Endophytic bacteria with allelopathic potential regulate gene expression and metabolite production in host Casuarina equisetifolia.

Introduction: Casuarina equisetifolia is a common protective forest in coastal areas. However, artificial C. equisetifolia forests cannot self-renew, mainly due to the accumulation of allelochemicals. Endophytic bacteria may alleviate the root growth inhibition caused by allelochemicals in C. equisetifolia seedlings. B. amyloliquefaciens and B. aryabhattai were endophytic bacteria with strong allelopathy in C. equisetifolia root. The allelopathy mechanism of these two endophytes and their interaction with C. equisetifolia remains to be studied. Methods: Whole-genome sequencing of B. amyloliquefaciens and B. aryabhattai isolated from the roots of allelochemical-accumulating C. equisetifolia was performed using Illumina Hiseq and PacBio single-molecule sequencing platforms. Sterile seedlings of C. equisetifolia were treated with either individual or mixed bacterial cultures through root drenching. Transcriptional and metabolomics analyses were conducted after 3 days of infection. Results and discussion: Whole-genome sequencing of Bacillus aryabhattai and Bacillus amyloliquefaciens showed that the two strains contained various horizontal gene transfer elements such as insertion sequence, prophage and transposon. In addition, these two strains also contain numerous genes related to the synthesis and catabolism of allelochemicals. After these two strains of bacteria were individually or mixed infected with C. equisetifolia, metabolomics and transcriptomic analysis of C. equisetifolia showed the 11 important secondary metabolite biosynthesis among them alkaloids biosynthesis, phenylpropanoid and terpenes biosynthesis and related genes were putatively regulated. Correlation analysis revealed that 48 differentially expressed genes had strong positive correlations with 42 differential metabolites, and 48 differentially expressed genes had strong negative correlations with 36 differential metabolites. For example, CMBL gene showed positive correlations with the allelochemical (-)-Catechin gallate, while Bp10 gene showed negative correlations with (-)-Catechin gallate. Conclusion: The intergenerational accumulation of allelochemicals may induce horizontal gene transfer in endogenic bacteria of Casuarina equisetifolia root. Endophytic Bacillus plays an allelopathic role by assisting the host in regulating gene expression and the production and/or variety of allelochemicals. This comprehensive study sheds light on the intricate genetic and metabolic interactions between Bacillus endophytes and C. equisetifolia. These findings provide insights into endophyte-mediated allelopathy and its potential uses in plant biology and forest sustainability.

Multi-omics analyses reveal the mechanisms underlying the responses of Casuarina equisetifolia ssp. incana to seawater atomization and encroachment stress.

Casuarina equisetifolia trees are used as windbreaks in subtropical and tropical coastal zones, while C. equisetifolia windbreak forests can be degraded by seawater atomization (SA) and seawater encroachment (SE). To investigate the mechanisms underlying the response of C. equisetifolia to SA and SE stress, the transcriptome and metabolome of C. equisetifolia seedlings treated with control, SA, and SE treatments were analyzed. We identified 737, 3232, 3138, and 3899 differentially expressed genes (SA and SE for 2 and 24 h), and 46, 66, 62, and 65 differentially accumulated metabolites (SA and SE for 12 and 24 h). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SA and SE stress significantly altered the expression of genes related to plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism pathways. The accumulation of metabolites associated with the biosynthetic pathways of phenylpropanoid and amino acids, as well as starch and sucrose metabolism, and glycolysis/gluconeogenesis were significantly altered in C. equisetifolia subjected to SA and SE stress. In conclusion, C. equisetifolia responds to SA and SE stress by regulating plant hormone signal transduction, plant-pathogen interaction, biosynthesis of phenylpropanoid and amino acids, starch and sucrose metabolism, and glycolysis/gluconeogenesis pathways. Compared with SA stress, C. equisetifolia had a stronger perception and response to SE stress, which required more genes and metabolites to be regulated. This study enhances our understandings of how C. equisetifolia responds to two types of seawater stresses at transcriptional and metabolic levels. It also offers a theoretical framework for effective coastal vegetation management in tropical and subtropical regions.

