Evaluating the effect of various bacterial consortia isolated from arid wild legumes on heat stress tolerance of Pisum sativum

High-temperature stress affects the growth and developmental process of cool-season grain legumes. We hypothesized that endophytic bacteria associated with arid plants could be a potential resource to ensure the tolerance of cold-season legumes to high temperature stress events. To test our hypothesis, Phyllobacterium salinitolerans (PH), Starkeya sp. (ST) and Pseudomonas turukhanskensis (PS) endophytes of different spontaneous legumes localised in Tunisian arid regions were selected to evaluate their potential in improving Pisum sativum growth and pea-rhizobia symbiosis under a heat stress event. Three consortia (containing different combinations of endophytes) were used along with the pea microsymbiont Rhizobium leguminosarum 128C53 (WT) or with its ΔacdS mutant derivative (MT) (Ma et al., 2003). Uninoculated plants without or with nitrogen supplement were used as negative (NC) or positive controls (PC), respectively. The heat stress event was applied 2 weeks after sowing for a period of 2 weeks with consecutive cycles of 30-35°C/16h and 20°C/8h. Interestingly, the shoot dry weight (SDW) of all plants co-inoculated with WT and any of the consortia containing PH increased significantly compared to that of plants inoculated with WT alone. A similar effect was observed on the root dry weight (RDW) in the treatments WT+ST+PH and WT+PS+PH. On the other hand, the best results either in terms of SDW or RDW with the mutant strain was the treatment that included all endophytes (MT+ST+PS+PH), even overcoming all treatments inoculated with WT and equalling the PC. As expected, plants inoculated with the MT had a lower number of nodules (NN) compared to plants inoculated with WT, except for MT+ST+PS+PH with similar NN. A significant increase in the NN was observed in plants co-inoculated with WT+ST+PH and WT+PS+PH compared to those in WT. The highest total chlorophyll content was in WT+ST+PS, which was significantly different from all other treatments while no differences were observed in phenolic compounds content among the inoculated treatments. Overall, our results suggest that endophytic isolates from arid leguminous plants are good candidates for increasing the resilience of plants not adapted to heat stress

Endophytic bacteria associated with spontaneous legumes in arid zones of Tunisia: Genetic diversity, metabolic functionalities and potential application to mitigate the impact of climate change

Legumes naturally adapted to harsh climate environments represent a new source of plant growth promoting (PGP) bacteria that can be used to improve crop resilience to climate change. However, the diversity and functionality of endophytic bacteria associated with endemic wild legumes in Tunisia are almost unknown. To study the taxonomic and functional diversity of these species, we conducted a study on root nodules of 15 spontaneous legumes, some of which studied for the first time, growing in three different Tunisian bioclimates (arid, semi-arid and Saharan). More than 210 strains, corresponding to 78 different phylotypes, were isolated. Sequencing of the 16S rDNA revealed the presence of rhizobial isolates belonging to the genera: Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium, Neorhizobium, Agrobacterium, Phyllobacterium, Cupriavidus and Burkholderia. Other non-rhizobial bacteria assigned to Microbacterium, Pseudomonas, Paenibacillus, Starkeya, Kocuria among others, were also obtained. In addition, the nucleotide similarity of housekeeping genes suggested the presence of new species in our collection. Indeed, the genomic sequencing analysis identified a new species of Mesorhizobium (PRJNA800673), a microsymbiont of Retama raetam, which is one of the most important Tunisian shrubs. Regarding PGP-screening abilities, several strains stood out for possessing several PGP traits, phosphate solubilization, indole acetic acid and siderophores production, along with high tolerance to abiotic stress (> 45°C; > 0.4M NaCl). For rhizobia, whole genome sequencing of the Saharan isolate Sinorhizobium meliloti IRAM:0087 (PRJNA842649) revealed the existence of several gene clusters coding for different PGP activities, rhizosphere competitiveness and stress tolerance, some of which were confirmed in vitro assays. Based on the evaluation of the PGP potential of non-rhizobial strains, three bacteria P. salinitolerans, P. turukhanskensis and Starkeya sp. were selected to improve Pisum sativum, a cold season crop, tolerance to a heat stress event. Our preliminary results suggest that endophytic isolates from arid legumes represent a promising resource of biofertilizers/biostimulants to increase plant resilience to heat stress. Future studies to explore the adaptative responses of these endophytes to different types of stress will allow to better understand their stress tolerance mechanisms and contribute to select strains for nature-based solutions towards the establishment of new agricultural technologies in drylands

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

\ua9 2024 The Authors.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\ub0C and pH values ranging from 5.5 to 9.0, with optima at 28–35\ub0C 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

<em>Blastococcus carthaginiensis</em> 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 \ub0C, 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

