Rhizobium Species Research Articles

Rhizobial-Host Interactions and Symbiotic Nitrogen Fixation in Legume Crops Toward Agriculture Sustainability.

Symbiotic nitrogen fixation (SNF) process makes legume crops self-sufficient in nitrogen (N) in sharp contrast to cereal crops that require an external input by N-fertilizers. Since the latter process in cereal crops results in a huge quantity of greenhouse gas emission, the legume production systems are considered efficient and important for sustainable agriculture and climate preservation. Despite benefits of SNF, and the fact that chemical N-fertilizers cause N-pollution of the ecosystems, the focus on improving SNF efficiency in legumes did not become a breeder’s priority. The size and stability of heritable effects under different environment conditions weigh significantly on any trait useful in breeding strategies. Here we review the challenges and progress made toward decoding the heritable components of SNF, which is considerably more complex than other crop allelic traits since the process involves genetic elements of both the host and the symbiotic rhizobial species. SNF-efficient rhizobial species designed based on the genetics of the host and its symbiotic partner face the test of a unique microbiome for its success and productivity. The progress made thus far in commercial legume crops with relevance to the dynamics of host–rhizobia interaction, environmental impact on rhizobial performance challenges, and what collectively determines the SNF efficiency under field conditions are also reviewed here.

ANTAGONISTIC ACTIVITY OF RHIZOBIUM SPECIES AGAINST PHYTOPATHOGENIC MICROORGANISMS

ABSTRACT Green gram (Vignaradiata L.) is a popular edible legume of India. The plant is cultivated by the local farmer as cash crops. Rhizobium sp. was isolated from the root nodules of green gram. The pure culture of the isolated bacterium was carried out at the laboratory with Yeast- Extract Mannitol (YEM) Agar medium. The characterization of the bacterium was made by morphological and biochemical tests. Production of siderophores by the Rhizobium bacterium was qualitatively measured by Chrome Azurol S (CAS) agar plate method. Antagonistic behaviour of the Rhizobium isolates against two phytopathogenic microorganisms such as Rhizoctonia solani and Rhizoctonia oryzae were studied by the agar co-cultivation method. The zone of inhibition was recorded for both the pathogens are 46% and 51% respectively. The results indicated that Rhizobium species are capable to resist the growth of both the studied phytopathogens at in vitro condition. Therefore, Rhizobium sp. is a good candidate to control the growth of both the studied phytopathogenic microorganisms and can be used as a biocontrol agent for the same. Keywords: Biocontrol; Rhizobium species; phytopathogens; legume plant

Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia

Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this study further suggest that the Tunisian Rhizobium community is more diverse than previously reported.

Peteryoungia gen. nov. with four new species combinations and description of Peteryoungia desertarenae sp. nov., and taxonomic revision of the genus Ciceribacter based on phylogenomics of Rhizobiaceae.

A novel bacterial strain designated as ADMK78T was isolated from the saline desert soil. The cells were rod-shaped, Gram-stain-negative, and non-motile. The strain ADMK78T grows best at 28°C. Phylogeny of 16S rRNA gene placed the strain ADMK78T with the members of genera Ciceribacter and Rhizobium, while the highest sequence similarity was with Rhizobium wuzhouense W44T (98.7%) and Rhizobium ipomoeae shin9-1T (97.9%). Phylogenetic analysis based on 92 core-genes extracted from the genome sequences and average amino acid identity (AAI) revealed that the strain ADMK78T forms a distinct cluster including five species of Rhizobium, which is separate from the cluster of the genera Rhizobium and Ciceribacter. We propose re-classification of Rhizobium ipomoeae, R. wuzhouense, R. rosettiformans and R. rhizophilum into the novel genus Peteryoungia. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of ADMK78T were less than 82 and 81%, respectively, among all type strains included in the genus Peteryoungia. The strain ADMK78T showed differences in physiological, phenotypic, and protein profiles estimated by MALDI-TOF MS to its closest relatives. Based on the phenotypic, chemotaxonomic properties, and phylogenetic analyses, the strain ADMK78T represents a novel species, Peteryoungia desertarenae sp. nov. The type strain is ADMK78T (= MCC 3400T; KACC 21383T; JCM 33657T). We also proposed the reclassification of Rhizobium daejeonense, R. naphthalenivorans and R. selenitireducens, into the genus Ciceribacter, based on core gene phylogeny and AAI values.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Rhizobium flavescens sp. nov., Isolated from a Chlorothalonil-Contaminated Soil.

