Comparative Analysis of Sucrose-Regulatory Genes in High- and Low-Sucrose Sister Clones of Sugarcane.

Sugarcane is a significant primitive source of sugar and energy worldwide. The progress in enhancing the sugar content in sugarcane cultivars remains limited due to an insufficient understanding of specific genes related to sucrose production. The present investigation examined the enzyme activities, levels of reducing and non-reducing sugars, and transcript expression using RT-qPCR to assess the gene expression associated with sucrose metabolism in a high-sucrose sugarcane clone (GXB9) in comparison to a low-sucrose sister clone (B9). Sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), sucrose synthase (SuSy), cell wall invertase (CWI), soluble acid invertase (SAI), and neutral invertase (NI) are essential enzymes involved in sucrose metabolism in sugarcane. The activities of these enzymes were comparatively quantified and analyzed in immature and maturing internodes of the high- and low-sucrose clones. The results showed that the higher-sucrose-accumulating clone had greater sucrose concentrations than the low-sucrose-accumulating clone; however, maturing internodes had higher sucrose levels than immature internodes in both clones. Hexose concentrations were higher in immature internodes than in maturing internodes for both clones. The SPS and SPP enzymes activities were higher in the high-sucrose-storing clone than in the low-sucrose clone. SuSy activity was higher in the low-sucrose clone than in the high-sucrose clone; further, the degree of SuSy activity was higher in immature internodes than in maturing internodes for both clones. The SPS gene expression was considerably higher in mature internodes of the high-sucrose clones than the low-sucrose clone. Conversely, the SuSy gene exhibited up-regulated expression in the low-sucrose clone. The enhanced expression of SPS in the high-sucrose clone compared to the low-sucrose clone suggests that SPS plays a major role in the increased accumulation of sucrose. These findings provide the opportunity to improve sugarcane cultivars by regulating the activity of genes related to sucrose metabolism using transgenic techniques.

Molecular mechanism of endophytic bacteria DX120E regulating polyamine metabolism and promoting plant growth in sugarcane.

Introduction: Sugarcane endophytic nitrogen-fixing bacterium Klebsiella variícola DX120E displayed broad impact on growth, but the exact biological mechanism, especially polyamines (PAs) role, is still meager. Methods: To reveal this relationship, the content of polyamine oxidase (PAO), PAs, reactive oxygen species (ROS)-scavenging antioxidative enzymes, phytohormones, 1-aminocyclopropane-1-carboxylic synthase (ACS), chlorophyll content, and biomass were determined in sugarcane incubated with the DX120E strain. In addition, expression levels of the genes associated with polyamine metabolism were measured by transcriptomic analysis. Results: Genomic analysis of Klebsiella variícola DX120E revealed that 39 genes were involved in polyamine metabolism, transport, and the strain secrete PAs in vitro. Following a 7-day inoculation period, DX120E stimulated an increase in the polyamine oxidase (PAO) enzyme in sugarcane leaves, however, the overall PAs content was reduced. At 15 days, the levels of PAs, ROS-scavenging antioxidative enzymes, and phytohormones showed an upward trend, especially spermidine (Spd), putrescine (Put), catalase (CAT), auxin (IAA), gibberellin (GA), and ACS showed a significant up-regulation. The GO and KEGG enrichment analysis found a total of 73 differentially expressed genes, involving in the cell wall (9), stimulus response (13), peroxidase activity (33), hormone (14) and polyamine metabolism (4). Discussion: This study demonstrated that endophytic nitrogen-fixing bacteria stimulated polyamine metabolism and phytohormones production in sugarcane plant tissues, resulting in enhanced growth. Dual RNA-seq analyses provided insight into the early-stage interaction between sugarcane seedlings and endophytic bacteria at the transcriptional level. It showed how diverse metabolic processes selectively use distinct molecules to complete the cell functions under present circumstances.

Corrigendum: Effect of endophytic diazotroph Enterobacter roggenkampii ED5 on nitrogen-metabolism-related microecology in the sugarcane rhizosphere at different nitrogen levels.

[This corrects the article DOI: 10.3389/fmicb.2023.1132016.].

Genome analysis of a halophilic Virgibacillus halodenitrificans ASH15 revealed salt adaptation, plant growth promotion, and isoprenoid biosynthetic machinery.

