Search Results (22)

Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI₃ produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI₃ produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI₃ had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI₃ were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection. Full article

Herbicide-tolerant soybeans are the most extensively cultivated genetically modified (GM) crop globally. The effects of GM soybean and associated agronomic practices on soil microbial communities remain poorly understood. This study aimed to investigate the impact of planting GM soybeans with a glyphosate application on soil microbial diversity. The main bacterial and fungal community compositions (phylum level) were consistent for GM and non-GM soybeans. The alpha diversity analysis indicated that the bacterial Shannon index was significantly higher in GM rhizosphere soil during flowering compared to non-GM soil. There were no significant differences in the Shannon, Simpson, or ACE indices of the soil fungal communities between GM and non-GM soybeans in the same period. The PCoA analysis showed no significant differences in community structure between the GM and non-GM soybean soil for either fungi or bacteria during the same period. Although the relative abundance of Bradyrhizobium at the seedling stage was significantly lower in those GM than in those non-GM, it did not affect the final number of root nodules in either soybean type. The relative abundance of Frankia was significantly lower in GM rhizosphere soil during the seedling and flowering stages, whereas that of Thelebolus was significantly higher during flowering and pod filling. The abundance and ecological functions of these taxa warrant continuous monitoring. Full article

Planting nitrogen-fixing plants in post-mining sites and similar degraded areas is a common approach to speed up soil development and buildup of the nitrogen pool in soil organic matter. The aim of this study was to explore if slower growth of alder seedlings in initial post-mining sites results from adverse soil conditions or lack of microbial symbionts. To address this question, we sampled young soil (age 15 years) and more developed soil (age 70 years) from heaps after coal mining near Sokolov (Czech Republic). Soil samples were sterilized and not inoculated or inoculated with arbuscular mycorrhizal fungi (AMF) or AMF + Frankia, followed by planting with alder (Alnus glutinosa) seedlings germinated and precultured under sterile conditions. The effect of soil age on alder growth appeared to be non-significant. The only significant growth effect was seen with Frankia inoculation, implicating this inoculum as a key factor in later succession in post-mining soils. When the soil was fully inoculated, alder biomass was higher in developed soil supplied with iron (Fe) and phosphorus (P), indicating that iron and phosphorus availability may affect alder growth. In young soil, alder growth was highest with a combination of iron, phosphorus, and sulfur (S), and a positive effect of sulfur in young soil may correspond with a reduced, alkaline soil pH and increased phosphorus and iron availability. Full article

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO₂ levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO₂ levels in N₂-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH₄⁺. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles’ pH to prevent the escape of NH₃ and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages. Full article

Nitrogen is an essential element needed for plants to survive, and legumes are well known to recruit rhizobia to fix atmospheric nitrogen. In this widely studied symbiosis, legumes develop specific structures on the roots to host specific symbionts. This review explores alternate nodule structures and their functions outside of the more widely studied legume–rhizobial symbiosis, as well as discussing other unusual aspects of nodulation. This includes actinorhizal-Frankia, cycad-cyanobacteria, and the non-legume Parasponia andersonii-rhizobia symbioses. Nodules are also not restricted to the roots, either, with examples found within stems and leaves. Recent research has shown that legume–rhizobia nodulation brings a great many other benefits, some direct and some indirect. Rhizobial symbiosis can lead to modifications in other pathways, including the priming of defence responses, and to modulated or enhanced resistance to biotic and abiotic stress. With so many avenues to explore, this review discusses recent discoveries and highlights future directions in the study of nodulation. Full article

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for “Alnus-infective strains” (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within “Alnus-infective strains”, Sp+ Frankia genomes were compared to Sp− genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp− strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients). Full article

As the oldest species of Betulaceae, Nepalese alder (Alnus nepalensis) shows a high capacity for nitrogen fixation, rapid growth rate, and strong adaptability to stress environments, and it plays an important role in maintaining the structure and function of forest and agroforestry ecosystems. We explored its geographic distribution and the corresponding environmental drivers through collecting specimen records and published literature for Nepalese alder over the world during the past 40 years. The research trends, the growth limiting factors, the physiological characteristics, and ecological functions were all summarized as well. In terms of geographical distribution and limiting factors, Nepalese alder is mainly distributed in southern mountainous areas of the Himalayas and southwest China. Since it presented a clear northern limit of distribution and an upper limit of elevation, temperature is assumed to be the main environmental limiting factor. According to historical development, the research history of Nepalese alder could be divided into three main periods: the initial development (before 2001), the fast development (2002–2015), and the high-quality development (2016–2022), with the two key points in 2002 and 2015 relating to the conversion of cropland to a forest project that the government conducted and the application from theory to practice, respectively. As can be seen from the ecological functions, Nepalese alder could form symbiotic nodules with Frankia, which plays an important role in improving soil physical and chemical properties and facilitating vegetation secondary succession. Overall, the present review provides a reference for further studies on ecological adaptability and sustainable utilization of Nepalese alder under climate change, and also for regional ecosystem service, forestry production practice, and vegetation restoration. Full article

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N₂-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO₃⁺) or nodulated by Frankia (NOD⁺) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD⁺) to 314 (600 mM NaCl/KNO₃⁺), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species. Full article

