Nodulation status of native woody legumes and phenotypic characteristics of associated rhizobia in soils of southern Ethiopia

The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.     

Phosphorus requirements and nitrogen accumulation by N2-fixing and non-N2-fixing leguminous trees growing in low P soils

The variation in P uptake and use efficiency and N accumulation by Gliricidia sepium (N₂-fixing tree), Senna siamea and S. spectabilis (leguminous non-N₂-fixing trees) were examined in the field at Fashola (savanna zone), southwestern Nigeria, using four P rates, 0, 20, 40 and 80 kg P ha^-1. Growth of G. sepium and S. spectabilis responded to P application at 24 weeks after planting (WAP) and average yield increases of 58% and 145% were observed by the application of 40 kg P ha^-1 for the two species, respectively. Such a P response was not found in S. siamea at 24 WAP and for any of the species at 48 WAP. G. sepium accumulated more P (on average 162%) than S. siamea and S. spectabilis at 24 WAP and had greater root length and a higher percentage of mycorrhizal infection. However, at 48 WAP S. siamea had 2.5 times more P than G. sepium. Differences in the physiological P use efficiency (PPUE) between G. sepium and the non-N₂-fixing trees were significant at the 0 P level, being higher for S. siamea (average, 0.61 g shoot mg^-1 P) than for G. sepium (0.27 g shoot mg^-1 P). G. sepium had a consistently lower atom % ¹⁵N than S. spectabilis, while that of S. siamea for most of the time did not differ from that of G. sepium. The reference plant affected N₂ fixation extimates, with negative values and a higher variability (CV 60%) associated with S. siamea than with S. spectabilis (CV<20%). Consequently, S. spectabilis was selected as a better reference plant for measuring N₂ fixation in G. sepium. G. sepium fixed on average 35% and 54% of its N at 24 and 48 WAP, respectively. Except at the lowest P rate, percentage and amount of N fixed were not generally enhanced by P application.     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Rhizobium sp. exopolysaccharide production,nodulation, and acetylene reduction activity (N2-fixation) in pigeon pea (Cajanus cajan L.)

Summary There was no correlation between the quantity of exopolysaccharide produced and acetylene reduction activity by Rhizobium spp. or by Bradyrhizobium spp. (Cajanus). The exopolysaccharide-defective mutants of Rhizobium sp. strain P 116 either failed to nodulate or showed a decrease in effectiveness. The deficiency in exopolysaccharide production was corrected by the addition of purified exopolysaccharide from the parent strain, or from Bradyrhizobium sp. strain P 149 or S24 isolated from pigeonpea (Cajanus cajan) and mungbean (Vigna radiata), respectively. However, the nodules so formed were not fully effective compared to those formed by the parent strain.     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

Burr medic (Medicago polymorpha L.) selections for improved N2 fixation with naturalised soil rhizobia

Burr medic (Medicago polymorpha L.) is an annual pasture legume that is widely distributed in southern Australian farming systems. Burr medic is nodulated by rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae) that reside in many Australian soils, but the symbioses that develop are often sub-optimal in their rate of N₂ fixation. We attempted to identify burr medic lines, which are able to form effective symbioses with the naturalised Sinorhizobium in Australian field soils, as potential parents for a breeding program. There were three glasshouse experiments. Initially, 222 lines (including the M. polymorpha cvv. Santiago, Serena and Circle Valley) were inoculated with extracts of two soils that had been collected near Waikerie (soil S109) and Lochiel (soil S142) in South Australia. These soils were used because they contained numerically large communities of naturalised Sinorhizobium spp. that produced sub-optimal rates of N₂ fixation with cv. Santiago. None of the 222 lines of burr medic were able to form an effective symbiosis with the rhizobia from soil S109. However, when nodulated by the rhizobia from soil S142, some lines (e.g. SA8194) formed a very effective symbiosis, producing up to double the shoot dry matter (DM) of Santiago and eight times the DM of uninoculated plants. Seven promising lines were selected for further testing (with extracts of nine soils). Subsequently, two lines (SA20056 and SA8194) were selected and their symbiotic performance compared with that of Santiago, using extracts from 28 soils. While soil treatment had a major effect on mean shoot DM (soil N103=120 mg, soil N105=17 mg), the three medic lines performed similarly. Santiago, SA20056 and SA8914 all formed ineffective symbioses with the rhizobia in at least half of the 28 soils, even though >95% of the plants were nodulated. These experiments confirm that ineffective symbioses are common between burr medics and the rhizobia that have become naturalised in many Australian soils. Although some lines of burr medic were identified that were able to form more effective symbioses with the rhizobia in individual soils, none were able to form effective symbioses with a wide range of soil rhizobia. If a plant breeding approach is to be used to improve symbiotic performance of burr medic we propose that its hybridisation with other medic species, that have less specific rhizobial needs, will be required.     