A Gram-negative, facultative anaerobic, non-lagellated and rod-shaped bacterium FML-4T was isolated from a chlorothalonil-contaminated soil in Nanjing, China. Phylogenetic analyses of 16S rRNA genes revealed that the strain FML-4T shared the highest sequence similarity of 97.1% with Ciceribacter thiooxidans KCTC 52231T, followed by Rhizobium rosettiformans CCM 7583T (97.0%) and R. daejeonense KCTC 12121T (96.8%). Although the sequence similarities of the housekeeping genes thrC, rceA, glnII, and atpD between strain FML-4T and C. thiooxidans KCTC 52231T were 83.8%, 88.7%, 86.2%, and 92.0%, respectively, strain FML-4T formed a monophyletic clade in the cluster of Rhizobium species. Importantly, the feature gene of the genus Rhizobium, nifH gene (encoding the dinitrogenase reductase), was detected in strain FML-4T but not in C. thiooxidans KCTC 52231T. In addition, strain FML-4T contained the summed feature 8 (C18:1ω7c and/or C18:1ω6c), C19:0 cyclo ω8c and C16:0 as the major fatty acids. Genome sequencing of strain FML-4T revealed a genome size of 7.3 Mbp and a G+C content of 63.0mol%. Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization, average nucleotide identity (ANI, similarity 77.3-75.4%), and digital DNA-DNA hybridization (dDDH, similarity 24.5-22.3%), it was concluded that strain FML-4T represented a novel species of the genus Rhizobium, for which the name Rhizobium flavescens sp. nov. was proposed (type strain FML-4T = CCTCC AB 2019354T = KCTC 62839T).

Rhizobium and Mycorrhizal Fungal Species Improved Soybean Yield Under Drought Stress Conditions

Food insecurity is a serious threat due to the increasing human population particularly in developing countries and may be minimized by the use of microbial inoculants. Also, the problems of excessive use of chemical fertilizers including the fact that most of the fertilizers are relatively non-affordable and that they also contaminate underground and surface water, which can increase the risk of blue baby syndrome in infants and stomach cancer in adults. There is therefore the need to harness a more cost-effective, eco-friendly and beneficial biological agents to improve crops productivity especially under drought conditions. Thus, in this study, the ability of rhizobia species and arbuscular mycorrhizal fungi (AMF) to enhance soybean tolerance to drought stress under water regimens of 100, 70 and 40% field capacity (FC) was investigated. It was observed that co-inoculation of soybean with Rhizobium spp. (R1+R3) as well as with Rhizobium spp. and mycorrhizal consortium (R1+R3MY) had significant impacts (P < 0.05) on soybean leaf relative water content and electrolyte leakage, respectively. The levels of proline increased mainly in microbially amended soybean exposed to drought stress. Plants inoculated with R1+R3MY showed the highest number of spore and % mycorrhization in all the water regimes. At 40% FC, R1+R3MY treatment was found to promote soybean growth compared to the non-inoculated plants. Similarly, at 40% FC, R1+R3MY inoculum had the greatest impacts on soybean pod number, seed number, seed fresh weight, highest seed number per pod and seed dry weight while at 70% water stress, significant impacts of R1MY inoculation were observed on pod number, pod fresh weight and seed dry weight. These results revealed that co-inoculation of rhizobia and mycorrhizal fungi can be harnessed biotechnologically to proffer solution to food insecurity.

Assessment of Shelf Life and Quality of Biofertilizers using Tricalcium Phosphate as an Anticaking Agent and Aluminium Silicate as the Inert Carrier

