of relatively immobile nutrients such as P, and BS2PS2 (64.67 days). In second year, the minimum Zn, Cu, Mn, Fe, etc. Increased number of primary and number of days taken for bud initiation (61.67 days) was secondary branches per plant in fenugreek was also recorded with inoculation of BS1PS3, which was at par reported by Shivran et al. (2013) with the application of with BS3PS2 (62.00 days), BS2PS2 (63.33 days), BS3PS3 different bioformulations. Similar findings have also been (64.00 days), and BS0PS2 (64.67 days) treatment reported by Prasad et al. (2012) in Chrysanthemum combinations. The maximum number of days taken for indicum and Jayamma et al. (2008) in jasmine. bud initiation (76.00 and 74.33 days) was found with control during both the years, respectively. The earliness of bud initiation in biofertilizers inoculated plants may be Number of days taken for bud initiation ascribed to easy uptake of nutrients and simultaneous transport of growth promoting substances like cytokinins The data regarding number of days taken for bud to the axillary buds, resulting in breakage of apical initiation are presented in Table 3. It is apparent from the dominance (Jayamma et al., 2008). The present results data that number of days taken for bud initiation was are in confirmation with the findings of Singh (2009) who significantly reduced by the inoculation of Pseudomonas reported that combined application of Bacillus (SYB101) strains. The minimum number of days taken for bud and Pseudomonas (CPS63) strains caused early spike initiation (64.92 and 65.25 days) was recorded with PS2 initiation in gladiolus. The results are also in line with the inoculation, which was at par with PS3 (65.58 and 65.50 findings of Salma et al. (2013) and Pandey et al. (2013) days) in the year 2011-2012 and 2012-2013, respec- in gladiolus. tively. The maximum number of days taken for bud initiation (72.17 and 71.92 days) was observed with control during both the years, respectively. The effect of Number of days taken for first flowering from bud Bacillus strains was also found to be significant with initiation respect to number of days taken for bud initiation. The minimum number of days taken for bud initiation (69.67 A close examination of data shown in Table 4 indicates and 65.33 days) was recorded with the inoculation of BS3 that number of days taken for first flowering from bud treatment in the year 2011-2012 and 2012-2013, initiation was significantly influenced due to different respectively, but in first year, it was at par with BS1 (71.00 strains of Pseudomonas. The minimum number of days days) and BS2 (72.00 days) treatments. The maximum taken for first flowering from bud initiation (13.92 and number of days taken for bud initiation (76.00 and 70.08 13.67 days) was recorded with PS2 application and it was days) was found in control during both the years, recorded maximum (18.17 and 17.48 days) in control respectively. during both the years, respectively. The response of Kumari et al. 1901
Table 4. Response of single and co-inoculation of PGPR on number of days taken for flowering from bud initiation in chrysanthemum.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 20.67 17.67 16.00 17.67 18.00 19.67 17.33 14.00 16.67 16.92
BS1-SYB101 19.33 16.67 15.00 13.33 16.08 19.57 16.67 17.00 14.00 16.81
BS2-SB155 18.00 15.00 12.33 15.67 15.25 16.33 15.00 12.00 17.00 15.08
BS3-SB127 14.67 16.33 12.33 13.33 14.17 14.33 15.67 11.67 12.33 13.50 Mean 18.17 16.42 13.92 15.00 17.48 16.17 13.67 15.00 C.D. at 5% Pseudomonas 0.78 0.84 Bacillus 0.78 0.84 Pseudomonas 1.56 1.69 x Bacillus
Table 5. Response of single and co-inoculation of PGPR on number of days taken for 50% flowering in chrysanthemum.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS3- PS - PS - PS3- PS -Control PS -WPS73 2 Mean PS -Control 1 2 Mean 0 1 CPA152 P20 0 WPS73 CPA152 P20
BS0-Control 112.67 109.33 99.67 103.67 106.33 113.33 111.67 100.67 106.67 108.09
BS1-SYB101 111.00 107.67 99.00 106.33 106.00 111.00 108.00 97.00 99.33 103.83
BS2-SB155 108.67 106.00 97.33 100.67 103.17 108.67 106.33 97.33 105.00 104.33
BS3-SB127 106.33 105.00 95.67 100.00 101.75 104.00 106.67 93.67 99.33 100.92 Mean 109.67 107.00 97.92 102.67 109.25 108.17 97.17 102.58 C.D. at 5% Pseudomonas 2.38 1.67 Bacillus 2.38 1.67 Pseudomonas NS 3.33 x Bacillus
Bacillus to number of days taken for flowering from bud respectively. The probable reason for earlier flowering initiation was also found significant in both the years. The may be that the hormone secretion by Bacillus and minimum number of days taken for first flowering from Pseudomonas strains which enhance early bud initiation bud initiation (14.17 and 13.50 days) was recorded with and flowering. Singh et al. (2010) reported that number of BS3 application, whereas, the maximum number of days days taken for spike initiation reduced with the application taken for flowering from bud initiation was seen in control of Bacillus and Pseudomonas strains in gladiolus. (18.00 and 16.92 days) during both the years, respectively. Barman et al. (2003) also observed that application of Among the various combinations of Bacillus and Bacillus reduced days required for flower opening in Pseudomonas strains, the minimum number of days tuberose. taken for first flowering from bud initiation (12.33 days) was recorded with BS2PS2 and BS3PS2 treatment combinations in first year, which was at par with BS1PS3 Number of days taken for 50% flowering and with BS3PS3 (13.33 days) treatments. In the second year, it was recorded minimum (11.67 days) with The data pertaining to response of different strains of application of BS3PS2, which were at par with BS2PS2 Pseudomonas and Bacillus and their interaction on (12.00 days) and BS3PS3 (12.33 days) treatment number of days taken for 50% flowering are presented in combinations. The maximum number of days taken for Table 5. The observation reveals that the minimum first flowering from bud initiation (20.67 and 19.67 days) number of days taken to 50% flowering (97.92 and was found with control during both the years, 97.17 days) was found in PS2 inoculation and maximum 1902 Afr. J. Microbiol. Res.
