Impact of two Erwinia sp. on the response of diverse Pisum sativum genotypes under salt stress.

Currently, salinization is impacting more than 50% of arable land, posing a significant challenge to agriculture globally. Salt causes osmotic and ionic stress, determining cell dehydration, ion homeostasis, and metabolic process alteration, thus negatively influencing plant development. A promising sustainable approach to improve plant tolerance to salinity is the use of plant growth-promoting bacteria (PGPB). This work aimed to characterize two bacterial strains, that have been isolated from pea root nodules, initially called PG1 and PG2, and assess their impact on growth, physiological, biochemical, and molecular parameters in three pea genotypes (Merveille de Kelvedon, Lincoln, Meraviglia d'Italia) under salinity. Bacterial strains were molecularly identified, and characterized by in vitro assays to evaluate the plant growth promoting abilities. Both strains were identified as Erwinia sp., demonstrating in vitro biosynthesis of IAA, ACC deaminase activity, as well as the capacity to grow in presence of NaCl and PEG. Considering the inoculation of plants, pea biometric parameters were unaffected by the presence of the bacteria, independently by the considered genotype. Conversely, the three pea genotypes differed in the regulation of antioxidant genes coding for catalase (PsCAT) and superoxide dismutase (PsSOD). The highest proline levels (212.88 µmol g-1) were detected in salt-stressed Lincoln plants inoculated with PG1, along with the up-regulation of PsSOD and PsCAT. Conversely, PG2 inoculation resulted in the lowest proline levels that were observed in Lincoln and Meraviglia d'Italia (35.39 and 23.67 µmol g-1, respectively). Overall, this study highlights the potential of these two strains as beneficial plant growth-promoting bacteria in saline environments, showing that their inoculation modulates responses in pea plants, affecting antioxidant gene expression and proline accumulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01419-8.

Correction to: Salt stress mitigation in Lathyrus cicera by combining different microbial inocula.

[This corrects the article DOI: 10.1007/s12298-022-01205-4.].

Rhizobium acaciae sp. nov., a new nitrogen-fixing symbiovar isolated from root nodules of Acacia saligna in Tunisia.

Three bacterial strains, 1AS11T, 1AS12 and 1AS13, members of the new symbiovar salignae and isolated from root nodules of Acacia saligna grown in Tunisia, were characterized using a polyphasic approach. All three strains were assigned to the Rhizobium leguminosarum complex on the basis of rrs gene analysis. Phylogenetic analysis based on 1734 nucleotides of four concatenated housekeeping genes (recA, atpD, glnII and gyrB) showed that the three strains were distinct from known rhizobia species of the R. leguminosarum complex and clustered as a separate clade within this complex. Phylogenomic analysis of 92 up-to-date bacterial core genes confirmed the unique clade. The digital DNA-DNA hybridization and blast-based average nucleotide identity values for the three strains and phylogenetically related Rhizobium species ranged from 35.9 to 60.0% and 87.16 to 94.58 %, which were lower than the 70 and 96% species delineation thresholds, respectively. The G+C contents of the strains were 60.82-60.92 mol% and the major fatty acids (>4 %) were summed feature 8 (57.81 %; C18 : 1 ω7c) and C18 : 1 ω7c 11-methyl (13.24%). Strains 1AS11T, 1AS12 and 1AS13 could also be differentiated from their closest described species (Rhizobium indicum, Rhizobium laguerreae and Rhizobium changzhiense) by phenotypic and physiological properties as well as fatty acid content. Based on the phylogenetic, genomic, physiological, genotypic and chemotaxonomic data presented in this study, strains 1AS11T, 1AS12 and 1AS13 represent a new species within the genus Rhizobium and we propose the name Rhizobium acaciae sp. nov. The type strain is 1AS11T (=DSM 113913T=ACCC 62388T).

Different species of Bradyrhizobium from symbiovars genistearum and retamae nodulate the endemic Retama dasycarpa in the High Atlas Mountains.