Background: Bio-fertilizers are the substances which contain living microorganisms, when applied to soil, seeds and plant root these fertilizers increases soil fertility and promote growth of the plant. Biofertilizers help plants to utilize important mineral resources, phosphorous and nitrogen. Microorganisms like Rhizobacteria, fungi and algae which provide nutrient to the soil and which are produced commercially are known as biofertilizers. The microorganisms which present in biofertilizers are Rhizobium species, Pseudomonas species and Azospirillum species etc. These biofertilizers have potential to replace conventional chemical fertilizers. The quality of biofertilizers is utmost important as they have to be used by farmers and should work well when applied to the soil. It should not form clumps after preparation. In this study, anticaking property provided by tricalcium phosphate (TCP) to individual biofertilizer containing Pseudomonas, Rhizobium and Azospirillum respectively (each separately) was studied. Methods: In our study, we have used serial dilution and direct count method (CFU) for checking viability of live microorganism for 15, 30 and 90 days duration in respective biofertilizers in our laboratory. Different percentage viz 5%, 10%, 15% and 20% of tricalcium phosphate (TCP) was used in addition to aluminium silicate as an inert carrier.Conclusion: Our study has validated that all percentage (5%, 10%, 15% and 20%) of tricalcium phosphate (TCP) is reducing clump formation as compared to control with no TCP added. On the basis of plate count method (CFU result) 10% TCP is found to be optimum to be used as an anticaking agent for biofertilizer containing Pseudomonas, Rhizobium and Azospirillum respectively.

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N2 ) into ammonia (NH3 ) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a '1+n-N' model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where '1' refers to appreciate rhizobium; '+n' means the addition of multiple trace elements and PGPR bacteria; and '-N' implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.

Act of Malondialdehyde and Total Phenolic Content Under Bean Yellow Mosaic Virus Infection and Biostimulants Application

Bean yellow mosaic virus represents a stress effect on fava bean plants. This study was conducted to assess the lipid peroxidation and total phenolic content (TPC) of fava bean plants under bean yellow mosaic virus infection and its management by the biostimulants; Rhizobium and Bacillus species spraying application. The obtained data revealed that virus-infected plants maximized stress factors, the content of malondialdehyde (MDA), while it decreased in other applications. TPC significantly increased in Bacillus, and Rhizobium isolates spraying treatments with 20.99 and 22.44 mg/100g extract, respectively, compared with control. The current work provides bio-resistance by Bacillus or Rhizobium isolates effectively minimizes the adverse effects of challenges such as stressing viral infection and representing an environmentally friendly commercial value for plant growers.

Rhizobium rhizolycopersici sp. nov., Isolated from the Rhizosphere Soil of Tomato Plants in China.

During characterization of rhizobacteria, strain DBTS2T was isolated from the rhizosphere soil samples of healthy tomato plants and characterized using a polyphasic taxonomic approach. Phylogenetic analysis using 16S rRNA gene sequences showed this strain belonged to the genus Rhizobium and was most closely related to Rhizobium subbaraonis JC85T (99.1%) and Rhizobium daejeonense CCBAU 10050T (97%). Cells of strain DBTS2T were Gram-negative, short rod, aerobic and non-motile. This novel strain was found to grow at 20-45°C (optimum 25-37°C), pH 5-9 (optimum 8) and in the presence of 4% NaCl. It was positive for catalase and oxidase. The predominant cellular fatty acids were Summed Feature 8 (52.7%) and C19:0 cyclo ω8c (23.3%). The polar lipids of strain DBTS2T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified aminophospholipid, unidentified aminolipid, four unidentified phospholipids, unidentified lipid, phosphatidylcholine, unknown glycolipid and unknown aminophosphoglycolipids. Q-10 was the major quinone. The DNA-DNA hybridization similarity values between the strain DBTS2T and R. subbaraonis JC85T, R. daejeonense CCBAU 10050T and Rhizobium azooxidifex DSM100211T were 46.4%, 20.7% and 25.5%, respectively. The ANI value was 91.96% between strain DBTS2T and R. subbaraonis JC85T and 75.18% between strain DBTS2T and R. daejeonense CCBAU 10050T. The DNA G+C content of the genomic DNA was 63.1mol%. Based on these results, it was concluded that the isolate represents a novel species of the genus Rhizobium, for which the name Rhizobium rhizolycopersici sp. nov. is proposed, with DBTS2T (= CICC 24887T = ACCC61707 = JCM 34245) as the type strain.

Genetic diversity and distribution of rhizobia associated with soybean in red soil in Hunan Province.