Globally, due to widespread dispersion, intraspecific diversity, and crucial ecological components of halophilic ecosystems, halophilic bacteria is considered one of the key models for ecological, adaptative, and biotechnological applications research in saline environments. With this aim, the present study was to enlighten the plant growth-promoting features and investigate the systematic genome of a halophilic bacteria, Virgibacillus halodenitrificans ASH15, through single-molecule real-time (SMRT) sequencing technology. Results showed that strain ASH15 could survive in high salinity up to 25% (w/v) NaCl concentration and express plant growth-promoting traits such as nitrogen fixation, plant growth hormones, and hydrolytic enzymes, which sustain salt stress. The results of pot experiment revealed that strain ASH15 significantly enhanced sugarcane plant growth (root shoot length and weight) under salt stress conditions. Moreover, the sequencing analysis of the strain ASH15 genome exhibited that this strain contained a circular chromosome of 3,832,903 bp with an average G+C content of 37.54%: 3721 predicted protein-coding sequences (CDSs), 24 rRNA genes, and 62 tRNA genes. Genome analysis revealed that the genes related to the synthesis and transport of compatible solutes (glycine, betaine, ectoine, hydroxyectoine, and glutamate) confirm salt stress as well as heavy metal resistance. Furthermore, functional annotation showed that the strain ASH15 encodes genes for root colonization, biofilm formation, phytohormone IAA production, nitrogen fixation, phosphate metabolism, and siderophore production, which are beneficial for plant growth promotion. Strain ASH15 also has a gene resistance to antibiotics and pathogens. In addition, analysis also revealed that the genome strain ASH15 has insertion sequences and CRISPRs, which suggest its ability to acquire new genes through horizontal gene transfer and acquire immunity to the attack of viruses. This work provides knowledge of the mechanism through which V. halodenitrificans ASH15 tolerates salt stress. Deep genome analysis, identified MVA pathway involved in biosynthesis of isoprenoids, more precisely "Squalene." Squalene has various applications, such as an antioxidant, anti-cancer agent, anti-aging agent, hemopreventive agent, anti-bacterial agent, adjuvant for vaccines and drug carriers, and detoxifier. Our findings indicated that strain ASH15 has enormous potential in industries such as in agriculture, pharmaceuticals, cosmetics, and food.

Effect of endophytic diazotroph Enterobacter roggenkampii ED5 on nitrogen-metabolism-related microecology in the sugarcane rhizosphere at different nitrogen levels.

Sugarcane is an important sugar and energy crop worldwide, requiring a large amount of nitrogen (N). However, excessive application of synthetic N fertilizer causes environmental pollution in farmland. Endophytic nitrogen-fixing bacteria (ENFB) provide N nutrition for plants through biological N fixation, thus reducing the need for chemical fertilizers. The present study investigated the effect of the N-fixing endophytic strain Enterobacter roggenkampii ED5 on phytohormone indole-3-acetic acid (IAA), N-metabolism enzyme activities, microbial community compositions, and N cycle genes in sugarcane rhizosphere soil at different N levels. Three levels of 15N-urea, such as low N (0 kg/ha), medium N (150 kg/ha), and high N (300 kg/ha), were applied. The results showed that, after inoculating strain ED5, the IAA content in sugarcane leaves was significantly increased by 68.82% under low N condition at the seedling stage (60 days). The nitrate reductase (NR) activity showed a downward trend. However, the glutamine synthase (GS) and NADH-glutamate dehydrogenase (NADH-GDH) activities were significantly enhanced compared to the control under the high N condition, and the GS and NR genes had the highest expression at 180 and 120 days, respectively, at the low N level. The total N content in the roots, stems, and leaves of sugarcane was higher than the control. The 15N atom % excess of sugarcane decreased significantly under medium N condition, indicating that the medium N level was conducive to N fixation in strain ED5. Metagenome analysis of sugarcane rhizosphere soil exhibited that the abundance of N-metabolizing microbial richness was increased under low and high N conditions after inoculation of strain ED5 at the genus level, while it was increased at the phylum level only under the low N condition. The LefSe (LDA > 2, p < 0.05) found that the N-metabolism-related differential microorganisms under the high N condition were higher than those under medium and low N conditions. It was also shown that the abundance of nifDHK genes was significantly increased after inoculation of ED5 at the medium N level, and other N cycle genes had high abundance at the high N level after inoculation of strain ED5. The results of this study provided a scientific reference for N fertilization in actual sugarcane production.

Complete genome analysis of sugarcane root associated endophytic diazotroph Pseudomonas aeruginosa DJ06 revealing versatile molecular mechanism involved in sugarcane development.

