Blastococcus brunescens sp. nov., a member of the Geodermatophilaceae isolated from sandstone collected from the Sahara Desert in Tunisia.

The taxonomic position of strain BMG 8361T, isolated from sandstone collected in the Sahara Desert of Southern Tunisia, was refined through a polyphasic taxonomic investigation. Colonies of BMG 8361T were pale-orange coloured, irregular with a dry surface and produced a diffusible pink or brown pigment depending on media. The Gram-positive cells were catalase-positive and oxidase-negative. The strain exhibited growth at 10-40 °C and pH values ranging from 5.5 to 9.0, with optima at 28-35 °C and pH 6.5-8.0. Additionally, BMG 8361T demonstrated the ability to grow in the presence of up to 1 % NaCl (w/v) concentration. The peptidoglycan of the cell wall contained meso-diaminopimelic acid, glucose, galactose, xylose, ribose, and rhamnose. The predominant menaquinones consisted of MK-9(H4) and MK-9. The main polar lipids were phosphatidylcholine, phosphatidylinositol, glycophosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, and two unidentified lipids. Major cellular fatty acids were iso-C16 : 0, iso-C16 : 1 h, and C17 : 1 ω8c. Phylogenetic analyses based on both the 16S rRNA gene and whole-genome sequences assigned strain BMG 8361T within the genus Blastococcus. The highest pairwise sequence similarity observed in the 16S rRNA gene was 99.5 % with Blastococcus haudaquaticus AT 7-14T. However, when considering digital DNA-DNA hybridization and average nucleotide identity, the highest values, 48.4 and 86.58 %, respectively, were obtained with Blastococcus colisei BMG 822T. These values significantly undershoot the recommended thresholds for establishing new species, corroborating the robust support for the distinctive taxonomic status of strain BMG 8361T within the genus Blastococcus. In conjunction with the phenotyping results, this compelling evidence leads to the proposal of a novel species we named Blastococcus brunescens sp. nov. with BMG 8361T (=DSM 46845T=CECT 8880T) as the type strain.

Enhancing Pisum sativum growth and symbiosis under heat stress: the synergistic impact of co-inoculated bacterial consortia and ACC deaminase-lacking Rhizobium.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a crucial bacterial trait, yet it is not widely distributed among rhizobia. Hence, employing a co-inoculation approach that combines selected plant growth-promoting bacteria with compatible rhizobial strains, especially those lacking ACC deaminase, presents a practical solution to alleviate the negative effects of diverse abiotic stresses on legume nodulation. Our objective was to explore the efficacy of three non-rhizobial endophytes, Phyllobacterium salinisoli (PH), Starkeya sp. (ST) and Pseudomonas turukhanskensis (PS), isolated from native legumes grown in Tunisian arid regions, in improving the growth of cool-season legume and fostering symbiosis with an ACC deaminase-lacking rhizobial strain under heat stress. Various combinations of these endophytes (ST + PS, ST + PH, PS + PH, and ST + PS + PH) were co-inoculated with Rhizobium leguminosarum 128C53 or its ΔacdS mutant derivative on Pisum sativum plants exposed to a two-week heat stress period.Our findings revealed that the absence of ACC deaminase activity negatively impacted both pea growth and symbiosis under heat stress. Nevertheless, these detrimental effects were successfully mitigated in plants co-inoculated with ΔacdS mutant strain and specific non-rhizobial endophytes consortia. Our results indicated that heat stress significantly altered the phenolic content of pea root exudates. Despite this, there was no impact on IAA production. Interestingly, these changes positively influenced biofilm formation in consortia containing the mutant strain, indicating synergistic bacteria-bacteria interactions. Additionally, no positive effects were observed when these endophytic consortia were combined with the wild-type strain. This study highlights the potential of non-rhizobial endophytes to improve symbiotic performance of rhizobial strains lacking genetic mechanisms to mitigate stress effects on their legume host, holding promising potential to enhance the growth and yield of targeted legumes by boosting symbiosis.

Bacterial Endophytes from Legumes Native to Arid Environments Are Promising Tools to Improve Mesorhizobium-Chickpea Symbiosis under Salinity.

