Symbiotic performance of selectedCyclopia Vent. (honeybush) rhizobia under nursery and field conditions

Three newly selected strains ofCyclopia rhizobia together with an inoculant strain, which has never been tested in the field with adequate experimental design, was assessed under both nursery and field conditions for symbiotic performance. The three new test strains were initially selected for their superior N₂-fbcing abilities under glasshouse conditions, and then evaluated in this study for field performance.Cyclopia subternata Vogel andCyclopia genistoides (L.) R. Br., which have the potential for producing high quality honeybushtea, were used as host plants in both the nursery and field studies. The effect of seedling inoculation at the nursery level was also examined for the four test strains under nursery conditions. The inoculation of cuttings under nursery conditions produced.significant increases in shoot biomass, shoot %N and shoot N content. More specifically, inoculatingC. subternata with strains UCT44b and UCT61a significantly increased shoot biomass and N content relative to strain PPRICI3. Strains UCT44b and UCT61a also showed better nodulation withC. subternata cuttings compared to strains UCT40a and PPRICI3. Field inoculation ofCyclopia increased all growth parameters relative to the uninoculated control, except for leaf %N.Cyclopia subternata inoculated with strains UCT44b, UCT40a and UCT61a produced significantly lower δ¹⁵N values than the uninoculatedC. subtemata reference plant. Using the¹⁵N natural abundance method,C. subternata was estimated to be gaining about half of its N from N₂ fixation, whileC. genistoides obtained less than half of its N from symbiotic nutrition.     

Overcoming non-selective nodulation of Lessertia by soil-borne rhizobium in the presence of inoculant mesorhizobium

Background and aims

Legumes of the South African genus Lessertia, along with their microsymbionts, were introduced into the Western Australia wheatbelt. They achieved poor establishment followed by weak summer survival. This was caused in part by low levels of nodulation with the inoculant strains, and by ineffective nodulation with naturalized strains –an example of non-selective nodulation. The aims of this work were to assess Lessertia spp. symbiotic promiscuity, to study the effect of increased doses of an effective inoculant strain (WSM3565) with L. herbacea, and to study the competitive ability and symbiotic performance of different Mesorhizobium strains nodulating L. diffusa.

Methods

A glasshouse experiment was set up to evaluate the ability of L. diffusa, L. capitata, L. herbacea and L. excisa to nodulate with inoculants under current use in Western Australia. To assess competitive ability two field experiments were set up at Karridale, Western Australia. L. herbacea was inoculated with the strain WSM3565 at different doses and L. diffusa was inoculated with ten different Mesorhizobium strains. Rhizobia were re-isolated from nodules and their identity confirmed through PCR fingerprinting and sequencing of their partial dnaK.

Results

There were differences in promiscuity between different Lessertia spp., where L. herbacea proved to be highly promiscuous under controlled conditions. Increasing the inoculation dose of L. herbacea with WSM3565 did not improve establishment and survival of the legume in the field. Although WSM3565 nodule occupancy improved from 28 to 54 % with higher doses of inoculation, none of the treatments increased L. herbacea yield over the inoculated control. The inoculation of L. diffusa with the strains WSM3598, 3636, 3626 and 3565 resulted in greater biomass production than the uninoculated control. These strains were able to outcompete resident rhizobia and to occupy a high (>60 %) proportion of lateral root nodules. The naturalised strains that achieved nodulation were identified as R. leguminosarum.

Conclusion

The high numbers of resident rhizobia and their ability to rapidly nodulate Lessertia spp. are likely to be the main reasons for the low nodule occupancy achieved by some effective inoculant strains with L. diffusa and L. herbacea. Strains WSM 3636 and 3598 were very competitive on nodule occupancy and together with WSM 3565, WSM 3612 and WSM3626, effective on nodule formation and plant growth of L. diffusa despite the high numbers of resident soil rhizobia. These strains and L. diffusa have potential to be introduced as exotic legumes species and rhizobia strains to Western Australia.     