Table 6. Response of single and co-inoculation of PGPR on size of flower (cm) in chrysanthemum.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 3.91 4.36 4.55 4.51 4.33 3.87 4.33 4.42 4.40 4.26
BS1-SYB101 4.01 4.28 4.37 4.43 4.27 4.06 4.22 4.38 4.54 4.30
BS2-SB155 4.23 4.34 4.57 4.54 4.42 4.12 4.28 4.53 4.32 4.31
BS3-SB127 4.22 4.19 4.62 4.72 4.44 4.27 4.31 4.46 4.41 4.36 Mean 4.09 4.29 4.53 4.55 4.08 4.28 4.45 4.42 C.D. at 5%
Pseudomonas 0.07 NS Bacillus 0.07 0.09 Pseudomonas 0.14 0.19 x Bacillus
in control (109.67 and 109.25 days) during the year response of Pseudomonas strains was found to be non- 2011-12 and 2012-2013, respectively. It is evident from significant. The smallest flower (4.09 and 4.08 cm) was the data that the response of Bacillus strain was found recorded with control during both the years, respectively. to be significant and the minimum number of days taken Further, it is cleared from the data that application of BS3 to 50% flowering (101.75 and 100.92 days) was recorded the biggest flower (4.44 and 4.36 cm) in the recorded with BS3 inoculation in the year 2011-2012 and year 2011-2012 and 2012-2013, respectively, which was 2012-2013, respectively, but in the year 2011-2012, it at par with BS2 application (4.42 cm) in first year, was at par with application of BS2 (103.17 days). The whereas, in second year, it was at par with BS2 (4.31 cm)
maximum number of days taken for 50% flowering and BS1 (4.30 cm) treatments. The smallest flower (4.27 (106.33 and 108.09 days) was found with control during cm) was recorded in BS1 inoculation during first year, both the years, respectively. whereas, in second year, it was recorded smallest (4.26 The interaction effect of different strains of biofertilizers cm) with control. on number of days taken for 50% flowering was found to The interaction effect of Bacillus and Pseudomonas be non-significant in the first year, while it was significant strains on flower size was found to be significant in both in the second year, and the minimum number of days the years. In the year 2011-2012, the biggest flower (4.72 taken for 50% flowering (93.67 days) was recorded with cm) was recorded with treatment receiving BS3PS3 inoculation of BS3PS2 in the year 2012-2013, which was strains, which was at par with BS3PS2 (4.62 cm), while in
at par with BS1PS2 (97.00 days) inoculation. The maximum second year, it was biggest with BS1PS3 (4.54 cm) number of days required for 50% flowering (112.67 and treatment combination, which remained at par with 113.33 days) was found in control in the year 2011-2012 BS2PS2 (4.53 cm), BS3PS2 (4.46 cm), BS0PS2 (4.42 cm), and 2012-2013, respectively. The results of our experiment BS3PS3 (4.41 cm), BS0PS3 (4.40 cm) and BS1PS2 (4.38 are in confirmation with the findings of Shivran et al. (2013) cm) combinations. The smallest flower (3.91 and 3.87 who reported that application of PGPR significantly cm) was recorded with control during both the years, decreased the number of days taken to 50% flowering respectively. The increased flower size might be due to fenugreek. Jayamma et al. (2008) also observed that the increased availability of nitrogen and phosphorus for plant receiving 50 per cent recommended dose of NPK flower development as a result of greater solubility and fertilizers + biofertilizers took significantly lesser number absorption of nutrients. Singh et al. (2010) also reported of days for 50 per cent flowering as comparison to 100 beneficial effect of different biofertilizers and their strains per cent recommended dose of fertilizers. on floret diameter of gladiolus. These findings are in line to that of Pandey et al. (2013) who observed that
Size of flower (cm) application of Bacillus subtilis + vermicompost registered maximum diameter of floret in gladiolus.
The data on size of flowers as influenced by different strains of Bacillus and Pseudomonas and their Flower yield per plant (g) interactions are furnished in Table 6. The biggest flower (4.55 cm) was recorded with the application of PS3 The data pertaining to flower yield per plant have been treatment, which was at par with PS2 (4.53 cm) in the presented in Table 7. The perusal of data elucidated year 2011-2012, whereas, in the year 2012-2013, the significant differences amongst the different strains of Kumari et al. 1903
Table 7. Response of single and co-inoculation of PGPR on flower yield per plant (g) in chrysanthemum.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 17.69 24.57 47.65 39.60 32.38 20.22 27.20 42.93 34.21 31.14
BS1-SYB101 28.27 39.00 47.28 50.93 41.37 27.60 38.23 51.00 59.87 44.18
BS2-SB155 32.40 41.10 69.07 51.58 48.54 47.17 50.00 67.23 54.00 53.10
BS3-SB127 44.07 47.80 60.63 61.67 53.53 47.20 55.77 65.43 70.93 54.83 Mean 30.61 38.12 56.16 50.95 34.05 42.80 56.65 54.75 C.D. at 5%
Pseudomonas 3.13 3.64 Bacillus 3.13 3.64 Pseudomonas 6.27 7.27 x Bacillus
Figure 3. Effect of different combinations of Bacillus and Pseudomonas strains on chrysanthemum.
Pseudomonas and Bacillus. The maximum flower yield was maximum with BS3PS3 (70.93g), which was at par per plant (56.16 and 56.65 g) was recorded with PS2 with BS2PS2 (67.23 g) and BS3PS2 (65.43g) treatment inoculation in both the years, respectively, which was at combinations. The minimum flower yield per plant (17.69 par with PS3 (54.75 g) in second year. The minimum and 20.22 g) was recorded with control during both the flower yield per plant (30.61 and 34.05 g) was recorded years, respectively. A large body of evidence suggests with control during the year 2011-12 and 2012-13, that PGPR enhance the growth and crop yield, and respectively. Amongst the Bacillus strains, the maximum contribute to the protection of plants against certain flower yield per plant was obtained with inoculation of BS3 pathogens and pests (Kaushal et al., 2011; Dey et al., (53.53 and 54.83 g) during both the years, respectively, 2004; Kloepper et al., 2004; Herman et al., 2008 and which was at par with BS2 (53.10 g) application in the Minorsky, 2008). year 2012-2013. The minimum flower yield per plant (32.38 and 31.14 g) was obtained in control during both the years, respectively. Nutrient content of plant The interaction effects of Pseudomonas and Bacillus strains were found to be significant in both the years and Nitrogen content (%) in plant (g) the maximum flower yield per plant (69.07 g) was recorded with treatment receiving (Figure 3) BS2PS2 in The data on N content are presented in Table 8, which the year 2011-2012, whereas in the year 2012-2013, it reveal that the different strains of Bacillus and 1904 Afr. J. Microbiol. Res.
Table 8. Response of single and co-inoculation of PGPR on N content (%) in chrysanthemum plant.
2011-2012 2012-2013 Biofertilizer strain Biofertilizer strain Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 0.51 1.74 2.20 2.33 1.69 0.92 2.50 2.34 2.51 2.07
BS1-SYB101 2.07 2.85 3.45 2.79 2.79 1.94 2.34 2.61 3.15 2.51
BS2-SB155 2.51 3.46 3.29 3.51 3.19 2.51 2.90 2.05 2.60 2.51
BS3-SB127 2.77 3.23 3.15 2.94 3.02 2.90 2.17 2.46 2.59 2.53 Mean 1.96 2.82 3.02 2.89 2.07 2.48 2.36 2.71 C.D. at 5%
Pseudomonas 0.53 0.35 Bacillus 0.53 0.35 Pseudomonas NS 0.69 x Bacillus
Pseudomonas have significant effect on N content in Table 9. The perusal of data elucidates significant plants. In the year 2011-2012, the maximum N content differences amongst the Pseudomonas strains for the (3.02 %) was recorded with application of PS2, which was year 2011-2012 but non-significant for the year 2012- at par with PS1 (2.82%) and PS3 (2.82 %) treatments, 2013. The maximum P content (0.36%) was recorded and the minimum N content (1.96%) was found with with PS3 treatment, which was at par with PS1 and PS2 control. In the year 2012-13, the maximum N content (0.32%), whereas, it was minimum (0.25%) in control (2.71%) was recorded with PS3, which was at par with during the year 2011-2012. The response of different PS1 (2.48%) and PS2 (2.36%) applications. Goel et al. strains of Bacillus on P content was found to be non- (2002) reported that co-inoculation chickpea with significant for both the year. The interaction effect of Pseudomonas strains increase the nitrogen content in different strains of Bacillus and Pseudomonas was found plants. to be significant and the maximum P content (0.43%) was It is inferred from the table that the maximum N content recorded with the treatment combination of BS1PS3, (3.19%) was observed with treatment receiving BS2 in which was at par with BS0PS2 (0.37%) and BS3PS1 first year, which remained at par with BS3 (3.02%) and (0.35%) in the year 2011-2012, whereas, it was non- BS1 (2.79%) treatments, whereas, in second year, the significant in the second year. The minimum N content maximum N content (2.53%) was recorded with BS3 (0.20%) was recorded in control in the year 2011-2012. treatment, which remained at par with BS1 and BS2 The beneficial role by biofertilizers may be ascribed as (2.51%). The minimum N content (1.69 and 2.07%) was the cumulative activity of P-solubilization and phyto- found in control during both the years, respectively. The hormones production. Barman et al. (2003) observed that increase in concentration of nitrogen in the plant is due to NPK + FYM + PSB increased phosphorus content in the uptake of nitrogen by plants. The findings of Singh leaves of tuberose from 0.04 to 0.06%. Similarly, (2009) also revealed that nitrogen content in plant Karishma et al. (2013) also reported that gerbera plants increased significantly with application of PGPR. The inoculated with mix culture of Glomus mosseae + interaction effect was found to be non-significant in first Acaulospora laevis + Pseudomonas fluorescens showed year, whereas, it was significant in second year. The maximum phosphorus content at lower concentration of maximum N content (3.15%) was found with BS1PS3, superphosphate. which was at par with PS0BS3 and BS2PS1 (2.90%),
BS1PS2 (2.61%), BS2PS3 (2.60%), BS3PS3 (2.59%), BS PS and PS BS (2.51%) and BS PS (2.46%) Potassium content (%) in plant 0 3 0 2 3 2 treatment combinations, whereas, the minimum N The data of K content in plants as influenced by different content (0.92%) was noticed in control in the year 2012- strains of Pseudomonas and Bacillus and their 2013. interactions are presented in Table 10, which reveals that
the effect of Pseudomonas was found to be non- Phosphorus content (%) in plant significant for both the years, whereas the effect of Bacillus strains on K content was found to be significant The data pertaining to P content (%) are presented in for both the years. The maximum K content (1.69 and Kumari et al. 1905
Table 9. Response of single and co-inoculation of PGPR on P content (%) in chrysanthemum plant.
