Philippine Journal of Crop Science 2004, 29(2): 45-58 Copyright 2005, Crop Science Society of the Philippines Released 31 March 2005 N2 FIXATION IN SUGARCANE AND POPULATION OF N2-FIXING ENDOPHYTES IN STEM APOPLAST SOLUTION CONSTANCIO A ASIS JRl, KATSUKI ADACHI2 & SHOICHIRO AKA03 Supervising Science Research Specialist, Philippine Rice Research Institute, Maligaya, Science City of Mufioz, 3119 Nueva Ecija, Philippines; cajrtony@yahoo.com. 2 Senior Researcher, Department of Upland Farming Research, National Agricultural Research Center for Kyushu Okinawa Region, Miyakonojo, Miyazaki 885-0091 Japan. 3 Professor, Department of Biochemistry and Applied BioScience, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192 Japan. Research supported by the Ministry of Education, Culture, Science and Technology (MEXT) and Japanese Society for the Promotion of Science (JSPS), Japan 1Studies were conducted to determine the population of N2-fixing endophytes in the sugarcane stem, analyze the carbon compounds (organic acid and sugar) in the stem apoplast solution, and estimate the contribution of N2-fixation to the nitrogen uptake of sugarcane (%Ndfa). The data gathered showed that the number of endophytic bacteria in the stem of sugarcane cultivars ranged from 104 to 109 cells / mL juice and varied among sites and cultivars. Moreover, not all of the stem apoplast solutions of sugarcane species and clones were colonized by N2-fixing endophytes. Among the acetylene reduction activity (ARA)+ samples, the number of N2-fixing endophytes was high in Saccharum robustum clone Robustum 16, S. sinense clone Kouchi, and S. hybrid cv. NCo310. The %Ndfa in sugarcane cv. NiF-8 ranged from 14.1 to 26.0% based on 15N dilution technique and 26.15 to 37.5% using the 615N abundance method. The identified organic acid compounds in stem apoplast solutions were aconitate, malate, and citrate, while the sugar compounds were sucrose, glucose, and fructose. Aconitate and sucrose were predominantly organic acid and sugar compounds, respectively. Moreover, the N2-fixing endophytes Gluconacetobacter diazotrophicus ATTCC 49037 and Herbaspirillum rubrisubalbicans PA18 differed in their carbon source, with G. diazotrophicus preferring sucrose and H. rubrisubalbicans preferring aconitate. biological nitrogen fixation, diazotrophs, endophytic bacteria, Gluconacetobacter diazotrophicus, Herbaspirillum rubrisubalbicans, N2-fixing endophytes, stem apoplast solution, sugarcane INTRODUCTION Sugarcane is one of the most important agricultural crops in the Philippines (Espino 1961), in most tropical countries (Nickell 1983), and even in the subtropical region of Japan (DAFF 1977). Sugarcane is grown not only for the raw or refined sugar but also as source of more than 150 by- products (Paturao 1988). Thus, improved sugarcane yields can contribute much to the economy of a country. In this area, even as a non-legume, sugarcane can benefit from biological nitrogen fixation (BNF). BNF, which is the conversion of atmospheric nitrogen (N2) into plant-available form by N2-fixing bacteria, helps sustain crop production by providing the host plant an inexpensive source of nitrogen (N). The enzyme nitrogenase, contained in the cells of N2-fixing bacteria (diazotrophs), catalyzes the reduction of the inert N2 molecule, which comprises 78% of the air, into an available form of N (Postgate 1998). The host plant obtains N from BNF directly in association or symbiosis with diazotrophs, or indirectly, after the death and mineralization of those diazotrophs (Hurek & Hurek 1998). The forms of BNF occurring in nature are Comment [TAM Tony