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RhizobiumClassification
Beijerinck in Holland was the first to isolate and cultivate a microorganism from the nodules of legumes in 1888. He named it Bacillus radicicola which is now placed in Bergey's Manual of Determinative Bacteriology under the genus Rhizobium.
Bacteria belonging to the genus Rhizobium live freely in soil and in the root region of both leguminous and non-leguminous plants.
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However, they can enter into symbiosis only with leguminous plants, by infecting their roots and forming nodules on them, the only exception being root nodulation in Trema (Parasponia) by a Rhizobium sp. The term symbiosis generally denotes a mutual beneficial partnership between two organisms. In legume root nodule symbiosis, the legume is the bigger partner while the Rhizobium is the smaller partner, often referred to as the 'microsymbiont'.
When a nodule becomes senescent after a period of nitrogen fixation, decay of tissue sets in liberating motile forms of Rhizobium into soil which normally serve as a source of inoculum for the succeeding crop of a given species of legume. The genus Rhizobium has been placed in Bergey's Manual of Determinative Bacteriology in such diverse families as Azotobacteriaceae, Myxobacteriaceae, Myxobacteriaceae and Pseudomonadaceae.
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Speciation of Rhizobium based on the Linnaean concept has proved difficult and therefore, the cross-inoculation grouping based on the classical studies of Fred, Baldwin and McCoy is being generally followed.
The principle of cross-inoculation grouping is based on the ability of an isolate of Rhizobium to form nodules in a limited number of species of legumes related to one another.
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All rhizobia that could form nodules on roots of certain legume types have been collectively taken as a species. This system of classification has provided a workable basis for the agricultural practice of legume inoculation. Under this scheme, seven species are generally recognized.
The system of cross-inoculation grouping of rhizobia is not perfect since bacteria have been found to cross-infect or interchange between groups. However, until a better system of classification has been perfected, it appears as if we have to be content with the cross-inoculation grouping as a convenient and workable method of classifying root nodule bacteria into species.
Cross-inoculation grouping Rhizobium
Rhizobium spp. |
Cross-inoculation grouping |
Legume types |
R. leguminosarum |
Pea group |
Pisum, Vicia, Lens |
R. phaseoli |
Bean group |
Phaseolus |
R. trifolii |
Cover group |
Trifolium |
R. meliloti |
Alfalfa group |
Melilotus, Medicago, Trigone |
R. lupini |
Lupini group |
Lupinus, Orinthopus |
R. japonicum |
Soybean group |
Glycine |
R. sp. |
Cowpea group |
Vigna, Arachis |
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Root nodule bacteria have been differentiated, on the basis of growth on a defined substrate, as fast growers and slow growers. Computer analysis of one hundred morphological and physiological characters (colonial character, vitamin, carbohydrate and nitrogen nutrition, antibiotic sensitivities and infective attributes) of nodule bacteria has revealed a new trend in their classification.
Fast-growing pea and bean rhizobia (R. trifolii, R. leguminosarum and R. phaseoli) have been united under a common species name, R. leguminosarum Frank (the type species in Bergey's classification) while the fast-growing R. meliloti Dangeard retains a distinctive status as a species. The agrobacteria (A.radiobacter and A. tumefaciens) have been clubbed to a species, Rhizobium radiobacter (Beijerinck and Van Delden Lohnis).
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The slow-growing lupin, soybean and cowpea rhizobia, R. lupini, R. japonicum and R. sp. (cowpea group) have been assigned to a separate genus Phytomyxa and given a new name Phytomyxa japonicum Kirchner. In this way, root nodule bacteria could be delimited into two genera, Rhizobium and Phytomyxa, the former having three species, R. leguminosarum, R. meliloti and R. radiobacter and the latter with only one species, R. japonicum.
After re-examination of the above proposal, other workers have suggested that the genus Rhizobium need not be split at the generic level and that Agrobacterium need not necessarily be merged with Rhizobium. In other words, a status quo has been proposed and we are back to the cross-inoculation grouping, of rhizobia as the only practical approach to the problem for the present.
