Exploring the antibacterial potential of plant extracts and essential oils against Bacillus thermophilus in beet sugar for enhanced sucrose retention: a comparative assessment and implications.

Sugar beet is one of the greatest sources for producing sugar worldwide. However, a group of bacteria grows on beets during the storage process, leading to a reduction in sucrose yield. Our study focused on identifying common bacterial species that grow on beets during manufacturing and contribute to sucrose loss. The ultimate goal was to find a potential antibacterial agent from various plant extracts and oils to inhibit the growth of these harmful bacteria and reduce sucrose losses. The screening of bacterial species that grow on beet revealed that a large group of mesophilic bacteria, such as Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas fluorescens, Escherichia coli, Acinetobacter baumannii, Staphylococcus xylosus, Enterobacter amnigenus, and Aeromonas species, in addition to a dominant thermophilic species called Bacillus thermophilus, were found to be present during the manufacturing of beets. The application of 20 plant extracts and 13 different oils indicated that the extracts of Geranium gruinum, Datura stramonium, and Mentha spicata were the best antibacterials to reduce the growth of B. thermophilus with inhibition zones equal to 40, 39, and 35 mm, respectively. In contrast, the best active oils for inhibiting the growth of B. thermophilus were Mentha spicata and Ocimum bacilicum, with an inhibitory effect of 50 and 45 mm, respectively. RAPD-PCR with different primers indicated that treating sugar juice with the most effective oils against bacteria resulted in new recombinant microorganisms, confirming their roles as strong antibacterial products. The characterization of Mentha spicata and Ocimum bacilicum oils using GC/MS analysis identified cis-iso pulegone and hexadecanoic acid as the two main bioactive compounds with potential antibacterial activity. An analysis of five genes using DD-PCR that have been affected due to antibacterial activity from the highly effective oil from Mentha spicata concluded that all belonged to the family of protein defense. Our findings indicate that the application of these pure antibacterial plant extracts and oils would minimize the reduction of sucrose during sugar production.

Definition of the symbiovar viciae in the species Rhizobium azibense and biogeographic implications.

Vicia faba L. (faba bean) is a legume cultivated worldwide which commonly establishes effective symbiosis with the symbiovar viciae of species from the Rhizobium leguminosarum phylogenetic group. However, on the basis of the rrs, recA, and atpD gene phylogenies, in this work we identified a strain named EFBRI 42 nodulating V. faba as Rhizobium azibense. This is the first report on the nodulation of Vicia by R. azibense which commonly nodulates P. vulgaris and to date encompasses strains harboring the nodC genes typical of the symbiovars gallicum and phaseoli. However, the strain EFBRI 42 carries a nodC gene typical of the symbiovar viciae for which we report here by the first time this symbiovar in R. azibense. This finding showed the existence of symbiotic genes horizontal transfer events during the coevolution of R. azibense with P. vulgaris and V. faba in their respective distribution centers of Mesoamerica and the Middle East.

Future Outlook of Transferring Biological Nitrogen Fixation (BNF) to Cereals and Challenges to Retard Achieving this Dream.

BNF is a fascinating phenomenon which contributes to protect the nature from environmental pollution that can be happened as a result of heavy nitrogen applications. The importance of BNF is due to its supply of the agricultural lands with about 200 million tons of N annually. In this biological process, a specific group of bacteria collectively called rhizobia fix the atmospheric N in symbiosis with legumes called symbiotic nitrogen fixation and others (free living) fix nitrogen gas from the atmosphere termed asymbiotic. Several trials were done by scientists around the world to make cereals more benefited from nitrogen gas through different approaches. The first approach is to engineer cereals to form nodulated roots. Secondly is to transfer nif genes directly to cereals and fix N without Rhizobium partner. The other two approaches are maximizing the inoculation of cereals with both of diazotrophs or endophytes. Recently, scientists solved some challenges that entangle engineering cereals with nif genes directly and they confirmed the suitability of mitochondria and plastids as a suitable place for better biological function of nif genes expression in cereals. Fortunately, this article is confirming the success of scientists not only to transfer synthetic nitrogenase enzyme to Escherichia coli that gave 50% of its activity of expression, but also move it to plants as Nicotiana benthamiana. This mini review aims at explaining the future outlook of BNF and the challenges limiting its transfer to cereals and levels of success to make cereals self nitrogen fixing.

The promiscuity of Phaseolus vulgaris L. (common bean) for nodulation with rhizobia: a review.