Nitrogenase activity and nitrogen-fixing bacteria associated with the roots ofAtriplex spp. growing in saline sodic soils of Pakistan

Summary TwoAtriplex spp. growing in low-fertility saline sodic soils were assayed for root-associated nitrogenase activity. The excised washed and unwashed root of the two species.A. lentiformis andA. amnicola, showed high root-associated nitrogenase activity. Acetylene-reducing activity seemed to be directly influenced by moisture. The highest number of diazotrophs, enumerated using a most probable number technique was observed on the root surface. Most of the isolated diazotrophs were identified asEnterobacter agglomerans. Root-associated nitrogenase activity inAtriplex spp. may explain the high protein and biomass content of these plants growing in low-fertility saline sodic soils.     

Nodulation and estimation of symbiotic nitrogen fixation by herbaceous and shrub legumes in Guinea savanna in Nigeria

Twelve herbaceous and shrub legume species were grown in pot and field experiments in five sites representing three agroecological zones in moist savanna in Nigeria. The objectives were to: (1) assess natural nodulation of the legumes and characterize their indigenous rhizobia, (2) determine their need for rhizobia inoculation and (3) estimate the amount of N₂ fixed by each of these legumes. At 4 weeks after planting (WAP), Crotolaria verrucosa was not nodulated at any of the sites while Centrosema pascuorum had the highest number of nodules in all sites. At 8 WAP, all legumes were nodulated, with Mucuna pruriens having the least number of nodules and Stylosanthes hamata the highest. The number of nodules, however, was inversely correlated to the mass of nodules. Significant differences in nodulation of the legume species grown in the field also occurred between and within sites. Mucuna pruriens and Lablab purpureus produced more shoot and nodule biomass than the other legumes in all sites. Growth of most of these legumes responded to fertilizer application, except for C. verrucosa and Aeschynomene histrix. Except for C. verrucosa, average proportion of N₂ fixed was about 80% and this was reduced by about 20% with N fertilizer application. The majority of rhizobia isolates (60%) were slow growing, belonging to the Bradyrhizobia spp. group. Selected rhizobia isolates evaluated on Cajanus cajan, C. pascuorum, M. pruriens and Psophocarpus palustris varied from ineffective to highly effective in Leonard jar conditions. However, only growth of M. pruriens responded to inoculation in potted soils, whereas it was lower than that obtained with N fertilizer application. This indicated the need to screen more rhizobia in order to improve N₂ fixation and growth of legume species such as M. pruriens when it is introduced in soils deficient in N.     

Physiological enhancement of early growth of rice seedlings (Oryza sativa L.) by production of phytohormone of N2-fixing methylotrophic isolates

Three plant-growth promoting, N₂-fixing methylotrophic strains isolated from rice cultivars (Oryza sativa L.), viz, Methylobacterium sp. CBMB20, Enterobacter sp. CBMB30, Burkholderia sp. CBMB40, were selected, and their activities in promoting the early growth of rice were studied. Seeds treated with the methylotrophic strains improved seed germination, seedling vigor index (SVI), and biomass of rice seedlings. The methylotrophic population in the treated seedlings increased in the vegetative stages when compared to seeding stages. Treated seedlings showed a higher accumulation of plant hormones viz trans-zeatin riboside, isopentenyladenosine, and indole-3-acetic acid than untreated seedlings. Plant hormones were detected immunologically using the phytodetek kit. Conformational evidence suggested that cytokinins were produced by the epiphytic bacteria colonizing the plants rather than by the plants themselves. In addition, the inoculated early stage rice seedlings also exhibited a wide range of acetylene reduction activity. The results suggest the potential use of these bacteria to stimulate germination, SVI, and biomass production, which is mediated by production of plant hormone accumulation and nitrogen fixation.     

Enhanced denitrification in plots of N2-fixing faba beans compared to plots of a non-fixing legume and non-legumes

Denitrification rates were studied using the C₂H₂ inhibition technique in a 2-year field experiment within plots of nodulated and non-nodulated faba beans, ryegrass, and cabbage. Denitrification rates ranged from 14.40 to 0.02 ng N₂O–N g^–1 soil dry weight h^–1. Mean denitrification increased fourfold in plots of N₂–fixing Vicia faba compared to non-nodulated V. faba mutant F48, Lolium perenne, and Brassica oleracea. The results with and without C₂H₂ treatment indicate that in the field the major part of this enhanced denitrification led to the endproduct N₂ rather than to the ozone-degrading N₂O. Higher denitrification rates of plots with N₂–fixing plants in September seemed to be caused by an increase in soil NO _inf3 ^sup- of about 20 kg ha^–1 found between July and August. Soil NO _inf3 ^sup- and soil moisture explained 67% of the variation in denitrification rates of the different soil samples over the growing seasons in the 2 years. Soil moisture explained 44% of the variation for soil planted with N₂–fixing plants and 62% for soil planted with non-fixing plants. Positive exponential relationships were obtained between denitrification rates and soil nitrate (r=0.71) and soil moisture (r=0.82).     

