Acacia koa is a large, evergreen nitrogen fixing tree in Hawaii but very little is known about the nature of its root nodule bacteria. The project will determine genetic diversity in bradyrhizobia nodulating Acacia koa trees in different Hawaiian Islands. The project will isolate and identify a few most effective Bradyrhizobium isolates for nodulation of A. koa.

OBJECTIVES

1. Acquire and characterize Bradyrhizobium or Rhizobium isolates from nodules of Acacia koa, determine the nitrogen-fixing potential of the Bradyrhizobium isolates on A. koa, and determine important physiological, symbiotic and molecular characteristics of selected isolates.
   
   

APPROACH

1. Approximately 200 to 300 isolates of root nodule bacteria will be obtained from the nodules of koa growing in different environments in Hawaii. Nodules will be sampled from juvenile plants by carefully excavating around the base of the stem until roots with attached nodules are located. Isolates will be evaluated for growth rate on YEM medium and acid or alkaline reaction on YEM containing bromothymol blue. Isolates that grow slowly on YEM agar and show alkaline reaction will be presumed to be Bradyrhizobium. All Bradyrhizobium isolates selected for further characterization will be authenticated as Bradyrhizobium by inoculating root systems of Macroptilium atropurpureum with a suspension of each isolate and examining their root systems for the presence of nodules 7 to 10 days after inoculation. General colony morphology resistance to a number of common antibiotics and the ability to utilize different carbon and nitrogen sources by the isolates will be determined. The results of these assays will establish the general characteristics of the isolates that nodulate A. koa and their differences from those that nodulate other Acacia species. The Bradyrhizobium isolates will be grouped into phylogenetic clusters on the basis of RFLP of the 16S rRNA gene and through RAPD analysis using GC rich primers. Heterogeneity among the isolates will also be examined through RAPD analysis using GC rich primers. It is expected that the root nodule isolates from koa will be distinct from other strains of bradyrhizobia and rhizobia. Based on the RFLP and RAPD analyses, the Bradyrhizobium isolates from koa nodules will be classified into groups. In this way, we may be able to classify 200 isolates into 50 or fewer number of groups. One representative isolate from each group will be subject to further molecular analyses. The isolates that are identified as diverse at the end of molecular analyses, will be tested for nodulation and nitrogen fixing potential on A. koa. The plants will be grown for ten weeks and the effectiveness of nodulation will be judged from number and weight of nodules, total dry weight of plant, and total nitrogen content. Based on these results, we will select 10 to 15 of the most effective strains for A. koa. The 10 to 15 isolates that are selected on the basis of the inoculation experiment will be characterized in detail for physiological, symbiotic and molecular characteristics. These isolates include the 3 to 5 best isolates selected at the end of the inoculation experiment. A well characterized B. japonicum strain such as USDA110 and a B. elkanii strain such as USDA74 will be included in these experiments.