G.W. O'Hara

Mineral constraints to nitrogen fixation

Mineral nturient defiencies are a major constraint limiting legume nitrogen fixation and yield. In this review general techniques for assessing nutrient involvement in symbiotic nitrogen fixation are described and specific methods are outlined for determining which developmental phase of the symbiosis is most sensitive to nutrient deficiency.The mineral nutrition of the Rhizobium component of the symbiosis is considered both as the free living organism in the soil and as bacteroids in root nodules. Rhizobial growth and survival in soils is not usually limited by nutrient availability. Multiplication of rhizobia in the legume rhizosphere is limited by low Ca availability. Nodule initiation is affected by severe Co deficiency through effects on rhizobia. Nodule development is limited by severe B deficiency via an effect on plant cell growth. Fe deficiency limits nodule development by affecting rhizobia and strains of rhizobia differ widely in their ability to acquire sufficient Fe for their symbiotic development. Nodule function requires more Mo than does the host plant, and in some symbioses nitrogen fixation may be specifically limited by low availability of Ca, Co, Cu and Fe. The importance of the peribacteriod membrane in determining nutrient availability to bacteroids is considered.It is concluded that the whole legume-Rhizobium symbiosis should be considered when improving legume growth and yield under nutrient stress conditions. Differences among rhizobial strains in their ability to obtain mineral nutrients from their environment may be agronomically important.

Nutritional constraints on root nodule bacteria affecting symbiotic nitrogen fixation: a review

Root nodule bacteria require access to adequate concentrations of mineral nutrients for metabolic processes to enable their survival and growth as free-living soil saprophytes, and in their symbiotic relationship with legumes. Essential nutrients, with a direct requirement in metabolism of rhizobia are carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, calcium, magnesium, iron, manganese, copper, zinc, molybdenum, nickel, cobalt and selenium. Boron does not seem to be required by rhizobia, but is essential for the establishment of effective legume symbioses. Nutrient constraints can affect both free-living and symbiotic forms of root nodule bacteria, but whether they do is a function of a complex series of events and interactions. Important physiological characteristics of rhizobia involved in, or affected by, their mineral nutrition include nutrient uptake, growth rate, gene regulation, nutrient storage, survival, genetic exchange and the viable non-culturable state. There is considerable variation between genera, species and strains of rhizobia in their response to nutrient deficiency. The effects of nutrient deficiencies on free-living rhizobia in the soil are poorly understood. Competition between strains of rhizobia for limiting phosphorus and iron in the rhizosphere may affect their ability to nodulate legumes. Processes in the development of some legume symbioses specifically require calcium, cobalt, copper, iron, potassium, molybdenum, nickel, phosphorus, selenium, zinc and boron. Limitations of phosphorus, calcium, iron and molybdenum in particular, can reduce legume productivity by affecting nodule development and function. The effects of nutrient deficiencies on rhizobia-legume signalling are not understood. The supply of essential inorganic nutrients to bacteroids in relation to nutrient partitioning in nodule tissues and nutrient transport to the symbiosome may affect effectiveness of nitrogen fixation. An integration of molecular approaches with more traditional biochemical, physiological and field-based studies is needed to improve understanding of the agricultural importance of rhizobia response to nutrient stress.

Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with ≥ 96.1% sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFB(T), with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8% similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Microvirga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), WSM3557(T) and WSM3693(T) from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6(T) was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100% identical nodA sequences of WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), WSM3557(T) and WSM3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6(T) =LMG 26460(T) =HAMBI 3236(T)), Microvirga lotononidis sp. nov. (type strain WSM3557(T) =LMG 26455(T) =HAMBI 3237(T)) and Microvirga zambiensis sp. nov. (type strain WSM3693(T) =LMG 26454(T) =HAMBI 3238(T)).

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.

Rapid In Situ Evolution of Nodulating Strains for Biserrula pelecinus L. through Lateral Transfer of a Symbiosis Island from the Original Mesorhizobial Inoculant

ABSTRACT Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.

The symbiotic requirements of different medicago spp. Suggest the evolution of sinorhizobium meliloti and S. medicae with hosts differentially adapted to soil pH

Nitrogen fixing rhizobia associated with the Medicago L. genus belong to two closely related species Sinorhizobium medicae and S. meliloti. To investigate the symbiotic requirements of different Medicago species for the two microsymbionts, 39 bacterial isolates from nodules of eleven Medicago species growing in their natural habitats in the Mediterranean basin plus six historical Australian commercial inocula were symbiotically characterized with Medicago hosts. The bacterial species allocation was first assigned on the basis of symbiotic proficiency with M. polymorpha. PCR primers specific for 16S rDNA were then designed to distinguish S. medicae and S. meliloti. PCR amplification results confirmed the species allocation acquired in the glasshouse. PCR fingerprints generated from ERIC, BOXA1R and nif-directed RPO1 primers revealed that the Mediterranean strains were genetically heterogenous. Moreover PCR fingerprints with ERIC and BOX primers showed that these repetitive DNA elements were specifically distributed and conserved in S. meliloti and S. medicae, clustering the strains into two divergent groups according to their species. Linking the Sinorhizobium species with the plant species of origin we have found that S. medicae was mostly associated with medics well adapted to moderately acid soils such as M. polymorpha, M. arabica and M. murex whereas S. meliloti was predominantly isolated from plants naturally growing on alkaline or neutral pH soils such as M. littoralis and M. tornata. Moreover in glasshouse experiments the S. medicae strains were able to induce well-developed nodules on M. murex whilst S. meliloti was not infective on this species. This feature provides a very distinguishing characteristic for S. medicae. Results from the symbiotic, genotypic and cultural characterization suggest that S. meliloti and S. medicae have adapted to different Medicago species according to the niches these medics usually occupy in their natural habitats.

Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards

Aims:  Deleterious rhizosphere inhabiting bacteria (DRB) have potential to suppress plant growth. This project focuses on the isolation of DRB with potential for development as commercial products for weed control.

The interactions of Rhizobium leguminosarum biovar trifolii in nodulation of annual and perennial Trifolium spp. from diverse centres of origin

The release of effective inocula for new perennial clovers into cropping zones where subterranean clover is important might compromise N2 fixation by this valuable annual clover if symbiosis between the new inoculants and subterranean clover is not optimal. To assist our understanding of the interactions between clovers and their microsymbionts, rhizobial strains and clovers from South and equatorial Africa, North and South America, and the Euro–Mediterranean regions were tested. Glasshouse-based studies of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum bv. trifolii associated with 38 genotypes of annual and perennial Trifolium spp. from these world centres of diversity were undertaken. Less than 7.5% of the perennial clover symbioses were effective whereas 40% of associations were effective for many of the annual clover species of Euro–Mediterranean origin. There was substantial specificity within the African clovers for effective nodulation. Rhizobial strains from the South American perennial T. polymorphum or from the African clovers were unable to nodulate subterranean clover effectively. Also, 7 of the 17 strains from these regions were unable to form nodules with the less promiscuous Mediterranean annual clovers, T. glanduliferum and T. isthmocarpum. Fifty-three of about 400 cross-inoculation treatments examined, which included annual and perennial clovers, were incapable of forming nodules, while only 65 formed effective nodules. There are 2 barriers to effective nodulation: a ‘geographic’ barrier representing the broad centres of clover diversity, across which few host-strain combinations were effective; and, within each region, a significant ‘phenological’ barrier between annual and perennial species. Clovers and their rhizobia from within the Euro–Mediterranean region of diversity were more able to cross the phenological barrier than genotypes from the other regions. It appears that only the relatively promiscuous clovers, whether annual or perennial, have been commercialised to date. The data indicate that, for perennial clovers, it will be a substantial challenge to develop inocula that do not adversely affect N2 fixation by subterranean clover and other annual clovers available commercially, especially if the perennial clovers were originally from Africa or America. Some future strategies for development of inoculants for clovers are proposed.

Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov., isolated from Biserrula pelecinus L. in Australia

Biserrula pelecinus L. is a pasture legume that was introduced to Australia from the Mediterranean basin in 1993. Although the native rhizobial population could not nodulate B. pelecinus at the time of its introduction, recent research has shown the emergence of a diversity of strains (novel isolates) that are able to do so. Three novel isolates, WSM2073T, WSM2074 and WSM2076, had nearly identical 16S rRNA gene sequences, and clustered separately with all recognized species of the genus Mesorhizobium. Conversely, the novel isolate WSM2075T had >23 nt mismatches with the above three isolates. All four novel isolates shared 97-99% 16S rRNA gene sequence similarity with the type strains of all recognized Mesorhizobium species. However, strains WSM2073T, WSM2074 and WSM2076 showed <95.2% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species, and <92.9% to WSM2075T (which also shared <95.5% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species). Results for GSII gene sequencing were consistent with those for the dnaK gene. The fatty acid profiles of the novel isolates were diagnostic of root-nodule bacteria, but did not match those of recognized bacterial species. Strain WSM2075T had a significantly different fatty acid profile from the other three isolates. The above results indicated that strains WSM2073T, WSM2074 and WSM2076 represent the same species. Strain WSM2073T showed <45% DNA-DNA relatedness and WSM2075T<5% DNA-DNA relatedness with the type strains of recognized Mesorhizobium species; these two novel isolates shared 59% DNA-DNA relatedness. Collectively, these data indicate that strains WSM2073T, WSM2074 and WSM2076, and strain WSM2075T belong to two novel species of the genus Mesorhizobium, for which the names Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov. are proposed, respectively. The type strain of Mesorhizobium australicum sp. nov. is WSM2073T (=LMG 24608T=HAMBI 3006T) and the type strain of Mesorhizobium opportunistum sp. nov. is WSM2075T (=LMG 24607T=HAMBI 3007T).

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

BACKGROUND AND AIMS The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. METHODS Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. KEY RESULTS Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. CONCLUSIONS Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.