Sugarcane is an important sugar and bioenergy source and a significant component of the economy in various countries in arid and semiarid. It requires more synthetic fertilizers and fungicides during growth and development. However, the excess use of synthetic fertilizers and fungicides causes environmental pollution and affects cane quality and productivity. Plant growth-promoting bacteria (PGPB) indirectly or directly promote plant growth in various ways. In this study, 22 PGPB strains were isolated from the roots of the sugarcane variety GT42. After screening of plant growth-promoting (PGP) traits, it was found that the DJ06 strain had the most potent PGP activity, which was identified as Pseudomonas aeruginosa by 16S rRNA gene sequencing. Scanning electron microscopy (SEM) and green fluorescent protein (GFP) labeling technology confirmed that the DJ06 strain successfully colonized sugarcane tissues. The complete genome sequencing of the DJ06 strain was performed using Nanopore and Illumina sequencing platforms. The results showed that the DJ06 strain genome size was 64,90,034 bp with a G+C content of 66.34%, including 5,912 protein-coding genes (CDSs) and 12 rRNA genes. A series of genes related to plant growth promotion was observed, such as nitrogen fixation, ammonia assimilation, siderophore, 1-aminocyclopropane-1-carboxylic acid (ACC), deaminase, indole-3-acetic acid (IAA) production, auxin biosynthesis, phosphate metabolism, hydrolase, biocontrol, and tolerance to abiotic stresses. In addition, the effect of the DJ06 strain was also evaluated by inoculation in two sugarcane varieties GT11 and B8. The length of the plant was increased significantly by 32.43 and 12.66% and fresh weight by 89.87 and 135.71% in sugarcane GT11 and B8 at 60 days after inoculation. The photosynthetic leaf gas exchange also increased significantly compared with the control plants. The content of indole-3-acetic acid (IAA) was enhanced and gibberellins (GA) and abscisic acid (ABA) were reduced in response to inoculation of the DJ06 strain as compared with control in two sugarcane varieties. The enzymatic activities of oxidative, nitrogen metabolism, and hydrolases were also changed dramatically in both sugarcane varieties with inoculation of the DJ06 strain. These findings provide better insights into the interactive action mechanisms of the P. aeruginosa DJ06 strain and sugarcane plant development.

Nitrogen fixation and phytohormone stimulation of sugarcane plant through plant growth promoting diazotrophic Pseudomonas.

Diazotrophic microorganisms are free-living groups of organisms that can convert atmospheric nitrogen (N) into bioavailable nitrogen for plants, which increases crop development and production. The purpose of the current study was to ascertain how diazotrophic plant growth promoting (PGP) Pseudomonas strains (P. koreensis CY4 and P. entomophila CN11) enhanced nitrogen fixation, defense activity, and PGP attributes of sugarcane varieties; GT11 and G×B9. A 15N isotope-dilution study was conducted to confirm the sugarcane strains' capacity to fix nitrogen, and the results indicated that between 21 to 35% of plant, nitrogen is fixed biologically by selected rhizobacteria. In comparison to the control, after 30, 60, and 90 days, both CY4 and CN11 strains significantly increased defense-related enzymes (catalase, peroxidase, phenylalanine ammonia-lyase, superoxide dismutase, glucanase, and chitinase) and phytohormones (abscisic acid, ABA, cytokinin, etc.) in GT11 and GXB. Additionally, the expression of SuCHI, SuGLU, SuCAT, SuSOD, and SuPAL genes was found to be elevated in Pseudomonas strains inoculated plants using real-time quantitative polymerase chain reaction (RT-qPCR). Both bacterial strains increased all physiological parameters and chlorophyll content in sugarcane plants more than their control. The effects of P. koreensis CY4 and P. entomophila CN11 strains on sugarcane growth promotion and nitrogen fixation under greenhouse conditions are described here for the first time systematically. The results of confirmation studies demonstrated that P. koreensis CY4 and P. entomophila are PGP bacterial strains with the potential to be employed as a biofertilizer for sugarcane growth, nitrogen nutrient absorption, and reduced application of chemical nitrogenous fertilizers in agricultural fields. .

Cloning, Prokaryotic Expression and Functional Characterization of NifH Gene from the Associative Nitrogen-Fixing Bacteria Klebsiella Variicola DX120E.

Background: Biological nitrogen fixation (BNF) is a unique mechanism in which microorganisms utilize the nitrogenase enzyme to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Fe protein, encoded by the nifH gene, is an essential component of the nitrogenase in Klebsiella variicola DX120E. However, the function of this gene in regulating nitrogen fixing activity is still unclear. Objectives: The objective of this study was to reveal the function of nifH gene in associative nitrogen-fixing bacteria Klebsiella variicola DX120E and micro-sugarcane system by immunoassay and gene editing. Materials and Methods: In the current investigation, the nifH gene was cloned in a pET-30a (+) vector and expressed in Escherichia coli. The NifH protein was purified and used to immunize rabbit, and then the serum was collected and purified to obtain rabbit anti-NifH polyclonal antibodies. The CRISPR-Cas9 system was applied to produce nifH mutant strains, and the nitrogen-fixing enzyme activity, gene, and protein expression were analyzed. Results: Both in vitro and in vivo NifH proteins were detected by Western blotting, which were 43 and 32 kDa respectively. The expression of nifD and nifK genes was decreased, and nitrogenase activity was reduced in the nifH mutant strain. Conclusion: The nifH gene mutant weakened the nitrogenase activity by regulating the expression of Fe protein, which suggests a potential strategy to study the nitrogen fixation-related genes and the interactions between endophytic nitrogen-fixing bacteria and sugarcane.

