Besma Ettoumi

A drought resistance-promoting microbiome is selected by root system under desert farming

Background Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress. Methodology/Principal Findings We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress. Conclusions/Significance Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.

Improved plant resistance to drought is promoted by the root‐associated microbiome as a water stress‐dependent trait

Although drought is an increasing problem in agriculture, the contribution of the root-associated bacterial microbiome to plant adaptation to water stress is poorly studied. We investigated if the culturable bacterial microbiome associated with five grapevine rootstocks and the grapevine cultivar Barbera may enhance plant growth under drought stress. Eight isolates, over 510 strains, were tested in vivo for their capacity to support grapevine growth under water stress. The selected strains exhibited a vast array of plant growth promoting (PGP) traits, and confocal microscopy observation of gfp-labelled Acinetobacter and Pseudomonas isolates showed their ability to adhere and colonize both the Arabidopsis and grapevine rhizoplane. Tests on pepper plants fertilized with the selected strains, under both optimal irrigation and drought conditions, showed that PGP activity was a stress-dependent and not a per se feature of the strains. The isolates were capable of increasing shoot and leaf biomass, shoot length, and photosynthetic activity of drought-challenged grapevines, with an enhanced effect in drought-sensitive rootstock. Three isolates were further assayed for PGP capacity under outdoor conditions, exhibiting the ability to increase grapevine root biomass. Overall, the results indicate that PGP bacteria contribute to improve plant adaptation to drought through a water stress-induced promotion ability.

Diversity and phylogeny of culturable spore-forming Bacilli isolated from marine sediments.

Members of the genus Bacillus and related genera are ubiquitous in nature. However, Bacillus species isolated from marine sediments have attracted less interest respect to their terrestrial relatives. Here, we report the phylogenetic diversity of a collection of 96 Bacilli, isolated from 17 distinct stations of 5 oceanographic campaigns. The diversity was analysed by phenotypic and molecular approaches based on the amplified rDNA restriction analysis (ARDRA), amplification of the internal transcribed spacers (ITS‐PCR) and on 16S rRNA sequencing. Intra‐specific polymorphism was efficiently detected by biochemical analysis and ARDRA while results of ITS‐PCR were in agreement with 16S rRNA sequencing. The identification results assigned 68% of the isolates to the species B. subtilis, B. licheniformis, B. pumilus and B. cereus. Phylogenetic analysis allowed the separation of 9 isolates in a clade that may represent a group of obligate marine Bacillus since they clustered with B. firmus, B. foraminis and marine isolates with metal oxidation and bioaccumulation capabilities. The remaining isolates showed a close affiliation to the genera Virgibacillus, Gracilibacillus and Paenibacillus. The widespread of Bacilli and their high diversity level observed in this work point out the need of more extensive studies to understand their distribution and ecology in deep‐sea environments. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches.

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade (n=53) and to Halomonas aquamarina (n=14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.

Cultivation-Dependant Assessment, Diversity, and Ecology of Haloalkaliphilic Bacteria in Arid Saline Systems of Southern Tunisia

Haloalkaliphiles are polyextremophiles adapted to grow at high salt concentrations and alkaline pH values. In this work, we isolated 122 haloalkaliphilic bacteria upon enrichments of 23 samples from 5 distinct saline systems of southern Tunisia, growing optimally in media with 10% salt and at pH 10. The collection was classified into 44 groups based on the amplification of the 16S–23S rRNA internal transcribed spacers (ITS-PCR). Phylogenetic analysis and sequencing of the 16S rRNA genes allowed the identification of 13 genera and 20 distinct species. Three gram-positive isolates showing between 95 and 96% of 16S rRNA sequence homology with Bacillus saliphilus could represent new species or genus. Beside the difference in bacterial diversity between the studied sites, several species ecological niches correlations were demonstrated such as Oceanobacillus in salt crust, Nesterenkonia in sand, and Salinicoccus in the rhizosphere of the desert plant Salicornia. The collection was further evaluated for the production of extracellular enzymes. Activity tests showed that gram-positive bacteria were mostly active, particularly for protease, lipase, DNase, and amylase production. Our overall results demonstrate the huge phenotypic and phylogenetic diversity of haloalkaliphiles in saline systems of southern Tunisia which represent a valuable source of new lineages and metabolites.

A novel phenotype based on esterase electrophoretic polymorphism for the differentiation of Lactococcus lactis ssp. lactis and cremoris

Aims:  To evaluate the esterase phenotype in Lactococcus lactis strains isolated from traditional Tunisian dairy products.

