Legume Inoculants Research Articles

EARLY ACCUMULATION OF REACTIVE OXYGEN SPECIES HAS A PIVOTAL ROLE IN NON-HOST RESISTANCE MECHANISMS IN LEGUME AND CEREAL PLANTS TO THE INCOMPATIBLE PATHOGENS

Most of plants under normal conditions are resistant to most of the incompatible pathogens (viral, fungal and bacterial infections). This is called ״non-host resistance (NHR) phenomenon״. It is very important to answer the question, why non-host plants are resistant? As a result of inoculation of legume (pea and soybean) and cereal (barley and wheat) plants with compatible and incompatible pathogens, strong resistance symptoms were observed in the non-host/incompatible pathogen combinations as compared with host/compatible pathogen combinations which showed severe infection (susceptibility). Levels of reactive oxygen species (ROS) mainly hydrogen peroxide (H2O2) and superoxide (O2.-) were significantly increased early 6, 12, 24 and 36 hours after inoculation (hai) in the non-host plants as compared with host plants. Interestingly enough that the activities of the antioxidant enzymes such as catalase (CAT), dehydroascorbate reductase (DHAR) and peroxidase (POX) were not significantly increased at the same early time 6 - 36 hai in the non-host plants. However, these enzymes were significantly increased later on 48, 72 and 96 dai in the non-host plants as compared with host plants. It seems that early accumulation of H2O2 and O2.- could have a dual roles, first role is inhibiting or killing the pathogens early in the non-host plants, second immunization of the non-host plants by stimulating the activities of the antioxidant enzymes later on which thereby, neutralize the harmful effect of ROS and consequently suppressing disease symptoms. The author recommends giving more attention to these new mechanisms of non-host resistance particularly in relation to ROS levels and antioxidant activities which are very important for plant breeders and useful for finding alternative control strategies as well.

Response of Cowpea, Soya Beans and Groundnuts to Non-Indigenous Legume Inoculants

<p>The use of inoculants is a critical strategy in legume production. In Zambia, inoculants are particularly used for the production of non-promiscuous genotypes of soya beans, but rarely for cowpeas and groundnuts. This study evaluated the response of soya beans, cowpeas and groundnuts to Biofix legume inoculants. Seeds were inoculated at the recommended or double the recommended rate at planting. Plants were grown under greenhouse conditions in a Completely Randomized Design for 7 weeks. Control, non-inoculated seeds were also planted and plants grown under the same conditions. At 7 weeks, nodule number and fresh weight per plant, nodule effectiveness (pinkness/redness), and above ground biomass were determined. Biologically fixed nitrogen was determined using the Nitrogen Difference Method. Nodule number and fresh weight per plant were higher at the recommended rate of Biofix application for soya beans and at both rates for groundnuts, while there was no effect on nodule fresh weight at either rate in cowpeas. All representative nodules assessed were effective. There was no significant benefit in inoculating seeds of the three legumes with Biofix on above ground biomass and biological nitrogen fixation. These results could suggest that possibly, the introduced strains though with a stronger nodulation competitiveness, were not as effective at fixing nitrogen as the indigenous strains in the soils in which soya beans, cowpeas and groundnuts have been repeatedly grown before. This could be an indication that sufficient and appropriate effective strains are already present in this soil. In general, the results suggest that to obtain the full benefits of biological nitrogen fixation, legume growers need to be provided with the correct inoculant, where required. Further work under field conditions is recommended to confirm these findings.</p>

Nodulation competitiveness as a requisite for improved rhizobial inoculants of Trifolium pratense

