Symbiotic Nitrogen Fixation

The Symbiosis Between Legumes and Rhizobia

Groupings of rhizobia and their separation into species

Each rhizobial strain or isolate has a finite host range, nodulating certain legumes but not others. In early studies this led to the concept of cross-inoculation, with legumes grouped according to the different rhizobia with which they formed nodules. Thus rhizobia isolated from species of Medicago (e.g., alfalfa) would also nodulate Melilotus, and vice versa, but neither group of rhizobia were able to nodulate Trifolium spp. (clovers). More than 20 different cross-inoculation groups were identified, with the bacteria from the clover, medic, bean, lupin, pea and soybean groups named as separate species within the single genus Rhizobium (e.g., R.trifolii for clover and R.meliloti for medics). Host specificity is still important in the identification of rhizobia, but more recently, other traits have assumed greater significance in their classification. There were a number of reasons for this:

  • Initial studies involved mainly legumes of agricultural importance. Study of less-traditional legumes has blurred cross-inoculation boundaries. For example, the bacterial strain NGR234, originally isolated from Lablab purpureus (the hyacinth bean) can nodulate at least 34 different species of legume, and the non-legume, Parasponia andersonii (Stanley and Cervantes, 1991). Even now, though, less than 15% of the roughly 19,000 species of legumes have been evaluated for nodulation.
  • There have been many anomalous results, one study providing more than 500 examples where strains were either promiscuous (nodulating legumes from other cross-inoculation groups) or else failed to nodulate legumes from their own group. Currently, for example rhizobia from at least six different species are thought to nodulate Phaseolus beans. Table 1 shows cross inoculation patterns for some of these organisms.

Table 1. Cross inoculation patterns among rhizobia associated with Phaseolus beans, Dalea, Coronilla, and Onobrychis

Strains
6864
6874
6815
7205
6861
6863
1632
1899
6917
6918
Host/Source
Cv
Cv
Dp
Dp
Ov
Ov
Pv
Pv
Pv
Pv
C.varia
+
++
+
+
+
+
0
0
+
+
D. purpurea
+
0
++
+
+
+
0
+
+
+
L.leucocephala
+
0
+
+
++
+
0
+
+
+
M.atropurpureum
+
0
+
+
++
+
+/-
+
+
+
O.viciifolia
+
+
+
+
+
+
0
++
0
+
P.vulgaris
0
+
+
+
+
+
+
+
+
++
nodulation (+) or lack of nodulation (0) with a specific host
++most effective strain tested
  • Nodulation genes of rhizobia may be plasmid-borne, with strains losing this plasmid also losing the ability to form nodules. In soils from Mexico, rhizobia lacking the symbiotic plasmid can outnumber those capable of nodule formation by 40 to 1, but for many years could not be identified.
  • Taxonomic methods were developed which compared strains on the basis of many different traits. When computer-based numerical classifications, or methods based on differences in cell DNA or RNA were used, the groupings that resulted were often different from those obtained using host range.

Some of the traits now used in identifying rhizobia and in distinguishing among the different types of rhizobia are shown in Table 2.

Table 2. Characteristics used in the phenotypic and phylogenetic characterization of rhizobia.

Phenotypic traits
  • range of substrates usable as sources of energy (sugars, sugar alcohols, complex carbohydrates, etc.)
  • range of substrates usable as sources of nitrogen (amino acids, urea, nitrate, etc.)
  • resistance to specific antibiotics
  • electrophoretic mobility of different cell enzymes
  • tolerance to different stresses (salt, temperature, pH)
Phylogenetic traits
  • pattern of banding of DNA restriction fragments (RFLPs,)
  • pattern of banding using repetitive primers such as BoxA1R or ERIC
  • degree of hybridization with specific DNA probes
  • 16S rRNA sequence analysis
Phenotypic traits can be observed in culture:
Phylogenetic traits are related to cell DNA or RNA composition

Identification and classification of these organisms is still at issue (see Table 3). Thus, Martinez et al. (2000) divided the original genus Rhizobium into 6 genera and 29 species, as shown below, whereas Young et al. (2001) also include species of Agrobacterium and Allorhizobium within the genus Rhizobium. The former do not produce nodules, but include saprophytes such as A.radiobacter, and the organisms responsible for crown gall and hairy root disease. Some strains of slow-growing Bradyrhizobium produce bacteriochlorophyll, and appear closely related to the photoautotroph, Rhodopseudomonas palustris.

Table 3: Genera and species of root-nodule bacteria of legumes.

Martinez-Romero et al (2000)
Young et al (2001)
Scientific/Common name of host
Azorhizobium1,2
A. caulinodans

 

Sesbania
Bradyrhizobium
B. elkanii

 

Glycine, soybean
B. japonicum

 

Glycine
B. liaoningense

 

Glycine
Mesorhizobium
M.amorphae
 
Amorpha
M.chacoense3
 
Prosopis, mesquite
M.ciceri
 
Cicer, chickpea
M.huakuii
 
Astragalus, milkvetch
M.loti
 
Lotus
M.mediterraneum
 
Cicer
M.plurifarium
 
Acacia, Leucaena, Ipil-ipil
M.tianshanense
 
Glycyrrhiza, Sophora
Rhizobium
R. etli
 
Phaseolus vulgaris, bean
R. galegae
 
Galega, Leucaena
R.gallicum
 
Phaseolus, Dalea, Onobrychis, Leucaena
R.giardinii
 
Phaseolus
R.hainanense
 
Stylosanthes, Centrosema
R. huautlense
 
Sesbania
R. leguminosarum
 
 
biovar trifolii
 
Trifolium, clover
viciae
 
Pisum, peas; Vicia, field beans; Lathyrus; Lens, lentil
phaseoli
 
Phaseolus
R. mongolense
 
Medicago, Phaseolus
R. tropici
 
Phaseolus; Leucaena, Dalea, Macroptilium
Allorhizobium
A.undicola
R.undicola
Neptunia
R.radiobacter non-nodulating saprophyte
R.rhizogenes causes hairy root disease
R.rubi
R.vitis
Sinorhizobium
S.arboris4
   
S. fredii
 
Glycine
S.kostiense4
 
 
S.medicae
 
Medicago
S. meliloti
 
Melilotus, sweetclover; Medicago, alfalfa; and Trigonella, fenugreek
S. saheli
S.sahalense
Sesbania
S. terangae
 
Sesbania, Acacia, wattle
S.xinjiangense
 
Glycine
Genus names refer to better-known host legumes nodulated by each species of root-nodule bacteria. There are several examples in this list where different species of root-nodule bacteria will nodulate the same legume.
1 You may come across other genus and species names. Some predate the present species, others after detailed investigation have not been accepted as valid.
2 Strains of Rhizobium, Bradyrhizobium , etc. which have not yet been identified as belonging to any named species are usually identified by the host from which they were isolated—e.g Rhizobium spp. (Acacia) or Bradyrhizobium spp. (Lupinus).
3 From Velasquez et al. (2001).
4 From Nick et al. (1999).

Previous phylogenetic studies have grouped the rhizobia into three different branches of the a subclass of the Proteobacteria. Very recently, members of a fourth group of organisms, identified as Methylobacterium nodulans, have been shown to nodulate Sesbania, Aspalathus, Machaerium, Crotalaria and Lotononis. A phylogenetic tree showing relationships among the different branches of the root nodule bacteria is shown in Fig 1.

[      ]

Fig 1: Phylogenetic tree showing relationships among the different rhizobial branches in the a subdivision of the Proteobacteria.The tree was constructed using the neighbour-joining method and almost full length 16S rDNA sequences. From: Sy et al (2001).