only treatment that gave consistent responses in N2-fixation and grain yield (Ssali, 1988; Amijee and Giller, 1998; Gilleret al., 1998). In Tanzania, the improvement in growth ofPhaseoluswhen phosphorus was added revealed that the ability of the soil to supply potassium was chronically deficient, and small potassium additions doubled grain yields in farmers’ fields (Smithsonet al., 1993).
Where P. vulgaris has responded to inoculation this is where effective, compatible rhizobia were absent, or present only in small numbers (Amijee and Giller, 1998). Absence of compatible rhizobia is particularly unlikely in the case of P. vulgaris, due to its promiscuity of nodulation. Although for a long time it was presumed thatP. vulgarisnodulated only withR. leguminosarumbv.phaseolistrains, it is now known that it can nodulate with many different species of rhizobia (see above). The wide range of rhizobia able to infectP. vulgarisincreases the likelihood that nodulation may occur with strains that are ineffective or poorly effective in N2-fixation. Further research is required to clarify the particular reasons why nodulation is poor in different environments, but there is no clear evidence to suggest thatP. vulgarisis particularly unusual in its response to inoculation.
Potential for strain selection for grain legumes
The vast majority of rhizobial strains that are recommended for use as inoculants have not been subjected to rigorous selection in soil in competition with native strains. Thus inoculation is rarely beneficial if populations of effective, compatible rhizobia are already present in the soil (Singleton and Tavares, 1986) but, despite the poor record of success, there remains considerable scope for strain improvement in the future (Chapter 14). A large proportion of rhizobia indigenous to Kenyan soils were more effective in N2-fixation withP. vulgaris than the widely recommended strain CIAT899 (Anyangoet al., 1995). Selection of adapted strains forP. vulgaris sown directly in pots of soils containing large populations of indigenous, compatible rhizobia has resulted in many cases in yield increases when these strains were tested in the field (Pineda and Kipe-Nolt, 1990; Sylvester-Bradley and Kipe-Nolt, 1990).
In some cases particular strains have shown promising results with grain legumes considered to be highly promiscuous in nodulation with rhizobia. An interesting example is found with groundnut, where a particular cultivar (Robut 33–1) gave increased pod yields on inoculation with a specific strain, Bradyrhizobium sp.
(Arachis) strain NC92 (Nambiar et al., 1984). Several experiments at a range of locations in India indicated the advantage of this host/strain combination but later work failed to achieve significant increases in yield and interest in this combination appears to have waned.
capacity to nodulate and fix N2. For instance, large differences in nodulation in the field have been shown between genotypes of cowpea (Zaryet al., 1978),P. vulgaris (Graham, 1981), groundnut (Nambiar et al., 1982), chickpea (Rupela and Dart, 1980), pigeonpea (Kumar Rao and Dart, 1987; Rupela and Johansen, 1995) and soybean (Neuhausenet al., 1988). But apart from the case of breeding for promiscu- ity of nodulation in soybeans discussed above, there have been few concerted efforts to enhance the potential for N2-fixation in grain legumes through plant breeding.
In groundnut, the Virginia types were shown to have greater capacity to nodulate and fix N2than the Spanish or Valencia types (Arunachalam, 1984; Giller et al., 1987). Recombination of the ability to form a large mass of nodules was demonstrated in a series of crossing experiments at ICRISAT in India (Nigamet al., 1985) but this work has not been pursued. In Australia, the rates of N2-fixation in Virginia and Spanish types were similar and greatest amounts of N2 were fixed by long-duration genotypes (Bell et al., 1994). Greater N2-fixation in groundnut varieties was found to be due mainly to better light interception, suggesting that alterations of row spacing to optimize light interception would give more immediate benefits of increased N2-fixation than breeding (Williamset al., 1990). A breeding programme to combine the high N2-fixation ability of some cowpea genotypes into well-adapted, high-yielding varieties was established (Miller and Fernandez, 1985;
Milleret al., 1986) but appears not to have been pursued.
Breeding for enhanced N2-fixation inP. vulgaris
At CIAT a programme of crossing and recurrent selection was begun to increase the contribution from N2-fixation inP. vulgaris(Graham, 1981; Graham and Temple, 1984). The effort was concentrated on improving N2-fixation in small-seeded bush beans of a similar maturity group and materials were screened for nodulation, ARA, total N and grain yield under glasshouse conditions before promising materials were advanced for screening in the field. An evaluation of materials produced from this programme (designated RIZ lines) in the field in Colombia indicated that the RIZ lines generally nodulated better and fixed more N2than the early parents used in the breeding programme (Kipe-Nolt and Giller, 1993). However, when compared with otherP. vulgarisgenotypes from CIAT breeding programmes, the RIZ lines were no better in N2-fixation than several of the other promising lines that had not been selected for N2-fixation potential. In this comparison the same genotypes were grown at two different sites in the Cauca valley in Colombia in two seasons and the ranking of genotypes for N2-fixation was remarkably consistent, although the absolute amounts of N2 fixed varied enormously between the separate experiments (Kipe- Noltet al., 1993). The soils in which this bean breeding programme was carried out in Colombia are rich in N and one can only speculate as to whether it would have been more successful with a more severe selection pressure for N2-fixation. The same recurrent selection method gave marked improvements in N accumulation over three generations in N-limited soils (Barronet al., 1999). A programme to select varieties for adaptation to infertile soils in Africa (Wortmannet al., 1995) has been
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remarkably successful in improving yields in farmers’ fields, at least in part due to enhanced N2-fixation.