Red clover (Trifolium pratense L.) is used in the improvement of grasslands in Uruguay and has been inoculated with commercial strain U204 of Rhizobium leguminosarum bv trifolii since 1970s. Native-naturalized rhizobia strains present in soil are the basis for selecting and developing new inoculants. With this aim, we evaluated the diversity of red clover rhizobia in Uruguayan red clover pastures both historically inoculated with U204 and non-inoculated ones. Thirty-eight different enterobacterial repetitive intergenic consensus (ERIC) PCR genomic fingerprints were identified, albeit surprisingly only one of 80 isolates showed an ERIC profile similar to U204. Under controlled conditions, red clover plants inoculated with one of the native isolates, strain 317, produced more biomass than those inoculated with the commercial U204. ERIC-PCR was also used to show that strain 317 competed for nodulation better than U204 in a field with previous history of inoculation. Moreover, both U204 and 317 were tagged with a gusA reporter gene and their competitiveness for nodulation assessed in various soil types. Again, strain 317 appeared more competitive than U204, particularly in soils with previous history of inoculation. Our results reinforce the long-known idea of assessing the actual needs of inoculation of legumes in different soils and suggest that the indigenous isolate 317 is an effective and competitive strain that can be used for development of a new red clover inoculant.

Rhizobial counts in peat inoculants vary amongst legume inoculant groups at manufacture and with storage: implications for quality standards

Inoculation of legumes at sowing with rhizobia has arguably been one of the most cost-effective practices in modern agriculture. Critical aspects of inoculant quality are rhizobial counts at manufacture/registration and shelf (product) life. In order to re-evaluate the Australian standards for peat-based inoculants, we assessed numbers of rhizobia (rhizobial counts) and presence of contaminants in 1,234 individual packets of peat–based inoculants from 13 different inoculant groups that were either freshly manufactured or had been stored at 4 °C for up to 38 months to determine (a) rates of decline of rhizobial populations, and (b) effects of presence of contaminants on rhizobial populations. We also assessed effects of inoculant age on survival of the rhizobia during and immediately after inoculation of polyethylene beads. Rhizobial populations in the peat inoculants at manufacture and decline rates varied substantially amongst the 13 inoculant groups. The most stable were Sinorhizobium, Bradyrhizobium and Mesorhizobium with Rhizobium, particularly R. leguminosarum bv. trifolii the least stable. The presence of contaminants at the 10−6 level of dilution, i.e. >log 6.7 g−1 peat, reduced rhizobial numbers in the stored inoculants by an average of 37 %. Survival on beads following inoculation improved 2–3 fold with increasing age of inoculant. We concluded that the Australian standards for peat-based rhizobial inoculants should be reassessed to account for the large differences amongst the groups in counts at manufacture and survival rates during storage. Key recommendations are to increase expiry counts from log 8.0 to log 8.7 rhizobia g−1 peat and to have four levels of inoculant shelf life ranging from 12 months to 3 years.

Nitrogen fixation, hydrogen production and N2O emissions

Flynn, B., Scott, N. and Dong, Z. 2014. Nitrogen fixation, hydrogen production and N2O emissions. Can. J. Plant Sci. 94: 1037-1041. H2 is a by-product of the nitrogenase reaction. Exposure to H2 is linked to increased N2O production, increased CO2 fixation and plant growth promotion in soil. The effects of H2 exposure on soil were observed using controlled H2 gas treatments and field trials with legumes. In field trials, increased N2O production was observed in soil adjacent to legume nodules and inoculation of H2-oxidizing isolates led to increased N2O emissions in corn fields. Many H2-oxidizing isolates tested positive for key denitrification genes, indicating a connection between H2 uptake and N2O emissions. H2 treatment significantly increased copy number of the nitrite reductase (nirK) gene suggesting increased denitrification as the source of N2O. There was also a significant increase in copy number and expression of the RubisCO (cbbL) gene in soil. H2-oxidizing bacterial isolates (JM63 and JM162a) ...

