SECTION A
GENERAL MICROBIOLOGY RHIZOBIUM INTRODUCTION
The bacteria of the genus Rhizobium (family Rhizobiaceae) are a genetically diverse and physiologically heterogeneous group of microorganisms that are nevertheless classified together by virtue of their ability to nodulate groups of plants of the family Leguminosae. This classification scheme is usually referred to as “cross-inoculatin” grouping. A cross-inoculation group is a group of legumes in which one species of Rhizobium nodulates all the legumes within that group. In the old system, species of Rhizobium fall into two groups based on their growth characteristics.
Group I
Rhizobium leguminosarum – nodulates peas (pisum spp.),
vetch (Vicia spp.), lentils,
(Lens culinaris).
Rhizobium phaseoli - nodulates beans (Phaseolus
Vulgaris and the scarlet
runner bean) (Phaseolus
coccineus).
Rhizobium trifolii - nodulates the clovers, e.g.,
Trifolium subterraneum, T.
Semipilosum, T. repens, and
Other Trifolium spp.
Rhizobium meliloti - nodulates alfalfa (Medicago
sativa) and other Medicago
spp., Melilotus spp.,
fenugreek (Trigonella).
Group II
Rhizobium lupini - nodulates lupins (Lupinus
Spp.) and serradella
(Ornithopus spp.).
Rhizobium japonicum - nodulates soybean (Glycine
max).
Rhizobium spp. – nodulates members of the
“cowpea miscellany” group of
legumes, e.g., Vigna spp.,
peanut, Desmodium spp.,
Macroptilium spp., Lablab
sp., Lima bean, Stylosanthes
spp., etc.
In Group I are the fast-growing acid producers which develop pronounced turbidity in liquid media within 2-3 days and have a mean doubling time of 2-4 hours. The cells are rod-shaped to pleomorphic, 0.5 to 0.9 microns in diameter and 1.2 to 3.0 microns long, and are motile by 2-6 peritrichous flagella. They can grow on a wide range of carbohydrates, but usually grow best on glucose, mannitol, or sucrose. Rhizobia of this group are generally infective on temperate legumes.
In group II are the slow-growing, alkali producing rhizobia. They require 3-5 days to produce moderate turbidity in liquid media and have a mean doubling time of 6-7 hours. Most strains in this group grow best with pentoses as their carbon source. The cells are predominantly rod-shaped, and motile by a single polar or subpolar flagellum. This group nodulates tropical legume species.
Rhizobia are characteristically Gram-negative and do not form endospores. Uneven Gram-staining is frequently encountered with rhizobia depending on the age of the culture. Cells from a young culture and nodule bacteroids usually show even Gram-staining while older cells and longer cells show unstained areas along the cell giving a banded appearance. These unstained areas have been identified to be large granules of polymeric-hydroxybutyric acid (PHBA). These granules are refractile under phase contrast microscopy.
Though the host-dependent cross-inoculation group system of classifying rhizobia has been subjected to much criticism, because it is not a taxonomic one, it is the best practical system currently available. Classification of rhizobia is becoming increasingly complex because of new findings, e.g. some soybeans are now known to be nodulated by a distinct group of fast-growing acid-producing rhizobia. Thus, a new system has been formulated to classify rhizobia. This new system recognizes three genera for the Rhizobiaceae. In this new system Genus I and Genus II include the rhizobia while Genus III is for the agrobacteria. All fast-growing acid-producing rhizobia now fall under the new genus Rhizobium (Genus I) and all slow-growing alkali-producing rhizobia under the new genus Bradyrhizobium (Genus II). Also, under this system, R. trifolii, R. phaseoli, and R. leguminosarum are combined as one species, designated as Rhizobium leguminosarum, comprising three biovars (trifolii, phaseoli, and viceae). R. meliloti remains as before and R. loti has been assigned to the fast-growing Lotus rhizobia. Genetically related to R. loti are rhizobia from Lotus corniculatus, Lotus tenuis, Cicer arietinum, Leucaena leucocephala, Sophora microphylla etc. The soybean rhizobia are now in two genera, i.e. R. japonicum (fast-growing and acid-producing) and Bradyrhizobium japonicum (slow-growing and alkali-producing). Rhizobia from Vigna, Arachis, Desmodium, Macroptilium, Stylosanthes etc. are still unclassified, but grouped as Bradyrhizobia spp. The non-legume Parasponia (called Trema previously) is also nodulated by Bradyrhizobium sp. Besides Leucaena, whose rhizobia are now R. loti, there are other legumes (Sesbania, Neptunia, Calliandra, Acacia) which are nodulated by fast-growing acid-producing rhizobia and the taxonomic status of these organisms may be resolved in the future.
As predominantly aerobic chemoorganotrophs, rhizobia are relatively easy to culture. They grow well in the presence of oxygen and utilize relatively simple carbohydrates and amino compounds. With the exception of a few strains, they have not been found to fix nitrogen away from their host legume. Some strains of Rhizobium require vitamins for growth. Optimal growth for most strains occurs in a temperature range of 25-30oC, and at a pH of 6-7. Despite their usual aerobic metabolism, many strains are able to grow well at oxygen tensions less than 0.01 atmosphere (microaerophilic).
Rhizobia are somewhat unique among soil microorganisms I their ability to form nitrogen-fixing symbioses with legumes. To enjoy the benefits of this partnership, however, the rhizobia must not only exhibit saprophytic competence among other soil microorganisms, but also out-compete other rhizobia for infection sites on legume roots. Potential for physiological versatility is therefore an important trait contributing to their adaptation to the competitive and complex soil environment.