Recent developments inRhizobium taxonomy

Recent developments inRhizobium taxonomy are presented from a molecular and evolutionary point of view. Analyses of ribosomal RNA gene sequences provide a solid basis to infer phylogenies in the Rhizobiaceae family. These studies confirmed thatRhizobium andBradyrhizobium are only distantly related and showed thatRhizobium andBradyrhizobium are related to other groups of bacteria that are not plant symbionts.Rhizobium andAgrobacterium species are intermixed. Differences in plasmid content may explain to a good extent the different behavior ofRhizobium andAgrobacterium as symbionts or pathogens. Other approaches to identify and classify bacteria such as DNA-DNA hybridization, fatty acid analysis, RFLP and RPD-PCR techniques and phylogenies derived from other genes are in general agreement to the groupings derived by ribosomal sequences. Only a small proportion of nodulated legumes have been sampled for their symbionts and more knowledge is required on the systematics and taxonomy ofRhizobium andBradyrhizobium species.     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Antigenic affinities of the root-nodule bacteria of legumes

Antisera prepared against 58 strains of root-nodule bacteria and against 16 strains belonging to the genusAgrobacterium were tested against 113 strains ofRhizobium, 20 strains ofAgrobacterium and 20 strains of other, possibly related, bacteria.Three serologically distinct groups of root-nodule bacteria were noted: (1)Rh. trifolii, Rh. leguminosarum andRh. phaseoli; (2)Rh. lupini, Rh. japonicum andRhizobium spp.; (3)Rh. meliloti. Strains ofRh. meliloti showed serological affinities withA. radiobacter andA. tumefaciens. All groups showed wider flagellar than somatic agglutination, and many different serotypes were apparent.The groupings obtained from this investigation are compared with those derived from other taxonomic studies, and the use of serological methods in rhizobial classification is discussed.     

Host specificity of nodulation inRhizobium sp. (Cicer) as revealed by Tsr bioassay

Bioassay based on thick and short root (Tsr) and hair deformation (Had) phenotypes were used to test the activity of Nod factors produced byRhizobium sp. (Cicer) strains HS-1, Rcd-301, IC-59, IC-76 and Ca-181 on chickpea (Cicer arietinum) cv. ‘C-235’. Nod⁻ mutants ofRhizobium sp. (Cicer) did not produce Tsr⁺ and Had⁺ phenotypes on chickpea, indicating the requirement of nodulation genes for their appearance. The strain HS-1 treated with root exudates of pea (Pisum sativum), berseem (Trifolium alexandrinum) and lucerne (Medicago sativa) failed to produce the Tsr⁺ and Had⁺ phenotypes on chickpea. ConverselyR. leguminosarum bvs.viciae andtrifolii, R. meliloti, Rhizobium sp. (Sesbania), andRhizobium sp. (Cajanus) induced with chickpea root exudates did not show Tsr⁺ and Had⁺ phenotypes on chickpea. It appears that host specificity inRhizobium sp. (Cicer)-chickpea symbiosis is regulated by the production of host-specific factors which are not active on heterologous hosts.     

Investigations on the endogenous levels of abscisic acid in a range of parasitic phanerogams

The endogenous levels of abscisic acid in the parasitic phanerogamsArceuthobium oxycedri (DC) Bieb.,Cassytha filiformis L.,Lathraea squamaria L.,Melampyrum pratense L.,Orobanche hederae Duby., andViscum album L. were investigated. In general, the content of abscisic acid was high in parasites which deprive their hosts of both phloem- and xylem-transported substances and much lower in those that deprive their hosts of sap from the xylem only. Within the parasites studied in more depth (e.g.,Orobanche hederae, Lathraea squamaria, andMelampyrum pratense) the highest abscisic acid levels were found in their sink regions, especially in their inflorescences. It is suggested that a high concentration of abscisic acid is associated with plant tissues showing a high demand for phloem-transported substances. The possible role of abscisic acid in such tissues is discussed.     

