Rhizobia occurring naturally in soils may be able to infect legume roots and form nodules, however the effectiveness in terms of nitrogen fixation may be highly variable (Pepper, 1991). Somasegaran and Hoben (1994) stated that nodules with a pink colour infer an effective nodule, hence active leghaemaglobin, whereas white or greenish nodules infer ineffective symbiosis. The inoculation of seeds or legume roots prior to planting with effective and competitive rhizobia may help improve host/rhizobia interactions and nitrogen fixation. The development of effective inoculants for particular legumes is very important, however, the rhizobia need to be suited physiologically to the inherent soil conditions in which they will be expected to survive and persist (Pepper, 1991). Attention to the Rhizobiumllegume association
can improve the productivity of symbiosis in semiarid regions (Pepper and Upchurch, 1991).
Brockwell et al. (1982), and Somasegaran and Hoben (1994) described some of the
various methods that may be used for the selection of rhizobia as inoculants, from the use of growth cabinets to field trials. Wilson et al. (1995) stated that in glasshouse
experiments the differences between inoculated and un-inoculated plants could be clearly seen due to differences in nitrogen availability. However, the repetition of these findings in the field is difficult due to two main factors, the inability to remove all nitrogen from the soil and the interactions with other introduced and background rhizobia.
8.7.1Considerations for Rhizobial Studies
The investigation of populations of rhizobia has been driven by ecological and agricultural considerations, where the symbiotic functions of legumes are often
overlooked by agronomists when investigating aspects of legume growth in the field (Brockwell et al., 1995). Howieson et al. (2000c) stated that in order to effectively
harness biological nitrogen fixation (BNF), the interaction between plant and bacterial genotypes, and soil conditions needs to be understood. This is further complicated as the genotypes of both the symbiont and host need to be considered and therefore, the many interactions that can occur need to be understood. Howieson
et al. (2000c) described the notion of a G2 (genotype) X E (environment) interaction
for legumes and rhizobia introduced to new environments. This interaction considers the three way interaction between host genotype, bacterial genotype and the environmental conditions.
To gain a greater understanding of the ecology of rhizobia in soil colonisation studies are essential. The complexity of the relationship between plant and Rhizobium with
environmental factors (both soil and climatic) often means that each individual situation needs to be examined (Chatel and Greenwood, 1973). The determination of the best method of inoculation is necessary prior to planting, as poor inoculation may be reflected in poor seedling establishment (Wright, 1985). The successful nodulation and subsequent fixation of nitrogen following sowing is reliant on the inoculation of legumes with the appropriate strain of Rhizobium (Wills, 1986), where
an effective symbiosis generally confers a beneficial plant response (Allen and Allen, 1981). Brockwell et al. (1982) stated that when selecting rhizobial strains for testing,
strains associated with the host should be selected, as, in a mixed culture situation there will be preferential selection for more effective strains.
8.7.2Rhizobia Strain Selection
Knowledge of the hosts preferences for rhizobia might help in the selection of superior inoculum in the field and assist in avoiding competition between inoculum and background rhizobial populations. Strains could be selected so that nodules formed by legumes are dominated by effective rhizobia (Vincent and Waters, 1953). Howieson et al. (2000a) described a four stage strain selection process that is used
Stage 1: Strain Isolation
The first stage of strain selection involves the excavation of plants in their natural environment and removal of nodules. Specialised media is then used to culture bacteria isolated from the nodules. Colonies displaying the typical characteristics of rhizobia i.e. slightly raised, opaque and entire, are then selected for purification.
Stage 2: Strain Nitrogen Fixing Ability
The second stage of strain selection as described by Howieson et al. (2000a) involves
the determination of host/symbiont compatibility via the examination of nitrogen fixation. The undertaking of this screening process makes three assumptions; nitrogen is the only limiting factor in the screening environment, host/rhizobia interactions are expected and elite strains under examination must not compromise existing agricultural legumes. The detailed methodology is described by Howieson et al. (1995 and 2000a and b).
Stage 3: Field Evaluation
The third stage of strain selection involves the examination of strain survival, persistence and migration in soils of interest. This type of trial is commonly referred to as a cross row experiment. Inoculated plants are established in accessions with buffer rows and are supplied with all macro and micro-nutrients required, except nitrogen. Plants are grown through the winter and are removed in the following summer. Seeds are then planted the following autumn across the original rows running perpendicular (see Chapter 10, Figure 10.1). After 10-12 weeks, plant shoots and roots are excavated, with shoot DM determined and root nodulation patterns observed.
Stage 4: Rhizobial Survival
The final stage of strain selection is the in situ assessment of nitrogen fixation,
nodule formation and plant production. Following stage 3, a break crop is planted in order to reduce the soils nitrogen status and expose the rhizobia to the stresses of living as a saprophyte.
8.7.3Desirable Strain Characteristics
Howieson et al. (2000c) stated that the following characteristics are required for
quality rhizobial strains; 1: Host/rhizobia compatibility examined via nitrogen fixation studies as described by Howieson et al. (1995 and 2000a and b). 2: The
ability to produce consistent nodulation patterns in field soils using methods as described by (Howieson et al., 2000c). 3: Rhizobial strain genetic stability is a very
important and desirable characteristic. Genetic drift via subculturing using rich medium is highly undesirable, and therefore the production of genetically stable strains is desirable. 4: The survival of rhizobia during commercial production and the exposure to physiological stresses during culturing, such as acidity, can help improve acid tolerance in the field. 5: The ability to compete effectively with the indigenous soil micro-flora is also highly desirable.