Retama dasycarpa is an endemic Retama species native to the cold semi-arid bioclimates of the High Atlas Mountains in Morocco. In this work, we analyzed the diversity of the microsymbionts nodulating this plant and their different phenotypic and symbiotic characteristics. Phylogenetic analysis of the 16S rRNA gene revealed that the tested isolates clustered in the Bradyrhizobium genus. Multilocus sequence analyses of four housekeeping genes (recA, gyrB, glnII and atpD) for 12 selected strains grouped them into four clusters close to B. lupini USDA 3051T, B. frederickii CNPSo 3446T, B. valentinum LmjM3T and B. retamae Ro19T. The individual phylogenies of these core genes and the symbiotic genes nodC, nodA and nifH were congruent. These isolates showed a broad host range, being able to nodulate different legume hosts, such as R. sphaerocarpa, R. monosperma, Lupinus luteus, Cytisus grandiflorus andChamaecytisus albidus, but not Phaseolus vulgaris or Glycine max. They all had a similar metabolic capacity, using the majority of the carbohydrates and amino acids tested as sole sources of carbon and nitrogen. Furthermore, out of the 12 selected strains, some displayed plant growth-promoting features, with six of them solubilizing phosphate and three of them producing siderophores. The present work provides, for the first time, a detailed description about the microsymbionts associated with the endemic legume R. dasycarpa.

Salt stress mitigation in Lathyrus cicera by combining different microbial inocula.

Arid and semi-arid areas are considered vulnerable to various environmental constraints which are further fortified by climate change. Salinity is one of the most serious abiotic factors affecting crop yield and soil fertility. Till now, no information is available on the effect of salinity on development and symbiotic nitrogen (N2) fixation in the legume species Lathyrus cicera. Here, we evaluated the effect of different microbial inocula including nitrogen-fixing Rhizobium laguerreae, arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis, a complex mixed inoculum of AMF isolated from rhizospheric soil in "Al Aitha", and various plant growth-promoting bacteria (PGPB) including Bacillus subtilus, Bacillus simplex and Bacillus megaterium combined with Rhizobium, the AMF consortium, or R. irregularis on alleviating salt stress in this legume. A pot trial was conducted to evaluate the ability of different microbial inocula to mitigate adverse effects of salinity on L. cicera plants. The results showed that salinity (100 mM NaCl) significantly reduced L. cicera plant growth. However, inoculation with different inocula enhanced plant growth and markedly promoted various biochemical traits. Moreover, the combined use of PGPB and AMF was found to be the most effective treatment in mitigating deleterious effects of salinity stress on L. cicera. In addition, this co-inoculation upregulated the expression of two marker genes (LcHKT1 and LcNHX7) related to salinity tolerance. Our findings suggest that the AMF/PGPB formulation has a great potential to be used as a biofertilizer to improve L. cicera plant growth and productivity under saline conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01205-4.

Chemical Composition and Antimicrobial Activity of Essential Oils from Three Mediterranean Plants against Eighteen Pathogenic Bacteria and Fungi.

The chemical composition and antimicrobial activity of essential oils (EOs) obtained from three medicinal plants of the Moroccan flora were evaluated. The chemical composition of EOs of Thymus leptobotrys, Laurus nobilis and Syzygium aromaticum was determined using a gas chromatograph coupled with mass spectrometry. Carvacrol (75.05%) was the main constituent of T. leptobotrys EOs, while 1,8-cineole (31.48%) and eugenol (82.16%) were the predominant components of L. nobilis and S. aromaticum EOs, respectively. The antimicrobial activity of the EOs was evaluated qualitatively and quantitatively against 18 microbial strains pathogenic to humans by using the disc diffusion method, and by measuring the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC). The EOs of T. leptobotrys were the most active against the strains tested, with inhibitory zone values ranging from 7.00 to 45.00 mm, and MIC and MMC values ranging from 0.312 to 80.00 mg/mL. In many cases, these EOs exhibited higher antibacterial and antifungal activities than the chemical compounds ciprofloxacin and fluconazole, respectively. This high antimicrobial activity can be ascribed to their richness in carvacrol. The EOs of T. leptobotrys, L. nobilis, and S. aromaticum could be considered a promising alternative to replace chemical antimicrobials, and a readily available natural source of bioactive compounds.

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium.

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. "salignae" is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. "cyanophyllae" is proposed. Isolates formed effective nodules on A. saligna.

Application of Native or Exotic Arbuscular Mycorrhizal Fungi Complexes and Monospecific Isolates from Saline Semi-Arid Mediterranean Ecosystems Improved Phoenix dactylifera's Growth and Mitigated Salt Stress Negative Effects.