Whole-Genome Sequencing of Two Potentially Allelopathic Strains of Bacillus from the Roots of C. equisetifolia and Identification of Genes Related to Synthesis of Secondary Metabolites.

The coastal Casuarina equisetifolia is the most common tree species in Hainan's coastal protection forests. Sequencing the genomes of its allelopathic endophytes can allow the protective effects of these bacteria to be effectively implemented in protected forests. The goal of this study was to sequence the whole genomes of the endophytes Bacillus amyloliquefaciens and Bacillus aryabhattai isolated from C. equisetifolia root tissues. The results showed that the genome sizes of B. amyloliquefaciens and B. aryabhattai were 3.854 Mb and 5.508 Mb, respectively. The two strains shared 2514 common gene families while having 1055 and 2406 distinct gene families, respectively. The two strains had 283 and 298 allelochemical synthesis-associated genes, respectively, 255 of which were shared by both strains and 28 and 43 of which were unique to each strain, respectively. The genes were putatively involved in 11 functional pathways, including secondary metabolite biosynthesis, terpene carbon skeleton biosynthesis, biosynthesis of ubiquinone and other terpene quinones, tropane/piperidine and piperidine alkaloids biosynthesis, and phenylpropanoid biosynthesis. NQO1 and entC are known to be involved in the biosynthesis of ubiquinone and other terpenoid quinones, and rfbC/rmlC, rfbA/rmlA/rffH, and rfbB/rmlB/rffG are involved in the biosynthesis of polyketide glycan units. Among the B. aryabhattai-specific allelochemical synthesis-related genes, STE24 is involved in terpene carbon skeleton production, atzF and gdhA in arginine biosynthesis, and TYR in isoquinoline alkaloid biosynthesis. B. amyloliquefaciens and B. aryabhattai share the genes aspB, yhdR, trpA, trpB, and GGPS, which are known to be involved in the synthesis of carotenoids, indole, momilactones, and other allelochemicals. Additionally, these bacteria are involved in allelochemical synthesis via routes such as polyketide sugar unit biosynthesis and isoquinoline alkaloid biosynthesis. This study sheds light on the genetic basis of allelopathy in Bacillus strains associated with C. equisetifolia, highlighting the possible use of these bacteria in sustainable agricultural strategies for weed management and crop protection.

Genome-wide identification of WRKY transcription factors in Casuarina equisetifolia and the function analysis of CeqWRKY11 in response to NaCl/NaHCO3 stresses.

BACKGROUND: Casuarina equisetifolia (C. equisetifolia) is a woody species with many excellent features. It has natural resistance against drought, salt and saline-alkali stresses. WRKY transcription factors (TFs) play significant roles in plant response to abiotic stresses, therefore, molecular characterization of WRKY gene family under abiotic stresses holds great significance for improvement of forest trees through molecular biological tools. At present, WRKY TFs from C. equisetifolia have not been thoroughly studied with respect to their role in salt and saline-alkali stresses response. The current study was conducted to bridge the same knowledge gap. RESULTS: A total of 64 WRKYs were identified in C. equisetifolia and divided into three major groups i.e. group I, II and III, consisting of 10, 42 and 12 WRKY members, respectively. The WRKY members in group II were further divided into 5 subgroups according to their homology with Arabidopsis counterparts. WRKYs belonging to the same group exhibited higher similarities in gene structure and the presence of conserved motifs. Promoter analysis data showed the presence of various response elements, especially those related to hormone signaling and abiotic stresses, such as ABRE (ABA), TGACG (MeJA), W-box ((C/T) TGAC (T/C)) and TC-rich motif. Tissue specific expression data showed that CeqWRKYs were mainly expressed in root under normal growth conditions. Furthermore, most of the CeqWRKYs were up-regulated by NaCl and NaHCO3 stresses with few of WRKYs showing early responsiveness to both stresses while few others exhibiting late response. Although the expressions of CeqWRKYs were also induced by cold stress, the response was delayed compared with other stresses. Transgenic C. equisetifolia plants overexpressing CeqWRKY11 displayed lower electrolyte leakage, higher chlorophyll content, and enhanced tolerance to both stresses. The higher expression of abiotic stress related genes, especially CeqHKT1 and CeqPOD7, in overexpression lines points to the maintenance of optimum Na+/K+ ratio, and ROS scavenging as possible key molecular mechanisms underlying salt stress tolerance. CONCLUSIONS: Our results show that CeqWRKYs might be key regulators of NaCl and NaHCO3 stresses response in C. equisetifolia. In addition, positive correlation of CeqWRKY11 expression with increased stress tolerance in C. equisetifolia encourages further research on other WRKY family members through functional genomic tools. The best candidates could be incorporated in other woody plant species for improving stress tolerance.