Pisolithus albus (Sclerodermataceae), a new record for Tunisia

Pisolithus albus was recently collected for the first time in Tunisia. This ectomycorrhizal fungus is found associated with Eucalyptus occidentalis, a new symbiotic relationship, in the urban area of Tunis. The new record of this fungus on this tree permit to expand our knowledge on the ecology and distribution of P. albus in Tunisia. The finding is also important since this fungal symbiont has great potential in forestation efforts. Data on the morphology, molecular identification, distribution and, ecology for P. albus in Tunisia are presented for the first time

Macromycetes' diversity in Tunisia

In Tunisia, about 12% of the country area is covered by forests which have favorable climatic factors likely to harbor a high macrofungal biodiversity. However, this valuable bioresource remains understudied especially regarding their distribution, ecology, abundance and diversity. The initiation of the first national inventory of macrofungi in NorthWestern Tunisian forests was published by our group in 2016. The study covered six governorates, sixty seven sites and enabled the identification of one hundred and twenty-three specimens. Abiotic environmental factors such (as bioclimatic stage, rainfall and edaphic conditions) and biotic factors (plant association and nature and density of the forest) have been studied to determine the distribution and ecology of the collected macromycetes. The obtained results showed a promising diversity of macfungal resources in Tunisia. The present work aims to determine an updated checklist for specimens collected from January 2014 to March 2017. A total of 75 collection and prospecting missions carried out to 105 sites in 11 governorates of northern Tunisia allowed the collection of a total of 1815 specimens. Some of which are considered infrequent or rare. A herbarium at the Laboratory of Microorganisms and Active Biomolecules at the Faculty of Sciences of Tunis and in the Laboratory of Microorganisms of the National Genes Bank of Tunisia was created and an online database with all the photos and data collected was carried out. In addition, a molecular data base of the collected specimens was initiated which currently contains 250 ITS sequences and continues to be regularly enriched. This work will allow the mapping of macromycetes in Tunisia which will be useful to realise the national inventory of macrofungi in our country. Data will be available to interested mycologists, to the silviculturists and to the gatherers of edible mushroom. This may improve the exploitation of these valuable resources and contribute to their best management

Macromycetes' diversity in Tunisia

In Tunisia, about 12% of the country area is covered by forests which have favorable climatic factors likely to harbor a high macrofungal biodiversity. However, this valuable bioresource remains understudied especially regarding their distribution, ecology, abundance and diversity. The initiation of the first national inventory of macrofungi in NorthWestern Tunisian forests was published by our group in 2016. The study covered six governorates, sixty seven sites and enabled the identification of one hundred and twenty-three specimens. Abiotic environmental factors such (as bioclimatic stage, rainfall and edaphic conditions) and biotic factors (plant association and nature and density of the forest) have been studied to determine the distribution and ecology of the collected macromycetes. The obtained results showed a promising diversity of macfungal resources in Tunisia. The present work aims to determine an updated checklist for specimens collected from January 2014 to March 2017. A total of 75 collection and prospecting missions carried out to 105 sites in 11 governorates of northern Tunisia allowed the collection of a total of 1815 specimens. Some of which are considered infrequent or rare. A herbarium at the Laboratory of Microorganisms and Active Biomolecules at the Faculty of Sciences of Tunis and in the Laboratory of Microorganisms of the National Genes Bank of Tunisia was created and an online database with all the photos and data collected was carried out. In addition, a molecular data base of the collected specimens was initiated which currently contains 250 ITS sequences and continues to be regularly enriched. This work will allow the mapping of macromycetes in Tunisia which will be useful to realise the national inventory of macrofungi in our country. Data will be available to interested mycologists, to the silviculturists and to the gatherers of edible mushroom. This may improve the exploitation of these valuable resources and contribute to their best management

Updated checklist of macromycetes of Tunisia

A preliminary checklist of macromycetes has been previously published with reference to six governo-rates located in northern Tunisia. This paper provides an updated version of the inventory of macro-mycetes in Tunisia, which includes 268 species, of which seven are worldwide recognized as rare. Theinvestigation was carried out from September 2015 to December 2018 in 13 governorates of theTunisian territory. This updated checklist reports 145 new national species records (139 basidiomycetesand six ascomycetes) belonging to 57 genera and 13 families. Data related to distribution, ecologyand preferential hosts of each recorded taxon are also provided

First report of the rare tooth fungus Hericium erinaceus in North African temperate forests

The rare fungus Hericium erinaceus (Bull.) Pers. was collected from temperate forests in northwestern Tunisia and described for the first time in Africa. In this paper, we report data about the distribution, ecology, morphology and molecular identification of H. erinaceus. Collected data may help expand our knowledge on this critically endangered rare species worldwide