Establishment and survival of the South African legume Lessertia spp. and rhizobia in Western Australian agricultural systems

Background and aims

The South African herbaceous legume species Lessertia capitata, L. diffusa, L. excisa L. incana and L. herbacea were introduced to Australia to assess plant establishment and survival, as well as the saprophytic ability of their root nodule bacteria (RNB).

Methods

Five Lessertia spp., were inoculated with selected RNB strains and were sown in five different agroclimatic areas of the Western Australian wheat-belt during 2007 and 2008. Plant population and summer survival were evaluated in situ. Soil samples and nodules from host plants were also taken from each site. The re-isolated rhizobia were RPO1-PCR fingerprinted and their partial dnaK and nodA genes were sequenced to confirm their identity.

Results

Plants achieved only poor establishment followed by weak summer survival. More than 83 % of the rhizobia re-isolated from Lessertia did not correlate with the original inoculants’ fingerprints, and were identified as Rhizobium leguminosarum. The nodA sequences of the naturalised strains were also clustered with R. leguminosarum sequences, thus eliminating the likelihood of lateral gene transference from Mesorhizobium and suggesting a competition problem with indigenous rhizobia.

Conclusion

The stressful soil conditions and high numbers of resident R. leguminosarum strains in Western Australian soils, and their ability to rapidly nodulate Lessertia spp. but not fix nitrogen are likely to preclude the adoption of Lessertia as an agricultural legume in this region.     

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

Aims

In the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.

Methods

Glasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.

Results

Complex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.

Conclusions

Effective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.     

Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region

The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region.     

Biosynthesis of compatible solutes in rhizobial strains isolated from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> nodules in Tunisian fields

Background  

Associated 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 899^T.     

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

Background and aims

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.

Methods

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.

Results

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.

Conclusions

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.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

Improvement of cowpea productivity by rhizobial and mycorrhizal inoculation in Burkina Faso

Cowpea is one of the most important food legume crops in Burkina Faso. It is able to associate with arbuscular mycorrhizal fungi (AMF) and rhizobia. This dual symbiosis improves nitrogen and phosphorus nutrient uptake in cowpea. As the application of exotic inoculants frequently lacks positive responses in field experiments, this study set out to select well-adapted native symbiotic rhizobial and AMF strains. Soil samples were collected from six study sites in three different climatic zones of Burkina Faso to investigate their native symbiotic strains. Soil-extraction of native spores led to the identification of four AMF genera (Scutellospora, Gigaspora, Glomus and Entrophospora) by morpho-anatomical characterization. The two most effective cowpea fungal strains were selected after spore isolation from field-collected soils, multiplication on maize roots and inoculation on cowpea seedlings in a greenhouse experiment. Cowpea-nodulating rhizobial strains were trapped in the greenhouse by planting cowpea seeds in collected soil samples and the strains were characterized using molecular methods. This characterization led to the rhizobial isolates being classified in four clusters on the phylogenetic tree (using the Maximum-Likelihood Phylogenies method). All strains belonged to the Bradyrhizobium genus and most of them were included in the B. japonicum branch. Some groups were clearly distinct species already identified and may be new species. The two most effective strains for cowpea yield improvement in the field were selected after cowpea inoculation in a greenhouse experiment. The inoculation design in the field experiment consisted of four single inoculation treatments, either rhizobial or mycorrhizal, along with four dual inoculations, one treatment with chemical fertilizers, and one uninoculated control. The results showed that cowpea productivity was significatively improved by dual inoculation with native rhizobial and mycorrhizal strains, reaching the same level as the application of commonly used chemical fertilizers [Nitrogen, Phosphorus and Potassium fertilizers (NPK)]. In addition, dual inoculation resulted in the highest iron content in cowpea leaves.     

