Use of the Chrome Azurol S Agar Plate Technique To Differentiate Strains and Field Isolates of Rhizobium leguminosarum biovar trifolii

  • Ames Gottfred N
  • Christie B
  • Jordan2 D
  • 39


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Identification of Rhizobium and Bradyrhizobium strains and especially of indigenous isolates continues to be one of the major difficulties associated with competition studies. Because there is no universally accepted method, the method of choice depends on preference, experience, and equipment. Here, an agar plate technique was used to distinguish strains and field isolates of Rhizobium leguminosarum biovar trifolii to provide a basis for identifying nodule occupants in further competition studies. A rapid plate technique, based on differential growth characteristics, complements other techniques such as serological reactions, particularly when antisera cross-react with nonhomologous strains. The technique involves culturing strains and isolates on chrome azurol S agar. Although similar responses were observed among some strains, the response was highly reproducible and was considered an ideal complementary technique used in conjunction with serological procedures. Strains with similar responses could often be differentiated by varying media components, such as the source of carbon. Studies involving effectiveness of legume-Rhizobiuml Bradyrhizobium associations or bacterial strain competition are complicated by the problem of identification of strains present in nodules under both controlled and field condi-tions. For determinations of effectiveness and competitive ability of strains in mixed inoculum or with indigenous populations there is a need for suitable methods of identify-ing strains recovered from the nodules. Such methods must be reliable and rapid enough to be applied to a large number of strains. Various methodologies have been used to differentiate strains of the root nodule bacteria. Among these, serological techniques such as agglutination reactions, immunodiffu-sion, immunofluorescence, and enzyme-linked immunoab-sorbance have been widely used (1, 6, 7, 10, 11, 17). Since serological techniques rely on reactions with antisera raised against laboratory strains, they do not provide information on strains or isolates that do not react with the antisera available. Therefore, when dealing with large numbers of strains or unknown indigenous populations, serology alone can be limiting. Wide use has also been made of induced and intrinsic antibiotic resistance (2). However, loss of effective-ness (13) and competitive ability (3) has been associated with induced antibiotic markers, and intrinsic antibiotic resis-tance has been considered unreliable for some Rhizobium species (16). Modified intrinsic antibiotic resistance tech-niques have been applied successfully to a small number of Rhizobium strains (4, 14). However, these methods are not

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  • N P Ames Gottfred

  • B R Christie

  • D C Jordan2

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