Identification of Rhizobium strains on antibiotic concentration gradients

Antibiotic concentration gradients were used to characterise several cultures of R. phaseoli and Rhizobium spp. (isolated from Cicer arietinum) by differences in intrinsic antibiotic resistance (IAR). Differentiation between cultures was facilitated by use of cluster analyses. The method permitted 15/16 cultures of R. phaseoli to be distinguished on 14 antibiotics. Two cultures which exhibited similar IAR patterns were shown to be the same strain obtained from different collections. The validity of the technique for strain identification was demonstrated by fluorescent antibody tests which gave corresponding identity for 50 nodule isolates from plants inoculated with a mixture of three strains of R. phaseoli. The method was less suitable for characterising cultures of the slow-growing Rhizobium spp. because several antibiotics produced growth lacking a clearly defined boundary between resistance and susceptibility. Although 15/16 cultures of Rhizobium spp. could be differentiated, several isolates were distinguishable only by a difference on a single antibiotic. Similarity between stock cultures and derivative nodule isolates indicated that IAR on gradient plates was a stable property unaffected by plant passage.     

Composition of lipopolysaccharides fromRhizobium andAgrobacterium

Lipopolysaccharides were prepared from 9Agrobacterium and 16Rhizobium strains. Glucose and rhamnose were the only monosaccharides present in all preparations, but mannose, glucosamine and 4-O-methylglucuronic acid were common components. Fucose (present in 9 strains), galactose (10 strains), xylose (3 strains) and arabinose (1 strain) were also detected but there was no evidence of dideoxy sugars, of heptose, or of other hexosamines. The possible significance of the results in the serological classification ofRhizobium andAgrobacterium, and in host selection ofRhizobium is discussed.     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.     

Biocontrol of fungal root rot diseases of crop plants by the use of rhizobia and bradyrhizobia

Twenty-oneRhizobium andBradyrhizobium strains were testedin vitro against the mycelial growth of three pathogenic fungi on solid and liquid media. All tested rhizobia and bradyrhizobia significantly suppressed the growth of the three soil-borne root-infecting fungi (Fusarium solani, Macrophominia phasolina andRhizoctonia solani) either in the absence or presence of iron. This indicates that the siderophore played a minor role in the biocontrol potential ofRhizobium andBradyrhizobium against pathogenic fungi. Pot experiments revealed that the numbers of propagules causing disease after 4 weeks of planting varied with species and host plant. The three most activeRhizobium andBradyrhizobium strains (R. leguminosarum bv.phaseoli TAL 182,B. japonicum TAL 377 andBradyrhizobium sp. (lupin) WPBS 3211 D) tested under greenhouse conditions for their ability to protect one leguminous (soybean) and two non-leguminous (sunflower and okra) seedlings from root rot caused byFusarium solani, Macrophominia phaseolina andRhizoctonia solani provided significant suppression of disease severity compared with nonbacterized control in both leguminous and non-leguminous seedlings.Bradyrhizobium sp. (lupin) WPBS 3211 D provided the lowest degree of resistance against all the tested pathogens with all host plants. *** DIRECT SUPPORT *** A00EN058 00013     

Effect of dual inoculation ofRhizobium and vesicular arbuscular mycorrhizal fungi onPisum sativum

The interaction of two symbionts,Rhizobium (a bacterium) andGigaspora calospora (a VAM fungus) was studied inPisum sativum (a nodulating legume).Pisum sativum plants inoculated with VA mycorrhizal fungi andRhizobium singly and in combination responded favourably as compared to uninoculated control. Characteristically dual inoculation exerted a higher beneficial effect on shoot and root dry mass, phosphorus and nitrogen uptake, nodule formation and degree of mycorrhizal infection than either inoculum alone.     