Pathogenic and Comparative Genomic Analysis of Ralstonia pseudosolanacearum Isolated from Casuarina.

Casuarina equisetifolia is crucial in protecting coastal regions of China against typhoon attacks but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in the 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene-based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than the representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations that possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharide biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.

Effects of continuous monoculture on rhizosphere soil nutrients, growth, physiological characteristics, hormone metabolome of Casuarina equisetifolia and their interaction analysis.

Continuous planting is unavoidable in agricultural production, but continuous planting affects plant growth and physiological characteristics. In this study, we analyzed rhizosphere soil nutrients, physiological characteristics, hormone metabolome changes and their interactions of Casuarina equisetifolia (C. equisetifolia) with the increase of continuous planting number. The results found that C. equisetifolia root was significantly inhibited, the plant height was dwarfed and the biomass was significantly reduced as continuous planting number increased. Secondly, continuous planting caused a decrease in the rhizosphere soil nutrient transformation capacity, and a significant decrease in the total soil nutrient and available nutrient content. Analysis of physiological indexes showed that continuous planting resulted in a decrease in nitrogen, phosphorus, and potassium content, a decrease in the activity of physiological indexes of resistance, and a decrease in photosynthetic capacity of C. equisetifolia leaves. Hormone metabolome analysis showed that continuous planting critically affected the accumulation of five characteristic hormones in C. equisetifolia leaves, in which salicylic acid 2-O-ß-glucoside (SAG), 2-oxindole-3-acetic acid (OxIAA), trans-zeatin-O-glucoside (tZOG) and gibberellin A3 (GA3) content decreased significantly while abscisic acid (ABA) content increased significantly. In conclusion, continuous planting lowered the rhizosphere soil nutrient transformation capacity of C. equisetifolia, lowered the soil available nutrient content, inhibited their root growth, and hindered the nutrient uptake and transportation by the root, thus led to the decrease of the nutrient accumulation capacity in the leaves of C. equisetifolia, and the decrease of SAG, OxIAA, and tZOG, GA3 synthesis ability decreased, ABA accumulated in large quantities, C. equisetifolia resistance and photosynthesis ability decreased, and their growth was impeded. This study provides insights for the effective management of continuous planting in the cultivation of C. equisetifolia.

The hepato- and neuroprotective effect of gold Casuarina equisetifolia bark nano-extract against Chlorpyrifos-induced toxicity in rats.