To explore the genetic diversity and distribution of rhizobia in the rhizosphere of soybean grown in red soil, we have collected 21 soil samples from soybean fields across seven counties in Hunan province, China. MiSeq sequencing of rpoB gene was used to determine the intra-species diversity of rhizobia existing in soybean rhizospheres. Soil chemical properties were determined by routine methods. The Principal Coordinates Analysis (PCoA) plot indicated a clear biogeographical pattern characterizing the soybean rhizosphere across different sites. The Mantel test demonstrated that biogeographical pattern was significantly correlated with the geographical distance (Mantel statistic R 0.385, p < 0.001). There were obvious differences in the rhizobial communities among northeastern eco-region, southeastern eco-region and western eco-region. In general, Bradyrhizobium diazoefficiens was the most abundant rhizobial species in the soybean rhizosphere. At an intermediate (10-400km) spatial scale, the biogeographical pattern of rhizobial communities in soybean rhizosphere is associated with both soil properties and geographical distance. Redundancy analysis (RDA) showed that total potassium (TK), available potassium (AK), soil organic carbon (SOC), and available nitrogen (AN) were the main factors that influenced the α-diversity of rhizobial communities. Canonical correspondence analysis (CCA) showed that pH and exchangeable Ca and Mg had the greatest influence on the β-diversity of the rhizobial communities in the soybean rhizosphere. These findings characterize the distribution pattern and its influencing factors of soybean rhizobia in rhizosphere in Hunan province, which may be helpful in selecting suitable strains or species as inoculants for soybeans in red soil regions.

Distinctive Features of Rhizobia Species Sinorhizobium fredii and Bradyrhizobium japonicum Inhabiting Soils of the Russian Far East

In the framework of the comprehensive research of Far Eastern natural populations of soybean nodule bacteria, laboratory experiments have been conducted at the All-Russia Scientific Research Institute of Soybean (Blagoveshchensk) with the purpose to identify distinctive features of rhizobia species Sinorhizobium fredii (Scholla and Elkan, 1984) and Bradyrhizobium japonicum (Jordan, 1982) whose pure cultures were isolated from soils of Far Eastern regions practicing soybean cultivation. It is established that B. japonicum strains start growing in Petri dishes on the seventh to tenth and even on the 20th day after the inoculation, assimilate a limited number of carbon nutrition sources, release mostly alkaline metabolic products, and feature a relatively low osmotic resistance. Representatives of this species are susceptible to extreme environmental conditions; their growth sharply slows down on acidic and alkaline nutrient media and stops at high (37–42°C) temperatures. However, under the optimal conditions, this rhizobia species dominates in the nodulation of soybean plants due to its high and persistent virulence. The restriction analysis of the studied B. japonicum strains confirmed their identity. S. fredii strains start growing in Petri dishes on the second to fourth day after the inoculation, assimilate well a broad spectrum of carbon nutrition sources, and release acidic metabolic products. Most strains of this species feature high osmotic resistance. Cultures retaining universal growth capacity under extreme environmental conditions (high temperatures and low and high pH values) have been identified in the group of S. fredii strains. This rhizobia species can predominate in the formation of symbiotic mechanisms in years featuring extreme weather conditions. Enzymatic fermentation of gene 16S rRNA in the studied S. fredii strains was performed using restriction enzyme HaeIII; the analysis of the fermentation results confirmed the identity of these strains. The RAPD-PCR analysis has demonstrated the intraspecific specificity of the studied B. japonicum and S. fredii strains: these species feature high degrees of polymorphism reflecting their population heterogeneity.

Genetic Diversity and Characterization of Symbiotic Bacteria Isolated from Endemic Phaseolus Cultivars Located in Contrasting Agroecosystems in Venezuela.

Phaseolus vulgaris is a grain cultivated in vast areas of different countries. It is an excellent alternative to the other legumes in the Venezuelan diet and is of great agronomic interest due to its resistance to soil acidity, drought, and high temperatures. Phaseolus establishes symbiosis primarily with Rhizobium and Ensifer species in most countries, and this rhizobia-legume interaction has been studied in Asia, Africa, and the Americas. However, there is currently no evidence to show that rhizobia nodulate the endemic cultivars of P. vulgaris in Venezuela. Therefore, we herein investigated the phylogenetic diversity of plant growth-promoting and N2-fixing nodulating bacteria isolated from the root nodules of P. vulgaris cultivars in a different agroecosystem in Venezuela. In comparisons with other countries, higher diversity was found in isolates from P. vulgaris nodules, ranging from α- and β-proteobacteria. Some isolates belonging to several new phylogenetic lineages within Bradyrhizobium, Ensifer, and Mesorhizobium species were also specifically isolated at some topographical regions. Additionally, some isolates exhibited tolerance to high temperature, acidity, alkaline pH, salinity stress, and high Al levels; some of these characteristics may be related to the origin of the isolates. Some isolates showed high tolerance to Al toxicity as well as strong plant growth-promoting and antifungal activities, thereby providing a promising agricultural resource for inoculating crops.