A bradyrhizobial-<Emphasis Type="Italic">Penicillium</Emphasis> spp. biofilm with nitrogenase activity improves N<Subscript>2</Subscript> fixing symbiosis of soybean

A bradyrhizobial-fungal biofilm (i.e. Bradyrhizobium elkanii SEMIA 5019-Penicillium spp.) developed in vitro was assayed for its nitrogenase activity and was evaluated for N₂-fixing symbiosis with soybean under greenhouse conditions. The biofilm showed nitrogenase activity, but the bradyrhizobial strain alone did not. Shoot and root growth, nodulation and N accumulation of soybean increased significantly with an inoculum developed from the biofilm. This study concludes that such biofilmed inoculants can improve N₂-fixing symbiosis in legumes, and can also directly contribute to soil N fertility in the long term. Further studies should be conducted to investigate the performance of these inoculants under field conditions.     

Establishment and effectiveness of added pigeonpea (Cajanus cajan) rhizobia in different soils of narrow abiotic variability

Summary Pot and laboratory experiments were conducted to study the establishment and effectiveness of a streptomycin-sulphate-resistant (1 mg/ml of medium) pigeonpea rhizobia strain (RM₇) in sterile sand and non-sterile soils. Strain RM₇ increased the drymatter yield of pigeonpea plants (Cajanus cajan) by 106% over control plants under sterile conditions. However, when the rhizobia strain was introduced into 14 different non-sterile soils with a narrow abiotic variability, the comparable beneficial effect was observed only in one soil inoculated with log 6.70 cells/pot. At this inoculum rate, the percentage increase in yield over control plants varied from –1 to 140 in different soils. Rhizobium (RM₇), applied at log 3.70 cells/pot (3 kg soil), showed less than 5% establishment in four soils. However, establishment varied from 8% to 72% at a higher level of inoculation (log 6.70 cells/pot). Displacement of native rhizobia and creation of new sites for nodulation by the introduced rhizobia were also affected by soil properties. The increase in shoot dry-matter yield compared with control plants was positively correlated with the percent establishment of RM₇ (r = 0.60^*) in these soils. Experiments showed that some biotic stresses led to poor survival, proliferation and establishment of the added alien in the soil. Therefore, any culture that is efficient in one soil may not produce similar results under all situations.     

Competition among strains of Bradyrhizobium and vesicular-arbuscular mycorrhizae for groundnut (Arachis hypogaea L.) root infection and their effect on plant growth and yield

Summary Strains of Bradyrhizobium influenced root colonization by a species of vesicular-arbuscular mycorrhizae (VAM), and species of VAM influenced root nodulation by strains of Bradyrhizobium in pot experiments. In a field experiment, the effects of VAM on competition amongst inoculated bradyrhizobia were less evident, but inoculation with Bradyrhizobium strains increased root colonization by VAM. Certain VAM/Bradyrhizobium inoculum strain combinations produced higher nodule numbers. Plants grown without Bradyrhizobium and VAM, but supplied with ammonium nitrate (300 g ml^–1) and potassium phosphate (16 g ml^–1), produced higher dry-matter yields than those inoculated with both symbionts in the pot experiment. Inoculation with either symbiont in the field did not result in higher pod and haulm yields at harvest.ICRISAT Journal Article No. 886     

Co-selection of compatible rhizobia and vesicular-arbuscular mycorrhizal fungi for cowpea in sterilized and non-sterilized soils

Summary We selected two isolates of Rhizobium for cowpea (Vigna unguiculata) with sterilized soil tests and two different isolates by non-sterilized soil testing. The four rhizobia were then paired individually with either Glomus pallidum, Glomus aggregatum, or Sclerocystis microcarpa in separate, sterilized, or non-sterilized soil experiments. The purpose of the experiments was to determine the effect of soil sterilization on the selection of effective cowpea rhizobia, and to see whether these rhizobia differed in their effects on cowpea growth when paired with various vesicular-arbuscular mycorrhizal (VAM) fungi. Our experiments showed that the rhizobia selected in sterilized soil tests produced few growth responses in the cowpea compared to the other introduced rhizobia, irrespective of pairing with VAM fungi in sterilized or non-sterilized soil. In contrast, the two rhizobia initially selected by non-sterilized soil testing significantly improved cowpea growth in non-sterilized soil, especially when paired with G. pallidum. Our results suggest that it is important to select for effective rhizobia in non-sterilized soil, and that pairing these rhizobia with specific, coselected VAM fungi can significantly improve the legume growth response.     

