S.B.M. Chimphango

Symbiotic diversity, specificity and distribution of rhizobia in native legumes of the core Cape Subregion (South Africa)

Rhizobial diversity and host preferences were assessed in 65 native Fynbos legumes of the papilionoid legume tribes Astragaleae, Crotalarieae, Genisteae, Indigofereae, Millettieae, Phaseoleae, Podalyrieae, Psoraleeae and Sesbanieae. Sequence analyses of chromosomal 16S rRNA, recA, atpD and symbiosis-related nodA, nifH genes in parallel with immunogold labelling assays identified the symbionts as alpha- (Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium and Rhizobium) and beta-rhizobial (Burkholderia) lineages with the majority placed in the genera Mesorhizobium and Burkholderia showing a wide range of host interactions. Despite a degree of symbiotic promiscuity in the tribes Crotalarieae and Indigofereae nodulating with both alpha- and beta-rhizobia, Mesorhizobium symbionts appeared to exhibit a general host preference for the tribe Psoraleeae, whereas Burkholderia prevailed in the Podalyrieae. Although host genotype was the main factor determining rhizobial diversity, ecological factors such as soil acidity and site elevation were positively correlated with genetic variation within Mesorhizobium and Burkholderia, respectively, indicating an interplay of host and environmental factors on the distribution of Fynbos rhizobia.

Effects of UV-B radiation on plant growth, symbiotic function and concentration of metabolites in three tropical grain legumes

Vigna unguiculata (L.) Walp. (cowpea), Glycine max (L.) Merr (soybean) and Phaseolus vulgaris (L.) (common bean) plants were exposed to UV-B radiation at above- and below-ambient levels, and their effects on growth, symbiotic performance and root concentration of metabolites were assessed. Moderately and highly elevated UV-B exposures averaging 32 and 62% above ambient had no effect on plant total dry matter, nodule number, nodule mass, nodule size, N fixed or root concentration of flavonoids, anthocyanins, soluble sugars and starch in the three species studied. However, N concentrations were markedly reduced in roots of G. max and P.vulgaris, and in leaves of P. vulgaris, which contrasted with the significant increase in stems and leaves of V.unguiculata. Below-ambient UV-B exposures averaging 22% of ambient also altered growth and metabolism of these legumes. Total plant dry matter, nodule number, nodule dry mass, N fixed and root starch concentrations in V.unguiculata decreased relative to both visible and UV-A radiation controls, whereas in G. max and P. vulgaris, these parameters were not altered. Root concentrations of flavonoids and anthocyanins in all species tested were also unchanged with below-ambient UV-B exposures. Taken together, growth and symbiotic function of these species remained unaltered with exposure to above-ambient UV-B, but differed in their response to below-ambient UV-B radiation.

Effect of legume plant density and mixed culture on symbiotic N2 fixation in five cowpea (Vigna unguiculata L. Walp.) genotypes in South Africa

A field experiment involving two plant densities (83,333 and 166,666 plants per hectare), two cropping systems (monoculture and mixed culture) and five cowpea (Vigna unguiculata L. Walp.) genotypes (3 farmer-selected varieties: Bensogla, Sanzie and Omondaw, and 2 breeder-improved cultivars: ITH98-46 and TVuI509) was conducted for two years in 2005 and 2006 at Nietvoorbij (33°54S, 18°14E), Stellenbosch, South Africa, to evaluate the effect of these treatments on the growth and symbiotic performance of cowpea. The results showed that, of the five cowpea genotypes, plant growth and N2 fixation were significantly greater in the three farmer-selected varieties (Sanzie, Bensogla and Omondaw) relative to the two improved cultivars (ITH98-46 and TVuI509). Furthermore, plant growth and symbiotic performance (measured as tissue N concentration, plant N content,15N natural abundance and N-fixed) were significantly (P<-50.05) decreased by both high plant density and mixed culture (intercropping). However, the %Ndfa values were significantly (P<-50.05) increased by both high plant density and mixed culture compared to low plant density or monoculture (or monocropping). Whether under low or high plant density, the cv. Sanzie was found to accumulate significantly greater total N per plant in both 2005 and 2006, followed by the other two farmer varieties relative to the improved cultivars. Similarly, the actual amount of N-fixed was much greater in cv. Sanzie, followed by the other farmer varieties, under both low and high plant density. The data also showed better growth and greater symbiotic N yield in cowpea plants cultivated in monoculture (or low plant density) relative to those in mixed culture (or high plant density). Our data suggest that optimising legume density in cropping systems could potentially increase N2 fixation in cowpea, and significantly contribute to the N economy of agricultural soils in Africa.