Phaseolus vulgaris L. (common bean) is a legume indigenous to American countries currently cultivated in all continents, which is nodulated by different rhizobial species and symbiovars. Most of species able to nodulate this legume worldwide belong to the genus Rhizobium, followed by those belonging to the genera Ensifer (formerly Sinorhizobium) and Pararhizobium (formerly Rhizobium) and minority by species of the genus Bradyrhizobium. All these genera belong to the phylum alpha-Proteobacteria, but the nodulation of P. vulgaris has also been reported for some species belonging to Paraburkholderia and Cupriavidus from the beta-Proteobacteria. Several species nodulating P. vulgaris were originally isolated from nodules of this legume in American countries and are linked to the symbiovars phaseoli and tropici, which are currently present in other continents probably because they were spread in their soils together with the P. vulgaris seeds. In addition, this legume can be nodulated by species and symbiovars originally isolated from nodules of other legumes due its high promiscuity, a concept currently related with the ability of a legume to be nodulated by several symbiovars rather than by several species. In this article we review the species and symbiovars able to nodulate P. vulgaris in different countries and continents and the challenges on the study of the P. vulgaris endosymbionts diversity in those countries where they have not been studied yet, that will allow to select highly effective rhizobial strains in order to guarantee the success of P. vulgaris inoculation.

Genome-Wide Association Analyses in the Model Rhizobium Ensifer meliloti.

Genome-wide association studies (GWAS) can identify genetic variants responsible for naturally occurring and quantitative phenotypic variation. Association studies therefore provide a powerful complement to approaches that rely on de novo mutations for characterizing gene function. Although bacteria should be amenable to GWAS, few GWAS have been conducted on bacteria, and the extent to which nonindependence among genomic variants (e.g., linkage disequilibrium [LD]) and the genetic architecture of phenotypic traits will affect GWAS performance is unclear. We apply association analyses to identify candidate genes underlying variation in 20 biochemical, growth, and symbiotic phenotypes among 153 strains of Ensifer meliloti For 11 traits, we find genotype-phenotype associations that are stronger than expected by chance, with the candidates in relatively small linkage groups, indicating that LD does not preclude resolving association candidates to relatively small genomic regions. The significant candidates show an enrichment for nucleotide polymorphisms (SNPs) over gene presence-absence variation (PAV), and for five traits, candidates are enriched in large linkage groups, a possible signature of epistasis. Many of the variants most strongly associated with symbiosis phenotypes were in genes previously identified as being involved in nitrogen fixation or nodulation. For other traits, apparently strong associations were not stronger than the range of associations detected in permuted data. In sum, our data show that GWAS in bacteria may be a powerful tool for characterizing genetic architecture and identifying genes responsible for phenotypic variation. However, careful evaluation of candidates is necessary to avoid false signals of association.IMPORTANCE Genome-wide association analyses are a powerful approach for identifying gene function. These analyses are becoming commonplace in studies of humans, domesticated animals, and crop plants but have rarely been conducted in bacteria. We applied association analyses to 20 traits measured in Ensifer meliloti, an agriculturally and ecologically important bacterium because it fixes nitrogen when in symbiosis with leguminous plants. We identified candidate alleles and gene presence-absence variants underlying variation in symbiosis traits, antibiotic resistance, and use of various carbon sources; some of these candidates are in genes previously known to affect these traits whereas others were in genes that have not been well characterized. Our results point to the potential power of association analyses in bacteria, but also to the need to carefully evaluate the potential for false associations.

Draft Genome Sequences of Four Novel Thermal- and Alkaline-Tolerant Egyptian Rhizobium Strains Nodulating Berseem Clover.

Four Rhizobium strains were isolated from berseem clover in Egypt. The symbiotically effective, salt-tolerant, strain Rhiz950 was identified as new species, Rhizobium aegypticaum sv. trifolii (USDA 7124(T)). The other three thermal- and pH-tolerant strains were identified as Rhizobium bangladeshense sv. trifolii, the type strain is USDA 7125(T).

The symbiovar trifolii of Rhizobium bangladeshense and Rhizobium aegyptiacum sp. nov. nodulate Trifolium alexandrinum in Egypt.