Nitrogen-fixing potential of Acacia mangium and Acacia auriculiformis seedlings inoculated with Bradyrhizobium and Rhizobium spp.

Summary Two Australian Acacia species, A. mangium and A. auriculiformis were inoculated in vitro with eight strains of Bradyrhizobium spp. and two strains of Rhizobium spp. On the two plant species, only Bradyrhizobium spp. strains formed effective N₂-fixing nodules. A. mangium, which nodulates effectively with a restricted range of Bradyrhizobium spp. strains, is a specific host compared to A. auriculiformis. A. auriculiformis is assumed to be a promiscuous host because it nodulates effectively with a wide range of Bradyrhizobium spp. strains. Nodule efficiency as expressed by the ratio of N₂ fixed to nodule dry weight appeared to be higher in A. auriculiformis (0.44–0.81) than in A. mangium (0.23–0.55).     

Effect of temperature on flavonoid production in pigeonpea [Cajanus cajan (L) Millsp] in relation to nodulation

To ascertain the reasons for poor nodulation of pigeonpea, we studied the effect of high temperature on the production of flavonoids by the pigeonpea host. A high temperature affected flavonoid production by pigeonpea and mungbean. At 37°C pigeonpea root exudates contained four flavonoids and the root extract contained five. The proportion of the second flavonoid in the pigeonpea and the mungbean was higher and the proportion of the third flavonoid was lower at 37°C compared to 30°C. At the higher temperature the flavonoids exuded from pigeonpea roots were same those in the root homogenate.     

花生连作红壤芽孢杆菌的群落多样性及其生防效果研究

芽孢杆菌是土壤中常见的微生物类群,在土壤生态系统中占据重要地位。本研究发现低丘红壤区花生连作红壤中芽孢杆菌的数量为2.0×105 ~ 2.3×105 cfu/g 干土。通过构建3个样品的基因文库,共得到9个种类;16S rDNA 测序结果表明该地区芽孢杆菌的优势类型有短小芽孢杆菌、枯草芽孢杆菌、高地芽孢杆菌,分别占总克隆的54%、12% 和14%。并研究了施用不同微生物制剂对连作花生产量和生育性状的影响,结果显示,施用光合细菌和枯草芽孢杆菌能够在一定程度上提高花生的产量,枯草芽孢杆菌和光合细菌混合施用的增产效果最好,比对照提高了32.14%。     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

Relationship between total nodulation and nodulation at the root crown of peanut, soybean and common bean plants

The objective of this study was to simplify the procedure for evaluation of legume nodulation, by examining if there is a relationship between the nodulation of the whole root system and at the crown region. Roots of peanut, soybean and common bean plants growing in soils were split in two parts (crown and bottom) and assessed for nodulation (nodule number and dry weight). In general, most nodules were concentrated at the crown, and crown nodulation was generally positively correlated with total nodulation of all three legume crops. The results are highly applicable in studies such as strain selection for inoculants and assessment of inoculation technologies, among others, and is an important contribution to help reduce the time and labor required for the evaluation of nodulation parameters.     

N supply by groundnuts to maize in a maize plus groundnut intercropping system,as affected by the genotype

Summary Identification of legume genotypes with high N-supplying ability is important in improving and sustaining the productivity of low-input cropping systems. Hence, ¹⁵N-aided studies were made to ascertain the relative N-supplying ability of some cultivars of groundnut, a widely grown tropical legume. The study was conducted outdoors in 1991 at Kamburupitiya, Sri Lanka, in tanks filled with 64 kg soil which had been tagged by incorporating ¹⁵N-labelled plant material. Maize cv. Badra was grown as a monocrop and as an intercrop with five genotypes of groundnut, X-14, MI-1, Red Spanish, ICGV 87127, and a non-nodulating line. All the nodulating genotypes derived over 90% of their N from the atmosphere. Significant genotypic differences in N₂ fixation were observed. X-14 fixed the highest amount (1.95 g plant^-1), while Red Spanish the lowest (0.88 g plant^-1). Intercropping of maize with nodulating groundnut significantly decreased the ¹⁵N atom excess of maize, depending on the genotype. However, this decrease did not appear to be related to the amount of N₂ fixed, based on aboveground material. The per cent N derived by maize from the intercropped groundnuts varied from 17% (X-14) to 39% (Red Spanish), indicating a marked genotypic variability in N-suppling ability. X-14, which fixed the largest amounts of N₂, grew most vigorously compared to other genotypes, causing a growth depression in the maize. The genotype that fixes the most N₂ may therefore not necessarily have the greatest N-supplying ability. The transfer of N from the legume and the consequent improvement of N nutrition in the associated cereal in low-fertility situations is therefore expected to be high when the growth of the legume is intermediate and does not suppress the growth of the cereal.