Assessment of Genetic Diversity and Symbiotic Efficiency of Selected Rhizobia Strains Nodulating Lentil (Lens culinaris Medik.).

A total of 14 Rhizobium strains were isolated from lentil accessions grown at the ICARDA experimental research station at Marchouch in Morocco and used for molecular characterization and symbiotic efficiency assessment. Individual phylogenetic analysis using the 16S rRNA gene, house-keeping genes rpoB, recA, and gyrB, and symbiotic genes nodD and nodA along with Multilocus Sequence Analysis (MLSA) of the concatenated genes (16S rRNA-rpoB-recA-gyrB) was carried out for the identification and clustering of the isolates. The symbiotic efficiency of the strains was assessed on three Moroccan lentil cultivars (Bakria, Chakkouf, and Zaria) based on the number of nodules, plant height, plant dry weight, and total nitrogen content in leaves. The results showed that the individual phylogenetic analysis clustered all the strains into Rhizobium laguerreae and Rhizobium leguminosarum with sequence similarity ranging from 94 to 100%, except one strain which clustered with Mesorhizobium huakuii with sequence similarity of 100%. The MLSA of the concatenated genes and the related percentages of similarity clustered these strains into two groups of Rhizobium species, with one strain as a new genospecies when applying the threshold of 96%. For symbiotic efficiency, the Bakria variety showed the best association with 10 strains compared to its non-inoculated control (p-value ≤ 0.05), followed by Chakkouf and Zaria. The present study concluded that the genetic diversity and the symbiotic efficiency of Rhizobium strains appeared to be mainly under the control of the lentil genotypes.

Phylogenomics reveals the basis of adaptation of Pseudorhizobium species to extreme environments and supports a taxonomic revision of the genus

The family Rhizobiaceae includes many genera of soil bacteria, often isolated for their association with plants. Herein, we investigate the genomic diversity of a group of Rhizobium species and unclassified strains isolated from atypical environments, including seawater, rock matrix or polluted soil. Based on whole-genome similarity and core genome phylogeny, we show that this group corresponds to the genus Pseudorhizobium. We thus reclassify Rhizobium halotolerans, R. marinum, R. flavum and R. endolithicum as P. halotolerans sp. nov., P. marinum comb. nov., P. flavum comb. nov. and P. endolithicum comb. nov., respectively, and show that P. pelagicum is a synonym of P. marinum. We also delineate a new chemolithoautotroph species, P. banfieldiae sp. nov., whose type strain is NT-26T (=DSM 106348T=CFBP 8663T). This genome-based classification was supported by a chemotaxonomic comparison, with increasing taxonomic resolution provided by fatty acid, protein and metabolic profiles. In addition, we used a phylogenetic approach to infer scenarios of duplication, horizontal transfer and loss for all genes in the Pseudorhizobium pangenome. We thus identify the key functions associated with the diversification of each species and higher clades, shedding light on the mechanisms of adaptation to their respective ecological niches. Respiratory proteins acquired at the origin of Pseudorhizobium were combined with clade-specific genes to enable different strategies for detoxification and nutrition in harsh, nutrient-poor environments.

Determinants of Host Range Specificity in Legume-Rhizobia Symbiosis.

Leguminous plants possess the almost unique ability to enter symbiosis with soil-resident, nitrogen fixing bacteria called rhizobia. During this symbiosis, the bacteria physically colonize specialized organs on the roots of the host plant called nodules, where they reduce atmospheric nitrogen into forms that can be assimilated by the host plant and receive photosynthates in return. In order for nodule development to occur, there is extensive chemical cross-talk between both parties during the formative stages of the symbiosis. The vast majority of the legume family are capable of forming root nodules and typically rhizobia are only able to fix nitrogen within the context of this symbiotic association. However, many legume species only enter productive symbiosis with a few, or even single rhizobial species or strains, and vice-versa. Permitting symbiosis with only rhizobial strains that will be able to fix nitrogen with high efficiency is a crucial strategy for the host plant to prevent cheating by rhizobia. This selectivity is enforced at all stages of the symbiosis, with partner choice beginning during the initial communication between the plant and rhizobia. However, it can also be influenced even once nitrogen-fixing nodules have developed on the root. This review sets out current knowledge about the molecular mechanisms employed by both parties to influence host range during legume-rhizobia symbiosis.