Endophytic nitrogen-fixing bacteria DX120E inoculation altered the carbon and nitrogen metabolism in sugarcane.

Endophytic nitrogen-fixing bacteria are versatile and widely distributed in plants. Numerous strains of endophytic nitrogen-fixing bacteria are used as biofertilizers to minimize the utilization of chemical fertilizers, improve nutrient use efficiency, increase crop productivity, and reduce environmental pollution. However, the mechanism underlying the interaction between nitrogen-fixing bacteria and plants is still unclear. So, the present study was planned to assess the effects of endophytic nitrogen-fixing bacteria on sugarcane by analyzing the changes in physiological and biochemical activities. In the current study, Klebsiella variicola DX120E, an endophytic nitrogen-fixing bacterium, was inoculated on sugarcane varieties B8 and ROC22 to evaluate the effects on nitrogen and carbon metabolism-related enzymatic activity and biomass. Results showed that DX120E inoculation improved the enzymatic activities related to gluconeogenesis and nitrogen metabolism increased the sugarcane plant's height, cane juice Brix, biomass, chlorophyll, and soluble sugar content in sugarcane. Metabolomics analysis revealed that the metabolome modules were highly enriched in carbon and nitrogen metabolic pathways of strain-affected sugarcane than uninoculated control. The identified carbohydrates were associated with the glycolysis or gluconeogenesis and tricarboxylic acid (TCA) cycle in plants. Metabolomic profiling in the present investigation showed that carbohydrate metabolism is coordinated with nitrogen metabolism to provide carbon skeletons and energy to amino acid synthesis, and amino acid degradation results in several metabolites used by the citric acid cycle as an energy source. Moreover, differentially expressed metabolites of non-proteinogenic amino acids have a further complementary role to the action of endophytic nitrogen-fixing bacteria. Meanwhile, a significant difference in metabolites and metabolic pathways present in stems and leaves of B8 and ROC22 varieties was found. This study discovered the potential benefits of DX120E in sugarcane and suggested candidate regulatory elements to enhance interactions between nitrogen-fixing microbes and sugarcane.

Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth.

Sugarcane (Saccharum officinarum L.) is one of the world's highly significant commercial crops. The amounts of synthetic nitrogen (N2) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen to crops through the biological nitrogen fixation mediated nif gene. The nifH gene is the most extensively utilized molecular marker in nature for studying N2 fixing microbiomes. The present research intended to determine the existence of novel endophytic diazotrophs through culturable and unculturable bacterial communities (EDBCs). The EDBCs of different tissues (root, stem, and leaf) of five sugarcane cultivars (Saccharum officinarum L. cv. Badila, S. barberi Jesw.cv Pansahi, S. robustum, S. spontaneum, and S. sinense Roxb.cv Uba) were isolated and molecularly characterized to evaluate N2 fixation ability. The diversity of EDBCs was observed based on nifH gene Illumina MiSeq sequencing and a culturable approach. In this study, 319766 operational taxonomic units (OTUs) were identified from 15 samples. The minimum number of OTUs was recorded in leaf tissues of S. robustum and maximum reads in root tissues of S. spontaneum. These data were assessed to ascertain the structure, diversity, abundance, and relationship between the microbial community. A total of 40 bacterial families with 58 genera were detected in different sugarcane species. Bacterial communities exhibited substantially different alpha and beta diversity. In total, 16 out of 20 genera showed potent N2-fixation in sugarcane and other crops. According to principal component analysis (PCA) and hierarchical clustering (Bray-Curtis dis) evaluation of OTUs, bacterial microbiomes associated with root tissues differed significantly from stem and leaf tissues of sugarcane. Significant differences often were observed in EDBCs among the sugarcane tissues. We tracked and validated the plethora of individual phylum strains and assessed their nitrogenase activity with a culture-dependent technique. The current work illustrated the significant and novel results of many uncharted endophytic microbial communities in different tissues of sugarcane species, which provides an experimental system to evaluate the biological nitrogen fixation (BNF) mechanism in sugarcane. The novel endophytic microbial communities with N2-fixation ability play a remarkable and promising role in sustainable agriculture production.

Morphological, agronomical, physiological and molecular characterization of a high sugar mutant of sugarcane in comparison to mother variety.