Genetic diversity of elite rhizobial strains of subtropical and tropical legumes based on the 16S rRNA and <Emphasis Type="Italic">glnII</Emphasis> genes

Biodiversity of diazotrophic symbiotic bacteria in the tropics is a valuable but still poorly studied resource. The objective of this study was to determine if a second housekeeping gene, glnII, in addition to the 16S rRNA, can be employed to improve the knowledge about taxonomy and phylogeny of rhizobia. Twenty-three elite rhizobial strains, very effective in fixing nitrogen with twenty-one herbal and woody legumes (including species from fourteen tribes in the three subfamilies of the family Leguminosae) were selected for this study; all strains are used as commercial inoculants in Brazil. Complete sequences of the 16S rRNA and partial sequences (480 bp) of the glnII gene were obtained. The same primers and amplification conditions were successful for sequencing the glnII genes of bacteria belonging to five different rhizobial genera—Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium, Sinorhizobium)—positioned in distantly related branches. The analysis of the concatenated genes (16S rRNA + glnII) considerably improved information about phylogeny and taxonomy of rhizobia in comparison to the single analysis of the 16S rRNA. Nine strains might belong to new species. The complementary analysis of the glnII gene was successful with all strains and improved the phylogenetic clustering and clarified the taxonomic position of several strains. The strategy of including the analysis of glnII, in addition to the 16S rRNA, is cost- and time- effective for the characterization of large rhizobial culture collections or in surveys of many isolates.     

Nodulation competitiveness in the Rhizobium-legume symbiosis

Successful nodulation of legumes by rhizobia is a complex process that, in the open field, depends on many different environmental factors. Generally, legume productivity in an agricultural field may be improved by inoculation with selected highly effective N₂-fixing root nodule bacteria. However, field legume inoculation with Rhizobium and Bradyrhizobium spp. has often been unsuccessful because of the presence in the soil of native strains that compete with the introduced strain in nodule formation on the host plants. This ability to dominate nodulation is termed competitiveness and is critical for the successful use of inoculants.The author is with the Departmentode Microbiologia del Suelo y Sistemas Simbioticos, Estation Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, C/Professor Albareda 1, 18008 Granada, Spain     

Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis

Background and aims

Legumes of the genus Lessertia have recently been introduced to Australia in an attempt to increase the range of forage species available in Australian farming systems capable of dealing with a changing climate. This study assessed the diversity and the nodulation ability of a collection of Lessertia root nodule bacteria isolated from different agro-climatic areas of the Eastern and Western Capes of South Africa.

Methods

The diversity and phylogeny of 43 strains was determined via the partial sequencing of the dnaK, 16srRNA and nodA genes. A glasshouse experiment was undertaken to evaluate symbiotic relationships between six Lessertia species and 17 rhizobia strains.

Results

The dnaK and 16S rRNA genes of the majority of the strains clustered with the genus Mesorhizobium. The position of the strains at the intra-genus level was incongruent between phylogenies with few exceptions. The nodA genes from Lessertia spp. formed a cluster on their own, separate from the previously known Mesorhizobium nodA sequences. Strains showed differences in their nodulation and nitrogen fixation patterns that could be correlated with nodA gene phylogeny. L. diffusa, L. herbacea and L. excisa nodulated with nearly all the strains examined while L. capitata, L. incana and L. pauciflora were more stringent.

Conclusion

Root nodule bacteria from Lessertia spp. were identified mainly as Mesorhizobium spp. Their nodA genes were unique and correlated with the nodulation and nitrogen fixation patterns of the strains. There were marked differences in promiscuity within Lessertia spp. and within strains of root nodule bacteria.     

Rhizobacterial inoculants can improve nickel phytoextraction by the hyperaccumulator Alyssum pintodasilvae

Aim

Rhizobacteria can influence plant growth and metal accumulation. The aim of this study was to evaluate the effect of rhizobacterial inoculants on the Ni phytoextraction efficiency of the Ni-hyperaccumulator Alyssum pintodasilvae.

Method

In a preliminary screening 15 metal-tolerant bacterial strains were tested for their plant growth promoting (PGP) capacity or effect on Ni bioaccumulation. Strains were selected for their Ni tolerance, plant growth promoting traits and Ni solubilizing capacity. In a re-inoculation experiment five of the previously screened bacterial isolates were used to inoculate A. pintodasilvae in two contrasting Ni-rich soils (a serpentine (SP) soil and a sewage sludge-affected agricultural (LF) soil).