BACKGROUND: The bark of Casuarina equisetifolia contains several active phytoconstituents that are suitable for the biosynthesis of gold nanoparticles (Au-NPs). These nanoparticles were subsequently evaluated for their effectiveness in reducing the toxicity induced by Chlorpyrifos (CPF) in rats. RESULTS: Various hematological and biochemical measurements were conducted in this study. In addition, markers of oxidative stress and inflammatory reactions quantified in liver and brain tissues were evaluated. Histopathological examinations were performed on both liver and brain tissues. Furthermore, the native electrophoretic protein and isoenzyme patterns were analyzed, and the relative expression levels of apoptotic genes in these tissues were determined. The hematological and biochemical parameters were found to be severely altered in the group injected with CPF. However, the administration of Au-C. equisetifolia nano-extract normalized these levels in all treated groups. The antioxidant system markers showed a significant decrease (P ≤ 0.05) in conjunction with elevated levels of inflammatory and fibrotic markers in both liver and brain tissues of the CPF-injected group. In comparison, the pre-treated group exhibited a reduction in these markers when treated with the nano-extract, as opposed to the CPF-injected group. Additionally, the nano-extract mitigated the severity of histopathological lesions induced by CPF in both liver and brain tissues, with a higher ameliorative effect observed in the pre-treated group. Electrophoretic assays conducted on liver and brain tissues revealed that the nano-extract prevented the qualitative changes induced by CPF in the pre-treated group. Furthermore, the molecular assay demonstrated a significant increase in the relative expression of apoptotic genes in the CPF-injected rats. Although the nano-extract ameliorated the relative expression of these genes compared to the CPF-injected group, it was unable to restore their values to normal levels. CONCLUSION: Our results demonstrated that the nano-extract effectively reduced the toxicity induced by CPF in rats at hematological, biochemical, histopathological, physiological, and molecular levels, in the group pre-treated with the nano-extract.

Effect of continuous planting on Casuarina equisetifolia rhizosphere soil physicochemical indexes, microbial functional diversity and metabolites.

Continuous planting has a severe impact on the growth of Casuarina equisetifolia. In this study, the effects of three different long-term monocultures (one, two and three replanting) on the physicochemical indexes, microbial functional diversity, and soil metabolomics were analyzed in C. equisetifolia rhizosphere soil. The results showed that rhizosphere soil organic matter content, cation exchange capacity, total and available nitrogen, total and available phosphorus, and total and available potassium contents significantly decreased with the increasing number of continuous plantings. The evaluation of microbial functional diversity revealed a reduction in the number of soil microorganisms that rely on carbohydrates for carbon sources and an increase in soil microorganisms that used phenolic acid, carboxylic acid, fatty acid, and amines as carbon sources. Soil metabolomics analysis showed a significant decrease in soil carbohydrate content and a significant accumulation of autotoxic acid, amine, and lipid in the C. equisetifolia rhizosphere soil. Consequently, the growth of C. equisetifolia could hinder total nutrient content and their availability. Thus, valuable insights for managing the cultivation of C. equisetifolia and soil remediation were provided.

Exogenously applied Casuarina equisetifolia leaf extracts act as an osmoprotectant on proline accumulation under drought stress in local rice from Indonesia.

The effects of exogenously supplied osmoprotectants in crops have not yet been extensively studied. In this study, an osmoprotectant containing a high concentration of proline (2.5 g mol-1 FW) was obtained from a Casuarina equisetifolia leaf extract. The effect of the extract was evaluated in local Indonesian rice cultivars Boawae Seratus Malam (BSM), Gogo Jak (GJ), Situ Bagendit (SB) (drought-tolerant), Kisol Manggarai (KM) and Ciherang (drought-susceptible) cultivars under drought at the morphological, physiological, and genetic levels. Under drought, the KM showed an increased level of OsWRKY, OsNAC, OsDREB1A, and OsDREB2A expression after application of the osmoprotectant, leading to the activation of proline synthesis genes including OsP5CS1, OsP5CR, and OsProDH, while the tolerant cultivars (BSM, GJ, and SB) showed no difference. The content of chlorophyll, carotenoids, anthocyanins, ascorbate peroxidase, catalase, and superoxide dismutase activities also increased in GJ and KM, during drought stress and applied osmoprotectants, but remained low in the BSM. We conclude that the foliar application of osmoprotectants derived from C.equisetifolia caused an accumulation of proline in susceptible plants. The existence of these extracts stabilizes leaf cells and supports photosynthetic compartments and carbon assimilation in plants, leading to growth.