An assessment of a method based on intrinsic antibiotic resistance for identifying Rhizobium strains

A method based on intrinsic antibiotic resistance (IAR) for identifying large numbers of Rhizobium strains was assessed and found to be unsatisfactory for R. phaseoli and isolates from Cicer arietinum (Rhizobium spp.). Our data showed that the number of different IAR patterns always exceeded the number of strains tested. With 90 nodule isolates from plants inoculated with a mixture of three strains of R. Phaseoli, the technique gave 18 different resistance patterns. When 24 strains of Rhizobium spp., each replicated three times, were examined 68 different resistance patterns were obtained. Single colony isolates from one strain also gave several different IAR patterns. All strains tested with fluorescent“ antibody were readily identified. Attempts to obtain correct strain identification with IAR by simplifying the scoring systems or allowing up to two differences in the resistance patterns were unsuccessful. We were unable to define the source of this variation although incubation time and inoculum concentration were shown to affect the IAR patterns     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product NaDDC

Summary The in-vitro effect of the seed dressing fungicide tetramethyl thiuram disulphide (TMTD, Thiram) and its degradation product sodium dimethyl dithiocarbamate (NaDDC) on the sensitivity of Rhizobium spp. and Azotobacter chroococcum isolates was studied at different pH values. The Azotobacter chroococcum isolates were more sensitive than Rhizobium spp. isolates at all pH values. The sensitivity of these isolates to TMTD and NaDDC had a maximum at pH 7, and it decreased on either side of this value. The Rhizobium isolates from urid (Phaseolus radiatus) were resistant to TMTD at pH 7 but proved sensitive to its degradation product. A difference between the power of the sensitive and resistant isolates to reduce TMTD was revealed.     

Genetic diversity of indigenous tropical fast-growing rhizobia isolated from soybean nodules

This study characterized genetically 30 fast-growing rhizobial strains isolated from nodules of Asian and modern soybean genotypes that had been inoculated with soils from disparate regions of Brazil. Analyses by rep-PCR (ERIC and REP) and RAPD indicated a high level of genetic diversity among the strains. The RFLP-PCR and sequencing analysis of the 16S rRNA genes indicated that none of the strains was related to Sinorhizobium (Ensifer) fredii, whereas most were related to Rhizobium tropici (although they were unable to nodulate Phaseolus vulgaris) and to Rhizobium genomic species Q. One strain was related to Rhizobium sp. OR 191, while two others were closely related to Agrobacterium (Rhizobium) spp.; furthermore, symbiotic effectiveness with soybean was maintained in those strains. Five strains were related to Bradyrhizobium japonicum and B. elkanii, with four of them being similar to strains carried in Brazilian inoculants, therefore modifications in physiological properties, as a shorter doubling time might have resulted from adaptation to local conditions. Phospholipid fatty acid analysis (PFLA) was less precise in delineating taxonomic relationships. The strains fit into eight Nod-factor profiles that were related to rhizobial species, but not to N₂-fixation capacity or competitiveness. The data obtained highlight the diversity and promiscuity of rhizobia in the tropics, being capable of nodulating exotic legumes and might reflect ecological strategies to survive in N-poor soils; in addition, the diversity could also represent an important source of efficient and competitive rhizobial strains for the tropics. Putative new rhizobial species were detected only in undisturbed soils. Three species (R. tropici, B. japonicum and B. elkanii) were found under the more sustainable management system known as no-till, while the only species isolated from soils under conventional till was R. tropici. Those results emphasize that from the moment that agriculture was introduced into undisturbed soils rhizobial diversity has changed, being drastically reduced when a less sustainable soil management system was adopted.     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)     

Drought acclimation of two deciduous tree species of different layers in a temperate forest canopy

