SECTION III Evaluating SYDlbiotic Potential of Rhizobia SIGNIFICANCE OF SYMBIOTIC NITROGEN FIXATION TO AGRICULTURE The value of legumes in improving and sustaining soil fertility was well known to agriculturalists, but it was the work of Lawes and Gilbert in 1891 which showed that legumes had the inherent ability to add nitrogen to the soil. In 1888, Hellriegel and Wilfarth demonstrated that nitrogen gains in peas (Pisum sativum) took place only in the presence of soil microorganisms and that the root nodules of legumes were necessary to the process. Finally, in 1888, Beijernick isolated the N 2-fixing bacteria in the root nodules. The names Rhizobium and Bradyrhizobium are now given to these organisms. The symbiotic association between legumes and rhizobia is by far the most important contributor to the world's supply of biologically fixed N2 to agriculture. Effective symbiosis can only be achieved when the nodules are formed by efficient and effective rhizobia. Researchers now accept the term symbiotic effectiveness to describe the ability of a nodulated legume to fix N 2, and this can be expressed qualitatively (high, moderate or intermediate, ineffective) or quantitatively (e.g., total plant N, shoot or nodule dry weight). Quantitative symbiotic effectiveness is measure<;l by comparison with the performance of standard rhizobial strains, with reference to the legume receiving adequate mineral N, or with noninoculated legumes. Efficiency is expressed as a qualified rate, such as milligrams of N2 fixed per gram of nodule weight. Effectively nodulated legumes can fix substantial amounts of N2. Estimates of N2 fixation have been made for various legume-rhizobial symbioses. Examples of the amounts of N2 fixed (kilograms of N2 fixed per hectare) by some legume-rhizobial symbioses are as follows: alfalfa (Medica go sativa), 125-335; red clover (Trifolium pratense), 85-190; pea (Pisum sativum), 80-150; soybean (Glycine max), 65-115; cowpea (Vigna unguiculata), 85; sweet clover (Melilotus sp.), 100-150; faba bean (Vida faba), 240-325; peanuts (Arachis hypogaea) 50; mung bean (Vigna radiata), 55; and lentils (Lens culinaris), 100. The symbiotic N2 fixation process and its potential for increasing protein production by legume inoculation is one of the means of providing improved nutrition in developing countries. Pigeon pea or red gram (Gajanus cajan) is a major food (pulse) legume in India, while chickpea (Gicer arietinum) is the third most widely grown grain legume in the world and is very important in the semiarid tropics. The common bean (Phaseolus vulgaris) is
166 EVALUATING SYMBIOTIC POTENTIAL OF RHIZOBIA an important source of dietary protein in many of the Latin American countries. Soybean has become a widely accepted crop in many parts of the world because of its food value, both for humans and animals. Besides their importance as a source of fodder and food, tree legumes play an important part in agroforestry and cropping systems. Species that are significant in agroforestry include Leucaena Ieucocephala, L. diversifolia, Gliricidia sepium, various species of Acacia, Sesbania rostrata, S. grandiflora, and numerous others. THE NEED TO INOCULATE LEGUMES The benefits of symbiotic N2 fixation by legumes can only be appreciated when the major crop legumes in a country show responses to inoculation, as shown by yield increases in experimental field trials and in farmers' fields. Demonstrating the inoculation response is the necessary first step in the adopting symbiotic N2 fixation technology for legume production, leading to the development of inoculant production capability. An economic analysis comparing costs and benefits of N fertilizer applications versus inoculation is also needed. The inoculation practice is warranted whenever a new legume is being introduced, especially in areas where there are no indigenous species belonging to the same crossinoculation group as the introduced legume. Inoculation is also recommended if a field has not been cropped to a legume in the past 3-4 years, or if temperature extremes or other soil conditions are likely to decrease rhizobial populations in the soil. SOME FACTORS AFFECTING INOCULATION RESPONSE Since N2 fixation by legumes is a symbiotic process, environmental factors that affect the host legume and the rhizobia must be optimal for establishing an effective N 2-fixing symbiosis. Soil ph is an important environmental factor. Many legumes respond to liming when grown in acid soils. Many legumes will grow and nodulate well at soil ph 5.6-6.8. For the rhizobia, optimal ph levels for growth in culture are variable (ph 5.8-7.2), depending on the rhizobial species. Generally, tropical rhizobia survive well in soils with ph 5.8-6.8. Aluminum and Mn toxicities are likely to be encountered in many tropical soils with soil ph 4.5 or less. Both plant roots and nodulation are adversely affected. Shortage of P will severely limit the formation of nodules and N2 fixation. Therefore, researchers selecting effective rhizobia in field soils must consider adequate P fertilization. Soil N2 (NO:;) has an inhibitory effect on the nodulation and N2 fixation of the legumerhizobial symbiosis. The size, effectiveness, and competitiveness of the native or indigenous rhizobial population are also important factors that influence the ability to achieve increased crop yield through inoculation.