2011-2012 2012-2013 Biofertilizer strains Biofertilizer strains Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 0.20 0.33 0.37 0.38 0.32 0.22 0.43 0.42 0.34 0.35
BS1-SYB101 0.28 0.30 0.32 0.43 0.33 0.33 0.35 0.37 0.46 0.38
BS2-SB155 0.25 0.30 0.34 0.29 0.29 0.27 0.35 0.39 0.34 0.34
BS3-SB127 0.28 0.35 0.28 0.33 0.31 0.33 0.36 0.38 0.38 0.36 Mean 0.25 0.32 0.32 0.36 0.29 0.37 0.39 0.38 C.D. at 5% Pseudomonas 0.04 NS Bacillus NS NS Pseudomonas 0.08 NS x Bacillus
Table 10. Response of single and co-inoculation of PGPR on K content (%) in chrysanthemum plant.
2011-2012 2012-2013 2011-2012 2012-2013 Treatment PS - PS - PS -Control PS -WPS73 PS -CPA152 3 Mean PS -Control PS -WPS73 PS -CPA152 3 Mean 0 1 2 P20 0 1 2 P20
BS0-Control 0.28 1.29 1.41 2.12 1.27 0.33 1.34 1.51 2.21 1.34
BS1-SYB101 1.39 1.89 1.86 1.61 1.69 1.89 2.39 2.01 2.11 2.10
BS2-SB155 1.71 1.55 1.62 1.15 1.51 2.21 1.85 1.77 1.30 1.78
BS3-SB127 1.39 1.66 1.67 1.56 1.57 1.54 1.86 1.87 1.81 1.77 Mean 1.19 1.60 1.64 1.61 1.49 1.86 1.79 1.86 C.D. at 5% Pseudomonas NS NS Bacillus 0.35 0.49 Pseudomonas NS NS x Bacillus
2.10%) was recorded with treatment BS1 which was at (BS3) and CPA152 (PS2) were found effective in par with BS2 (1.51 and 1.78%) and BS3 (1.57 and 1.77%) increasing growth, flowering and yield parameters. The for both the year, respectively. According to Kohler et al. BS2PS2 (SB155 + CPA152), BS3PS2 (SB127 + CPA152) (2007), inoculation with Bacillus subtilis increased and BS3PS3 (SB127 + P20) combinations showed best significantly the urease, protease and phosphatase result with respect to growth, flowering and yield activities of the rhizosphere soil of the lettuce plants and parameters of chrysanthemum. increased foliar P and K contents. The interaction effect of Pseudomonas and Bacillus strains on K content was found to be non-significant during both the years of Conflict of interests investigation. The authors did not declare any conflict of interest.
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Vol. 9(32), pp. 1907-1916, 12 August, 2015 DOI: 10.5897/AJMR2015.7610 Article Number: F478D0655011 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR
Full Length Research Paper
Isolation and genetic characterization of endophytic and rhizospheric microorganisms from Butia purpurascens Glassman
Cintia Faria da Silva1, Jaqueline Alves Senabio2, Liliane Círa Pinheiro1, Marcos Antônio Soares2 and Edson Luiz Souchie1*
1Laboratório de Microbiologia Agrícola, Instituto Federal de Educação, Ciência e Tecnologia Goiano – Câmpus Rio Verde, Rod. Sul Goiana, Km 01, Cx. P. 66, 75901-970, Rio Verde, Goiás, Brasil. 2Laboratório de Biotecnologia e Ecologia Microbiana, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, 2367 Boa Esperança 78060-900, Cuiabá, Mato Grosso, Brasil.
Received 15 March, 2015; Accepted 10 August, 2015
Interactions between plants and microorganism are complex and can affect the establishment of plant communities and change ecosystem properties. These interactions are of interest for researchers and have many ecological and biotechnological applications. The present study is the first report on the microbial diversity associated with Butia purpurascens Glassman, and the goal was to isolate and identify the genetic diversity of endophytic and rhizospheric growable microorganisms from B. purpurascens using molecular biology techniques. Endophytic and rhizospheric microorganisms were isolated from the roots and rhizospheric soil of the purple yatay palm. DNA was extracted, and the 16S region for bacteria and internal transcribed spacer (ITS) region for fungi were amplified and sequenced. The resulting sequences were compared with known sequences from GenBank by similarity search using BLASTn. The rhizosphere and roots of B. purpurascens harbor a diverse set of microorganism groups. Fourteen (14) genera of endophytic and 12 genera of rhizospheric bacteria were identified that belonged to three phyla (Proteobacteria, Firmicutes and Actinobacteria), and 11 endophytic and six rhizospheric genera of fungi were identified that belonged to the phylum Ascomycota. The most frequent isolated genera were Enterobacter and Pseudomonas for endophytic bacteria, Gibberella and Codinaeopsis for endophytic fungi, Bacillus and Enterobacter for rhizospheric bacteria, and Ceratocystis for rhizospheric fungi. Differences were observed between the endophytic and rhizospheric microbial communities of B. purpurascens, with some microorganisms only detected in one environment. Further studies with a higher number of individuals are required to confirm these results.
Key words: Purple yatay palm, genetic diversity, molecular biology, microbial ecology.
INTRODUCTION
The purple yatay palm (Butia purpurascens Glassman) is the region of Jataí), Minas Gerais Triangle, state of Mato a native species to the Brazilian Cerrado. It is restricted Grosso do Sul and in neighboring regions of the state of to southwest regions of the state of Goiás (especially in Goiás. This species presents pronouncedly arched leaves, 1908 Afr. J. Microbiol. Res.