Transcriptome and metabolome analysis reveals key genes and secondary metabolites of Casuarina equisetifolia ssp. incana in response to drought stress.

Casuarina equisetifolia is drought tolerant, salt tolerant, and able to grow in barren environments. It is often used to reduce wind damage, to prevent sand erosion, and to help establish plant communities in tropical and subtropical coastal zones. To determine the basis for its drought tolerance, we conducted transcriptomic and metabolic analyses of young branchlets under a non-drought treatment (D_0h) and 2-, 12-, and 24-h-long drought treatments (D_2h, D_12h, and D_24h). A total of 5033 and 8159 differentially expressed genes (DEGs) were identified in D_2h/D_0h and D_24h/D_0h. These DEGs were involved in plant hormone signal transduction, jasmonic acid (JA) biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis. A total of 148 and 168 differentially accumulated metabolites (DAMs) were identified in D_12h/D_0h and D_24h/D_0h, which were mainly amino acids, phenolic acids, and flavonoids. In conclusion, C. equisetifolia responds to drought by regulating plant hormone signal transduction and the biosynthesis of JA, flavonoid, and phenylpropanoid. These results increase the understanding of drought tolerance in C. equisetifolia at both transcriptional and metabolic levels and provide new insights into coastal vegetation reconstruction and management.

Is allelochemical synthesis in Casuarina equisetifolia plantation related to litter microorganisms?

Productivity decline of Casuarina equisetifolia plantation and difficulty in natural regeneration remains a serious problem because of allelopathy. Previous studies have confirmed that 2,4-di-tert-butylphenol (2,4-DTBP) are the major allelochemicals of the C. equisetifolia litter exudates. The production of these allelochemicals may derive from decomposition of litter or from the litter endophyte and microorganisms adhering to litter surfaces. In the present study, we aimed to evaluate the correlation between allelochemicals in litter and endophytic and epiphytic fungi and bacteria from litter. A total of 100 fungi and 116 bacteria were isolated from the interior and surface of litter of different forest ages (young, half-mature, and mature plantation). Results showed that the fermentation broth of fungal genera Mycosphaerella sp. and Pestalotiopsis sp., and bacterial genera Bacillus amyloliquefaciens, Burkholderia-Paraburkholderia, and Pantoea ananatis had the strongest allelopathic effect on C. equisetifolia seeds. Allelochemicals, such as 2,4-DTBP and its analogs were identified in the fermentation broths of these microorganisms using GC/MS analysis. These results indicate that endophytic and epiphytic fungi and bacteria in litters are involved in the synthesis of allelochemicals of C. equisetifolia. To further determine the abundance of the allelopathic fungi and bacteria, Illumina MiSeq high-throughput sequencing was performed. The results showed that bacterial genera with strong allelopathic potential were mainly distributed in the young and half-mature plantation with low abundance, while the abundance of fungal genera Mycosphaerella sp. and Pestalotiopsis sp. were higher in the young and mature plantations. In particular, the abundance of Mycosphaerella sp. in the young and mature plantations were 501.20% and 192.63% higher than in the half-mature plantation, respectively. Overall, our study demonstrates that the litter fungi with higher abundance in the young and mature plantation were involved in the synthesis of the allelochemical 2,4-DTBP of C. equisetifolia. This finding may be important for understanding the relationship between autotoxicity and microorganism and clarifying the natural regeneration problem of C. equisetifolia.

Ecological niche selection shapes the assembly and diversity of microbial communities in Casuarina equisetifolia L.

