UNCORRECTED PROOF Systematic and Applied Microbiology ] (]]]]) ]]]–]]] Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice Ramachandran Muthukumarasamy a,Ã , Ilse Cleenwerck b , Gopalakrishnan Revathi a , Muthaiyan Vadivelu a , D. Janssens b , B. Hoste a , Kang Ui Gum c , Ki-Do Park c , Cho Young Son c , Tongmin Sa d , Jesus Caballero-Mellado e a Main Bio-control Research Laboratory (Unit of Tamilnadu Co-operative Sugar Federation), Good Will Avenue, Venpakkam, Chengalpattu 603 111, Tamilnadu, India b BCCM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University, Belgium c National Yeongnam Agricultural Experiment Station, Milyang 627-130, Republic of Korea d Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea e Centro de Investigacio´n sobre Fijacio´n de Nitro´geno, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos, Mexico Received 3 November 2004 Abstract The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotro- phicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N 2 -fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans. r 2005 Published by Elsevier GmbH. Keywords: Wetland rice; Isolation; N 2 fixation; Gluconacetobacter diazotrophicus; Acetobacter peroxydans; nifH; 16S rDNA; DNA–DNA hybridization Introduction Nitrogen is known as an important element in plant growth since it is often the primary nutrient limiting the plant growth in many ecosystems. In agriculture, crop productivity is mainly supported by the use of inorganic N fertilizers, which are expensive and damaging for the environment. From ecological and economical perspec- tives plant-associated biological nitrogen fixation is desirable especially for economically important crops 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 ARTICLE IN PRESS www.elsevier.de/syapm 3B2v8:06a=w ðDec 5 2003Þ:51c XML:ver:5:0:1 SYAPM : 34 Prod:Type:FTP pp:1210ðcol:fig::NILÞ ED:Ashim PAGN:Uday SCAN:Roopa 0723-2020/$ - see front matter r 2005 Published by Elsevier GmbH. doi:10.1016/j.syapm.2005.01.006 Ã Corresponding author. +91 4114 231393; fax: +91 44 24348024. E-mail address: mutindia@yahoo.com (R. Muthukumarasamy).