Sugarcane is a significant crop plant with the capability of accumulating higher amount of sucrose. In the present study, a high sucrose content sugarcane mutant clone, GXB9, has been studied in comparison to the low sucrose mother clone B9 on morphological, agronomical and physiological level in order to scrutinize the variation because of mutation in GXB9 in field under normal environmental condition. The results showed that GXB9 has less germination, tillering rate, stalk height, leaf length, leaf width, leaf area, number of internodes, internode length and internode diameter than B9. Qualitative traits of leaf and stalk displayed significant variation between GXB9 and B9. Endogenous hormones quantity was also showed variation between the two clones. The relative SPAD reading and chlorophyll a, b concentrations also showed variation between GXB9 and B9. The photosynthetic parameter analysis indicated that the GXB9 has significantly higher net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr) than B9. The qRT-PCR analysis of genes encoding enzymes like SPS, SuSy, CWIN, and CeS showed upregulation in GXB9 and downregulation in B9. However, these genes were significantly differentially expressed between the immature and maturing internodes of GXB9. The cane quality trait analysis showed that GXB9 had higher juice rate, juice gravity purity, brix, juice sucrose content and cane sucrose content than B9. The yield and component investigation results indicated that GXB9 had lower single stalk weight, however higher number of millable stalks per hectare than B9, and GXB9 had lower theoretical cane yield than B9. SSR marker analysis showed genetic variation between GXB9 and B9. This study has shown significant variation in the traits of GXB9 in comparison to B9 which advocates that GXB9 is a high sugar mutant clone of B9 and an elite source for future breeding.

High-Throughput Sequencing-Based Analysis of Rhizosphere and Diazotrophic Bacterial Diversity Among Wild Progenitor and Closely Related Species of Sugarcane (Saccharum spp. Inter-Specific Hybrids).

Considering the significant role of genetic background in plant-microbe interactions and that most crop rhizospheric microbial research was focused on cultivars, understanding the diversity of root-associated microbiomes in wild progenitors and closely related crossable species may help to breed better cultivars. This study is aimed to fill a critical knowledge gap on rhizosphere and diazotroph bacterial diversity in the wild progenitors of sugarcane, the essential sugar and the second largest bioenergy crop globally. Using a high-throughput sequencing (HTS) platform, we studied the rhizosphere and diazotroph bacterial community of Saccharum officinarum L. cv. Badila (BRS), Saccharum barberi (S. barberi) Jesw. cv Pansahi (PRS), Saccharum robustum [S. robustum; (RRS), Saccharum spontaneum (S. spontaneum); SRS], and Saccharum sinense (S. sinense) Roxb. cv Uba (URS) by sequencing their 16S rRNA and nifH genes. HTS results revealed that a total of 6,202 bacteria-specific operational taxonomic units (OTUs) were identified, that were distributed as 107 bacterial groups. Out of that, 31 rhizobacterial families are commonly spread in all five species. With respect to nifH gene, S. barberi and S. spontaneum recorded the highest and lowest number of OTUs, respectively. These results were validated by quantitative PCR analysis of both genes. A total of 1,099 OTUs were identified for diazotrophs with a core microbiome of 9 families distributed among all the sugarcane species. The core microbiomes were spread across 20 genera. The increased microbial diversity in the rhizosphere was mainly due to soil physiochemical properties. Most of the genera of rhizobacteria and diazotrophs showed a positive correlation, and few genera negatively correlated with the soil properties. The results showed that sizeable rhizospheric diversity exists across progenitors and close relatives. Still, incidentally, the rhizosphere microbial abundance of progenitors of modern sugarcane was at the lower end of the spectrum, indicating the prospect of Saccharum species introgression breeding may further improve nutrient use and disease and stress tolerance of commercial sugarcane. The considerable variation for rhizosphere microbiome seen in Saccharum species also provides a knowledge base and an experimental system for studying the evolution of rhizobacteria-host plant association during crop domestication.

Impact of carbendazim on cellular growth, defence system and plant growth promoting traits of Priestia megaterium ANCB-12 isolated from sugarcane rhizosphere.