The date, the palm tree (Phoenix dactylifera L.) is an important component of arid and semi-arid Mediterranean ecosystems, particularly in Morocco where it plays a considerable socio-economic and ecological role. This species is largely affected by desertification, global warming, and anthropic pressure. Salinity is a very worrying problem that negatively affects the growth and the physiological and biochemical activities of the date palm. In these arid zones, the main challenge is to develop new environmentally friendly technologies that improve crop tolerance to abiotic restraints including salinity. In this sense, Arbuscular mycorrhizal fungi (AMF) have received much attention due to their capability in promoting plant growth and tolerance to abiotic and biotic stresses. It is thus fitting that the current research work was undertaken to evaluate and compare the effects of native AMF on the development of the growth and tolerance of date palm to salt stress along with testing their role as biofertilizers. To achieve this goal, two complexes and two monospecific isolates of native and non-native AMF were used to inoculate date palm seedlings under saline stress (0 g·L-1 Na Cl, 10 g·L-1, and 20 g·L-1 Na Cl). The obtained results showed that salinity drastically affected the physiological parameters and growth of date palm seedlings, whilst the application of selected AMF significantly improved growth parameters and promoted the activities of antioxidant enzymes as a protective strategy. Inoculation with non-native AMF complex and monospecific isolates showed higher responses for all analyzed parameters when compared with the native complex and isolate. It therefore becomes necessary to glamorize the fungal communities associated with date palm for their use in the inoculation of Phoenix dactylifera L. seedlings.

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.

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia.

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.

Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological control against Phoma medicaginis infection.

Among nine chitinase-producing strains isolated from Tunisian soil, one isolate called S213 exhibited a potent chitinolytic activity. S213 strain was identified as Bacillus licheniformis by API 50CH system and sequence analysis of its partial 16S ribosomal DNA. Chitinolytic activity was induced either by colloidal chitin or fungal cell walls, and the highest chitinase activity reached at the late stationary phase exhibiting optimal temperature and pH of 50-60 °C and pH 6.0, respectively. SDS-PAGE analysis of the secreted colloidal chitin-induced proteins showed a major protein of about 65 kDa. This protein was identified as chitinase on the basis of its peptide sequences which displayed high homology with chitinase sequence of B. licheniformis ATCC 14580. Moreover, chitinolytic activity containing supernatant inhibited the growth of several phytopathogenic fungi including Phoma medicaginis. Interestingly, S213 strain reduced efficiently the damping-off disease caused by P. medicaginis in Medicago truncatula and should be envisaged in enzyme-based biopesticides against phytopathogen application.

Rhizobium azibense sp. nov., a nitrogen fixing bacterium isolated from root-nodules of Phaseolus vulgaris.

Three microbial strains isolated from common beans, 23C2T (Tunisia), Gr42 (Spain) and IE4868 (Mexico), which have been identified previously as representing a genomic group closely related to Rhizobium gallicum, are further studied here. Their 16S rRNA genes showed 98.5-99% similarity with Rhizobium loessense CCBAU 7190BT, R. gallicum R602spT, Rhizobium mongolense USDA 1844T and Rhizobium yanglingense CCBAU 71623T. Phylogenetic analysis based on recA, atpD, dnaK and thrC sequences showed that the novel strains were closely related and could be distinguished from the four type strains of the closely related species. Strains 23C2T, Gr42 and IE4868 could be also differentiated from their closest phylogenetic neighbours by their phenotypic and physiological properties and their fatty acid contents. All three strains harboured symbiotic genes specific to biovar gallicum. Levels of DNA-DNA relatedness between strain 23C2T and the type strains of R. loessense, R. mongolense, R. gallicum and R. yanglingense ranged from 58.1 to 61.5%. The DNA G+C content of the genomic DNA of strain 23C2T was 59.52%. On the basis of these data, strains 23C2T, Gr42 and IE4868 were considered to represent a novel species of the genus Rhizobium for which the name Rhizobium azibense is proposed. Strain 23C2T (=CCBAU 101087T=HAMBI3541T) was designated as the type strain.

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field.

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.

Diversity of nodule-endophytic agrobacteria-like strains associated with different grain legumes in Tunisia.

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella.

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia.

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.

Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti.

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR-RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR-RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N(2)-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.

Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum.

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.