Evaluating Symbiotic Potential of Rhizobia 167 Molybdenum is an essential micronutrient to all plants and is required for the formation and function of the nitrogenase enzyme complex. Soils deficient in Mo produce poor and ineffectively nodulated legumes. SPECIFICITY IN THE SYMBIOSIS The legume-rhizobial symbiosis exhibits widely differing degrees of specificity. In some instances, the symbiosis is highly specific in that a particular species or strain of Rhizobium or Bradyrhizobium can form an effective symbiotic association with only one particular legume species or variety. This category includes the temperate legumes Trifolium, Cicer, Phaseolus, and Medicago, and tropical species like Glycine max, Leucaena, and Lotononis. There are also intermediate cases that exhibit varying degrees of crossinoculation capability, as in Centrosema, Phaseolus acutifolius, P. lunatus, some Desmodium spp., and Acacia spp. At the opposite extreme are the promiscuous associations, in which diverse legumes may be infected by one or more of several rhizobia. This condition is more prevalent in the tropical legumes than in the temperate species. Because the earlier studies of symbiotic N2 fixation were initiated in temperate regions, the taxonomy of the genera Rhizobium and Bradyrhizobium was based on a host-dependent classification system that emphasizes temperate associations (see Section I). Several tropical rhizobia that form symbiotic associations with Vigna, Macroptilium, Arachis, Cajanus, Lablab, and other genera of legumes are simply labeled as the "cowpea miscellany" or Bradyrhizobium spp. In some cases, it is desirable to select a strain for a wide range of hosts. An example would be the Bradyrhizobium sp. (CB756; TAL 309) isolated from the nodule of Macrotyloma africanum. This strain effectively nodulates approximately 40 of the promiscuous tropical legumes. This broad-spectrum-strain characteristic would be advantageous if this superior strain of Bradyrhizobium sp. were to be introduced to locations where those diverse legumes are to be grown. In a different situation, it might be advisable to work with a very specific symbiosis to ensure infection by a particular inoculant strain that competes with native soil rhizobia. Due to these and other considerations, characterizing rhizobia I associations is of utmost importance when a legume cultivar is being developed through breeding or when a legume is being introduced into a new environment. STRAIN SELECTION After rhizobial strains have been isolated from nodules, they must be evaluated for their ability to form nodules and fix N2 with targeted legumes. The source of rhizobial strains for a strain selection program can range from local isolates, to strains already tested in other parts of the region or country, to cultures from various overseas collections. Preliminary screening is performed in the greenhouse, where numerous strains can be tested
168 EVALUATING SYMBIOTIC POTENTIAL OF RHIZOBIA on several host varieties. If the inoculated plants form nodules and produce healthy green leaves when grown in N-free media, it can be assumed that an effective symbiosis has been established. Rhizobia selected in greenhouse trials, where conditions are usually optimal, must then be evaluated in the field. Rhizobia that adapt to the agronomic conditions under which the host legumes will be cultivated and that enhance crop production through N2 fixation can then be selected for inoculant production. Field evaluation of effective rhizobia is critical because the symbiosis may be affected by many environmental factors discussed earlier in this section. The ability of an inoculant strain to persist in a particular environment, while in some cases competing against a resident soil population of rhizobia, is of critical importance. A combination of the these factors should be anticipated in the selection process to ensure good performance at different geographical locations. The task of introducing superior strains into soils that are already inhabited by effective rhizobia is difficult, and evaluation methods are an important key to success. The standard approach for isolating rhizobia from nodules is to seek out legumes that appear successful in native pastures and/or stable natural ecosystems. Nodules collected and desiccated in glass vials are later used in isolating