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This study identified the phenotypic and genotypic characteristics of the bacteria that nodulate wild Lathyrus and Vicia species natural distribution in the Gaziantep province of Turkey. Principle component analysis of phenotypic features revealed that rhizobial isolates were highly resistant to stress factors such as high salt, pH and temperature. They were found to be highly sensitive to the concentrations (mg/mL) of the antibiotics neomycin 10, kanamycin, and tetracycline 5, as well as the heavy metals Ni 10, and Cu 10, and 5. As a result of REP-PCR analysis, it was determined that the rhizobial isolates were quite diverse, and 5 main groups and many subgroups being found. All of the isolates nodulating wild Vicia species were found to be related to Rhizobium sp., and these isolates were found to be in Clades II, III, IV, and V of the phylogenetic tree based on 16S rRNA. The isolates that nodulated wild Lathyrus species were in Clades I, II, IV, V, VI, VII, and VIII, and they were closely related to Rhizobium leguminasorum, Rhizobium sp., Phyllobacterium sp., Serratia sp., and Pseudomonas sp. According to the genetic analyses, the isolates could not be classified at the species level, the similarity ratio was low, they formed a distinct group that was supported by strong bootstrap values in the phylogenetic tree, and the differences discovered in the network analysis revealed the diversity among the isolates and gave important findings that these isolates may be new species., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

In a jujube orchard, cropping withgrass may influence bacterial diversity and ecological networks due to changes of physicochemical properties in soil, which has a serious effect on the stability of soil ecosystems. The aim of this study was to analyze the effects of different cultivation methods (CK: cleaning tillage; NG: cropping with native grass; VV: cropping with Vicia villosa ) on the soil's bacterial structure and its co-occurrence network in a jujube orchard. The results showed that the highest moisture content, total nitrogen, and organic matter in the rhizosphere soil of a jujube orchard was found in the VV group. The soil's moisture content, total nitrogen, and organic matter in the VV group were 2.66%, 0.87 g kg -1 , and 5.55 mg kg -1 higher than that found in the CK group. Compared to the CK group, the number of unique species in the rhizosphere soil in the NG and the VV groups increased by 7.33% and 21.44%. The PICRUSt and FAPROTAX analysis showed that sown grass had a greater influence on the ecological function of the soil's bacteria. Cropping with Vicia villosa and native grass significantly increased aerobic chemoheterotrophy, nitrogen respiration, nitrate reduction related to biochemical cycles, and the relative abundance of genes related to carbohydrate metabolism and the biodegradation of xenobiotics. The bacterial network complexity in the NG group was higher than that in the CK and VV groups and was greatest in the hub nodes (OTU42, Bacteroidota ; OTU541, Nitrospiraceae ). In this study, the ecological benefit seen in the soil's microbial function provides support to the theory that cropping with grass ( Vicia villosa ) increases the sustainable development of a jujube orchard., Competing Interests: The authors declare there are no competing interests., (©2024 Wang et al.)

Soil microbial transformations of nitrogen (N) can be affected by soil health management practices. Here, we report in situ seasonal dynamics of the population size (gene copy abundances) and functional activity (transcript copy abundances) of five bacterial genes involved in soil N cycling (ammonia-oxidizing bacteria [AOB] amoA , nifH , nirK , nirS , and nosZ ) in a long-term continuous cotton production system under different management practices (cover crops, tillage, and inorganic N fertilization). Hairy vetch ( Vicia villosa Roth), a leguminous cover crop, most effectively promoted the expression of N cycle genes, which persisted after cover crop termination throughout the growing season. Moreover, we observed similarly high or even higher N cycle gene transcript abundances under vetch with no fertilizer as no cover crop with N fertilization throughout the cover crop peak and cotton growing seasons (April, May, and October). Further, both the gene and transcript abundances of amoA and nosZ were positively correlated to soil nitrous oxide (N 2 O) emissions. We also found that the abundances of amoA genes and transcripts both positively correlated to field and incubated net nitrification rates. Together, our results revealed relationships between microbial functional capacity and activity and in situ soil N transformations under different agricultural seasons and soil management practices. IMPORTANCE Conservation agriculture practices that promote soil health have distinct and lasting effects on microbial populations involved with soil nitrogen (N) cycling. In particular, using a leguminous winter cover crop (hairy vetch) promoted the expression of key functional genes involved in soil N cycling, equaling or exceeding the effects of inorganic N fertilizer. Hairy vetch also left a legacy on soil nutrient capacity by promoting the continued activity of N cycling microbes after cover crop termination and into the main growing season. By examining both genes and transcripts involved in soil N cycling, we showed different responses of functional capacity (i.e., gene abundances) and functional activity (i.e., transcript abundances) to agricultural seasons and management practices, adding to our understanding of the effects of soil health management practices on microbial ecology., (Copyright © 2021 Hu et al.)