The plant microbiome profoundly affects many aspects of host performance; however, the ecological processes by which plant hosts govern microbiome assembly, function, and dispersal remain largely unknown. Here, we investigated the bacterial and fungal communities in multiple compartment niches (bulk soil, rhizosphere soil, root endosphere, phylloplane, and leaf endosphere) of Casuarina equisetifolia L. at three developmental stages in Hainan Province, China. We found that microbiome assemblages along the soil-plant continuum were shaped by the compartment niches. Bacterial diversity and richness decreased from the soils to roots to leaves, with the highest network complexity found in the roots and the lowest found in the phylloplane. However, fungal diversity gradually increased from the soils to roots to phyllosphere, whereas fungal richness decreased from the soils to roots but increased from the roots to phyllosphere; the greatest network complexity was found in bulk soils and the lowest was found in the roots. Different biomarker taxa occurred in the different ecological niches. Bacterial and fungal communities exhibited distinct ecological functions; the former played important roles in maintaining plant growth and providing nutrients, whereas the latter predominantly decomposed organic matter. The bacterial community of C. equisetifolia mostly originated from bulk soil, whereas the fungal community was mainly derived from rhizosphere soil and air. Leaf endophytes were positively correlated with organic carbon, and root and soil microorganisms were positively correlated with total nitrogen, total phosphorus, and total potassium. Our findings provide empirical evidence for plant-microbiome interactions and contribute to future research on non-crop management and the manipulation of non-crop microbiomes.

Metabolite Profiling and Biological Activity Assessment of Casuarina equisetifolia Bark after Incorporating Gold Nanoparticles.

OBJECTIVE: Casuarina equisetifolia bark is rich in various active metabolites and selected to be studied due to limitation of the synthetic antioxidants that have adverse side effects. The present study aimed to enhance efficiency of the most effective extract by incorporating gold nanoparticles (Au-NPs). METHODS: The phytochemical and biological measurements were carried out in total methanolic extract and its successive fractions. Moreover, these measurements were assayed in the most effective extract after incorporating Au-NPs. RESULTS: The study revealed that total methanolic extract exhibited the highest biological and cytotoxic activities as compared to other fractions. Therefore, it is considered as good candidate for nano-extract preparation. The methanolic extract incorporated with Au-NPs showed higher antioxidant, scavenging and cytotoxic activities in addition to higher inhibitory effect against α-amylase activity as compared to native extract itself. To pinpoint active agents in total methanolic extract, the secondary metabolite profiling via HPLC-MS showed that 33 and 17 metabolites were annotated in the extract before and after incorporating Au-NPs, respectively. The median lethal dose (LD50) showed that gold total methanolic nano-extract is safer than total methanolic extract. CONCLUSION: This study concluded that total methanolic C. equisetifolia bark extract is a valuable bioresource to synthesize an eco-friendly Au-NPs with health-enhancing effect as antioxidant, antidiabetic and cytotoxic agents. The present study is considered as the first report on utilization of C. equisetifolia bark in synthesis of Au-NPs by mean of green nanotechnology and investigation of its biological activity in relation to its metabolite fingerprint.

Root exudates and chemotactic strains mediate bacterial community assembly in the rhizosphere soil of Casuarina equisetifolia L.

Rhizosphere bacterial diversity and community structure are important factors involving in plant growth. However, the exact process of how plant rhizosphere bacterial community structures is assembled remains unclear. To investigate the role of bacterial chemotaxis to rhizosphere secretions in the establishment of rhizosphere microbial community in Casuarina equisetifolia, we screened bacteria strains derived from the rhizosphere of Casuarina equisetifolia L. using top three chemicals of the plant root exudates (2,4-di-tert-butylphenol, methyl stearate, and arginine) as chemoattractant. Among 72 bacterial strains, five showed strong chemotaxis to 2,4-di-tert-butylphenol, six to methyl stearate, and eleven to arginine, with the highest bacterial chemotaxis occurring at a concentration of 60 µM. This indicates that arginine is a more important chemoattractant than 2,4-di-tert-butylphenol, methyl stearate in the establishment of rhizosphere microbial community in Casuarina equisetifolia. Bacterial community assembly analysis using different chemoattractants and chemoattractants-plus-bacteria combinations were then performed by burying laboratory prepared bags of sterlized soil into C. equisetifolia forest. Bacteria diversity and enrichment analyses using 16S rDNA sequencing at 7 and 14 days after burying showed that arginine-plus-Ochrobactrum sp. and Pantoea sp. treatment exhibited the greatest similarity to the natural forest bacterial community. Our date provides new insights into how chemoattractants and chemotactic bacteria strains shape the rhizosphere microbial community of C. equisetifolia, which constitutes foundational information for future management of these communities.