A different approach to the improvement of N2-fixation in P. vulgaris was adopted with the aim of improving N2-fixation in ‘Sanilac’, a poorly nodulating, white-seeded, determinate (Type I) variety that is widely grown in the USA. A back-cross method was used to combine the characters of ‘Sanilac’ with ‘Puebla 152’, an abundantly nodulating, black-seeded, indeterminate bush bean (Type IIIB) from Mexico. Selection criteria used were visual nodule scores and ARA. The lines selected were of intermediate character: all formed more nodules and showed greater capacity to fix N2than the poorly nodulating parent ‘Sanilac’, but none nodulated or fixed N as well as ‘Puebla 152’ (Dubois and Burris, 1986; Rosas and Bliss, 1986; Pereiraet al., 1989). Thus N2-fixation was increased by crossing two genotypes with widely differing abilities to nodulate and fix N2 but, as might be expected, when the two parental genotypes were only slightly different in their ability to fix N2, success in improvement of N2-fixation was limited (St Clair et al., 1988). The back-cross method is, however, a useful method to improve N2-fixation in genotypes that nodulate poorly but otherwise have good agronomic characteristics (Bliss, 1993).
After 15 years of screening in Queensland, Australia, in which a total of 1500 genotypes of P. vulgaris were evaluated, two were identified that fixed roughly 30% more N than commercial check cultivars (Redden and Herridge, 1999). These genotypes, however, lacked disease resistance and responded strongly to N fertilizer, which indicated that the supply of N from N2-fixation was inadequate. On the basis of these results, Redden and Herridge (1999) concluded that prospects for reliance on N2-fixation in P. vulgaris were limited in the short term and that recommendations for use of N fertilizer should remain.
Breeding for enhanced N2-fixation in soybean
A programme designed to improve the ability of soybean to fix N2 in fertile Australian soils rich in nitrate was established in 1980 (Herridge and Rose, 2000). Of some 500 soybean varieties that were screened in glasshouse and field experiments, a group of varieties from Korea was able to form many more nodules and fix N2when grown in soils with an abundant nitrate supply (Betts and Herridge, 1987; Herridge and Betts, 1988). Screening of progeny from crosses between the nitrate-tolerant genotypes and other high-yielding varieties, using a non-destructive ureide assay on part of the plant shoots, led to the identification of lines with clearly enhanced nitrate tolerance (Herridge and Rose, 1994). The effectiveness of this approach in improving the efficiency of N use in cropping systems depends on any nitrate that is not used by the legume crop being retained in the soil. It can be argued that a legume that is able to utilize mineral N when it is available, but rely on N2-fixation when soil N is limiting, is ideal, and this questions the rationale for selecting legume varieties that are able to fix N2in the presence of large concentrations of nitrate (Chapter 14).
Selection criteria
Obviously, to conduct a breeding programme, clear selection criteria are required.
The acetylene reduction assay was frequently used as a selection criterion in breeding for improved N2-fixation in the past, but the method is now known to be inaccurate.
The extent of nodulation, assessed as nodule number, nodule mass or simple nodule scores, has also been used as a criterion and is sometimes correlated with total N2
fixed, at least in soils poor in N. Total N accumulation, which is a good indication of the total amount of N2fixed, at least in soils with a poor capacity to supply combined N (Chapter 4), is probably the best broad criterion for selection programmes in the tropics. In soybean, ability to fix N2appears to be closely related to early formation of nodules, a simple criterion which could be used in breeding (Pazderniket al., 1996, 1997a,b).
A large number of plant characters contribute to N2-fixation and it is thus important to clarify whether the plant selection can discriminate between plants with a real ability to nodulate and fix N2better consistently in the field and plants that are simply more vigorous, and thus nodulate better, under a given environment. Genetic adaptation to specific environments can complicate the selection of genotypes with enhanced N2-fixation. This is particularly apparent in P. vulgaris. Rio Tibagi, a genotype considered to have a poor capacity to nodulate and fix N2in Brazil (Duque et al., 1985), nodulates and fixes N2 as well as some of the genotypes specifically selected for N2-fixation in Colombia (Kipe-Nolt and Giller, 1993). The genotype Puebla 152, which nodulates profusely in Colombia and has been used as a parent line with good nodulation in breeding for enhanced N2-fixation (Rosas and Bliss, 1986), was found to be among the poorest nodulators when many genotypes of P. vulgariswere compared in Queensland, Australia (Reddenet al., 1990). This is perhaps not surprising given the enormous breadth of environmental adaptation withinP. vulgaris, but it emphasizes the need either to breed specifically for local environments, or to screen genotypes across the wide range of environmental conditions that they are likely to encounter in the field. This is especially true given the unpredictability of the climate in many parts of the tropics.