Ronald J Yates

In this chapter, we discuss methodologies to select rhizobial strains for their ad¬aptation to field conditions as this is an important trait when developing inocu¬lant-quality strains. Techniques to assess nitrogen fixation per se are described in Chapters 5 and 10, and traits related to manufacturing in Chapter 9. For many legume symbioses, the greatest challenge in developing inoculants is to select strains that confer consistent nodulation upon the legume in the target soil environment. Edaphic stresses after inoculation, such as dry heat and acidity, can be detrimental to rhizobial survival, so selecting strains tolerant of these stresses is very useful. Some legumes regenerate annually from hard (impermeable) seeds in the soil and therefore, like their rhizobia, are only sown once in several dec¬ades. Hence, assessment of the ability of strains to persist in the field environment through several seasons is required. This trait cannot be reliably predicted without field experimentation (Howieson et al. 2000). Many legume evaluation programs over the years have been compromised because inoculants suitable to the field conditions have not been available.

After strains of rhizobia have been isolated from nodules (Chapter 3), and (ide¬ally) before long-term preservation (Chapter 4), the strains should be examined to ensure they retain the essential features of nodule bacteria. The first step in this process is termed ‘authentication’, which examines the ability of the strain to in¬fect a legume to form a nodule. Following this, strains may be evaluated for their ability to fix nitrogen. This latter characteristic is sometimes termed ‘effectiveness’; it is an assessment of the genetic compatibility between the host plant and the rhizobium strain for nitrogen fixation. If a strain can nodulate a legume and fix N2 effectively in the glasshouse environment, the researcher may wish to proceed further, to assessment in the field. However, if the strain is to be released to the field, then ‘duty of care’ requires that we have an understanding of its host-range (Section 5.2). This is because releasing strains into the general environment that might be detrimental to existing legumes either agricultural or natural would be negligent. The techniques described in this chapter allow a researcher to compare strain symbiotic performance across a spectrum of plant genotypes to fulfil this duty of care.

For the legumes widely used in commerce, rhizobial strains that are well matched to these for nitrogen fixation (i.e. highly effective at N2 fixation) are usually avail¬able from manufacturers or from gene banks. Some of these sources are listed in Table 2.1. However, new acquisitions of rhizobia may be required for many reasons, for example: to overcome poor N2 fixation from an existing symbiotic relationship as described in Case 2, Chapter 1; to select well-adapted strains for a difficult environment (e.g. acid soil); to assist in legume domestication programs; to match with sequenced legumes for genetic studies of N2 fixation (e.g. Terpolilli et al. 2008); or to undertake biodiversity studies. If researchers feel that currently available strains may not satisfy their research program, then a broader range of rhizobium germplasm must be sought. Strains of rhizobia for many legumes have long been collected from their natural envi¬ronments for this purpose. These environments represent in situ repositories of rhizobium genetic resources (Date 1982). After the collection, isolation and eval¬uation of new strains they should be deposited in curated gene banks. The bulk of this chapter covers methods to acquire nodule bacteria from in situ sources.