Diversidade e potencial de utilização dos rizóbios isolados de nódulos de Gliricidia sepium

Research involving the selection of N2 fixing rhizobia for inoculation of forage legumes can contribute to improve the nutritional quality, and promote the management and conservation of pastures. Thus, the aim of this study was to analyze the morphological, phenotypic and symbiotic diversity of rhizobia derived from 30 root nodules of legume Gliricidia sepium. Thirty three bacterial isolates were obtained in medium 79, of which eight were selected for tolerance studies in different conditions of acidity and salinity and the symbiotic properties in G. sepium legumes, Phaseolus vulgaris and Crotalaria spectabilis. Bacterial isolates showed similar morphological characteristics, but differed in regard to phenotypic and symbiotic properties. Regarding G. sepium, all isolates inoculated including inoculant strain, BR 8802, showed similar ability to provide nitrogen for plant growth. However, some of the isolates showed greater ability to survive in different pH and salinity compared to BR 8802, suggesting that these should be selected for further studies.

Potential of indigenous bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. walp.) and green gram (Vigna radiata L. wilczek.) yields in Kenya

Limited information is available on reduced cowpea (Vigna unguiculata L. Walp.) and green gram (Vigna radiata L.Wilczek.) yields in Kenya. Declining soil fertility and absence or presence of ineffective indigenous rhizobia in soils are assumptions that have been formulated but still require to be demonstrated. In this study, soils were collected from legume growing areas of Western (Bungoma), Nyanza (Bondo), Eastern (Isiolo), Central (Meru) and Coast (Kilifi) provinces in Kenya to assess indigenous rhizobia in soils nodulating cowpea and green gram under greenhouse conditions. Our results showed that highest nodule fresh weights of 4.63 and 3.32 g plant−1 for cowpea and green gram were observed in one soil from Isiolo and another from Kilifi, respectively, suggesting the presence of significant infective indigenous strains in both soils. On the other hand, the lowest nodule fresh weights of 2.17 and 0.72 g plant−1 were observed in one soil from Bungoma for cowpea and green gram, respectively. Symbiotic nitrogen (N) fixation by cowpea and green gram was highest in Kilifi soil with values of 98% and 97%, respectively. A second greenhouse experiment was undertaken to evaluate the performance of commercial rhizobial inoculants with both legumes in Chonyi soil (also from Coast province) containing significant indigenous rhizobia [>13.5 × 103 Colony Forming Units (CFU) g−1]. Rhizobial inoculation did not significantly (P < 0.05) affect nodulation, biomass yield and shoot N content in cowpea and green gram compared with controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer (IGS) region analysis of nodules revealed six groups of which only IGS Group IV corresponded with those from commercial inoculants applied, indicating a lower competitiveness of inoculated strains. In cowpea, IGS III was dominant in nodules of plants inoculated with Biofix and Rizoliq commercial inoculants, and the uninoculated control treatment (63.2, 60 and 52.9%, respectively). Similarly, in green gram, IGS Group III was dominant in nodules of plants inoculated with Biofix 704 and Rizoliq commercial inoculants, and the uninoculated control treatment (75, 73.7 and 61.1%, respectively). Our results suggest that the systematic inoculation of both legumes with current available commercial inoculants to improve biomass yields is not necessary in these regions of Kenya. Also, according to our study, it would make sense to promote the utilization of indigenous strains performing well with both legumes.

Rhizobium Promotes Non-Legumes Growth and Quality in Several Production Steps: Towards a Biofertilization of Edible Raw Vegetables Healthy for Humans

The biofertilization of crops with plant-growth-promoting microorganisms is currently considered as a healthy alternative to chemical fertilization. However, only microorganisms safe for humans can be used as biofertilizers, particularly in vegetables that are raw consumed, in order to avoid sanitary problems derived from the presence of pathogenic bacteria in the final products. In the present work we showed that Rhizobium strains colonize the roots of tomato and pepper plants promoting their growth in different production stages increasing yield and quality of seedlings and fruits. Our results confirmed those obtained in cereals and alimentary oil producing plants extending the number of non-legumes susceptible to be biofertilized with rhizobia to those whose fruits are raw consumed. This is a relevant conclusion since safety of rhizobia for human health has been demonstrated after several decades of legume inoculation ensuring that they are optimal bacteria for biofertilization.