is used as an ornamental plant, and has a great potential through the production of plant hormones or their for use in landscaping and urban afforestation in tropical precursors, and they can affect both the production and regions (Lorenzi et al., 2010; Guilherme and Oliveira, degradation of more than one group of hormones, such 2011). as abscisic acid, auxins, cytokinins, ethylene, gibberellin, The absence of sustainable management practices has jasmonic acid, and salicylic acid, and minimize contributed to the extinction of this species because of agricultural environmental impacts (Dodd et al., 2010). the use of its leaves in the manufacturing of broom Endophytic microorganisms colonize plant tissues (Oliveira, 2010). In addition, its seeds have slow and without damaging the host (Reinhold-Hurek and Hurek, heterogeneous germination and low germination percent- 2011). Their symbiotic life strategy can vary between tages because of the physical dormancy imposed by the facultative saprophyte, parasitism, and mutualism (Schulz seed-coat, which is considerably lignified (Lorenzi et al., and Boyle, 2005). The benefits depend on the balance 2010; Rubio Neto, 2010; Rubio Neto et al., 2012). between the microorganism and plant response. If the Novel uses of microorganisms may benefit integrations interaction becomes unbalanced, symptoms of disease between plants and microorganisms in natural and can appear or the microorganism may be excluded agricultural ecosystems (Philippot et al., 2013). Plant through induced defense reactions by the host plant growth promotion by microorganisms is commonly used (Kogel et al., 2006). to improve crop productivity, and microorganisms have a Plant colonization by endophytes may occur through great potential for remediation of environmental natural openings and wounds, such as fissures problems, such as in phytoremediation for decontamina- (especially at the root region) that are caused by lateral tion of soils and water (Bashan et al., 2012). root emergence or abrasions resulting from root growth Microorganisms are essential components of and soil penetration, or through stomata, hydathodes, ecosystems that participate in 80 to 90% of soil openings caused by insects, and pathogenic fungi processes, such as the carbon and nitrogen biogeoche- structures (Santos and Varavalho, 2011). mical cycles, and are efficient and dynamic indicators of Independent of the microorganisms' location on the soil quality (Bresolin et al., 2010). Different types of soils plant, they can promote plant growth through several and plants possess specific microbial communities, and mechanisms that are important to the host plant (Dodd et each plant species is colonized by a different microbial al., 2010). In endophytic interactions, such mechanisms population that is mostly controlled by edaphic variables, include the supply of nutrients to the host plant, a especially pH (Berg and Smalla, 2009). decrease in abiotic stress, competition between microbes The rhizosphere is the narrow region of soil (Schulz and Boyle, 2005), changes to plant physiology, surrounding the roots. This microenvironment is inhabited and production of phytohormones. The latter mechanism by countless microorganisms and invertebrates and is is potentially useful for agriculture and industry, especially considered one of the most dynamic interfaces on the for pharmaceutical and pesticide production (Santos and earth (Philippot et al., 2013). The rhizosphere can vary Varavallo, 2011). according to factors related to soil, age, plant species, In addition, microorganisms can potentially substitute etc. (Campbell and Greaves, 1990). Complex biological chemicals and contribute to environmental sustainability and ecological processes occur through the release of and soil productivity, thus providing strategic alternatives exudates by the plant in which the microbiota can have a in agricultural and ecological systems because of their direct influence on the plant's growth, development, biotechnological value (Esitken et al., 2010; Santos and nutrition and health (Bais et al., 2006; Philippot et al., Varavallo, 2011). 2013). Determining the ecological and functional aspects of The composition of root exudates varies according to microbial communities is of great importance because the the plant species and affects microbial diversity because development of molecular biology techniques has it can supply nutrients to the microorganisms (in certain resulted in a considerable increase in information on their plant species) and may contain unique antimicrobial diversity, structure and functionality. These new metabolites (Berg and Smalla, 2009). The released techniques are able to analyze the high complexity of compounds are organic acids, amino acids, proteins, microbial communities, especially because a minority of sugars, phenols, and other secondary metabolites that microbial groups cannot be grown in vitro (Bresolin et al., are readily used by microorganisms (McNear Jr., 2013). 2010). Plant growth promoting rhizobacteria (PGPR) are The goal of the present study was to isolate and commonly found in the rhizosphere and can also be identify the genetic diversity of endophytic and isolated from plant tissues (endophytic bacteria). PGOR rhizospheric microorganisms from B. purpurascens using can be used as inoculants for plant growth promotion molecular biology techniques.
*Corresponding author. E-mail:[email protected].
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Da Silva et al. 1909
MATERIALS AND METHODS Molecular identification
Butia purpurascens root collection, processing, and Molecular biology methods were used to identify bacterial and microorganism isolation fungal isolates and genetic sequencing was performed to determine the genus as well as the species when possible. Extraction, The roots and rhizosphere soil of an individual B. purpurascens amplification and purification of the microbial DNA were performed (average 10 years old) were collected close to the Rio Verde at the “Laboratório de Biotecnologia e Ecologia Microbiana do municipality in the state of Goiás (GO) at the following geographic Instituto de Biociências da Universidade Federal de Mato Grosso”. coordinates: latitude 17° 35’ 10.51” S, longitude 50° 59' 13.06” W DNA samples of each isolate were sent to the “Laboratório and 822 m altitude. The chosen plant did not present apparent Multiusuário Centralizado para Sequenciamento de DNA em Larga symptoms of disease. The roots and rhizosphere soil were collected Escala e Análise de Expressão Gênica da UNESP” at Jaboticabal (0-15 cm depth) using a mattock, placed in a plastic receptacle, for sequencing. labeled, and transported to the “Laboratório de Microbiologia Agrícola (IF Goiano – Câmpus Rio Verde)”. For isolation of rhizospheric microorganisms, 10 g of the root Bacteria fragments was chosen randomly and stirred in sterile peptone water containing 0.1% Tween 80 (Polysorbate 80) for 30 min at 70 rpm at The extraction of total genome DNA from purified bacteria was room temperature. A dilution series of the supernatant was performed according to Cheng and Jiang (2006), and the evaluation prepared in saline solution, and 50 µL aliquots of each dilution were of bacterial genetic diversity was performed by enterobacterial cultured in approximately 20 mL GELP growth medium according to repetitive intergenic consensus (ERIC)-PCR using the the pour plate technique (Sylvester-Bradley et al., 1982). The plates oligonucleotides ERIC1 (5'-ATGTAAGCTCCCTGGGGATTCAC-3') were incubated at room temperature. and ERIC2 (5'-AAGTAA GTGACTGGGGTGAGCG-3') (Tian-Xing et The growth of microorganisms and appearance of CaHPO4 al., 2011). ERIC-PCR was conducted in a final volume of 25 μL that solubilization halos were evaluated following four days of growth. contained 3 μL DNA, 14.45 μL MilliQ water, 2.5 μL 10x PCR The total and phosphate solubilizing colony forming units (CFU) buffer, 0.75 μL MgCl2, 2 μL dNTP, 1 μL primer ERIC1, 1 μL were quantified, and the necessary dilution corrections were primer ERIC2 and 0.3 μL Taq polymerase (Invitrogen). The PCR performed to express the results in CFU/g root. CaHPO4 solubilizing program consisted of an initial denaturation at 94°C for 2 min, colonies were isolated for purification and stored in cold followed by 30 cycles at 94°C for 1 min, 50°C for 1 min 30 s, and temperatures. 68°C for 4 min, and final extension at 68°C for 10 min. For endophyte isolation, the root fragments were washed in ERIC-PCR products were separated by electrophoresis in 1.2% running water to remove the adhering soil and stirred in 1% neutral agarose gel, stained with ethidium bromide in tris-borate-EDTA detergent at 70 rpm for 10 min to decrease the number of epiphytic (TBE) 0.5x buffer. The amplicon size was determined using the 123 microorganisms. The fragments were surface sterilized in 70% bp DNA Ladder molecular weight marker (Sigma-Aldrich, Inc.). ethanol solution for 1 min, sodium hypochlorite solution (2.5 active Images were recorded using a gel documentation system (Loccus chlorine) for 5 min, and 70% ethanol solution for 30 sand then Biotecnologia, Brasil) for subsequent analysis. Diversity was washed three times in distilled and autoclaved water to remove the evaluated through visual assessment of the gel and considered all excess chemicals. A 500 µL aliquot of water from the last washing visible amplicons. All of the isolates of the same species with the was used for evaluation of the sterilization. same amplification profile were considered, and one isolate Sterilized fragments were cut in approximately 1 cm long representative of each morphotype was selected for molecular fragments and placed in Petri dishes containing potato dextrose identification. agar medium (PDA). The growth of endophytic microorganisms was Amplification of gene 16S rDNA was performed using primers monitored for 10 days, and the colonization frequency was 27F (5′-AGA GTT TGA TCM TGG CTCAG-3′) and 1492R (5′-TAC quantified based on the percentage of fragments with at least one GGY TAC CTT GTT ACG ACT T-3′) (Weisburg et al., 1991). The endophyte relative to the total analyzed fragments. reaction was conducted in a final volume of 25 μL that contained 1 μL DNA, 17.45 μL MilliQ water, 2.5 μL PCR 10x buffer, 0.75 μL Number of fragments MgCl2, 2 μL dNTP, 1 μL primer 27 F, 1 μL primer 1492 R and 0.3 Colonization frequency = x 100 μL Taq polymerase (Invitrogen). The PCR program consisted of an Total fragments initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 40 s, 58°C for 35 s, and 72°C for 1 min 20 s, and final extension at 72°C for 10 min. Purification and storage of microorganism lines The amplification products were purified (Dunn and Blattner, 1987), and the DNA was quantified using 1 μL of the product in The bacterial isolates were purified using microbial culture streaking 0.8% agarose gel electrophoresis. Sequencing was performed in nutrient agar medium (NA). Purification was confirmed through a according to the Sanger method using the Kit Big Dye from Gram staining test. The isolated colonies were cultured in penicillin ABI3100 Applied Biosystems. 16S sequences were compared with jars containing NA and stored in cold temperatures. The lines were known sequences from the GenBank database reactivated every 45 days and stored again. (http://www.ncbi.nlm.nih.gov) through a BLASTn similarity search. Sporulating fungi were isolated through monospore purification, and fungi without reproductive structure differentiation were isolated through the removal of fragments from the outer edge of the Fungi mycelium; these fragments were placed in penicillin jars containing PDA medium under cold temperatures, and new cultures were The fungi were grown in BD liquid medium (400 mL potato infusion, established every 60 days. 20 g dextrose) at room temperature for seven days. Fungal mycelia A replicate of each isolate was stored in an ultrafreezer at an were washed in distilled water using a sieve for complete removal average of -80°C for long-term preservation of the cultures. The of culture medium. The sample was then dried with a paper towel bacteria were stored in 80% glycerol (v/v), and fungi were stored in and stored in a freezer at -20 °C for further analysis. 30% glycerol (v/v), thus establishing a microbial germplasm bank The extraction of the fungal genomic DNA was performed using a for this tree species. DNA extraction kit according to the manufacturer's instructions 1910 Afr. J. Microbiol. Res.