Micropropagation, encapsulation, physiological, and genetic homogeneity assessment in Casuarina equisetifolia.

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.

Characterization of HAK protein family in Casuarina equisetifolia and the positive regulatory role of CeqHAK6 and CeqHAK11 genes in response to salt tolerance.

The potassium transporter group of the HAK/KUP/KT (high-affinity K+)/KUP (K+ uptake)/KT (K+ transporter) family plays a crucial role in plant growth and development as well as in environmental adaptation such as tolerance to salt stress. HAK/KUP/KT genes and their functions have been characterized for a number of plant species, but they remain unknown for Casuarina equisetifolia, an important tree species for coastal protection in southern China and many other countries. In this study, 25 HAK genes were identified in the C. equisetifolia genome. Their gene structure, conserved motif, phylogeny, and expression were comprehensively and systematically analyzed to understand their functions. All HAK genes were relatively conserved and could be divided into four clusters. The expression level of two particular genes, CeqHAK11 and CeqHAK6, increased significantly with the duration of salt treatment. To further elucidated their function in response to salt stress, subcellular localization, and their functional analysis were developed. Results revealed that CeqHAK11 and CeqHAK6 were localized on the plasma membrane, which mainly mediated high-affinity K+ uptake. Overexpression of CeqHAK6 or CeqHAK11 in Arabidopsis showed higher germination and survival rates and longer root length than wild-type (WT) under salt stress, suggesting that both genes improve tolerance to salt stress. Moreover, CeqHAK6 and CeqHAK11 improved their ability to tolerate salt stress by increasing the K+/Na+ ratio and antioxidant enzyme activities (CAT, POD, and SOD), and decreasing reactive oxygen species (ROS) accumulation. Consequently, CeqHAK6 and CeqHAK11 were verified as potassium transport proteins and could be applied for further molecular breeding for salt tolerance in C. equisetifolia or other crops to increasing salt tolerance.

[Effects of exogenous microorganisms on seedling growth and soil microbial community of Casuarina equisetifolia]. / 外源微生物对木麻黄幼苗生长和土壤微生物群落的影响.

With a pot experiment, the Biolog microplate and phospholipid fatty acid (PLFA) technology were used to explore whether the application of bacteria, Bacillus amyloliquefaciens (YB706) and Burkholderia (BK8), could improve the soil nutrient, microbial community and growth of Casuarina equisetifolia. The results showed that the concentrations of soil alkali-hydrolyzed nitrogen and available phosphorus of C. equisetifolia treated with YB706 and BK8 increased significantly compared with the control (CK), but the concentrations of total nitrogen, total phosphorus, total potassium and available potassium changed little, plant height increased by 59.1% and 63.9%, respectively, and the chlorophyll content of plant treated with BK8 increased by 81.9%. The average well color development values showed a pattern of YB706>CK>BK8. The utilization rate of different carbon sources showed the same trend except the amino acids. Both YB706 and BK8 treatments significantly increased the richness and quantity of soil microorganisms. The PLFA of all kinds of microorganisms was BK8>YB706>CK except actinomycetes. The ratio of soil fungi to bacteria was increased compared with CK. The Simpson, Shannon, Brillouin and McIntosh indices of rhizosphere soil microbial community in YB706 and BK8 treatments were significantly increased. Our results suggested that application of YB706 and BK8 could improve the growth rate of C. equisetifolia seedlings, effectively increase the contents of soil available nutrients, increase soil microbial diversity, and improve soil microbial environment.

The complete chloroplast genome sequence of Casuarina equisetifolia.