Symbiotic nitrogen fixation is a major contributor of N in agricultural ecosystems, but the establishment of legume-rhizobium symbiosis is highly affected by soil salinity. Our interest is focused on the use of non-rhizobial endophytes to assist the symbiosis between chickpea and its microsymbiont under salinity to avoid loss of production and fertility. Our aims were (1) to investigate the impact of salinity on both symbiotic partners; including on early events of the Mesorhizobium-chickpea symbiosis, and (2) to evaluate the potential of four non-rhizobial endophytes isolated from legumes native to arid regions (Phyllobacterium salinisoli, P. ifriqiyense, Xanthomonas translucens, and Cupriavidus respiraculi) to promote chickpea growth and nodulation under salinity. Our results show a significant reduction in chickpea seed germination rate and in the microsymbiont Mesorhizobium ciceri LMS-1 growth under different levels of salinity. The composition of phenolic compounds in chickpea root exudates significantly changed when the plants were subjected to salinity, which in turn affected the nod genes expression in LMS-1. Furthermore, the LMS-1 response to root exudate stimuli was suppressed by the presence of salinity (250 mM NaCl). On the contrary, a significant upregulation of exoY and otsA genes, which are involved in exopolysaccharide and trehalose biosynthesis, respectively, was registered in salt-stressed LMS-1 cells. In addition, chickpea co-inoculation with LMS-1 along with the consortium containing two non-rhizobial bacterial endophytes, P. salinisoli and X. translucens, resulted in significant improvement of the chickpea growth and the symbiotic performance of LMS-1 under salinity. These results indicate that this non-rhizobial endophytic consortium may be an appropriate ecological and safe tool to improve chickpea growth and its adaptation to salt-degraded soils.

Blastococcus carthaginiensis sp. nov., isolated from a monument sampled in Carthage, Tunisia.

A comprehensive polyphasic investigation was conducted to elucidate the taxonomic position of an actinobacterium, designated BMG 814T, which was isolated from the historic ruins of Carthage city in Tunisia. It grew as pink-orange pigmented colonies and displayed versatile growth capabilities, thriving within a temperature range of 20-40 °C, across a pH spectrum ranging from pH 5.5 to 10 and in the presence of up to 4 % NaCl. Chemotaxonomic investigations unveiled specific cell components, including diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, an unidentified aminoglycophospholipid, six unidentified aminolipids, two unidentified phospholipids and one unidentified lipid in its polar lipid profile. Furthermore, galactose, glucose and ribose were identified as the primary cell-wall sugars. Major menaquinones identified were MK-9(H4), MK-9(H2) and MK-9, while major fatty acids comprised iso-C15 : 0, iso-C16 : 0, C17 : 1 ω8c and C18 : 1 ω9c. Through phylogenetic analysis based on the 16S rRNA gene sequence, the strain was positioned within the genus Blastococcus, with Blastococcus capsiensis BMG 804T showing the closest relationship (99.1 %). In light of this, draft genomes for both strains, BMG 814T and BMG 804T, were sequenced in this study, and comparative analysis revealed that strain BMG 814T exhibited digital DNA-DNA hybridization and average nucleotide identity values below the recommended thresholds for demarcating new species with all available genomes of type strains of validly names species. Based on the polyphasic taxonomy assessment, strain BMG 814T (=DSM 46848T=CECT 8878T) was proposed as the type strain of a novel species named Blastococcus carthaginiensis sp. nov.

Decrypting phytomicrobiome of the neurotoxic actinorhizal species, Coriaria myrtifolia, and dispersal boundary of Frankia cluster 2 in soil outward compatible host rhizosphere.