Conditions Affecting Shelf-Life of Inoculated Legume Seed

Microbial inoculants are becoming more available as sustainable alternatives to fertilizers and other agrichemicals in broad-acre cropping. However, with the exception of legume inoculants little is understood about effective delivery and survival of the inoculum. Legume inoculants are applied to both seed and soil but seed inoculation is the most economical technique. Large quantities of pasture seed in Australia are inoculated by commercial seed coating companies, but the long-term survival of seed-applied inoculum is variable and monitoring of viability requires specialist microbiology skills and facilities. The aim of our research was to define optimum storage conditions for survival of rhizobia on legume seed and evaluate water activity as a means of monitoring shelf-life. The relationship between survival and water activity varied according to seed species, inoculum preparation, coating ingredients, initial water activity and time suggesting that storage conditions would need to be defined for each different combination. Although drying seeds after coating significantly reduced viable numbers of rhizobia, survival of rhizobia on dried commercially coated lucerne seed after 11 weeks was less variable than seeds that had not been dried. The highest numbers were maintained when seeds remained dry with water activities of between 0.47 and 0.38. The quality of inoculated seed could be improved by reducing the death rate of inoculum during preparation and providing optimum storage conditions for long-term survival.

Australian Legume Research – synthesis and future directions

The Australian Legumes Symposium was the first in a planned series of regular technical symposia organised by the Australian Grasslands Association. The aim was to provide researchers with the opportunity to interact, present up-to-date reviews on topics related to pasture legume science, present results of current research and participate in planning of future research and development relevant to pasture legumes. This paper is intended to be the key output of the forum – a summary of findings and highlights from review and contributed research papers as well as identifying key research priorities for the future. In terms of the former, reviews presented at the symposium provided an overview of the development and role of pasture legumes in temperate farming systems. Closely related topics – nitrogen (N) fixation, N balance of farming systems and management of legume inoculation provided a focus on the importance of legumes in terms of N input and overall productivity. International perspectives on lucerne – its improvement and adoption provided a noticeable contrast to the apparent paucity of research into this species in Australia – despite its widespread use in temperate farming systems. In terms of content, there were many other papers delivered dealing with a wide diversity of relevant issues. On one hand the diversity of work in pasture legume research and development may stem from the wide array of expertise available in Australia and New Zealand, while on the other it might suggest that research and development inputs are being thinly spread over a large number of species. With respect to determining research priorities, it was surprising that participants were most concerned with how research is funded and conducted and the need to address this by reconsidering current arrangements. A greater role for economic analysis in determining research priorities was foreshadowed. The identification and management of acid-tolerant perennial legumes for higher rainfall zone permanent pastures was nominated as a major research priority, as was the need to address problems related to pre-inoculation of legume seed. The clear message from the symposium was that there needs to be a reorganisation of pasture legume improvement in order for gains to be effectively realised, and to maintain research and development capacity.

THE VALUE OF KNOWLEDGE TRANSFER PARTNERSHIPS AS A TOOL FOR CAPACITY BUILDING WITHIN THE KENYAN HORTICULTURE SECTOR

Knowledge Transfer Partnerships are an established method of developing academic-commercial relationships to further competitiveness and productivity through the better use of knowledge, technology and skills. Building on the UK model, the British Council is currently piloting AKTP (Africa Knowledge Transfer Partnerships) in Uganda, Ghana, Nigeria and Kenya. AKTP facilitates a partnership between an African commercial organisation and African higher education institution; supported by UK academic partner institutions. The commercial organisation employs a recent graduate (“associate”) who works in the company to implement the project by transferring knowledge between the higher education institutions and the company. The programme is designed to help businesses improve their productivity and competitiveness using the scientific knowledge, technology and skills available from the higher education Institutions. Five AKTP pilot projects have a horticultural focus [three in Kenya, one in Nigeria, and one in Ghana]; Writtle College has participated as UK academic partner for the Kenyan projects. The first case study examines the AKTP between the University of Nairobi and MEA, a fertiliser production company, in order to commercialise a legume inoculant. The product was developed by the University of Nairobi in the late 1970’s but the University was able to achieve only minimal market penetration. The projected outcomes for the AKTP are reviewed against the actual achievements for the company, the associate and the academic partner. The second case study reviews the progress to date of a two-year AKTP launched in January 2010 to measure the carbon footprint of key horticultural exports from Kenya. This partnership between Sunripe Ltd, producers and exporters of fruit and vegetables to Europe, Asia and South Africa, and Jomo Kenyatta University of Agriculture and Technology seeks to build capacity in both academic and commercial sectors through knowledge transfer from the UK. The third project which is only recently started is on postharvest aspects between East Africa Growers and Egerton University.