Table 1. Community of rhizospheric microorganisms capable of CaHPO4 solubilization in vitro isolated from B. purpurascens at Rio Verde, GO.
Microorganism Population density Total rhizospheric microorganisms (CFU/g root) 1.39x10⁶
CaHPO4 solubilizing rhizospheric microorganisms (CFU/g root) 6.66x10⁴
Number of CaHPO4 solubilizing bacteria 79
Number of CaHPO4 solubilizing fungi 8 % solubilizing agents of rhizospheric microorganisms in total community 4.79
(Axygen Biosciences, USA). Morphotyping validation was RESULTS determined through a genetic variability analysis using the inter- simple sequence repeat (ISSR) amplification molecular marker. Isolation The amplification of the ISSR molecular marker was performed using oligonucleotide BH1 (5' - GTG GTG GTG GTG GTG - 3') (Smith and Bateman, 2002). The reaction was conducted in a final A community of microorganisms capable of CaHPO4 volume of 25 μL that contained 4 μL DNA, 14.45 μL MilliQ water, solubilization in vitro was isolated from the B. 2.5 μL PCR 10x buffer, 0.75 μL MgCl2, 2 μL dNTP, 1 μL primer BH1 purpurascens rhizosphere, and 79 bacteria and eight and 0.3 μL Taq polymerase (Invitrogen). The PCR program fungi were obtained (Table 1). consisted of an initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min, and The colonization frequency of the root fragments by final elongation at 72°C for 10 min (performed using an AmpliTherm endophytes was 100% after 10 days of incubation; 66 Thermal Cycler). bacteria and 29 fungi were isolated, which constituted the The ISSR-PCR products were subjected to electrophoresis in endophyte bank set for use in future experiments. 1.2% agarose gel in buffer TBE 0.5x. The amplicon size was Thirty-seven endophytic and rhizospheric fungi were determined using a 123 bp DNA Ladder molecular weight marker grouped in 26 morphotypes that were differentiated by (Sigma-Aldrich, Inc.). Images were recorded using a gel documentation system (Loccus Biotecnologia, Brazil) for macroscopic characteristics (outer edge, type and color subsequent analysis. Diversity was evaluated through a visual of mycelium) and microscopic characteristics (differen- assessment of the gel that considered all of the visible amplicons. tiation of reproductive structures). Isolates with the same amplification profile were considered as belonging to the same morphotype, and a representative line of each morphotype was selected for molecular identification. Molecular identification Identification was performed through partial sequencing of the rDNA internal transcribed spacer (ITS) region from representatives of each morphotype group. Oligonucleotides ITS 4 (5′-TCC TCC The molecular identification of 66 endophytic and 79 GCT TAT TGA TAT GC-3′) and ITS 5 (5`-GGA AGT AAA AGT CGT rhizospheric bacteria was performed; 29 endophytic and AAC AAG G-3') (White et al., 1990) were used for amplification of eight rhizospheric fungi were identified, and 145 the 18S-28S intergene region. The reaction was conducted in a endophytic and rhizospheric bacteria lines were final volume of 35 μL that contained 22.35 μL MilliQ water, 3.5 μL identified. The bacteria belonged to 17 genera distributed buffer PCR 10x, 1.5 μL MgCl2, 2.8 μL dNTP, 1 μL primer ITS 4, 1 over 38 taxa belonging to phylum Proteobacteria and μL primer ITS 5 and 0.3 μL Taq polymerase (Invitrogen). The PCR program consisted of an initial denaturation at 94°C for 2 min, classes α, β and γ (65.5%), phylum Firmicutes and class followed by 35 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for Bacilli (33.8%), and phylum Actinobacteria and class 1 min, and final elongation at 72°C for 10 min, using an AmpliTherm Actinobacteria (0.7%). The predominant genera were Thermal Cycler. Bacillus (31.0%), Enterobacter (20%), Pseudomonas The amplification products were purified (Dunn and Blattner, (13.8%), and Agrobacterium (8.3%). The remaining 1987), and DNA was quantified using 1 μL of the product in 0.8% agarose gel electrophoresis. Sequencing was performed according genera presented relative frequencies lower than 6.3% to the Sanger method using the Kit Big Dye and ABI3100 Applied (Table 2). Biosystems. The ITS sequences were compared with known Sixty-six endophytic bacterial lines were identified that sequences from the GenBank data base belonged to 14 genera distributed over 27 taxa belonging (http://www.ncbi.nlm.nih.gov) through a BLASTn similarity search to phylum Proteobacteria and classes α, β and γ (78.8%), (Altschul et al., 1990). phylum Firmicutes and class Bacilli (19.7%), and phylum Actinobacteria and class Actinobacteria (1.5%). The predominant genera were Enterobacter (28.8%), Data analysis Pseudomonas (18.2%), Bacillus (15.2%), and Agrobacterium (9.1%). The remaining genera presented The relative frequency of the observed species was calculated by dividing the number of isolates of each species by the total number relative frequencies lower than 6.2% (Table 2). of isolates. The Shannon diversity index (Hꞌ) was calculated Seventy-nine (79) rhizospheric bacterial lines were according to Kumar and Hyde (2004). identified belonging to 12 genera distributed over 25 taxa Da Silva et al. 1911
Table 2. Molecular identification of endophytic and rhizospheric bacteria isolates from B. purpurascens, based on sequencing of the 16S region.