The actinorhizal plant, Coriaria myrtifolia, is a neurotoxic plant species endemic to the western Mediterranean area, which forms a nitrogen-fixing symbiosis with members of Frankia cluster 2. Contrarily to other Frankia clusters, the occurrence and mode of dispersal for infective cluster 2 units outside of the host plant rhizosphere remains controversial. The present study was designed to investigate the structure of the microbiomes of C. myrtifolia phytosphere, rhizosphere, and soil samples extending outward linearly up to 1 km. Results showed that the epiphyte and endophyte communities were not significantly different from each other for most of the plant tissues. The communities associated with the below-ground tissues (nodule and root) were significantly different from those found on the above-ground tissues (fruit, leaves, and stems) and had a higher community richness. Coriaria myrtifolia phytomicrobiomes were dominated by Cyanobacteria for leaf, stem, and fruit while Actinobacteria and Proteobacteria were dominant in the root and nodule organelles. The nodule, a special niche for nitrogen fixation, was mainly inhabited by Frankia but contained several non-Frankia bacteria. Beside Frankia cluster 2, the presence of clusters 1, 4, and large numbers of cluster 3 strains have been detected in nodules, roots, and rhizospheres of C. myrtifolia. Despite Frankia being found in all plots using plant trapping bioassays with C. myrtifolia seedlings, Frankia cluster 2 was not detected in soil metagenomes showing the limits of detection by this approach. This result also suggests that in the absence of appropriate host plant species, Frankia cluster 2 has a reduced number of infective units present in the soil outward from the rhizosphere.

Root Nodule Microsymbionts of Native Coriaria myrtifolia in Algeria.

Coriaria myrtifolia occurs as natural flora of warm temperate climates of northern Algeria which commonly found in hedges, forest and ravine edges. This actinorhizal species was known to establish a mutualistic symbiosis with members of phylogenetic cluster 2 (including strains associated to Coriaria spp., Ceanothus, Datiscaceae, and Dryadoideae) within the genus Frankia. Attempts to isolate C. myrtifolia microsymbionts from native plants growing in 4 locations in Algeria permitted to only recover asymbiotic Frankia strains (unable to reestablish nodulation and to fix nitrogen) from phylogenetic cluster 4 and several non-Frankia actinobacteria including members of Micrococcus, Micromonospora, Nocardia, Plantactinospora, and Streptomyces genera. The biodiversity of Frankia microsymbionts of C. myrtifolia root nodules was assessed using PCR-amplification followed by partial nucleotide sequencing of glnA1 (glutamine synthetase type 1) gene. On the 12 different glnA1 gene sequences obtained in this study, 9 were detected for the first time, and were mainly closelyrelated to Mediterranean genotypes previously described in the Grand Maghreb countries (Morocco and Tunisia) and in Europe (France) but without clear separations from other cluster 2 genotypes.

Characterization of natural habitats and diversity of Libyan desert truffles.

Desert truffles have traditionally been used as food in Libya. Desert truffle grows and gives fruit sporadically when adequate and properly distributed rainfall occurs with existence of suitable soil and mycorrhizal host plant. The present study aimed to identify and characterize two kinds of wild desert truffles from ecological and nutritional points that were collected from the studied area. The truffle samples were identified as Terfezia (known as red or black truffle) and Tirmania (known as white truffle). The nutritional values (protein, lipid and carbohydrate) of both Libyan wild truffle (Terfezia and Tirmania) were determined on a dry weight basis and result showed that Tirmania and Terfezia contained 16.3 and 18.5% protein, 6.2 and 5.9% lipid, 67.2 and 65% carbohydrate, respectively, in ascocarp biomass. The soil pH of the upper and lower regions of the Hamada Al-Hamra ranged between 8.2 and 8.5 giving suitable conditions for fructification. The plants, Helianthemum kahiricum and Helianthemum lippii were the dominant plants in Hamada Al-Hamra region found to form a mycorrhiza with desert truffles. The phylogenetic analysis of the genomic rDNA ITS region showed that, out of five collections three represented Tirmania pinoyi (Maire) Malencon, one Tirmania nivea (Desf.) Trappe, and one Terfezia boudieri Chatin.

An integrated overview of the midgut bacterial flora composition of Phlebotomus perniciosus, a vector of zoonotic visceral leishmaniasis in the Western Mediterranean Basin.

BACKGROUND: The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect's digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission. METHODOLOGY/PRINCIPAL FINDINGS: The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species. CONCLUSION/SIGNIFICANCE: Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission.

Contrasted Genetic Diversity, Relevance of Climate and Host Plants, and Comments on the Taxonomic Problems of the Genus Picoa (Pyronemataceae, Pezizales).