Arbuscular mycorrhiza maintains nodule function during external NH 4 + supply in Phaseolus vulgaris (L.)

The synergistic benefits of the dual inoculation of legumes with nodule bacteria and arbuscular mycorrhizae (AM) are well established, but the effect of an external NH(4)(+) supply on this tripartite relationship is less clear. This effect of NH(4)(+) supply was investigated with regards to the growth and function of the legume host and both symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were grown in a sand medium with either 0 N, 1 mM or 3 mM NH(4)(+). Plants were harvested at 30 days after emergence and measurements were taken for biomass, N(2) fixation, photosynthesis, asparagine concentration, construction costs and N nutrition. The addition of NH(4)(+) led to a decline in the percentage AM colonization and nodule dry weights, although AM colonization was affected to a lesser extent. NH(4)(+) supply also resulted in a decrease in the reliance on biological nitrogen fixation (BNF); however, the AM roots maintained higher levels of NH(4)(+) uptake than their non-AM counterparts. Furthermore, the non-AM plants had a higher production of asparagine than the AM plants. The inhibitory effects of NH(4)(+) on nodule function can be reduced by the presence of AM at moderate levels of NH(4)(+) (1 mM), via improving nodule growth or relieving the asparagine-induced inhibition of BNF.

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields

BackgroundAssociated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T.ResultsThe most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the β-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined.ConclusionsThe soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

The biodiversity of beneficial microbe-host mutualism: the case of rhizobia

Symbiotic nitrogen fixation is the main route for sustainable input of nitrogen into ecosystems. Nitrogen fixation in agriculture can be improved by inoculation of legume crops with suitable rhizobia. Knowledge of the biodiversity of rhizobia and of local populations is important for the design of successful inoculation strategies. Soybeans are major nitrogen-fixing crops in many parts of the world. Bradyrhizobial inoculants for soybean are very diverse, yet classification and characterization of strains have long been difficult. Recent genetic characterization methods permit more reliable identification and will improve our knowledge of local populations. Forage legumes form another group of agronomically important legumes. Research and extension policies valorizing rhizobial germplasm diversity and preservation, farmer training for proper inoculant use and legal enforcement of commercial inoculant quality have proved a successful approach to promoting the use of forage legumes while enhancing biological N 2 fixation. It is worth noting that taxonomically important strains may not necessarily be important reference strains for other uses such as legume inoculation and genomics due to specialization of the different fields. This article points out both current knowledge and gaps remaining to be filled for further interaction and improvement of a rhizobial commons.

