Graham O'Hara

The use of rhizobiumRhizobium inoculants for improvement in nitrogen-fixationNitrogen and productivity of grainGrainlegumesLegume has been well established in developed countries. However, the practice is still under-utilized in NigeriaNigeria. NitrogenNitrogen (N) is the most frequently deficient nutrient for crop production, while nitrogenNitrogenfertilizersFertilizers are costly, inadequate, and may not be timely in supply. These make rhizobia inoculants a cheaper, easier and safer option to improve the N2-fixation and productivity of grainGrainlegumesLegume. Inoculant use in NigeriaNigeria was initiated in the 1970s, but still remains very limited. Studies conducted on inoculant use were initially on “US type” SoybeanSoybean (Glycine max (L.) Merrill), which has been found to require specific inoculation with Bradyrhizobim japonicum for optimum productivity. Studies were also conducted on inoculation of cowpeaCowpea (Vigna unguiculata (L.) Walp), but rarely on bambara groundnutGroundnut (Vigna subterranea (L.) Verdc.) and groundnutGroundnut or peanutPeanut (Arachis hypogaea L.). In the 1980s, the International Institute of TropicalTropicalAgricultureAgriculture (IITA) Ibadan, NigeriaNigeria, introduced promiscuous soybeanSoybeancultivarsCultivars; TropicalTropical Glycine Cross (TGx). These genotypesGenes nodulate freely with the indigenous rhizobiumRhizobium population, fix large amount of atmosphericAtmospherenitrogenNitrogen and produce higher grainGrainyieldsYields than the localLocal genotypes. However, some experiments indicated up to 40–45% increases in yieldYields by some of the genotypesGenes on inoculation. Hence, the ultimate solution remains the development of inoculants using highly effective indigenous rhizobia strains for particular crops. The recent efforts of the project “Putting NitrogenNitrogenfixationNitrogen fixation to work for smallholder farmersFarmers in Africa (N2Africa)Africa” towards the promotion of inoculants technology are highly welcomed in the country.

Experimentation with strains of rhizobia can last for many decades; hence there must be a reliable and efficient means of storing the bacteria. While many re¬positories of rhizobia have been developed since the symbiosis was scientifically understood, few remain available for exploitation. This is because strains were commonly stored on agar and remained the responsibility of an enthusiast, who may not necessarily have been replaced by his institution upon retirement. Agar slope-borne cultures have a relatively finite life. For this reason we recommend long-term preservation of valuable cultures lyophilized in glass tubes which will ensure survival over long periods of inattention.

The enumeration of rhizobia is valuable for the assessment of rhizobial populations in soil and how they vary, to follow the growth of cultures in the laboratory or to assess the number and viability of rhizobia in commercial inoculants for quality control. The number of rhizobia in soil is dynamic and varies within and between seasons, so any enumeration must be set in context. Enumeration of rhizobia is la¬bour and resource intensive and should only be undertaken when the information is vital. There are few new techniques with which to quantify rhizobia. Despite the mo¬lecular and genomic eras, there has emerged no robust DNA-based technology to replace serial dilutions and direct or indirect quantification. The qPCR and MISEQ methodologies cannot currently differentiate rhizobial strains reliably nor distinguish sufficiently between DNA from live cells and DNA from dead cells to make them effective substitutes for the standard viable counting techniques dis¬cussed below, although this may change. This chapter, therefore, reiterates appro¬priate and well-utilised approaches to enumeration of rhizobia in soil, inoculants and in vitro.

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.

This chapter describes basic techniques for the isolation and growth of rhizobia, some of which have been used for more than a century. While these techniques re¬tain their importance, the success of current and future rhizobiology studies and enterprises will depend on the training, skills and techniques described in this chapter. A note of caution: nodules (particularly those collected from the field) are not always occupied by a single rhizobial isolate nor even by a single micro-or¬ganism. Nodules of pea and lupin, for example, have been described as containing both the nitrogen-fixing symbiont and associative organisms such as Micromono¬spora (Trujillo et al. 2010). Hence, we must be prepared for a range of organisms to appear on growth plates during isolation procedures. Recognition of rhizobia when growing on a solid medium is an essential skill in rhizobiology.

The Medicago genus is of global importance to agriculture, with the perennial M. sativa being the most widely cultivated and studied member. After many years of studying this plant along with its microsymbiont Sinorhizobium meliloti, it became clear that another host was required to allow simultaneous study of the genetic determinants of both symbiotic partners.M. sativawas unsuited to this role as it is autotetraploid, allogamous and shows strong in-breeding depression, making the analysis of recessive mutations no easy task. Researchers identified the annual medic M. truncatulaas a viable alternative as this host is diploid, autogamous and possess a rapid generation time, among other traits. Consequently, this organism was chosen for sequencing.

The emergence of biodiversity in rhizobia after the introduction of exotic legumes and their respective rhizobia to new is a challenge for contemporary rhizobiology. Biserrula pelecinus L. is a pasture legume species that was introduced to Australia from the Mediterranean basm and which is having a substantial impact on agricultural productivity on acidic and sandy soils of Western Australia and New South Wales (Howieson et al., 2000). This deep-rooted plant is also valuable in reducing the development of dryland salinity This legume is nodulated by a specific group of root-nodule bacteria that belongs to Mesorhizobium (Nandasena et at , 2001, 2007).

The Shark Bay World Heritage Property (SBWHP) is a transition zone between the South West and Eremaean Biogeographic regions and was listed in 1991 due to its great geological, botanical and zoological importance (UNESCO, 2002). Shark Bay Salt is located within the SBWHP. Its lease area includes Useless Inlet and Useless Loop and there has been salt-mining production since 1965 (EPA, 1991). Over this time, borrow pits have been mined in areas surrounding the evaporation ponds, the majority of which were decommissioned over 15 years ago. Many of these pits remain in a highly disturbed state when compared to the surrounding undisturbed flora (Figure 1a, b).