The species concept within the genus Picoa Vittad. is here revisited in light of new molecular and ecological data obtained from samples collected throughout the Mediterranean basin. Two highly diverse widespread clades and four additional minor lineages were significantly supported by three genes dataset (ITS, 28s LSU and RPB2) inferences for 70 specimens. The two widespread clades occur in very different geographical and ecological areas associated with exclusive host plants in the genus Helianthemum. SEM study of spore surface morphology in these lineages revealed the existence of smooth ascospores in the majority of these clades. However the most frequent lineage in Europe and coastal North Africa displayed either smooth or verrucose spores. Hence this morphological criterion cannot be reliably used to discriminate between the different clades. In addition, SEM observations made on ascospores from several original collections of P. juniperi and P. lefebvrei supported the hypothesis that ornamentation depends on the degree of maturity in some of these lineages. Geographical and ecological, rather than morphological data are here suggested as the most useful characters to separate the different lineages in Picoa. Further studies focusing on these features are needed before the names P. juniperi and P. lefebvrei can be unambiguously linked with the genetic lineages observed.

Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains.

The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch's postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation.

Diversity and enzymatic profiling of halotolerant micromycetes from Sebkha El Melah, a Saharan salt flat in southern Tunisia.

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as ß-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (n = 3), Alternaria (n = 4), Aspergillus (n = 3), Penicillium (n = 5), Ulocladium (n = 2), and Engyodontium (n = 2). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkali-halotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45°C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium.

Contrasted reactivity to oxygen tensions in Frankia sp. strain CcI3 throughout nitrogen fixation and assimilation.

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixing Frankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions on Frankia sp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions. The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhC and hpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, the hup2 mRNA levels increased with hyperoxic environment, while hup1 mRNA levels remained relatively constant. Taken together, these results indicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection.

First report on the occurrence of the uncultivated cluster 2 Frankia microsymbionts in soil outside the native actinorhizal host range area.

The occurrence of uncultivated Frankia was evaluated in Tunisian soils by a plant-trapping assay using Coriaria myrtifolia seedlings. Despite the lack of this compatible host plant for more than two centuries, soil-borne Frankia cells were detected in one sampled soil as shown by the development of root nodules on 2-year-old seedlings. Based on glnA sequences, Tunisian trapped Frankia strains belong to the uncultivated cluster 2 strains that associate with other Coriaria species and also with Ceanothus, Datisca and Rosaceae actinorhizal species. This is the first report on the occurrence of Frankia cluster 2 strains in soils from areas lacking compatible host plant groups.

Genome sequence of Blastococcus saxobsidens DD2, a stone-inhabiting bacterium.

Members of the genus Blastococcus have been isolated from sandstone monuments, as well as from sea, soil, plant, and snow samples. We report here the genome sequence of a member of this genus, Blastococcus saxobsidens strain DD2, isolated from below the surface of a Sardinian wall calcarenite stone sample.

Diversity of the desert truffle Terfezia boudieri Chatin. in southern Tunisia.

This study reports the genetic diversity of Terfezia boudieri collected from southern Tunisia. The study was carried out using 135 truffle fruiting bodies harvested from seven different locations. Twenty-eight Terfezia claveryi fruiting bodies were also sampled from one of the seven locations. A PCR-based technique was used to amplify the internal transcribed spacer (ITS) region of the rDNA, including the ITS1-5.8S-ITS2. The PCR products were digested with the four restriction enzymes RsaI, HhaI, AluI, and HinfI. Based on the HinfI patterns, T. boudieri specimens were separated into two different haplotypes (I and II). Nucleotide sequences of some representative amplicons were also obtained. Based on the phylogenetic results, three T. boudieri genotypes could be differentiated. One sequence, SKtb1, retrieved from PCR-RFLP of haplotype I, was obtained from a low pH soil in association with Helianthemum kahiricum . Based on the results presented in the current study, this isolate may represent a novel taxa within the Terfezia genus.

Phylogenetic affiliation of the desert truffles Picoa juniperi and Picoa lefebvrei.

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.