Ecoregion—Rather Than Sympatric Legumes—Influences Symbiotic Bradyrhizobium Associations in Invasive Scotch Broom (Cytisus scoparius) in the Pacific Northwest

Plant-microbe mutualisms can determine the success of invasive plants. Legumes (Fabaceae) are particularly successful invaders in a variety of habitats. This is partly due to their ability to access atmospheric nitrogen through microbial mutualists (rhizobia) in their root systems, which allow them to colonize a wide variety of disturbed or nutrient-poor habitats. While many plant-rhizobia mutualisms are highly species-specific, plant promiscuity with different species of rhizobia can significantly enhance the success of invasive legumes, since the availability of suitable rhizobial mutualists in a new geographic area may serve as a limiting factor. Scotch broom (Fabaceae: Cytisus scoparius) is one of the most problematic invasive legumes in the Pacific Northwest (PNW), yet very little is known about the Scotch broom-rhizobia system. We explored the rhizobial communities of root nodules of Scotch broom and sympatrically occurring legumes across three major ecoregions (coast, valley, and mountain) in the western PNW (Washington, Oregon, and California) to better understand the Scotch broom-rhizobia system in nature. We found that bradyrhizobia are the exclusive rhizobial mutualists of Scotch broom but that there is promiscuity at the species level. While there was very little overlap with rhizobial communities of sympatric native and naturalized legumes, ecoregion did influence the species composition of Scotch broom-associated rhizobial communities. Our findings suggest that Scotch broom is not reliant on sympatric legumes to provide a source of suitable rhizobial mutualists, but instead forms spatially variable associations with a range of other bradyrhizobia.

The role of cytokinins in Legume-Rhizobium symbiosis

Legume plants have a unique ability to form associations with nitrogen-fixing rhizobial species. An exchange of specific signaling molecules between host and microsymbiont constitutes the first step required both for bacterial infection and the formation of new, root-derived organs, called nodules. Since these two events occur in different root tissues, namely in the rhizodermis and in the underlying cortical cells, their strict regulation and coordination have to exist. An essential role of plant hormones, especially cytokinins, in the modulation of nitrogen-fixing symbiosis has been widely postulated. Activation of the cytokinin signaling pathway in the root cortex, by an unknown signal, is thought to be a key event of the infection process. As a consequence bioactive cytokinins are biosynthesized in the root susceptible zone, and they are a part of a positive feedback loop to trigger cortical cell division and sustain nodule organogenesis. Understanding the genetic mechanisms underlying events that occur in the inner layers of the root is one of the key challenges emerging in the study of symbiotic processes.

Facile method of curing toxicity in large viral genomes by high-throughput identification and removal of cryptic promoters

Infectious plant virus clones are challenging to construct and manipulate due to the presence of cryptic promoter sequences that induce toxicity in bacteria. Common methods to overcome toxicity include intron insertion to interrupt toxic open reading frames and the use of Rhizobium or yeast species that do not recognize the same cryptic promoters. Unfortunately, intron insertion must be attempted on a trial and error basis within full-length clones and may change the infection characteristics of the virus. We have developed a facile method that can detect multiple cryptic bacterial promoters within large virus genomes. These promoters can then be silenced to obtain infectious clones that can be manipulated in E. coli. Our strategy relies on the generation of a viral library which is cloned upstream of either an eGFP open reading frame for low-throughput analysis or chloramphenicol for next generation sequencing. Pokeweed mosaic virus (PkMV), a 9.5 Kb ssRNA potyvirus, was used as a proof of concept. We found 16 putative promoter regions within 150-250 bp library fragments throughout the PkMV genome. 5’RACE allowed identification of the promoter sequence within each fragment, and subsequent silencing produced infectious clones. Our results indicate that cryptic promoters are ubiquitous within large viral genomes and that promoter screening is a desirable first step when constructing a viral clone. Our method can be applied to large plant and animal viruses as well as any DNA sequence for which low level of background transcriptional activity is required.