GenBank accession ID Relative frequency Isolate Environment/Quantity GenBank number (%) (%) BP1EB E (1) Herbaspirillum rubrisubalbicans AB681857 98 0.7 BP60EB E (6)/R (6) Pseudomonas putida JQ701740 99 8.3 BP61EB E (1) Enterobacter oryzae JF513179 99 0.7 BP276EB E (1) Burkholderia vietnamiensis KF114029 99 0.7 BP321EB E (6)/R (6) Agrobacterium tumefaciens AB826000 99 8.3 BP324AEB E (2)/R (1) Citrobacter amalonaticus KC689293 99 2.1 BP336EB E (2) Pseudomonas plecoglossicida KC469709 99 1.4 BP342EB E (1)/R (1) Burkholderia tropica HQ023259 97 1.4 BP3EB E (2) Rhizobium huautlense KC355318 99 1.4 BP7EB E (4)/R (4) Pantoea cypripedii JX556216 98 5.5 BP59EB E (10)/R (6) Enterobacter asburiae EU221358 99 11.0 BP46EB E (2)/R (5) Bacillus subtilis KF241533 99 4.8 BP318EB E (3)/R (2) Pectobacterium cypripedii JF430157 99 3.5 BP38EB E (1) Microbacterium oxydans EU714340 96 0.7 BP42EB E (1) Enterobacter cancerogenus HQ683957 98 0.7 BP35EB E (1) Bacillus methylotrophicus JF899259 99 0.7 BP339BEB E (1) Paenibacillus illinoisensis HQ683877 96 0.7 BP58EB E (1) Bacillus megaterium KC609020 99 0.7 BP343EB E (6)/R (20) Bacillus pumilus GU969599 99 17.9 BP371EB E (2) Pseudomonas mosselii KC293833 98 1.4 BP356EB E (1) Achromobacter insolitus JQ659574 99 0.7 BP15EB E (2)/R (1) Brevibacillus agri HM629394 99 2.1 BP23EB E (5)/R (2) Enterobacter cloacae HM854373 98 4.8 BP261EB E (1) Enterobacter aerogenes KF358443 97 0.7 BP323EB E (1)/R (2) Enterobacter ludwigii KF475838 98 2.1 BP334EB E (1)/R (1) Pseudomonas denitrificans NR_102805 99 1.4 BP8RB E (1)/R (1) Pseudomonas sp. AB461802 96 1.4 BP188RB R (3) Klebsiella pneumoniae KC153122 99 2.1 BP5RB R (1) Bacillus safensis KF318396 99 0.7 BP61RB R (4) Yokenella regensburgei AB681877 99 2.8 BP35RB R (1) Bacillus altitudinis KC414719 99 0.7 BP44RB R (1) Bacillus aerophilus KC414720 99 0.7 BP55RB R (6) Bacillus amyloliquefaciens KF483660 99 4.1 BP57ARB R (1) Bacillus cereus KF475814 99 0.7 BP16RB R (1) Pantoea sp. CP002433 96 0.7 BP187RB R (1) Serratia nematodiphila HF585498 99 0.7 BP212RB R (1) Klebsiella variicola JX968498 99 0.7 BP54RB R (1) Burkholderia sp. KF059273 95 0.7
E = endophytic/R = rhizospheric; ID (%) = % identity.
that belong to phylum Proteobacteria and classes α, β The morphotype grouping of rhizospheric and and γ (54.4%) and phylum Firmicutes and class Bacilli endophytic fungal isolates resulted in the formation of 26 (45.6%). The predominant genera were Bacillus (44.3%), groups based on macroscopic characteristics. Identical Enterobacter (12.7%), Pseudomonas (10.1%), and profiles were detected following molecular identification of Agrobacterium (7.6%). The remaining genera presented a representative of each group through amplification of relative frequencies lower than 6.4% (Table 2). the ISSR molecular marker (data not shown), which 1912 Afr. J. Microbiol. Res.
Table 3. Molecular identification of endophytic and rhizospheric fungi isolates from B. purpurascens based on the ITS region sequencing.
GenBank accession ID Isolate Environment/Quantity GenBank Relative frequency (%) number (%) BP328EF E (5) Codinaeopsis sp. EF488392 99 13.5 BP364EF E (1) Bionectria ochroleuca HQ607832 99 2.7 BP16EF E (1) Penicillium purpurogenum GU566198 97 2.7 BP5EF E (8) Gibberella moniliformis JN232122 99 21.6 BP375EF E (2) Phomopsis sp. EF488379 99 5.4 BP314BEF E (2) Fusarium proliferatum HQ332533 99 5.4 BP14EF E (1) Fusarium oxysporum AY669123 99 2.7 BP329BEF E (1) Periconia macrospinosa FJ536208 99 2.7 BP33EF E (1) Hamigera insecticola JQ425375 98 2.7 BP40EF E (1) Talaromyces amestolkiae JX965247 99 2.7 BP55EF E (1)/R (2) Fusarium concentricum HQ379635 99 8.1 BP332EF E (1) Viridispora diparietispora JN049838 97 2.7 BP341EF E (4) Diaporthe sp. EF423549 95 10.8 BP196RF R (2) Ceratocystis paradoxa JQ963886 99 5.4 BP62RF R (1) Curvularia affinis EF187909 99 2.7 BP191RF R (1) Neodeightonia phoenicum HQ443209 99 2.7 BP202RF R (1) Hypocreales sp. HQ248209 98 2.7 BP192RF R (1) Aspergillus brasiliensis JQ316521 97 2.7
E = Endophytic/R = rhizospheric; ID (%) = % identity.
allowed for the identification of 18 identical profiles. (62.5 %), Dothideomycetes (25.0%) and Eurotiomycetes Morphological groups were identified through (12.5 %) and six genera, predominantly Fusarium and sequencing of the ITS region of the large subunit rRNA. Ceratocystis (25%), Curvularia, Neodeightonia, Eighteen taxa were identified with high identity using the Hypocreales and Aspergillus (14.3%) (Table 3). ITS region identity above 97% as a similarity criterion at the species level (O’Brien et al., 2005). The sequences were then compared with the deposited sequences at the Diversity GenBank. Molecular analysis of the groups revealed taxa from the The Shannon-Wiener Hꞌ diversity indexes for the phylum Ascomycota and belonging to the three classes microbial populations of B. purpurascens were 2.97 for Sordariomycetes (83.8%), Eurotiomycetes (10.8%) and endophytic and 2.73 for rhizospheric bacteria and 2.23 Dothideomycetes (5.4%) as well as 16 genera, for endophytic and 1.73 for rhizospheric fungi. predominantly Gibberella (20.5%), Fusarium (15.4%), Codinaeopsis (12.8%), Diaporthe (10.3%), Ceratocystis (7.7%), Phomopsis and Hypocreales (5.1%), Curvularia, DISCUSSION Bionectria, Penicillium, Periconia, Hamigera, Talaromyces, Viridispora, Neodeightonia and Aspergillus Interactions between plants and microorganisms are (2.6%). complex, can influence the establishment of plant Twenty-nine (29) endophytic fungal lines were communities, and can change ecosystem properties. identified that belonged to 12 genera distributed over 13 These relations are interesting for researchers, and taxa in the classes Sordariomycetes (89.7%) and related research results may clarify interaction Eurotiomycetes (10.3%). The predominant genera were mechanisms and have relevant ecological and Gibberella (27.6%), Codinaeopsis (17.2%), Fusarium biotechnological applications. The present study is the (13.8%), Diaporthe (13.8%), Phomopsis (6.9%), first on the taxonomic and genetic diversity of endophytic Bionectria, Penicillium, Periconia, Hamigera, and rhizospheric growable microorganisms from B. Talaromyces and Viridispora (3.4%) (Table 3). purpurascens from the Brazilian Cerrado. Eight rhizospheric fungal lines were identified that Microbial diversity can be defined in terms of belonged to six taxa in the classes Sordariomycetes taxonomic, genetic, and functional diversity and used in Da Silva et al. 1913
the characterization of species richness and real At Rio Grande do Sul state, corn roots and rhizospheric communities. The metabolic versatility of a population is soil were found in association with 21 genera of based on its genetic variability and capacity to interact rhizobacteria, with Klebsiella and Burkholderia the with other populations of species with functional diversity dominant genera (Arruda et al., 2013). This result is in (Jha et al., 2010). contrast with the present study, where Bacillus and Factors such as the type of soil, climate and plant Enterobacter were found to be the dominant genera of species may affect the microbial community, and it is rhizospheric bacteria and Enterobacter was found to be important to understand interactions between plants and the dominant genus of endophytic bacteria. microorganisms because they play a fundamental Madhaiyan et al. (2013) studied endophytic bacteria ecological role in nutrient recycling and plant growth isolated from Jatropha curcas roots from Indonesia, promotion (Kumar et al., 2012). China and India and observed a predominance of strains Jha et al. (2010) studied the Jatropha curcas from genus Enterobacter, such as the species E. oryzae, rhizosphere in five different soil conditions and locations E. asburiae, E. cancerogenus, E. cloacae, E. aerogenes at the Indian state of Gujarat and observed Shannon (Hꞌ) and E. ludwigii, which were also found in the present indexes between 0.96 and 0.92, which indicated that the study, with several only found in the endophytic plants generate a strong selection pressure on the environment and others found in both environments. rhizosphere under the studied conditions and are The genus