The mining of the oil sands region of Canada’s boreal forest creates disturbed land with elevated levels of salts. Understanding how native plants respond to salt stress is critical in reclaiming these lands. The native species, Alnus alnobetula subsp. crispa forms nitrogen-fixing nodules with Frankia, and ectomycorrhizae with a number of fungal species. These relationships may make the plant particularly well suited for restoring disturbed land. We inoculated A. alnobetula subsp. crispa with Frankia and Hebeloma crustiliniforme and exposed the plants to 0, 50, or 100 mM NaCl for seven weeks. Frankia-inoculated plants had increased biomass regardless of salt exposure, even though salt exposure reduced nitrogen fixation and reduced the efficiency of nitrogen-fixing nodules. The nitrogen-fixing symbiosis also decreased leaf stress and increased root phosphatase levels. This suggests that N-fixing plants not only have increased nitrogen nutrition but also have increased access to soil phosphorus. Mycorrhizae did not affect plant growth but did reduce nodule numbers and nodule efficiency. These results suggest that the nitrogen-fixing trait is more critical than mycorrhizae. While salt stress inhibits nitrogen-fixing symbiosis, plants still benefit from nitrogen fixation when exposed to salt. Full article

The Korean fir (Abies koreana), a native coniferous tree species mainly found on Mt. Halla in Jeju, South Korea, is suffering from continuous population decline and has been declared an endangered species. Research efforts have focused on the possible abiotic causes behind this worrying decline. However, the potential link between tree vitality and the rhizosphere microbiome remains unclear. In this study, a comparative metagenomic 16S rRNA sequence analysis was used to investigate the composition of the rhizosphere microbiota of samples collected from healthy and die-back-affected trees on Mt. Halla. The results indicated a significant reduction in the richness and diversity of microbiota in the rhizosphere of die-back-affected trees. Moreover, the relative abundance of Proteobacteria, Actinobacteria, and Bacteroidetes were significantly higher in healthy trees than in standing dead trees. Many bacterial genera were significantly more abundant in the rhizosphere of healthy trees, including those known for promoting plant growth and tolerance to biotic and abiotic stresses (e.g., Bradyrhizobium, Rhizomicrobium, Caulobacter, Nitrosospira, Rhizobacter, Paraburkholderia, Rhizobium, Devosia, Caballeronia, Niveispirillum, Dyella, Herbaspirillum, Frankia, Streptomyces, Actinoallomurus, Lysobacter, Luteibacter, Mucilaginibacter, and Variovorax). To our knowledge, this is the first report on rhizosphere bacterial microbiome dysbiosis in die-back-affected Korean fir trees, suggesting that the influence of rhizosphere microbiota should be considered to save this endangered species by investigating possible intervention strategies in future work. Full article

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N-replete pure cultures with propionate as the carbon source and ammonium as the nitrogen-source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-replete pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis. Full article

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper. Full article

Many studies have found that future predicted CO₂ levels can increase plant mass but dilute N content in leaves, impacting antiherbivore compounds. Nitrogen-fixing plants may balance their leaf C:N ratio under elevated CO₂, counteracting this dilution effect. However, we know little of how plants respond to herbivores at the higher CO₂ levels that occurred when nitrogen-fixing plants first evolved. We grew Alnus incana ssp. rugosa was grown at 400, 800, or 1600 ppm CO₂ in soil collected from the field, inoculated with Frankia and exposed to herbivores (Orgyia leucostigma). Elevated CO₂ increased nodulated plant biomass and stimulated the nitrogen fixation rate in the early growth stage. However, nitrogen-fixing plants were not able to balance their C:N ratio under elevated CO₂ after growing for 19 weeks. When plants were grown at 400 and 1600 ppm CO_2, herbivores preferred to feed on leaves of nodulated plants. At 800 ppm CO₂, nodulated plants accumulated more total phenolic compounds in response to herbivore damage than plants in the non-Frankia and non-herbivore treatments. Our results suggest that plant leaf defence, not leaf nutritional content, is the dominant driver of herbivory and nitrogen-fixing plants have limited ability to balance C:N ratios at elevated CO₂ in natural soil. Full article

Land salinization is a major constraint for the practice of agriculture in the world. Considering the extent of this phenomenon, the rehabilitation of ecosystems degraded by salinization has become a priority to guarantee food security in semi-arid environments. The mechanical and chemical approaches for rehabilitating salt-affected soils being expensive, an alternative approach is to develop and utilize biological systems utilizing salt-tolerant plant species. Casuarina species are naturally halotolerant, but this tolerance has been shown to be improved when they are inoculated with arbuscular mycorrhizal fungi (AMF) and/or nitrogen-fixing bacteria (Frankia). Furthermore, Casuarina plantations have been proposed to promote the development of plant diversity. Thus, the aim of the current study was to evaluate the impact of a plantation comprising the species Casuarina inoculated with AMF and Frankia on the diversity of the sub-canopy and adjacent vegetation. Work was conducted on a plantation comprising Casurina equisetifolia and C. glauca variously inoculated with Frankia and Rhizophagus fasciculatus prior to field planting. The experimental area of 2500 m² was divided into randomized blocks and vegetation sampling was conducted below and outside of the Casuarina canopy in 32 m² plots. A total of 48 samples were taken annually over 3 years, with 24 taken from below the Casuarina canopy and 24 from outside the canopy. The results obtained show that co-inoculation with Frankia and Rhizophagus fasciculatus improves the height and survival rate of both species. After 4–5 years, there was greater species diversity and plant biomass in the sub-canopy environment compared with that of the adjacent environments. Our results suggest that inoculation of beneficial microbes can improve growth of Casuarina species and that planting of such species can improve the diversity of herbaceous vegetation in saline environments. Full article