Agrochemicals are consistently used in agricultural practices to protect plants from pathogens and ensure high crop production. However, their overconsumption and irregular use cause adverse impacts on soil flora and non-target beneficial microorganisms, ultimately causing a hazard to the ecosystem. Taking this into account, the present study was conducted to determine the high dosage of fungicide (carbendazim: CBZM) effects on the rhizobacteria survival, plant growth promoting trait and reactive oxygen species (ROS) scavenging antioxidant enzyme system. Thus, a multifarious plant growth promoting rhizobacteria (PGPR) isolate, ANCB-12, was obtained from the sugarcane rhizosphere through an enrichment technique. The taxonomic position of the isolated rhizobacteria was confirmed through 16S rRNA gene sequencing analysis as Priestia megaterium ANCB-12 (accession no. ON878101). Results showed that increasing concentrations of fungicide showed adverse effects on rhizobacterial cell growth and survival. In addition, cell visualization under a confocal laser scanning microscope (CLSM) revealed more oxidative stress damage in the form of ROS generation and cell membrane permeability. Furthermore, the increasing dose of CBZM gradually decreased the plant growth promoting activities of the rhizobacteria ANCB-12. For example, CBZM at a maximum 3,000 µg/ml concentration decreases the indole acetic acid (IAA) production by 91.6%, ACC deaminase by 92.3%, and siderophore production by 94.1%, respectively. Similarly, higher dose of fungicide enhanced the ROS toxicity by significantly (p < 0.05) modulating the stress-related antioxidant enzymatic biomarkers in P. megaterium ANCB-12. At a maximum 3,000 µg/ml CBZM concentration, the activity of superoxide dismutase (SOD) declined by 82.3%, catalase (CAT) by 61.4%, glutathione peroxidase (GPX) by 76.1%, and glutathione reductase (GR) by 84.8%, respectively. The results of this study showed that higher doses of the fungicide carbendazim are toxic to the cells of plant-beneficial rhizobacteria. This suggests that a recommended dose of fungicide should be made to lessen its harmful effects.

Differential Protein Expression Analysis of Two Sugarcane Varieties in Response to Diazotrophic Plant Growth-Promoting Endophyte Enterobacter roggenkampii ED5.

Plant endophytic bacteria have many vital roles in plant growth promotion (PGP), such as nitrogen (N) fixation and resistance to biotic and abiotic stresses. In this study, the seedlings of sugarcane varieties B8 (requires a low concentration of nitrogen for growth) and GT11 (requires a high concentration of nitrogen for growth) were inoculated with endophytic diazotroph Enterobacter roggenkampii ED5, which exhibits multiple PGP traits, isolated from sugarcane roots. The results showed that the inoculation with E. roggenkampii ED5 promoted the growth of plant significantly in both sugarcane varieties. 15N detection at 60 days post-inoculation proved that the inoculation with strain ED5 increased the total nitrogen concentration in the leaf and root than control in both sugarcane varieties, which was higher in B8. Biochemical parameters and phytohormones in leaf were analyzed at 30 and 60 days after the inoculation. The results showed that the inoculation with E. roggenkampii ED5 improved the activities of superoxide dismutase (SOD), catalase (CAT), NADH-glutamate dehydrogenase (NADH-GDH), glutamine synthetase (GS), and endo-ß-1,4-glucanase, and the contents of proline and indole acetic acid (IAA) in leaf, and it was generally more significant in B8 than in GT11. Tandem Mass Tags (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to perform comparative proteomic analysis in the sugarcane leaves at 30 days after inoculation with strain ED5. A total of 27,508 proteins were detected, and 378 differentially expressed proteins (DEPs) were found in the treated sugarcane variety B8 (BE) as compared to control (BC), of which 244 were upregulated and 134 were downregulated. In contrast, a total of 177 DEPs were identified in the treated sugarcane variety GT11 (GE) as compared to control (GC), of which 103 were upregulated and 74 were downregulated. The DEPs were associated with nitrogen metabolism, photosynthesis, starch, sucrose metabolism, response to oxidative stress, hydrolase activity, oxidative phosphorylation, glutathione metabolism, phenylpropanoid metabolic process, and response to stresses in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. To the best of our knowledge, this is the first proteomic approach to investigate the molecular basis of the interaction between N-fixing endophytic strain E. roggenkampii ED5 and sugarcane.

Insights into the Bacterial and Nitric Oxide-Induced Salt Tolerance in Sugarcane and Their Growth-Promoting Abilities.

Soil salinity causes severe environmental stress that affects agriculture production and food security throughout the world. Salt-tolerant plant-growth-promoting rhizobacteria (PGPR) and nitric oxide (NO), a distinctive signaling molecule, can synergistically assist in the alleviation of abiotic stresses and plant growth promotion, but the mechanism by which this happens is still not well known. In the present study, in a potential salt-tolerant rhizobacteria strain, ASN-1, growth up to 15% NaCl concentration was achieved with sugarcane rhizosphere soil. Based on 16S-rRNA gene sequencing analysis, the strain ASN-1 was identified as a Bacillus xiamenensis. Strain ASN-1 exhibits multiple plant-growth-promoting attributes, such as the production of indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, siderophores, HCN, ammonia, and exopolysaccharides as well as solubilized phosphate solubilization. Biofilm formation showed that NO enhanced the biofilm and root colonization capacity of the PGPR strain ASN-1 with host plants, evidenced by scanning electron microscopy. The greenhouse study showed that, among the different treatments, the combined application of PGPR and sodium nitroprusside (SNP) as an NO donor significantly (p ≤ 0.05) enhanced sugarcane plant growth by maintaining the relative water content, electrolyte leakage, gas exchange parameters, osmolytes, and Na+/K+ ratio. Furthermore, PGPR and SNP fertilization reduced the salinity-induced oxidative stress in plants by modulating the antioxidant enzyme activities and stress-related gene expression. Thus, it is believed that the acquisition of advanced information about the synergistic effect of salt-tolerant PGPR and NO fertilization will reduce the use of harmful chemicals and aid in eco-friendly sustainable agricultural production under salt stress conditions.