Pseudomonas is known for its habitat and associated with bacteria that provide beneficial effects for physiological diversity, and certain strains can degrade plant growth and health, thus resulting in low bacterial recalcitrant compounds, which makes these species diversity. In the present study, the rhizospheric bacteria important for the bioremediation of natural and xenobiotic presented high Hꞌ (2.73), indicating higher bacterial pollutants. In addition, this genus is able to stimulate diversity at the rhizosphere. plant growth through different mechanisms (Patel et al., A value of Hꞌ = 1.41 was reported for halotolerant 2011; Behera et al., 2014). bacteria from Suaeda fruticosa collected from a salt The genus Bacillus possesses remarkable properties desert, indicating that the community of organisms because of several mechanisms, and it produces a wide residing at a given niche is specific and dependent on range of compounds beneficial for the promotion of plant physical and environmental factors (Goswami et al., growth through phosphate solubilization, indole-3-acetic 2014). Based on the bacterial diversity indexes observed acid (IAA) production and nitrogen fixation (Idris et al., for B. purpurascens, it can be concluded that the 2007; Kavamura et al., 2013) and for biological control of endophytic (Hꞌ= 2.97) and rhizospheric (Hꞌ= 2.73) species many common phytopathogens, such as through the diversity was higher for this species than for S. fruticosa, production of hydrogen cyanide (HCN), siderophores, indicating lower selection pressure. hydrolytic enzymes and antibiotics (Kumar et al., 2012). In a similar study by Alberto (2013) of the endophytic Bacillus pumilus is known for the production of fungi from Anacardium othonianum from the Cerrado of enzymes such as proteases, which are widely used in the Goiás state, an Hꞌ value of 1.82 was observed, which is food, chemical, detergent and leather industries, as well lower than the value observed in the present study. as the production of antifungals, antibiotics and chitinase, Higher diversity (Hꞌ = 2.99) was observed by Bezerra et which are used in the biological control of plant diseases al. (2013) in a study performed with Cereus jamacaru in (Zhang et al., 2010). Higher relative frequencies were Brazilian tropical dry forest between September and observed for this species, and it is easily found in the B. November. purpurascens roots and rhizosphere. The microbial diversity observed in the present study Yadav et al. (2013) observed the capacity for in vitro indicated differences in the endophytic and rhizospheric phosphate solubilization in Brevibacillus sp. strains, communities because some of the described genera which is a thermotolerant bacteria isolated from deposits were only detected in one environment. Kumar et al. in India. In the present study, this genus was also (2012) studied the bacteria associated with the roots of isolated as an endophyte and at the rhizosphere. Ajuga bracteosa, a medicinal plant traditional in India and Ascomycetes are referred to as producers of active China, from the Kangra Valley in India and found the antimicrobial compounds. In a study of Vitis labrusca in phyla Proteobacteria (69.9%), Firmicutes (24.4%) and the São Paulo region, Brazil, 48.14% of the endophyte Actinobacteria (5.7%). The first two were also observed fungi isolates belonged to phylum Ascomycota (Brum et in the B. purpurascens rhizospheric community in the al., 2012). In the present study, all of the isolates present study. belonged to this phylum, suggesting that they may be Using 16S rRNA sequence analysis, a recent study involved in the protection of the host plant against fungal performed on soil from three Cerrado regions of Minas diseases. Gerais, Brazil identified a bacterial community formed by Alberto (2013) studied the endophytic fungi community genera such as Bacillus, Klebsiella, Enterobacter, of A. othonianum and isolated genera Penicillium, Pantoea, Escherichia and Leuconostoc (Mesquita et al., Fusarium, Periconia, Bionectria and Diaporthe, some of 2013). which were also found in the present study. This result 1914 Afr. J. Microbiol. Res.
indicates that these genera are common to plant hosts with this tree species, and this study enabled the from locations such as the Brazilian Cerrado. establishment of a microbial species bank that can be The fungus Penicillium purpurogenum is of great used to identify these microorganisms, assist in future interest for the food industry because it produces a red research to develop processes and/or products, pigment through the combined effects of pH and contribute to the improvement of soil quality, and promote temperature. These pigments are frequently more stable plant growth and productivity. and soluble than pigments from plant or animal sources; in addition, they may present high productivity and are used as additives, color intensifiers, antioxidants, etc. Conclusions (Méndez et al., 2011). This species was also detected in the present study. The rhizosphere and roots of B. purpurascens harbor a The endophytic fungi from cacti of the Brazilian tropical diverse set of growable organisms, including fungi, dry forest that were isolated by Bezerra et al. (2013) bacteria and actinobacteria. Fourteen endophytic and 12 belonged to genera Fusarium and Aspergillus. Srinivasan rhizospheric bacterial genera belonging to three phyla et al. (2012) also isolated these genera from salt-affected (Proteobacteria, Firmicutes and Actinobacteria) and 11 soils in India. Similarly, in the present study, these genera endophytic and six rhizospheric fungal genera were also detected in B. purpurascens in both endophytic (exclusively from phylum Ascomycota) were detected in and rhizospheric environments. B. purpurascens endophytic and rhizospheric environ- Genus Fusarium produces a wide variety of biologically ments. The most frequently isolated genera were active secondary metabolites, including mycotoxins and Enterobacter and Pseudomonas for the endophytic fumonisins, which are detrimental for humans and bacteria, Gibberella and Codinaeopsis for the endophytic animals (Matić et al., 2013). Although it is known for its fungi, Bacillus and Enterobacter for the rhizospheric pathogenicity, it also includes non-pathogenic species bacteria, and Ceratocystis for the rhizospheric fungi. A that are effective for biological control of phytopathogenic difference was observed between the endophytic and fungi. However, species such as Fusarium oxysporum rhizospheric microbial communities of B. purpurascens, can produce toxins and cause plant diseases, thereby with certain microorganisms only present in one of the invalidating the use of Fusarium for biocontrol because of environments. Further studies with a larger number of the risks of food contamination (Brum et al., 2012). In the individuals are required to confirm this result. present study, the phytopathogenic and biocontrol capacities of this genus were not evaluated. Conflict of interests Fungus Gibberella moniliformis corresponds to the Fusarium verticillioides anamorphic phase. It commonly The authors did not declare any conflict of interest. colonizes corn plants and can act as endophyte or pathogen depending on environmental and genetic factors which are not well understood (Desjardins et al., ACKNOWLEDGEMENTS 2007). Fusarium verticillioides can cause diseases such This work was supported by the Goiás State Foundation as root rot and stem and grain wilting, and it is a highly for Research Support - FAPEG (Chamadas Públicas toxigenic species because it produces of mycotoxins 01/2011 – PAPPE INTEGRAÇÃO and 12/2012 - Apoio a (Covarelli et al., 2012). 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Vol. 9(32), pp. 1917-1920, 12 August, 2015 DOI: 10.5897/AJMR2015.7651 Article Number: E817F8F55021 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR
Short Communication
Isolation and identification of Sparicotyle chrysophrii (Monogenea: Microcotylidae) from gilthead sea bream (Sparus aurata L.) in the Mediterranean Sea, Greece
Jin Woo Jun
Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea.
