Project description:Symbiotic nitrogen-fixing associations between Casuarina trees and the actinobacteria Frankia are widely used in agroforestry in particular for salinized land reclamation. The aim of this study was to analyze the effects of salinity on the establishment of the actinorhizal symbiosis between C. glauca and two contrasting Frankia strains (salt sensitive; CcI3 vs. salt tolerant; CeD) and the role of these isolates in the salt tolerance of C. glauca and C. equisetifolia plants. We show that the number of root nodules decreased with increasing salinity levels in both plants inoculated with CcI3 and CeD. Nodule formation did not occur in seedlings inoculated with CcI3 and CeD, at NaCl concentrations above 100 and 200 mM, respectively. Salinity also affected the early deformation of plant root hairs and reduced their number and size. In addition, expression of symbiotic marker Cg12 gene, which codes for a subtilase, was reduced at 50 mM NaCl. These data suggest that the reduction of nodulation in C. glauca under salt stress is in part due to inhibition of early mechanisms of infection. We also show that prior inoculation of C. glauca and C. equisetifolia with Frankia strains CcI3 and CeD significantly improved plant height, dry biomass, chlorophyll and proline contents at all levels of salinity tested, depending on the Casuarina-Frankia association. There was no correlation between in vitro salt tolerance of Frankia strains and efficiency in planta under salt-stressed conditions. Our results strongly indicate that increased N nutrition, photosynthesis potential and proline accumulation are important factors responsible for salt tolerance of nodulated C. glauca and C. equisetifolia.
Project description:Casuarina equisetifolia is an important tree of the forest, cultivated in tropical and subtropical regions, providing fuelwood, land reclamation, dune stabilization, paper production, and nitrogen fixation. We have developed a systematic in vitro propagation protocol in C. equisetifolia using nodal segments (NS). Murashige and Skoog (MS) medium augmented with BA (5.0 μM) and NAA (0.5 μM) gave rise to a maximum of 32.00 ± 0.31 shoots per explant (S/E) with shoot length (SL) of 3.94 ± 0.02 cm, and a maximum of 70% regeneration potential (RP) was recorded after 8 weeks of post inoculation. For root induction, in vitro derived shoots were transferred to the nutrient medium consisting of a half-strength (½) MS medium augmented with 2.5 μM NAA, which produced a maximum of 12.68 ± 0.33 roots/shoot (R/S) with 3.04 ± 0.50 cm root length (RL) in 60% of culture after 6 weeks. Micropropagated plants with healthy shoots and roots were successfully acclimatized in vermicompost + garden soil + sand (1:2:1) and a maximum survival percentage of 95.1% was recorded. NS was taken from a 6-weeks-old in vitro derived plant of C. equisetifolia for synthetic seed production, and it was reported that CaCl2 · 2H2O (100 mM) + Na2-alginate (4%) resulted in clear and uniform beads. Furthermore, the maximum conversion of synthetic seeds into plantlets occurred over a period of 4 weeks of storage at 4°C. Scanning Electron Microscopy (SEM) revealed the formation of direct shoot buds without any intermediate callus formation. In addition, the chlorophyll and carotenoid contents of the direct regenerated and mother plant were compared. Similarly, RAPD and ISSR primers were used for genetic homogeneity assessment of the direct regenerated plants, where a total of 18 and 19, respectively, clear and reproducible bands with 100% monomorphism were recorded. The developed micropropagation protocol can certainly be used for large-scale multiplication and germplasm preservation of C. equisetifolia. It will also help in meeting the growing demands of C. equisetifolia in the forest industry.
Project description:Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from (15)N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.
Project description:Ganoderma is a cosmopolitan white rot fungal genus, famous for its medicinal properties. In the present study, two new Ganoderma species were collected from south-eastern China and described on the basis of morphological characters and phylogenetic analyses of sequences of the internal transcribed spacer (ITS) region, the translation elongation factor 1-? gene (EF1-?) and the second subunit of RNA polymerase II (RPB2). Specimens of both species were found on living trees of Casuarina equisetifolia. Ganoderma angustisporumsp. nov. is characterised by its sessile basidiomata and almond-shaped, slightly truncate, narrow basidiospores (9-11.3 × 4-5.2 µm). Ganoderma casuarinicolasp. nov. is characterised by its strongly laccate reddish-brown pileal surface, luminous yellow to yellowish-brown cutis and ellipsoid, truncate basidiospores (9-10.2 × 5-6 µm). The two new species are compared with their related taxa. Phylogenetic analyses confirmed that G. angustisporum and G. casuarinicola are distinct species within Ganoderma.