Influence of Silicon on Biocontrol Strategies to Manage Biotic Stress for Crop Protection, Performance, and Improvement.

Silicon (Si) has never been acknowledged as a vital nutrient though it confers a crucial role in a variety of plants. Si may usually be expressed more clearly in Si-accumulating plants subjected to biotic stress. It safeguards several plant species from disease. It is considered as a common element in the lithosphere of up to 30% of soils, with most minerals and rocks containing silicon, and is classified as a "significant non-essential" element for plants. Plant roots absorb Si, which is subsequently transferred to the aboveground parts through transpiration stream. The soluble Si in cytosol activates metabolic processes that create jasmonic acid and herbivore-induced organic compounds in plants to extend their defense against biotic stressors. The soluble Si in the plant tissues also attracts natural predators and parasitoids during pest infestation to boost biological control, and it acts as a natural insect repellent. However, so far scientists, policymakers, and farmers have paid little attention to its usage as a pesticide. The recent developments in the era of genomics and metabolomics have opened a new window of knowledge in designing molecular strategies integrated with the role of Si in stress mitigation in plants. Accordingly, the present review summarizes the current status of Si-mediated plant defense against insect, fungal, and bacterial attacks. It was noted that the Si-application quenches biotic stress on a long-term basis, which could be beneficial for ecologically integrated strategy instead of using pesticides in the near future for crop improvement and to enhance productivity.

Foliar application of silicon boosts growth, photosynthetic leaf gas exchange, antioxidative response and resistance to limited water irrigation in sugarcane (Saccharum officinarum L.).

Plant cell and water relationship regulates morphological, physiological and biochemical characteristics to optimize carboxylation for enhanced biomass yield in sugarcane. Insufficient water irrigation is one of the serious problems to impair potential yield of agriculturally important sugarcane cash crop by loss in plant performance. Our study aims to reveal consequences of foliar spray of silicon (Si) using calcium metasilicate powder (Wollastonite, CaO.SiO2) to alleviate the adverse effects of limited water irrigation in sugarcane. Silicon (0, 50, 100 and 500 ppm) was applied as foliar spray on normally grown 45 days old sugarcane plants. Further, these plants were raised at half field capacity (50%) using water irrigation precisely up to 90 days under open environmental variables. Consequently, restricted irrigation impaired plant growth-development, leaf relative water content (%), photosynthetic pigments, SPAD unit, photosynthetic performance, chlorophyll fluorescence variable yield (Fv/Fm) and biomass yield. Notably, it has enhanced values of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), antioxidative defense enzyme molecules viz., catalase (CAT), ascorbate peroxidase (APx) and superoxide dismutase (SOD). The foliar spray of Si defended sugarcane plants from limited water irrigation stress as Si quenched harmful effect of water-deficit and also enhanced the operation of antioxidant defense machinery for improved sugarcane plant performance suitably favored stomatal dynamics for photosynthesis and plant productivity.

Whole Genome Analysis of Sugarcane Root-Associated Endophyte Pseudomonas aeruginosa B18-A Plant Growth-Promoting Bacterium With Antagonistic Potential Against Sporisorium scitamineum.