Photosynthesis, water-use efficiency and δ13C of five cowpea genotypes grown in mixed culture and at different densities with sorghum

A field experiment involving two planting densities (83,333 and 166,666 plants per ha), two cropping systems (monoculture and mixed culture) and five cowpea [Vigna unguiculata L. (Walp.)] genotypes was conducted at Nietvoorbij (33°54S, 18°14E), Stellenbosch, South Africa, to select cowpea material with superior growth and water-use efficiency (WUE). The results showed significantly higher photosynthetic rates, stomatal conductance and transpiration in leaves of plants at low density and in monoculture due to greater chlorophyll (Chl) levels relative to those at high density and in mixed culture. As a result, C concentration in leaves and the amount of C, P, K, Ca, Mg, Fe, Cu, Zn, Mn, and B accumulated in shoots at low density and under monoculture were also much higher. Even though no marked differences in photosynthetic rates were found between and among the five cowpea genotypes, leaf C concentration and shoot C, P, K, Ca, Mg, Fe, Cu, Zn, Mn, and B contents differed considerably, with Sanzie exhibiting the highest C concentration and C, P, K, Ca, Mg, Fe, Cu, Zn, Mn, and B contents in shoots, followed by Bensogla and Omondaw, while ITH98-46 and TVu1509 had the lowest shoot concentration and contents of C, P, K, Ca, Mg, Fe, Cu, Zn, Mn, and B. WUE (calculated as photosynthate produced per unit water molecule transpired) was significantly greater in plants at low density and monoculture relative to those at high density and in mixed culture. Isotopic analysis revealed significant differences in δ13C values of sorghum [Sorghum bicolor L. (Moench.)] and cowpea, with higher δ13C values being obtained for plants at low density and in monoculture relative to those at high density or in mixed culture. The five cowpea genotypes also showed significant differences in δ13C values, with Sanzie exhibiting the most negative value (i.e. low WUE) and ITH98-46, the least negative δ13C value (i.e. high WUE). Whether measured isotopically or from gas-exchange studies, sorghum (a C4 species) exhibited much higher WUE relative to cowpea (a C3 species). Both correlation and regression analyses revealed a positive relationship between WUE from gas-exchange studies and δ13C values from isotopic analysis of cowpea and sorghum shoots.

Biogeographical Patterns of Legume-Nodulating Burkholderia spp.: from African Fynbos to Continental Scales.

ABSTRACT Rhizobia of the genus Burkholderia have large-scale distribution ranges and are usually associated with South African papilionoid and South American mimosoid legumes, yet little is known about their genetic structuring at either local or global geographic scales. To understand variation at different spatial scales, from individual legumes in the fynbos (South Africa) to a global context, we analyzed chromosomal (16S rRNA, recA) and symbiosis (nifH, nodA, nodC) gene sequences. We showed that the global diversity of nodulation genes is generally grouped according to the South African papilionoid or South American mimosoid subfamilies, whereas chromosomal sequence data were unrelated to biogeography. While nodulation genes are structured on a continental scale, a geographic or host-specific distribution pattern was not detected in the fynbos region. In host range experiments, symbiotic promiscuity of Burkholderia tuberum STM678T and B. phymatum STM815T was discovered in selected fynbos species. Finally, a greenhouse experiment was undertaken to assess the ability of mimosoid (Mimosa pudica) and papilionoid (Dipogon lignosus, Indigofera filifolia, Macroptilium atropurpureum, and Podalyria calyptrata) species to nodulate in South African (fynbos) and Malawian (savanna) soils. While the Burkholderia-philous fynbos legumes (D. lignosus, I. filifolia, and P. calyptrata) nodulated only in their native soils, the invasive neotropical species M. pudica did not develop nodules in the African soils. The fynbos soil, notably rich in Burkholderia, seems to retain nodulation genes compatible with the local papilionoid legume flora but is incapable of nodulating mimosoid legumes that have their center of diversity in South America. IMPORTANCE This study is the most comprehensive phylogenetic assessment of root-nodulating Burkholderia and investigated biogeographic and host-related patterns of the legume-rhizobial symbiosis in the South African fynbos biome, as well as at global scales, including native species from the South American Caatinga and Cerrado biomes. While a global investigation of the rhizobial diversity revealed distinct nodulation and nitrogen fixation genes among South African and South American legumes, regionally distributed species in the Cape region were unrelated to geographic and host factors.