Received 30 June, 2015; Accepted 3 August, 2015
The gilthead sea bream is one of the main aquacultured fish species in the Mediterranean Sea. Sparicotyle chrysophrii is an ectoparasite specific for sea bream and able to cause mortality in the Mediterranean aquaculture system. In this study, a total of 20 gilthead sea bream were sampled for parasitological research at Heraklion Bay, Crete, Greece. Gills of the fish were dissected and examined for parasites using a light microscope. S. chrysophrii was identified by field emission scanning electron microscopy, PCR and sequencing.
Key words: Gilthead sea bream, Mediterranean Sea, Sparicotyle chrysophrii, field emission scanning electron microscopy, PCR, sequencing.
INTRODUCTION
Monogeneans are known to be mostly host-specific chrysophrii was originally called Microcotyle chrysophrii, through the life cycle (Desdevises et al., 2002). The belonging to genus Microcotyle (Microcotylidae, monophyly of the Monogenea comprises two groups: the Polypisthocotylea); however, it is now placed in the Monopisthocotylea and the Polyopisthocotylea (Mladineo monospecific genus Sparicotyle (Mladineo and Maršić- and Maršić-Lučić, 2007). Polypisthocotyleans are Lučić, 2007). Its clinical signs include lethargy due to pathogenic to economically important fish around the hypoxia, emaciation, histopathological damage, and world (Ogawa, 2002). There have been reports about severe anemia (Cruz e Silva et al., 1997). mortalities due to polyopisthocotyleans in several The gilthead sea bream, Sparus aurata L., is one of the cultured fish species (Silan et al., 1985; Ogawa, 2002). main cultured fish species in the Mediterranean Sea Among them, Sparicotyle chrysophrii is a gill (Melis et al., 2014). European Union (EU) is the largest monogenean specific for sea bream and able to cause producer of gilthead sea bream worldwide; Greece mortality in aquaculture systems (Sanz, 1992). S. dominates aquaculture production of this species (FAO,
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Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1918 Afr. J. Microbiol. Res.
2012). S. chrysophrii has been regarded as one of the propagation of parasites (Gannicott and Tinsley, 1998). most threatening ectoparasites for the Mediterranean Morphological examinations by FE-SEM showed aquaculture (Antonelli et al., 2010). The aim of this study elongated and sharp narrowing shape (Figure 1A). The was to isolate and identify S. chrysophrii from gilthead enlarged picture of head area of S. chrysophrii was taken sea bream in the Mediterranean Sea, Greece based on to observe a characteristic structure such as pit-like morphological and molecular analysis. invagination although the trial failed to observe it (Figure 1B). It is known that pit-like invaginations contain taxono- mic and functional significance: pit-like invaginations MATERIALS AND METHODS have been observed on other Microcotylidae such as P. luquei and Microcotyle labracis (Antonelli et al., 2010); Sampling pit-like invaginations play a role in absorption of organic nutrients directly from seawater, excretion, and From March to May 2015, a total of 20 gilthead sea bream (237 ± osmoregulation (Halton, 1978; Oliver, 1981; Willams and 83.1 g) were sampled for parasitological examinations. Fish were McKenzie, 1995). S. chrysophrii infecting gills (Figure 1C, purchased from fishermen at Heraklion Bay (35°20′N, 25°08′E), D) was found while examining gills of the gilthead Crete (South Greece). Sampled fish were packed on ice in separate plastic bags and then transported to the diagnostic facility within 1 seabream sampled during the study. Pathological effects h. Examination was carried out immediately after arrival. Skin, fins, are mainly related to its attachment to gills: blood loss and gills of the fish were examined. Gill arches were removed and and reduction of breathing surface (Repullés-Albelda et placed on a glass microscope slide. The slides were examined for al., 2011). parasites using a light microscope. Infected samples were The main characteristic of S. chrysophrii is the haptor immediately fixed in 2.5% Glutaraldehyde for scanning electron with its clamps (Repullés-Albelda et al., 2011), which was microscopy (SEM). the main purpose of the morphological examination by
FE-SEM in this study. However, there was no
Field emission scanning electron microscopy (FE-SEM) morphological characteristic. It may be because the samples were destroyed when they were removed from Specimens were fixed in modified Karnovsky’s fixative, post fixed in gills, which occurs quite often during the on-site disease 1% osmium tetroxide in 0.05 M sodium cacodylate buffer (pH 7.2), diagnostic process. It emphasizes the usefulness of dehydrated through an ethanol serial solutions of 50, 60, 70, 80, 90, molecular analysis although molecular analysis based on 95, 100, 100% for 1 h per each concentration. Specimen drying morphological examinations is considered as the most was performed at room temperature overnight and sputter coated desirable method for parasite identification (Perkins et al., with gold/palladium. Samples were examined using a JSM-6700F field emission scanning electron microscope (JEOL, Japan). 2010). For molecular identification, the 28S rRNA gene of S. chrysophrii was sequenced and compared with the 28S rRNA gene of another S. chrysophrii. The sequence Molecular analysis of parasites obtained in this study showed 99% identity with that of another S. chrysophrii. DNA extraction was conducted using the DNeasy® Blood and The present study revealed that the percentage of fish Tissue Kit (QIAGEN) following the manufacturer’s instruction. For infected by S. chrysophrii, prevalence value was 40%, amplification of the 28S rDNA, PCR was performed using C1/D2 mean intensity was 3.6 ± 1.4, and maximum abundance primer pair: C1: 5’-ACC CGC TGA ATT TAA GCA T-3’, D2: 5’-TGG TCC GTG TTT CAA GAC-3’. The amplification was carried out as was 6. According to the previous report, S. chrysophrii previously reported (Chisholm et al., 2001). The amplified PCR has produced mortalities in farmed fish and even higher product was sequenced using the BigDyeTM terminator cycle mortalities in sea cages due to its favor of high densities sequencing kit (Macrogen Genomic Division). The sequence and net biofouling (Sanz, 1992; Sitjà-Bobadilla et al., obtained in this study was aligned with that of the S. chrysophrii 2006). However, S. chrysophrii has been rarely isolated 28S ribosomal RNA gene (AF311719) available in the GenBank in wild sea bream from Mediterranean Sea (Paperna et database, and the percentage sequence similarity was determined. al., 1977; Mladineo and Marsic-Lucic, 2007). It is known that S. chrysophrii is commonly related to mixed infections with other parasites and secondary bacterial RESULTS AND DISCUSSION infections (Cruz e Silva et al., 1997). In addition, further studies of S. chrysophrii are required as it can switch Samplings in the present study were conducted from easily to the new host although Monogeneans are known March to May 2015 as S. chrysophrii exhibited seasonal to be dependent to their hosts (Mladineo and Maršić- changes in its prevalence (Antonelli et al., 2010). Lučić, 2007). Previous reports show that cyclic peaks in the abundance of parasite have been mainly recorded in Mediterranean Sea during spring and summer (Reversat et al., 1992; Conflict of interests Sanz, 1992). In general, high water temperature is regarded to promote hatching of monogenean eggs and The authors did not declare any conflict of interest. Jun 1919
Figure 1. Field emission scanning electron micrograph (FE-SEM) of S. chrysophrii from gilthead sea bream. A, FE-SEM of an entire specimen. B, Head area of the parasite. C, The parasite attached to the gills of affected fish. D, The enlarged picture of the parasite attachment. Scale bars: A = 1 mm; B, C, D = 100 μm.
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