Host plant as an organizer of microbial evolution in the beneficial symbioses

Evolution of beneficial plant–microbe symbioses is presented as a result of selective processes induced by hosts in the associated microbial populations. These processes ensure a success of “genuine mutualists” (which benefit the host, often at the expense of their own fitness) in competition with “symbiotic cheaters” (which consume the resources provided by host without expressing the beneficial traits). Using a mathematical model describing the cyclic microevolution of rhizobia–legume symbiosis, we suggest that the selective pressures in favor of N2-fixing (Fix+) strains operate within the in planta bacterial population due to preferential allocation of C resources into Fix+ nodules (positive partners’ feedbacks). Under the clonal infection of nodules, Fix+ strains (“genuine mutualists”) are supported by the group (inter-deme, kin) selection while under the mixed infections, this selection is ineffective since the Fix+ strains are over-competed by Fix− ones (“symbiotic cheaters”) in the nodular habitats. Nevertheless, under mixed infections, Fix+ strains may be supported due to the coevolutionary responses form plant population which induce the mutualism-specific types of natural (group, individual) selection including the frequency dependent selection implemented in rhizobia population during the competition for host infection. Using the model of multi-strain bacterial competition for inoculation of symbiotic (rhizospheric, nodular) habitats, we demonstrate that the individual selection in favor of host-specific mutualist genotypes is more intensive than in favor of non-host-specific genotypes correlating the experimental data on the coordinated increases of symbiotic efficiency and specificity in the rhizobia–legume coevolution. However, an overall efficiency of symbiotic system is maximal when the non-host-specific mutualists are present in rhizobia population, and selection in favor of these mutualists operating at the whole population level is of key importance for improving the symbiosis. Construction of the agronomically valuable plant–microbe systems should provide the optimization of host-specific versus non-host-specific mutualists’ composition in legume inoculants combined with the clonal penetration of these mutualists into the nodules.

Caracterização de rizóbios indicados para produção de inoculantes por meio de sequenciamento parcial do 16S rRNA

O objetivo deste trabalho foi confrontar as sequências parciais do gene 16S rRNA de estirpes padrão de rizóbios com as de estirpes recomendadas para a produção de inoculantes no Brasil, com vistas à verificação da confiabilidade do sequenciamento parcial desse gene para a identificação rápida de estirpes. Foram realizados sequenciamentos através de reação em cadeia da polimerase (PCR) com iniciadores relativos à região codificadora do gene 16S rRNA entre as bactérias estudadas. Os resultados foram analisados pela consulta de similaridade de nucleotídeos aos do "Basic Local Alignment Search Tool" (Blastn) e por meio da interpretação de árvores filogenéticas geradas usando ferramentas de bioinformática. A classificação taxonômica das estirpes Semia recomendadas para inoculação de leguminosas com base em propriedades morfológicas e especificidade hospedeira não foi confirmada em todas as estirpes. A maioria das estirpes estudadas, consultadas no Blastn, é consistente com a classificação proposta pela construção de árvores filogenéticas das sequências destas estirpes, com base na similaridade pelo sequenciamento parcial do gene considerado.

Are the rates of photosynthesis stimulated by the carbon sink strength of rhizobial and arbuscular mycorrhizal symbioses?

Rhizobial and arbuscular mycorrhizal (AM) symbioses each may consume 4–16% of recently photosynthetically-fixed carbon to maintain their growth, activity and reserves. Rhizobia and AM fungi improve plant photosynthesis through N and P acquisition, but increased nutrient uptake by these symbionts does not fully explain observed increases in the rate of photosynthesis of symbiotic plants. In this paper, we test the hypothesis that carbon sink strength of rhizobial and AM symbioses stimulates the rates of photosynthesis. Nutrient-independent effects of rhizobial and AM symbioses result in direct compensation of C costs at the source. We calculated the response ratios of photosynthesis and nutrient mass fraction in the leaves of legumes inoculated with rhizobial and/or AM fungi relative to non-inoculated plants in a number of published studies. On average, photosynthetic rates were significantly increased by 28 and 14% due to rhizobial and AM symbioses, respectively, and 51% due to dual symbiosis. The leaf P mass fraction was increased significantly by 13% due to rhizobial symbioses. Although the increases were not significant, AM symbioses increased leaf P mass fraction by 6% and dual symbioses by 41%. The leaf N mass fraction was not significantly affected by any of the rhizobial, AM and dual symbioses. The rate of photosynthesis increased substantially more than the C costs of the rhizobial and AM symbioses. The inoculation of legumes with rhizobia and/or AM fungi, which resulted in sink stimulation of photosynthesis, improved the photosynthetic nutrient use efficiency and the proportion of seed yield in relation to the total plant biomass (harvest index). Sink stimulation represent an adaptation mechanism that allows legumes to take advantage of nutrient supply from their microsymbionts without compromising the total amount of photosynthates available for plant growth.

Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 10 9 cfu ml −1 of Sinorhizobium ( Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.

Response to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum

Flavonoids play a crucial role as signal molecules in promoting the formation of nodules by symbiotic bacteria commonly known as rhizobia. The early interaction between flavonoids and NodD regulatory protein activates nod gene transcription and the synthesis of Nod factor that initiates nodule primordium. In this study, we assessed response to flavonoids as factors influencing competitiveness of rhizobia and their symbiotic activity. Rhizobium leguminosarum nodule isolates belonging to three biovars, trifolii, viciae and phaseoli characterized earlier as competitive or uncompetitive relative to native rhizobia, were used. Investigating nodA promoter induction using plasmid lacZ fusion, we found that competitive strains more readily responded to a wide range of synthetic flavonoids and seed exudates in comparison to uncompetitive strains, albeit some exceptions were noticed. Of all the synthetic flavonoids and seed exudates studied, naringenin, hespertin and clover and vetch exudates were the most effective inducers of nodA promoter in competitive strains. Only one of the nine examined uncompetitive strains was highly induced by clover seed exudate. Subsequently, the effect of preinduction of R. leguminosarum bv. trifolii with clover exudate was assessed. Out of 18 pre-activated strains, nine strains (including competitive ones) increased clover wet mass of shoots and nodule number when used as inoculants. Our results demonstrate a plausible approach of isolating and characterizing flavonoid-responsive field isolates that could be further developed into relevant legume inoculants.

Desiccation tolerance of rhizobia when protected by synthetic polymers

Survival of rhizobia applied to the surface of legume seeds is poor due to factors such as desiccation. Poor survival of rhizobia results in poor nodulation and yield of legumes. Selecting polymeric adhesives for inoculation of legume seed with rhizobia that provide protection during desiccation may improve survival and increase the potential for maximum legume yields. Vacuum-drying cells after suspension in selected polymers proved an effective method for screening the potential of polymers to improve the desiccation tolerance of rhizobia. The effect of different polymers on survival of desiccated rhizobia could be attributed to their different chemical and physical properties. The specific protective properties of polymers have been difficult to determine due to the variation in the chemical nature of polymers often compared. In this research polyvinyl alcohol (PVA) with varying degrees of hydrolysis provided a useful range of measurable physical properties against which bacterial survival could be measured. PVA with a percent hydrolysis in the range 86.5–89% was better able to protect desiccated cells of a range of rhizobial strains than polymers with higher (98.5%) or lower (78.5–82%) degrees of hydrolysis. The percent hydrolysis affected the moisture properties of PVA and survival of rhizobia was not maximised with high moisture sorption or low water activity by the polymer but rather when moisture properties were at an intermediate level. In comparison, survival was poorest in highly hygroscopic polymers methyl cellulose (MC) and polyvinyl pyrrolidone (PVP). The survival profile of desiccated rhizobia stored at different relative humidities was altered when cells were embedded in different polymers and is probably related to moisture sorption by those polymers. The percent hydrolysis also affects the extent to which PVA is able to stabilise colloids against the precipitating action of KCl. The colloid-stabilising property and survival was highest at 86.5–89% indicating that this property may be manipulated to achieve better survival. There is an indication that highly stabilising PVA may lead to more evenly dispersed cells providing more colony forming units rather than better survival. However, survival was not strongly correlated to the colloid-stabilising properties of the other polymers and was very poor after suspension in highly stabilising MC indicating a strong interaction between factors. Synthetic polymers designed to improve survival of rhizobia exposed to desiccation stress should include properties that combine high stabilisation and optimum moisture sorption properties.