Genomic diversity and relationship of Bacillus thuringiensis and Bacillus cereus by multi-REP-PCR fingerprinting

The genomic diversity and relationship among 56 Bacillus thuringiensis and Bacillus cereus type strains were investigated by multi-REP-PCR fingerprinting consisting of three PCR reactions targeting the enterobacterial ERIC1 and ERIC2 and the streptococcal BOXA1R consensus sequences. A total of 113 polymorphic bands were generated in the REP-PCR profiles that allowed tracing of a single dendrogram with three major groups. Bacillus cereus strains clustered together in the A and B groups. Most of the B. thuringiensis strains clustered in group C, which included groups of serovars with a within-group similarity higher than 40% as follows: darmstadiensis, israelensis, and morrisoni; aizawai, kenyae, pakistani, and thompsoni; canadensis, entomocidus, galleriae, kurstaki, and tolworthi; alesti, dendrolimus, and kurstaki; and finitimus, sotto, and thuringiensis. Multi-REP-PCR fingerprinting clustered B. thuringiensis serovars in agreement with previously developed multilocus sequence typing schemes, indicating that it represents a rapid shortcut for addressing the genetic relationship of unknown strains with the major known serovars.

Diversity, ecological distribution and biotechnological potential of Actinobacteria inhabiting seamounts and non-seamounts in the Tyrrhenian Sea

In the present study, the ecological distribution of marine Actinobacteria isolated from seamount and non-seamount stations in the Tyrrhenian Sea was investigated. A collection of 110 isolates was analyzed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and 16S rRNA gene sequencing of representatives for each ARISA haplotype (n=49). Phylogenetic analysis of 16S rRNA sequences showed a wide diversity of marine isolates and clustered the strains into 11 different genera, Janibacter, Rhodococcus, Arthrobacter, Kocuria, Dietzia, Curtobacterium, Micrococcus, Citricoccus, Brevibacterium, Brachybacterium and Nocardioides. Interestingly, Janibacter limosus was the most encountered species particularly in seamounts stations, suggesting that it represents an endemic species of this particular ecosystem. The application of BOX-PCR fingerprinting on J. limosus sub-collection (n=22), allowed their separation into seven distinct BOX-genotypes suggesting a high intraspecific microdiversity among the collection. Furthermore, by screening the biotechnological potential of selected actinobacterial strains, J. limosus was shown to exhibit the most important biosurfactant activity. Our overall data indicates that Janibacter is a major and active component of seamounts in the Tyrrhenian Sea adapted to low nutrient ecological niche.

A novel thermally stable heteropolysaccharide-based bioflocculant from hydrocarbonoclastic strain Kocuria rosea BU22S and its application in dye removal

ABSTRACT A new bioflocculant named pKr produced by hydrocarbonoclastic strain Kocuria rosea BU22S (KC152976) was investigated. Gas chromatography–flame ionization detector (GC-FID) analysis confirmed the high potential of the strain BU22S in the degradation of n-alkanes. Plackett–Burman experimental design and response surface methodology were carried out to optimize pKr production. Glucose, peptone and incubation time were found to be the most significant factors affecting bioflocculant production. Maximum pKr production was about 4.72 ± 0.02 g/L achieved with 15.61 g/L glucose, 6.45 g/L peptone and 3 days incubation time. Chemical analysis of pKr indicated that it contained 71.62% polysaccharides, 16.36% uronic acid and 2.83% proteins. Thin layer chromatography analysis showed that polysaccharides fraction consisted of galactose and xylose. Fourier transform infrared analysis revealed the presence of many functional groups, hydroxyl, carboxyl, methoxyl, acetyl and amide that likely contribute to flocculation. K. rosea pKr showed high flocculant potential using kaolin clay at different pH (2–11), temperature (0–100°C) and cation concentrations. The bioflocculant was particularly effective in flocculating soluble anionic dyes, Reactive Blue 4 and Acid Yellow, with a decolorization efficiency of 76.4% and 72.6%, respectively. The outstanding flocculating performances suggest that pKr could be useful for bioremediation applications. GRAPHICAL ABSTRACT

Esterase electrophoretic polymorphism ofBacillus thuringiensis andBacillus cereus reference strains

The genomic diversity and relationship among 61Bacillus thuringiensis andBacillus cereus reference strains were investigated by electrophoretic analysis of esterase enzymes on native polyacrylamide gel. Polymorphism of the esterolytic bands revealed seven esterases, designed as Est A to Est G in order of decreasing anodal migration. Each esterase showed two to three mobility variants that assigned the analysed strains into 35 electrophoretic types (ETs). This high diversity allowed the identification of several serovar or strain-specific ETs. Cluster analysis of ETs showed three major groups in which the strains belonging to the serovartolworthi were the most distant. The ETs distribution showed thatB. thuringiensis andB. cereus are intermingled in the dendrogram with the resolution of some common serovars ofB. thuringiensis in tight phylogenetic lineages. These results indicate that the esterase enzyme electrophoresis, applied as a sole typing method for the closely related speciesB. thuringiensis andB. cereus is suitable to highlight the intraspecific genetic diversity.