Bio-encapsulation of microbial cells for targeted agricultural delivery.

Biofertilizers, namely Rhizobium and biocontrol agents such as Pseudomonas and Trichoderma have been well established in the field of agricultural practices for many decades. Nevertheless, research is still going on in the field of inoculant production to find methods to improve advanced formulation and application in fields. Conventionally used solid and liquid formulations encompass several problems with respect to the low viability of microorganisms during storage and field application. There is also lack of knowledge regarding the best carrier in conventional formulations. Immobilization of microorganisms however improves their shelf-life and field efficacy. In this context, microencapsulation is an advanced technology which has the possibility to overcome the drawbacks of other formulations, results in extended shelf-life, and controlled microbial release from formulations enhancing their application efficacy. This review discusses different microencapsulation technologies including the production strategies and application thereof in agricultural practices.

Tolerance to agricultural pesticides of strains belonging to four genera of Rhizobiaceae.

In order to determine their tolerance to pesticides, 122 strains of rhizobia isolated from different geographical regions, and belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium and Bradyrhizobium were tested against eight herbicides, four fungicides and five insecticides. Sensitivity to the pesticides was measured by using the filter paper disk method at four concentrations, 0.45, 4.5, 45 and 450 µg per disk. When the pesticides were used at 0.45 µg per disk, a concentration similar to that found when pesticides are applied under field conditions, no inhibition was observed. Strains growth was affected at concentrations of 45 and 450 µg pesticide per disk. These higher concentrations can be encountered when seeds are treated with pesticides. Pesticides tolerance level was correlated to pesticide function, i.e rhizobial strains were more tolerant to insecticides, followed by herbicides and then fungicides. Two fungicides, captan and mancozeb, inhibited the highest number of strains. Only one insecticide, carbaryl, affected the growth of some rhizobial strains. Strains isolated from the arctic (Mesorhizobium spp. and R. leguminosarum bv. viciae), a putative pesticides-free environment, were either less or equally affected by pesticides compared to strains isolated from agricultural regions.

Isolation of free-living dinitrogen-fixing bacteria and their activity in compost containing de-inking paper sludge.

Knowledge of the microbiology of dinitrogen (N2)-fixing bacteria in compost rich in de-inking paper sludge (DPS) is limited. Dinitrogen (N2)-fixing bacteria from DPS composts were isolated and studied for their N2-fixing activity in vitro and in vivo. Two Gram-negative N2-fixing isolates were identified as Pseudomonas. At 20 degrees C, both isolates revealed that N2-fixing activity was higher than that of three arctic Pseudomonas strains. Their N2-fixing activity was found to occur between 18 and 25 degrees C, a pattern that was similar to the reference isolate Azotobacter ATCC 7486. Composts successfully showed N2-fixing activity after carbohydrate amendments both with and without inoculation of a N2-fixing isolate. These results suggest that DPS composts support N2-fixing bacteria and that N2-fixing activity is dependent on a usable carbohydrate source.

The effect of different temperatures on the fatty acid composition of Rhizobium leguminosarum bv. viciae in the faba bean symbiosis.

Fatty acid composition was determined in cells of strains CBhS and CBp7 of Rhizobium leguminosarum bv. viciae grown at four temperatures (10, 15, 22 and 30°C), and in bacteroids and nodules formed with faba bean (Vicia faba L.) grown at two day/night temperature regimes (22/15 and 15/10°C). Growth temperature markedly affected the fatty acid composition of free-living bacteria in both strains studied, and both showed similar variations at each temperature. The proportion of unsaturated fatty acids increased significantly with lowering of temperature. The major fatty acid found in bacteria and bacteroids was cis-vaccenic (C18: 1Δ11 ), which comprised up to c. 78 (bacteria) and 56% (bacteroids) of total fatty acids. The presence of polyunsaturated fatty acids (linoleic (C18:Δ,9,12 ) and linolenic (C18:3Δ9,12,15 ) acids) was noted only in bacteroids, indicating changes following the differentiation of bacteria into bacteroids in the nodules. The fatty acid composition of nodules was similar to that of bacteroids, although major differences were found in their proportions. The different day/night temperature regimes had contrasting effects in bacteroids and in nodules. In bacteroids of both strains, the proportions of stearic (C18:0) and linoleic (C18:Δ9,12 ) acids decreased at the lower temperature regime. In nodules, the proportion of stearic (C18:0) acid decreased, while that of linolenic (C18:3Δ9,12,15 ) acid increased at the lower temperature regime. However, those of cis-vaccenic (C18:1Δ11 ), linoleic (C18:Δ9,12,15 ) and palmitic (C16:0) acids increased or decreased depending on the rhizobial strain. The proportion of unsaturated fatty acids increased with the lowering of temperatures in bacteroids of both strains, and varied in whole nodules depending on the strain. Strain CBp7 showed a greater symbiotic efficiency (dry matter yield) than strain CBh5 under both temperature regimes, but no relationship was found with the proportion of unsaturated fatty acids of bacteria, bacteroids or nodules.