Characterization of the papilionoid-Burkholderia interaction in the Fynbos biome: The diversity and distribution of beta-rhizobia nodulating Podalyria calyptrata (Fabaceae, Podalyrieae).

The South African Fynbos soils are renowned for nitrogen-fixing Burkholderia associated with diverse papilionoid legumes of the tribes Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae. However, despite numerous rhizobial studies in the region, the symbiotic diversity of Burkholderia has not been investigated in relation to a specific host legume and its geographical provenance. This study analyzed the diversity of nodulating strains of Burkholderia from the legume species Podalyria calyptrata. Diverse lineages were detected that proved to be closely related to Burkholderia taxa, originating from hosts in other legume tribes. By analyzing the genetic variation of chromosomal (recA) and nodulation (nodA) sequence data in relation to the sampling sites we assessed the geographical distribution patterns of the P. calyptrata symbionts. Although we found a degree of genetically differentiated rhizobial populations, a correlation between genetic (recA and nodA) and geographic distances among populations was not observed, suggesting high rates of dispersal and rhizobial colonization within Fynbos soils.

Recombination and horizontal transfer of nodulation and ACC deaminase (acdS) genes within Alpha- and Betaproteobacteria nodulating legumes of the Cape Fynbos biome

The goal of this work is to study the evolution and the degree of horizontal gene transfer (HGT) within rhizobial genera of both Alphaproteobacteria (Mesorhizobium, Rhizobium) and Betaproteobacteria (Burkholderia), originating from South African Fynbos legumes. By using a phylogenetic approach and comparing multiple chromosomal and symbiosis genes, we revealed conclusive evidence of high degrees of horizontal transfer of nodulation genes among closely related species of both groups of rhizobia, but also among species with distant genetic backgrounds (Rhizobium and Mesorhizobium), underscoring the importance of lateral transfer of symbiosis traits as an important evolutionary force among rhizobia of the Cape Fynbos biome. The extensive exchange of symbiosis genes in the Fynbos is in contrast with a lack of significant events of HGT among Burkholderia symbionts from the South American Cerrado and Caatinga biome. Furthermore, homologous recombination among selected housekeeping genes had a substantial impact on sequence evolution within Burkholderia and Mesorhizobium. Finally, phylogenetic analyses of the non-symbiosis acdS gene in Mesorhizobium, a gene often located on symbiosis islands, revealed distinct relationships compared to the chromosomal and symbiosis genes, suggesting a different evolutionary history and independent events of gene transfer. The observed events of HGT and incongruence between different genes necessitate caution in interpreting topologies from individual data types.