Casuarina equisetifolia, as windbreaks, soil erosion, and sand dune stabilization with high resistant to typhoon force winds, drought and salinization, belongs to the Casuarinaceae family. In this study, the complete chloroplast genome of C. equisetifolia was sequenced by Illumina sequencing platform and annotated by Geneious Prime. The complete chloroplast genome size is 156,128 bp in length, with a large single copy region (LSC: 86,192 bp) and a small single-copy region (SSC: 18,462 bp), which was separated by a pair of 25,737 bp inverted repeated regions (IRs). The chloroplast genome of C. equisetifolia encodes total 127 genes, including 82 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenomic relationship analysis suggested that the Casuarinaceae family, which includes C. equisetifolia, was more closely related to the family of Betulaceae.

Genome-wide analysis of MYB transcription factors and their responses to salt stress in Casuarina equisetifolia.

BACKGROUND: MYB transcription factors are a kind of DNA binding protein that can specifically interact with the promoter region. Members of MYB TFs are widely involved in plant growth and development, secondary metabolism, stress response, and hormone signal transduction. However, there is no report of comprehensive bioinformatics analysis on the MYB family of Casuarina equisetifolia. RESULTS: In this study, bioinformatics methods were used to screen out 182 MYB transcription factors from the Casuarina equisetifolia genome database, including 69 1R-MYB, 107 R2R3-MYB, 4 R1R2R3-MYB, and 2 4R-MYB. The C. equisetifolia R2R3-MYB genes were divided into 29 groups based on the phylogenetic topology and the classification of the MYB superfamily in Arabidopsis thaliana, while the remaining MYB genes (1R-MYB, R1R2R3-MYB, and 4R-MYB) was divided into 19 groups. Moreover, the conserved motif and gene structure analysis shown that the members of the CeqMYBs were divided into the same subgroups with mostly similar gene structures. In addition, many conserved amino acids in the R2 and R3 domains of CeqMYBs by WebLogo analysis, especially tryptophan residues (W), with 3 conserved W in R2 repeat and 2 conserved W in R3 repeat. Combining promoter and GO annotation analysis, speculated on the various biological functions of CeqMYBs, thus 32 MYB genes were selected to further explore its response to salt stress by using qPCR analysis technique. Most CeqMYB genes were differentially regulated following multiple salt treatments. CONCLUSIONS: Seven genes (CeqMYB164, CeqMYB4, CeqMYB53, CeqMYB32, CeqMYB114, CeqMYB71 and CeqMYB177) were assigned to the "response to salt stress" by GO annotation. Among them, the expression level of CeqMYB4 was up-regulated under various salt treatments, indicating CeqMYB4 might participated in the response to salt stress. Our results provide important information for the biological function of C. equisetifolia, as well as offer candidate genes for further study of salt stress mechanism.

Kinetics, equilibrium and thermodynamic investigations of methylene blue dye removal using Casuarina equisetifolia pines.

Casuarina equisetifolia pines are degradable biopolymeric substance with dye-sequestering property was utilized as biosorbent to expel a cationic dye; methylene blue dye from simulated wastewater. The prepared adsorbent material was characterized for their structural, morphological and elemental features to understand their suitability in augmenting in dye-wastewater remediation. The results infer that 0.5 g/L biosorbent was proficient in removing 100 mg/L methylene blue (pH 7.0 ± 0.2) when agitated at 150 rpm for 120 min. Isothermal behavior were evaluated using non-linear isotherm models like Temkin, Langmuir and Freundlich models while the rate-limiting steps were found using kinetic models. Temkin isotherm and pseudo-first order model explained the removal mechanism among the models evaluated, which infers that the biosorption followed physisorption with the maximum adsorption capacity of 41.35 mg/g. Thermodynamic behavior of methylene blue removal by C. equisetifolia pines powder described the feasibility of biosorption as well as the type of heat involved. Equilibrium sorption capacities, rate constants and correlation coefficients explains that MB dye removal by C. equisetifolia pines is presumably physisorption, spontaneous and endothermic in nature.