Based on the ability of rhizobia to produce acid or alkali on yeast extract mannitol agar medium, the fast-growing R. phaseoli, R. trifolii, R. leguminosarum and R. meliloti have been grouped as acid producers whereas the slow-growing R. japonicum, R. lupini and R. sp. (cowpea) have been grouped as non-acid producers. The slow-growing non-acid producing rhizobia have been considered to be the ancestral forms of rhizobia since they are associated with primitive tropical legumes growing in acidic environment.
Attempts have been made to find the base composition of pure DNA (expressed as molar percentage of guanine and cytosine) of several rhizobia. Based on such studies, a regrouping of species has been suggested in which the fast-growing peritrichous strains having a low % (G + C) composition in the range 58.6-63.1% belong to R. leguminosarum and R. meliloti.
On the other hand, the subpolarly flagellated, slow-growing strains having a somewhat higher % (G + C), mostly in the range of 62.865.5% correspond to R. japonicum. Since it is now believed that a knowledge of the base composition of DNA would clarify the present classification of bacteria in general, more investigations on these lines intended to clarify the ticklish problems in the taxonomy of Rhizobium are bound to be useful in future.
The following revision has been proposed by the international committee on systematic bacteriology and included in the ninth edition of Bergey's Manual of Determinative Bacteriology.
The genus Rhizobium will consist of three reorganized species: R. leguminosarum, which will contain three biovars (biovar trifolii, biovar phaseoli, and biovar viceae); R. meliloti; and R. loti. The reorganization combines into one the former species of R. leguminosarum, R. trifolii, and R. phaseoli. The fast-growing members of the cowpea rhizobia and the former species R. lupinus have been included in the species R. loti. The new genus, Bradyrhizobium, is made up of one species, B. japonicum, which consists of the former species R. japonicum, plus the slow-growing members of the cowpea rhizobia. The newly proposed classification of Rhizobium is as follows (from Elkan, 1984).
GENUS I:
Rhizobium
R. leguminosarum
biovar trifolii
biovar phaseoli
biovar viceae
R. meliloti
R. loti-fast-growing, sub-polar flagellated strains from Lotus and Lupinus with strong affinity for L. corniculatus, L. densiflorus, and Anthyllis vul neraria (but also nodulates Ornithopus sativum). Includes the fast-growing strains nodulating Cicer, Sesbania, Leucaena, Mimosa, and Lablab.
GENUS II: Bradyrhizobium
Slow-growing, polar or sub-polar flagellated strains nodulating soybean, Lotus uliginosus, L. pendutulatus, and Vigna. Includes those slow-growing strains nodulating Cicer, Sesbania, Leucaena, Mimosa, Lablab, and Acacia. The possibility exists that other species will eventually be defined within this genus, but for the present it is suggested that, other than B. japonicum (the type species), the various cultures be designated ex. Bradyrhizobium sp. (Vigna), Bradyrhizobium sp. (Cicer), etc.
Recently, two more genera have been added to the family Rhizobiaceae. They are Sinorhizobium and Azorhizobium, nodulating soybean and Sesbania, respectively (1993).
The application of serological methods has helped in the delineation of strains among different rhizobia. There are at least two distinct kinds of antigens associated with the rhizobial cell-on the main body of the cell (somatic) and on the flagella. Agglutinations of a suspension of specific Rhizobium due to flagellar antigens can be distinguished from those due to somatic ones by the nature of its reaction. By this means, it has been shown that rhizobia are serologically heterogenous.
The combined results of both somatic and flagellar reactions have served to distinguish strains within a cross-inoculation group. Serological methods can be used as a means of obtaining information on the distribution of strains that can be recognised within an area, on widely separated areas, on the plant or within a nodule. Serologically, it is known that a single nodule contains a homogeneous population of a single strain of Rhizobium, although it is not uncommon to find more than one strain on the same plant.
Serological heterogeneity of isolates from the same locality has also been recorded. Serological methods have helped to distinguish nodules formed by inoculum applied to seeds from those formed by strains of Rhizobium already present in soil.
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