The aim of the present study was to isolate novel lactic acid bacteria (LAB) from hairy vetch forage crop and characterize their probiotic and fermentative potential for preparing Korean cabbage kimchi. First, functional characterization of isolated strains such as antagonistic property, auto-aggregation, antibiotic susceptibility, and extracellular enzyme production was performed. The isolated Lactobacillus plantarum KCC-41 strain was able to inhibit pathogenic fungal spore formation. It showed susceptibility to common commercial antibiotics drugs. The selected LAB strain was then subjected to microencapsulation with alginate biopolymer. Its ability to survive in in vitro simulated gastro-intestinal fluid was evaluated. It was also used in the fermentation of cabbage kimchi samples. The encapsulated KCC-41 strain could effectively lead to kimchi fermentation in terms of reducing its pH and dominating bacterial count. It also significantly increased organic acid production than non-encapsulated LAB (KCC-41) for cabbage kimchi samples.

We introduce an easy, fast and effective method to analyze the influence of genetically modified (GM) plants on soil and model organisms in the laboratory to substitute laborious and time consuming field trials. For the studies described here we focused on two GM plants of the so-called 3rd generation: GM plants producing pharmaceuticals (PMP) and plant made industrials (PMI). Cyanophycin synthetase (cphA) was chosen as model for PMI and Choleratoxin B (CTB) as model for PMP. The model genes are expressed in transgenic roots of composite Vicia hirsuta plants grown in petri dishes for semi-sterile growth or small containers filled with non-sterile soil. No significant influence of the model gene expression on root induction, growth, biomass, interaction with symbionts such as rhizobia (number, size and functionality of nodules, selection of nodulating strains) or arbuscular mycorrhizal fungi could be detected. In vitro, but not in situ under field conditions, structural diversity of the bulk soil microbial community between transgenic and non-transgenic cultivars was determined by PLFA pattern-derived ratios of bacteria: fungi and of gram + : gram - bacteria. Significant differences in PLFA ratios were associated with dissimilarities in the quantity and molecular composition of rhizodeposits as revealed by Py-FIMS analyses. Contrary to field trials, where small effects based on the transgene expression might be hidden by the immense influence of various environmental factors, our in vitro system can detect even minor effects and correlates them to transgene expression with less space, time and labour., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea ( Pisum sativum ) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants ( nodC and nifH ), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective ( nifH ) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo -inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a γ-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch ( Vicia hirsuta ), producing similar results. In addition, vetch exhibited a local increase of nod gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere., (© 2017 The author(s). All Rights Reserved.)

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

We introduce an easy, fast and effective method to analyze the influence of genetically modified (GM) plants on soil and model organisms in the laboratory to substitute laborious and time consuming field trials. For the studies described here we focused on two GM plants of the so-called 3rd generation: GM plants producing pharmaceuticals (PMP) and plant made industrials (PMI). Cyanophycin synthetase (cphA) was chosen as model for PMI and Choleratoxin B (CTB) as model for PMP. The model genes are expressed in transgenic roots of composite Vicia hirsuta plants grown in petri dishes for semi-sterile growth or small containers filled with non-sterile soil. No significant influence of the model gene expression on root induction, growth, biomass, interaction with symbionts such as rhizobia (number, size and functionality of nodules, selection of nodulating strains) or arbuscular mycorrhizal fungi could be detected. In vitro, but not in situ under field conditions, structural diversity of the bulk soil microbial community between transgenic and non-transgenic cultivars was determined by PLFA pattern-derived ratios of bacteria: fungi and of gram + : gram - bacteria. Significant differences in PLFA ratios were associated with dissimilarities in the quantity and molecular composition of rhizodeposits as revealed by Py-FIMS analyses. Contrary to field trials, where small effects based on the transgene expression might be hidden by the immense influence of various environmental factors, our in vitro system can detect even minor effects and correlates them to transgene expression with less space, time and labour., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.