Sugarcane smut is a significant fungal disease that causes a major loss in sugar yield and quality. In this study, we isolated an endophytic strain B18 from a sugarcane root, which showed plant growth-promotion, hydrolytic enzyme production, antifungal activity against sugarcane pathogens (Sporisorium scitamineum, Ceratocystis paradoxa, Fusarium verticillioides), and the presence of nifH, acdS, and antibiotic genes (hcn, prn, and phCA) under in vitro conditions. BIOLOG(R) phenotypic profiling of B18 established its ability to use various carbon and nitrogen sources and tolerate a range of pH and osmotic and temperature stresses. Whole-genome analysis of B18, identified as Pseudomonas aeruginosa, showed that it consists of a single circular chromosome of 6,490,014 bp with 66.33% GC content. Genome annotation has identified 5,919 protein-coding genes, and 65 tRNA, and 12 rRNA genes. The P. aeruginosa B18 genome encodes genes related to ethylene, nitrogen (nifU, norBCDERQ, gltBDPS, and aatJMPQ), and phosphate (pstABCS and phoBDHRU) metabolism and produce indole-3-acetic acid and siderophores. This also includes genes encoding hydrolases and oxidoreductases, those associated with biocontrol mechanisms (hcnABC, phzA_B, phzDEFGMS, and pchA), colonization (minCDE and lysC), and biofilm formation (efp, hfq, flgBCDEFGHI, and motAB), and those associated with metabolism of secondary metabolites. Collectively, these results suggest a role for P. aeruginosa B18 in plant growth enhancement and biocontrol mechanisms. The P. aeruginosa B18 strain was found to be an efficient colonizer in sugarcane; it can improve growth through modulation of plant hormone production and enhanced host-plant resistance to smut pathogen S. scitamineum in a smut-susceptible sugarcane variety (Yacheng71-374). These biocontrol and plant growth promotion properties of P. aeruginosa B18 area are discussed in this report.

Predication of Photosynthetic Leaf Gas Exchange of Sugarcane (Saccharum spp) Leaves in Response to Leaf Positions to Foliar Spray of Potassium Salt of Active Phosphorus under Limited Water Irrigation.

Sufficient water and fertilizer inputs in agriculture play a major role in crop growth, production, and quality. In this study, the response of sugarcane to limited water irrigation and foliar application of potassium salt of active phosphorus (PSAP) for photosynthetic responses were examined, and PSAP's role in limited water irrigation management was assessed. Sugarcane plants were subjected to limited irrigation (95-90 and 45-40% FC) after three months of germination, followed by a foliar spray (0, 2, 4, 6, and 10 M) of PSAP. The obtained results indicated that limited water irrigation negatively affected sugarcane growth and reduced leaf gas exchange activities. However, the application of PSAP increased the photosynthetic activities by protecting the photosynthetic machinery during unfavorable conditions. Mathematical modeling, a Skewed model, was developed and compared with the existing Gaussian model to describe the photosynthetic responses of sugarcane leaves under the limited irrigation with and without PSAP application. The models fitted well with the observed values, and the predicted photosynthetic parameters were in close relationship with the obtained results. The Skewed model was found to be better than the Gaussian model in describing the photosynthetic parameters of plant leaves positioned over a stem of limited water irrigation and applied PSAP application and is recommended for further application.

Root-Derived Endophytic Diazotrophic Bacteria Pantoea cypripedii AF1 and Kosakonia arachidis EF1 Promote Nitrogen Assimilation and Growth in Sugarcane.

Excessive, long-term application of chemical fertilizers in sugarcane crops disrupts soil microbial flora and causes environmental pollution and yield decline. The role of endophytic bacteria in improving crop production is now well-documented. In this study, we have isolated and identified several endophytic bacterial strains from the root tissues of five sugarcane species. Among them, eleven Gram-negative isolates were selected and screened for plant growth-promoting characteristics, i.e., production of siderophores, indole-3-acetic acid (IAA), ammonia, hydrogen cyanide (HCN), and hydrolytic enzymes, phosphorus solubilization, antifungal activity against plant pathogens, nitrogen-fixation, 1-aminocyclopropane-1-carboxylic acid deaminase activity, and improving tolerance to different abiotic stresses. These isolates had nifH (11 isolates), acdS (8 isolates), and HCN (11 isolates) genes involved in N-fixation, stress tolerance, and pathogen biocontrol, respectively. Two isolates Pantoea cypripedii AF1and Kosakonia arachidis EF1 were the most potent strains and they colonized and grew in sugarcane plants. Both strains readily colonized the leading Chinese sugarcane variety GT42 and significantly increased the activity of nitrogen assimilation enzymes (glutamine synthetase, NADH glutamate dehydrogenase, and nitrate reductase), chitinase, and endo-glucanase and the content of phytohormones gibberellic acid, indole-3-acetic acid, and abscisic acid. The gene expression analysis of GT42 inoculated with isolates of P. cypripedii AF1 or K. arachidis EF1 showed increased activity of nifH and nitrogen assimilation genes. Also, the inoculated diazotrophs significantly increased plant nitrogen content, which was corroborated by the 15N isotope dilution analysis. Collectively, these findings suggest that P. cypripedii and K. arachidis are beneficial endophytes that could be used as a biofertilizer to improve plant nitrogen nutrition and growth of sugarcane. To the best of our knowledge, this is the first report of sugarcane growth enhancement and nitrogen fixation by Gram-negative sugarcane root-associated endophytic bacteria P. cypripedii and K. arachidis. These strains have the potential to be utilized as sugarcane biofertilizers, thus reducing nitrogen fertilizer use and improving disease management.