In the present work we analyzed the taxonomic status of several Rhizobium strains isolated from Trifolium alexandrinum L. nodules in Egypt. The 16S rRNA genes of these strains were identical to those of Rhizobium bangladeshense BLR175(T) and Rhizobium binae BLR195(T). However, the analyses of recA and atpD genes split the strains into two clusters. Cluster II strains are identified as R. bangladeshense with >98% similarity values in both genes. The cluster I strains are phylogenetically related to Rhizobium etli CFN42(T) and R. bangladeshense BLR175(T), but with less than 94% similarity values in recA and atpD genes. DNA-DNA hybridization analysis showed 42% and 48% average relatedness between the strain 1010(T) from cluster I with respect to R. bangladeshense BLR175(T) and R. etli CFN42(T), respectively. Phenotypic characteristics of cluster I strains also differed from those of their closest related Rhizobium species. Analysis of the nodC gene showed that the strains belong to two groups within the symbiovar trifolii which was identified in Egypt linked to the species R. bangladeshense. Based on the genotypic and phenotypic characteristics, the group I strains belong to a new species for which the name Rhizobium aegyptiacum sp. nov. (sv. trifolii) is proposed, with strain 1010(T) being designated as the type strain (= USDA 7124(T)=LMG 29296(T)=CECT 9098(T)).

Isolation and identification of efficient Egyptian malathion-degrading bacterial isolates.

Bacterial isolates degrading malathion were isolated from the soil and agricultural waste water due to their ability to grow on minimal salt media amended with malathion as a sole carbon source. Efficiencies of native Egyptian bacterial malathion-degrading isolates were investigated and the study generated nine highly effective malathion-degrading bacterial strains among 40. Strains were identified by partial sequencing of 16S rDNA analysis. Comparative analysis of 16S rDNA sequences revealed that these bacteria are similar with the genus Acinetobacter and Bacillus spp. and RFLP based PCR of 16S rDNA gave four different RFLP patterns among strains with enzyme HinfI while with enzyme HaeI they gave two RFLP profiles. The degradation rate of malathion in liquid culture was estimated using gas chromatography. Bacterial strains could degrade more than 90% of the initial malathion concentration (1000 ppm) within 4 days.

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.).

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan=4200m(2)) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5kb) and whole ribosomal DNA (5kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.

Use of DNA marker to select well-adapted Phaseolus-symbionts strains under acid conditions and high temperature.

Soil acidity and high temperature contribute to the failure of nodulation in the common bean. It is therefore urgent to select strains with a high competitive ability under these stress conditions. Two Egyptian Rhizobium etli strains, EBRI 2 and EBRI 26, were examined against Rhizobium tropici CIAT 899G labeled with the gus (beta-glucuronidase) reporter gene. EBRI 2 and EBRI 26 were less competitive than CIAT 899G under acid conditions with both the Egyptian cultivar Giza 3 and the Colombian cultivar Rab 39. However, EBRI 2 and EBRI 26 gave higher nodule occupancy (78% and 62.5, respectively) than the nodule occupancy (18.5% and 35%) obtained by CIAT 899G at 35 degrees C with cultivar Giza 3. Soil acidity (pH 5.8) was less detrimental to the nodule occupancy of EBRI 2 than EBRI 26 when they tested in competition with CIAT 899G.

A proteomic approach towards the analysis of salt tolerance in Rhizobium etli and Sinorhizobium meliloti strains.

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.

High salt and high pH tolerance of new isolated Rhizobium etli strains from Egyptian soils.

Saline and alkaline soils are major problems contributing to the low productivity of common bean (Phaseolus vulgaris) in arid and semi-arid regions such as Egypt. Therefore our study was directed toward selecting strains more tolerant to these environmental stresses. Among seven Rhizobium etli strains isolated from Egyptian soils, we found a high degree of diversity. Strains EBRI 21 and EBRI 26 are highly tolerant to a salt concentration up to 4% NaCl. A positive correlation was found between the salt tolerance and the adaptation to alkaline pH (9). Strains EBRI 2 and EBRI 26 were adapted to elevated temperatures (42 degrees C). The minimum level of low pH for the majority of Rhizobium etli strains from Egypt was pH 4.7 while the Colombian strain Rhizobium tropici CIAT 899 survived well at pH 4. At 0.4% NaCl, the symbiotic efficiency of the salt-tolerant strain EBRI 26 was superior in cultivar Giza 6 compared with the salt-sensitive strain EBRI 2 (18.2 compared with 13.9 nM: C2H4 h(-1) mg(-1) nodule fresh weight). In the bean cultivar Saxa, nitrogen fixation was much more affected by high salt concentration (0.4% NaCl) than in the cultivar Giza 6 with both strains (3.9 and 3.8 nM: C2H4 h(-1) mg(-1) nodule fresh weight, respectively). In general, stress of alkalinity had a less detrimental effect on nodulation and N2 fixation than stress of salinity.