Ravi Tiwari

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.

Working with DNA is now a fundamental skill in working with rhizobia. It is necessary for typing strains using PCR methods and for sequencing activities ap¬plied to understanding genomes; their structure, how they function, and their taxonomic position. Nucleic acid purification is the separation of nucleic acids from proteins, cell wall debris and polysaccharide after lysis of cells. For rhizobia, we provide here a num¬ber of commonly used methods for the extraction of genomic and plasmid DNA. Methods for extraction of total RNA are presented in Chapter 13. The CTAB method (Protocol 11.1.1) has been used extensively for extraction of total genom¬ic DNA for DNA sequencing while Protocol 11.1.2 gives higher yields but gener¬ally with slightly lower purity. Plasmid DNA can be differentially displayed using Protocol 11.2.1 for determination of replicon number. This method allows locali¬sation of genes to replicons, confirmation of genome assemblies and identification of genetic changes. The plasmids can subsequently be purified from low melting point gels using GELase (Epicentre, http://www.epibio.com/item.asp?id=297). Protocol 11.2.2 presents a method to recover introduced plasmids from rhizobia (i.e. complementing plasmids) for transformation into Escherichia coli prior to restriction analysis. Protocol 11.2.3 provides an alternative method to the GELase procedure for purifying plasmids but has not been tested as extensively.

The aim of this chapter is to provide a selection of specialised genetic techniques which are widely used to interrogate gene functions in root nodule bacteria (RNB). The reader is referred to reviews of the applicability of these techniques to understand biological processes occurring in RNB (i.e. Long 1989; Stanley and Cervantes 1991). The techniques presented here cover the ability to randomly mutate genes, select mutants of interest based on their phenotype and identify the gene affected, verify that the gene mutation caused the observed phenotypic defect, examine expression of a target gene, perform genome structural and func¬tional studies and conduct comparative analyses with other RNB.

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).

Biological nitrogen fixation, especially via the legume-Rhizobium symbiosis, is important for world agriculture. The productivity of legume crops and pastures is significantly affected by soil acidity; in some cases it is the prokaryotic partner that is pH sensitive. Growth of Rhizobium is adversely affected by low pH, especially in the acid stress zone. Rhizobia exhibit an adaptive acid tolerance response (ATR) that is influenced by calcium concentration. Using Tn5-mutagenesis, gusA fusions and proteome analysis, we have identified a range of genes that are essential for growth at low pH (such as actA, actP, exoR, actR and actS). At least three regulatory systems exist. The two-component sensor-regulator system, actSR, is essential for induction of the adaptive ATR. Two other regulatory circuits exist that are independent of ActR. One system involves the low pH-induced regulator gene, phrR, which may control other low pH-regulated genes. The other circuit, involving a regulator that is yet unidentified, controls the expression of a pH-regulated structural gene (lpiA). We have used pH-responsive gusA fusions to identify acid-inducible genes (such as lpiA), and then attempted to identify the regulators of these genes. The emerging picture is of a relatively complex set of systems that respond to external pH.