A total of 212 symbiotic bacteria were isolated from nodules of Vicia ervilia, a traditional crop cultivated in Northern Morocco. The isolates were recovered from 10 different sites, trapped each time with the local cultivar grown in the same field. Four loci were sequenced in order to characterize the isolates, including two housekeeping genes (recA and glnII), one plasmidic symbiotic gene (nodC) and one locus from another plasmid (prL11). In several isolates, two different copies of glnII were detected and sequenced, suggesting a unique duplication event, which has never been reported previously. There was no correlation between the genetic differentiation among cultivars and among bacteria, showing that the evolution of the bacterial population was independent, at least partially, from the host plant. By placing the haplotypes in a wide-ranging phylogenetic reconstruction, it was shown that the diversity detected in Morocco was spread throughout the different clades detected worldwide. The differentiation between areas relied on frequency variations of haplotypes rather than a presence/absence pattern. This finding raises new questions concerning bacterial genetic resource preservation, and confirms the old tenet "everything is everywhere but the environment selects"., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

It is known that the genetic diversity of conspecific rhizobia present in root nodules differs greatly among populations of a legume species, which has led to the suggestion that both dispersal limitation and the local environment affect rhizobial genotypic composition. However, it remains unclear whether rhizobial genotypes residing in root nodules are representative of the entire population of compatible symbiotic rhizobia. Since symbiotic preferences differ among legume populations, the genetic composition of rhizobia found within nodules may reflect the preferences of the local hosts, rather than the full diversity of potential nodulating rhizobia present in the soil. Here, we assessed whether Vicia cracca legume hosts of different provenances select different Rhizobium leguminosarum genotypes than sympatric V. cracca hosts, when presented a natural soil rhizobial population. Through combining V. cracca plants and rhizobia from adjacent and more distant populations, we found that V. cracca hosts are relatively randomly associated with rhizobial genotypes. This indicates that pre-infection partner choice is relatively weak in certain legume hosts when faced with a natural population of rhizobia., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

MgtE is predicted to be a Rhizobium leguminosarum channel and is essential for growth when both Mg²⁺ is limiting and the pH is low. N₂was only fixed at 8% of the rate of wild type when the crop legume Pisum sativum was inoculated with an mgtE mutant of R. leguminosarum and, although bacteroids were present, they were few in number and not fully developed. R. leguminosarum MgtE was also essential for N₂fixation on the native legume Vicia hirsuta but not when in symbiosis with Vicia faba. The importance of MgtE and the relevance of the contrasting phenotypes is discussed.

The genetic diversity and population structure of about 350 Rhizobium leguminosarum biovar viciae isolates from Vicia cracca were analysed. A hierarchical sampling design was used covering three regions, one region in Belgium and two in France, in which multiple local V. cracca populations were sampled. Rhizobium isolates were genotyped using RAPD and by sequencing two chromosomal housekeeping genes (glnII and recA) and one plasmid-borne gene (nodC). Twenty-six nodC types and sixty-seven chromosomal types were identified, many of which appeared to be regional or local endemics. We found strong genetic differentiation both among V. cracca populations that are separated by only a few kilometres, and among regions that are 50 to 350km apart. Despite significant plasmid exchange, chromosomal and nod types were similarly structured among host populations and regions. We found two lineages of which one prevailed in the Belgian region while the other dominated the French regions. Although a significant correlation between genetic differentiation and geographic distance was found, it is deemed more likely that the observed biogeographic patterns are rather due to coevolutionary interactions and environmental pressures. Furthermore, the impact of recombination on the chromosomal differentiation was found to be considerable., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum.

Polymorphism analysis was performed in Rhizobium leguminosarum bv. viceae populations isolated from geographically distant regions of Ukraine and Middle Asia. Examination of cultural, biochemical, and symbiotic traits revealed interpopulation differences, which were attributed to the difference in conditions between natural ecosystems and agrocenoses. Vetch has high species diversity and is not cultivated in Middle Asia, and the corresponding rhizobial population displayed higher genetic diversity and higher polymorphism of adaptive traits ensuring saprophytic development in soil and the rhizosphere, including melanin synthesis (35%) and active exopolysaccharide production (90%). Strains of the Ukrainian population had a lower exopolysaccharide production (10%), did not produce melanin, had higher herbicide resistance, and utilized glucose and succinate (main components of plant root exudation) as carbon sources. Strains capable of efficient symbiosis with Vicia villosa Roth. had a higher frequency in the Middle Asian than in the Ukrainian population, especially among strains isolated from soil (80 and 35%, respectively). In addition, strains of the Middle Asian population better competed for nodulation. It was assumed that the formation of rhizobial populations in vetch cultivation regions (Ukraine) is aimed at adaptation to ectosymbiotic (rhizospheric) interactions with plants and anthropogenic stress factors, while strains of the vetch original center (Middle Asia) are mostly adapted to the endosymbiotic interaction and to natural edaphic stress factors.