rhizobia in the laboratory. The standard process of selecting a strain involves evaluating authenticated rhizobia in step-by-step screening experiments in controlled environments, greenhouses, and under field conditions. Considerable time is involved before a rhizobial strain finally gets an approval for use in inoculant production. It is not uncommon to encounter inoculation failure attributable to the selected strain in spite of all the careful methods of strain evaluation. Such failures may be avoided if the rhizobial strains made available for inoculant production are genetically compatible with the intended host legume. This is often not possible if the standard collection, isolation, and strain testing procedures are followed. Recent approaches to isolating rhizobia and selecting a strain have resorted to screening site soil containing indigenous rhizobia for symbiotic potential as a preliminary step before a decision is made to isolate rhizobia from nodules. This approach has been tentatively described as the whole-soil inoculation technique, and has recently been successfully applied in demonstrating the need to inoculate Trifolium subterraneum and Medicago sativa under field conditions. The whole-soil inoculation technique has also been adapted for strain selection work for soybean using soils from the center of origin of the soybean. The results of the research demonstrated clearly that soils containing indigenous populations of B. japonicum vary in their symbiotic potential. Further, it was shown that highly effective and competitive strains of B. japonicum (compared with widely used inoculant strains) can be found in the center of origin of the soybean. The conclusion from this work is that the symbiotic potential of indigenous rhizobia needs to be compared alongside recommended or imported exotic strains in screening experiments before embarking on inoculant production. This approach (Le., whole-soil inoculation) presents excellent potential for strain
Evaluating Symbiotic Potential of Rhizobia 169 selection work for new legumes (e.g., tree legumes) for which effective strains are not yet available from various rhizobial germ plasm resources. Another application of the technique is its ability to map out the symbiotic potentials of indigenous rhizobial populations of the sites under legume cultivation or projected for future planting. Such information can yield useful information and aid in isolating highly effective rhizobia that are available locally for inoculant production. PARAMETERS FOR MEASURING SYMBIOTIC NITROGEN FIXATION Final evaluation of the symbiosis will be based on several measurable parameters. Shortterm trials with Leonard jars or sterile sand culture pots can provide an adequate basis for gross comparison of strains. The shoot dry weight of plants harvested at floral initiation or after significant plant biomass accumulation is the generally accepted criterion for N2-fixing effectiveness, but nodule dry weight may also be employed. Nodule number is a less reliable indicator of strain effectiveness. The measurement of activity in the nodules by the N2-fixing enzyme, nitrogenase, may also be done. This is accomplished by means of the acetylene reduction assay, which is a measure of ethylene production and indicates nitrogenase activity. However, the results of this assay should not be used to conclude on the actual amounts of N2 fixed. This assay requires the availability of a gas chromatograph and other rather sophisticated equipment and materials. Total N accumulation in the shoot can be measured by the Kjeldahl method. Since total N content and nodule dry weight frequently correlate well with shoot dry weight, the latter parameter provides an acceptable basis for strain comparison. In recent years, the ureide technique has been developed for measuring N2 fixation. Ureides are a group of nitrogenous compounds including allantoin and allantoic acid. Some legumes produce large quantities of ureides when N is fixed symbiotically, but not when assimilated from soil mineral sources. Isotopic techniques using Nt5 are also used for measuring N2 fixation, but the analysis is costly. The final proof of inoculation response must come from the field, when the seed and N yields at harvest are determined for grain legumes or from the dry matter production for forage legumes.