Results

Plant growth was greater in serpentine soil (where it grows naturally) than in the LF soil, probably due to Cd phytotoxicity. Rhizobacterial inoculants influenced plant growth and Ni uptake and accumulation, but the effect of the strains was dependent upon soil type. The increase in plant biomass and/or Ni accumulation significantly promoted shoot Ni removal.

Conclusion

One strain (Arthrobacter nicotinovorans SA40) was able to promote plant growth and phytoextraction of Ni in both soil types and could be a useful candidate for future field-based trials.     

Symbiotic effectiveness of indigenous arctic rhizobia on a temperate forage legume: Sainfoin (Onobrychis viciifolia)

Sainfoin (Onobrychis viciifolia), a temperate perennial forage legume, can be nodulated by rhizobia isolated from 3 arctic legume species:Astragalus alpinus, oxytropis maydelliana andOxytropis arctobia. Arctic rhizobia, which are adapted to growth at low temperatures, may be useful in improving symbiotic nitrogen fixation during cold phases of the growing season, if they are effective on a temperate legume. In this study, we report on the symbiotic effectiveness of arctic rhizobia on sainfoin, as appraised by the total shoot dry matter yield obtained from 2 harvests. Under N-free conditions, 5 arctic strains at the first harvest and 8 at the second harvest were as effective as temperate standard strains. In the presence of 30 mgl⁻¹ NO₃-N, 7 arctic strains gave significantly higher yields than temperate strains at the second harvest. These results indicate that effective arctic rhizobia have a potential for use as inoculants on sainfoin. Contribution no 325 of Agriculture Canada Research Station a Sainte-Foy.     

The symbiotic performance of lupin bacteria under glasshouse and field conditions

Summary Bacteria from the nodules ofLupinus angustifolius L.,L. digitatus Forsk., andL. luteus L. have been isolated, and the symbiotic relationships of nine of these strains and the three lupin species investigated in a glasshouse experiment. All symbioses were effective and no interaction or specificity was detected among the symbionts.Soil factors modified expression of the strains' symbiotic abilities in the field, but several strains have shown successful symbiosis with all three lupin species.One lupin species,L. digitatus, nodulates freely under natural conditions in Western Australia, whereasL. luteus andL. angustifolius do not. This lupin is more readily infected by native strains of rhizobia than the others.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013)

Background

Inoculation of plants to enhance yield of crops and performance of other plants is a century old, proven technology for rhizobia and a newer venue for plant growth-promoting bacteria and other plant symbionts. The two main aspects dominating the success of inoculation are the effectiveness of the bacterial isolate and the proper application technology.

Scope

An assessment of practical aspects of bacterial inoculants for contemporary agriculture and environmental restoration is critically evaluated from the point of view of their current technological status, current applications, and future use. This is done because there are windows of opportunity for new developments in applied research using renewable, non-contaminated natural resources and new venues for research. Special emphasis is given to formulations and polymeric carriers. This review concentrates on practical aspect of inoculation technology dating from 1998 to 2013. Earlier publications are mentioned only for clarification of a specific point.

Conclusions

This review discusses characteristics of a carrier for inoculants, formulations of inoculants including liquid, organic, inorganic, polymeric, and encapsulated formulations. Technical aspects include inoculation techniques (soil and seed application), mass culture production, bulk sterilization, seed coating, shelf-life, and effect of moisture. Future research venues needed are noted.     

Symbiotic incompatibility between two forage species of Hedysarum, grown in Morocco, and their homologous rhizobia

Abstract Symbiotic experiments in the glasshouse demonstrated that two species of sulla, Hedysarum coronarium and Hedysarum flexuosum , grown in Morocco were mutually incompatible in their requirements for effectively nitrogen-fixing strains of rhizobia: nitrogen-fixing Rhizobium strains isolated from H. coronarium nodulated H. flexuosum but did not fix nitrogen, conversely strains from H. flexuosum were ineffective for H. coronarium . The agronic implications are discussed.