Water-use strategies of Populus tremula and Tilia cordata, and the role of abscisic acid in these strategies, were analysed. P. tremula dominated in the overstorey and T. cordata in the lower layer of the tree canopy of the temperate deciduous forest canopy. Shoot water potential (), bulk-leaf abscisic acid concentration ([ABA]_leaf), abscisic acid concentration in xylem sap ([ABA]_xyl), and rate of stomatal closure following the supply of exogenous ABA (v) decreased acropetally through the whole tree canopy, and foliar water content per area (w), concentration of the leaf osmoticum (c), maximum leaf-specific hydraulic conductance of shoot (L), stomatal conductance (g_s), and the threshold dose per leaf area of the exogenous ABA (d_a) required to reduce stomatal conductance increased acropetally through the tree canopy (from the base of the foliage of T. cordata to the top of the foliage of P. tremula) in non-stressed trees. The threshold dose per leaf dry mass of the exogenous ABA (d_w) required to reduce stomatal conductance, was similar through the tree canopy. After a drought period (3 weeks), the , w, L, g_s, d_a and d_w had decreased, and c and v had increased in both species. Yet, the effect of the drought period was more pronounced on L, g_s, d_a, d_w and v in T. cordata, and on , w and c in P. tremula. It was concluded that the water use of the species of the lower canopy layer—T. cordata, is more conservative than that of the species of the overstorey, P. tremula. [ABA]_leaf had not been significantly changed in these trees, and [ABA]_xyl had increased during the drought period only in P. tremula. The relations between [ABA]_leaf, [ABA]_xyl and the stomatal conductance, the osmotic adjustment and the shoot hydraulic conductance are also discussed.     

Pollination ecology ofOxalis violacea (Oxalidaceae) following a controlled grass fire

Vernal grass fires may encourage profuse flowering in clonal, colonies ofOxalis violacea. Long-styled colonies appear to be more floriferous than short-styled colonies and set a greater number of capsules. Individual flowers of both morphs live one or two days, change position on their respective pedicels and advertise nectar concealed at the base of the floral throat. AlthoughDiptera, Hymenoptera, andLepidoptera forage for nectar, bees (Andrenidae,Anthophoridae, Halictidae, andMegachilidae) probably make the only effective pollen transfers between the two morphs. Both male and female bees may transport pollen of both morphs and short-tongued bees (e.g.,Augochlorella spp.,Dialictus spp.) may be more common but as effective as pollinators as long-tongued bees (e.g.,Calliopsis andreniformis andHoplitis spp.). The conversion rate of flowers into capsules is only 13–17%. The spreading style in the short-styled morph is interpreted as an adaptation restricting insect-mediated, self-pollination but encouraging bee-stigma contact during nectar foraging.     

A re-examination of the taxonomy of the genusRhizobium and related genera using numerical analysis

Different methods of clustering were applied to the results of a numerical study of the taxonomy ofRhizobium, Agrobacterium, Beijerinckia, Chromobacterium, andBacillus published by Graham (1964) (J. Gen. Microbiol.35: 511).The groupings obtained were compared with that of Graham and external information from published studies on flagellation and DNA-base-composition was used to judge the merits of the various groupings. It is shown that a more satisfactory grouping is obtained with furthest neighbour and flexible sorting than with either nearest neighbour sorting or with the modified method of Sneath (1957) used by Graham.The conclusions from this study are that (1)Agrobacterium radiobacter andA. tumefaciens belong to one taxon; (2)Agrobacterium is more closely related to fast-growingRhizobium than is slow-growingRhizobium; (3) fast growing and slow-growingRhizobium are more closely related to each other than toBeijerinckia andChromobacterium; (4)Bacillus, Beijerinckia andChromobacterium are more closely related to each other than toRhizobium. Of these conclusions (1) and (2) are in agreement with Graham.It is postulated that the genusRhizobum should not be split at the generic level and thatAgrobacterium should not be merged withRhizobium as was suggested by Graham. Instead it is proposed thatRhizobium be considered as consisting of two Sections, one withR. japonicum as the type species and the other withR. leguminosarum as the type species, and that taxonomic treatment at the species level be left until much more material has been studied, and thatAgrobacterium be retained as a generic name until more evidence has been produced.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum bv. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.