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan.

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.

Poly-beta-hydroxybutyrate production by fast-growing rhizobia cultivated in sludge and in industrial wastewater.

In our study, the potential of producing polyhydroxybutyrate (PHB) by cultivating fast-growing rhizobia (Sinorhizobium meliloti, Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli and R. leguminosarum bv. trifolii) in sludge and in industrial wastewater was evaluated. Results confirmed the possibility of using sludge as media for rhizobial growth. During growth, substantial quantity of PHB was accumulated and yields varied depending on the media and rhizobial species. Growing in sludge, PHB production did not exceed 3.7% w/w for all strains at the end of experiment (after 72 h). During the growth of S. meliloti, PHB yield varied and the maximum value reached 7.27% w/w after 60 h, with 1% Total Suspend Solid (TSS) sludge. Alkaline sludge pre-treatment affects rhizobial growth but did not improve the PHB accumulation. While growing S. meliloti in industrial wastewater, the PHB yields varied and the highest value was obtained with slaughterhouse wastewater (10.7% w/w) after 35 h of growth. Therefore, this work shows the potential of exploiting PHB production by rhizobia growing in wastewater or sludge which could be applied to bioplastic industry, and confirms the potential of these recyclable wastes for high production of rhizobial cells useable for legumes inoculants production. This study provides an environmentally sound way of sludge and wastewater management and use in diverse biotechnological applications.

Elevated atmospheric CO2 and strain of rhizobium alter freezing tolerance and cold-induced molecular changes in alfalfa (Medicago sativa).

BACKGROUND AND AIMS: The objective of the study was to assess the impact of elevated CO2 in interaction with rhizobial strains on freezing tolerance and cold-induced molecular changes in alfalfa. METHODS: Alfalfa inoculated with two different strains of rhizobium (A2 and NRG34) was grown and cold acclimated (2 weeks at 2 degrees C) under either 400 (ambient) or 800 micromol mol(-1) (elevated) CO2. KEY RESULTS: Plants acclimated under 400 micromol mol(-1) CO2 were more freezing tolerant than those maintained under 800 micromol mol(-1). Cryoprotective sugars typically linked with the acquisition of freezing tolerance such as sucrose, stachyose and raffinose increased in roots in response to low temperature but did not differ between CO2 treatments. Similarly high CO2 did not alter the expression of many cold-regulated (COR) genes although it significantly increased the level of transcripts encoding a COR gene homologous to glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). A significant effect of rhizobial strain was observed on both freezing tolerance and gene expression. Plants of alfalfa inoculated with strain A2 were more freezing tolerant than those inoculated with strain NRG34. Transcripts of COR genes homologous to a pathogenesis-related protein (PR-10) and to a nuclear-targeted protein were markedly enhanced in roots of alfalfa inoculated with strain A2 as compared with strain NRG34. Transcripts encoding the vegetative storage proteins (VSPs) beta-amylase and chitinase were more abundant in roots of non-acclimated plants inoculated with strain NRG34 than with strain A2. CONCLUSIONS: Taken together, the results suggest that elevated CO2 stimulates plant growth and reduces freezing tolerance. The acquisition of cold tolerance is also influenced by the rhizobial strain, as indicated by lower levels of expression of COR genes and sustained accumulation of VSP-encoding transcripts in alfalfa inoculated with strain NRG34 as compared with strain A2.

Toxicity of phenolic compounds extracted from bark residues of different ages.

In Quebec, Canada, industrial bark wastelands cover several hundred hectares of land. Bark residue that has piled up for decades tends to remain free of vegetation for years. To assess the revegetation potential of such sites, we sought to determine those factors responsible for poor plant growth. Phenolic compounds from fresh to 20-year-old bark residues were extracted with four solvents and quantified by high-performance liquid chromatography (HPLC). We simulated solutions (mixtures of standard phenolic compounds) to evaluate the potential toxic effects of phenolic compounds on the rhizobial growth, germination index, plant growth, nodule number, and nitrogen fixation activity of two legume species under laboratory conditions. The concentration of individual phenolic compounds varied from none detected to 350 microg/g bark residue. The extracted phenolic compounds differed among solvents and bark residues. The highest concentration of total phenolic compounds was from fresh bark; most of these were soluble in water or 0.1 M NaOH. For older bark residues, the total phenolic content depended on solvent strength, generally in the order of 2.0 M NaOH > 0.1 M NaOH = hot water > cold water. The biological activity of the simulated bark extracts was not established with the rhizobial growth inhibition test but with the germination index and rhizobium-legume symbiosis tests. With these, the toxicity of the simulated phenolic extracts decreased from fresh to the older bark residues. Plant dry weight, nodule number, and nitrogen fixation activity of vetch (Vicia sativa L.) were less negatively affected by high concentrations of phenolics than birdsfoot trefoil (Lotus corniculatus L.), although birdsfoot trefoil grew at lower concentrations. The rhizobium-legume symbiosis has potential for revegetating bark wastelands with less than 1 year old and older bark residues.