N and P colimitation of N2-fixing and N-supplied fynbos legumes from the Cape Floristic Region

Background and aimsLegume species in the fynbos vegetation of the Cape Floristic Region, that fix N2 in soils with low P, may have evolved for enhanced acquisition and efficient use of P. It was hypothesized that N2-fixing and combined-N supplied (N-supplied) A. linearis, P. calyptrata and C. genistoides are adapted to low P and would be relatively unresponsive to increased P of 100 μM.Methods18 legume species were evaluated for their nodulation response to low P availability. The N X P interaction was then examined in A. linearis, P. calyptrata and C. genistoides reliant on either N2-fixation or 300 μM N (NH4NO3), and receiving 0.1, 1.0, 10 and 100 μM P (NaH2PO4).ResultsIn the species selection experiment, A. linearis, P. calyptrata and C. genistoides, with the greatest nodule fresh weight (FW) and nodule FW to root FW ratio, were the most prolific nodulating species. In the N X P experiment, with low P supply, the biomass of N2-fixing P. calyptrata and C. genistoides was consistently greater than that of N-supplied plants. In contrast, with high P supply of 100 μM P, all N-supplied plants accumulated more biomass than the corresponding N2-fixing plants. High P-use efficiency, poor down-regulation of P uptake and P storage was evident in A. linearis and P. calyptrata.ConclusionThe growth response to P and the significant N X P interactions indicate that N2-fixing and N-supplied plants were not adapted to low P, but rather colimited by both N and P.

Elevated levels of acid and alkaline phosphatase activity in roots and rhizosphere of cowpea (vigna unguiculata L. Walp.) genotypes grown in mixed culture and at different densities with sorghum (Sorghum bicolor L.)

The aim of this study was to assess P acquisition efficiency in 5 cowpea genotypes in mixed culture and at different plant densities using assays of acid and alkaline phosphatase activity and measurement of P in organs. Five cowpea genotypes (2 improved cvv. ITH98-46 and TVu1509, and 3 farmer-selected varieties, namely Bensogla, Sanzie, and Omondaw) were grown in the field at 2 planting densities (83 333 and 166 666 plants/ha) under monoculture and mixed culture with sorghum during 2005 and 2006. Fresh plant roots and rhizosphere soils were collected during the 2 years of experimentation, and assayed for acid and alkaline phosphatase activity. P concentrations in root tissue and rhizosphere soil were also determined using inductively coupled plasma-mass spectrometry. The data for 2005 and 2006 were similar, and therefore pooled for statistical analysis. Our results showed that raising cowpea density from 83 333 to 166 666 plants/ha significantly increased both acid and alkaline phosphatase activity in the rhizosphere, just as mixed culture (or intercropping) also increased the acid and alkaline phosphatase activity in cowpea rhizosphere soil. High plant density and mixed culture (or intercropping) also raised the acid phosphatase activity in fresh roots of cowpea plants. The increased enzyme activity in roots and rhizosphere soil resulted in significantly improved P nutrition in cowpea, greater plant growth, and higher grain yield in the farmer-selected varieties, especially cv. Sanzie. This suggests that field-grown legumes can be screened for high P acquisition efficiency by assaying for acid and alkaline phosphatase activities.

Effects of UV-B radiation on seed yield of Glycine max and an assessment of F1 generation progeny for carryover effects

Glycine max (L.) Merr plants were grown outdoors in potted sand exposed to elevated ultraviolet-B (UV-B) radiation provided by filtered fluorescent lamps to determine the effects of UV-B on seed yield and UV-B-induced carryover effects in the F1 generation. Increased UV-B radiation had no detectable effects on reproductive parameters except for a reduction on seed number per plant and an increase in the number of unseeded pods per plant and dry weight of unseeded pods per plant in the field supplemental UV-B experiment. Studies on carryover effects in the greenhouse progeny growth trial also showed no effect of parental treatment with UV-B on biomass production, and most symbiotic-N traits and plant metabolite measured. However, the concentrations of N in nodules and starch in roots were significantly increased in the F1 generation progeny from elevated UV-B radiation relative to their F1 counterparts from ambient radiation. Assessing the effects of seed size on plant growth and symbiotic function in the F1 progeny showed that total biomass, dry matter yield of individual organs (leaves, stems, roots and nodules), total plant N and fixed-N rose with increasing seed size. Seed concentration of flavonoids was also enhanced with increasing seed size. These findings suggest that subtle changes did occur in the F1 generation progeny of parental plants exposed to elevated UV-B with potential to accumulate with further exposure to elevated UV-B radiation.