In this study a collection of rhizobial strains were isolated from effective nodules of Phaseolus vulgaris in a wide region of northern Spain, which is the major producer region of this legume in Spain. The analysis of their core genes, rrs, atpD, and recA, and the 16S-23S intergenic spacer showed that all isolates belong to the phylogenetic group of Rhizobium leguminosarum and some of them were identical to those of strains nodulating Vicia or Trifolium. None of the isolates was identified as Rhizobium etli; however, all of them carry the nodC alleles alpha and gamma harboured by American strains of this species. These alleles were also found in strains nodulating P. vulgaris in southern Spain identified as R. etli. These results suggest that R. etli was carried from America to Spain with common bean seeds, but that they could have found difficulties persisting in the soils of northern Spain, probably because of the climatic conditions. The symbiotic genes of this species could have been transferred, after the arrival of P. vulgaris, to strains of R. leguminosarum already present in northern Spanish soils.

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S-23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium-Vicia symbiosis.

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

In the present study, a total of 154 bacterial strains isolated from nodules of eighteen Vicia species mainly grown in the temperate Chinese provinces were characterized by ARDRA, ITS PCR-RFLP, BOX-PCR, sequencing of 16S rDNA, nodC, nifH, atpD and glnII, and nodulation tests. The results demonstrated that most of the R. leguminosarum strains were effective microsymbionts of the wild Vicia species, while genomic species related to Rhizobium gallicum, Mesorhizobium huakuii, Ensifer meliloti and Bradyrhizobium spp. were symbiotic bacteria occasionally nodulating with Vicia species. In addition, fourteen strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, as well as 22 strains of R. leguminosarum might be nodule endophytes without symbiotic genes. Diverse symbiotic gene lineages were found among the test strains and a strong association was found among the symbiotic gene types and genomic species, indicating the absence of lateral gene transfer. These results greatly enlarged the rhizobial spectrum of Vicia species.

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

Two experiments were conducted to investigate the effect of inoculating Vicia faba plants (broad beens) raised in clean and oily sand with nodule-forming rhizobia and plant-growth-promoting rhizobacteria (PGPR) on growth of these plants in sand and to test whether this can improve the phytoremediation potential of this crop for oily desert areas. It was found that crude oil in sand at concentrations < 1.0% (w/w) enhanced the plant heights, their fresh and dry weights, the total nodule weights per plant, and the nitrogen contents of shoots and fruits. Similar enhancing effects were recorded when roots of the young plants were inoculated with nodule bacteria alone, PGPR alone, or a mixture of one strain of nodule bacteria and one of the PGPR. Such plant growth effects were associated with a better phytoremediation potential of V. faba plants for oily sand. The total numbers of oil-utilizing bacteria increased in the rhizosphere and more hydrocarbons were eliminated in sand close to the roots. The nodule bacteria tested were two strains of Rhizobium leguminosarum and the PGPR were Pseudomonas aeruginosa and Serratia liquefaciens. The four strains were found to use crude oil, n-octadecane, and phenanthrene as sole sources of carbon and energy. It was concluded that coinoculation of V. faba plant roots in oily sand with nodule bacteria and PGPR enhances the phytoremediation potential of this plant for oily desert sand through improving plant growth and nitrogen fixation.

A technique was optimized for the in situ detection of nodulation (nod) gene activity in Rhizobium leguminosarum bv. viciae symbiosis with compatible plant hosts Vicia tetrasperma (L.) SCHREB. and Pisum sativum L. The transcription of nodABC-lacZ fusion was visualized as beta-galactosidase (beta-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the background of the indigenous beta-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment. V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model of V. hirsuta (L.) S.F. GRAY: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent of nod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the pea sym8 locus. Judging from the nod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.

Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent (CND) genes among 2839 B. cinerea genes. Among the co-regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug.