Silicon induces ROS scavengers, hormone signalling, antifungal metabolites, and silicon deposition against brown stripe disease in sugarcane.

Bipolaris setariae is known to cause brown stripe disease in sugarcane, resulting in significant yield losses. Silicon (Si) has the potential to enhance plant growth and biotic resistance. In this study, the impact of Si on brown stripe disease was investigated across susceptible and resistant sugarcane varieties, utilizing four Si concentrations (0, 15, 30, and 45 g per barrel of Na2SiO3·5H2O). Si significantly reduced the incidence of brown stripe disease (7.41-59.23%) and alleviated damage to sugarcane growth parameters, photosynthetic parameters, and photosynthetic pigments. Submicroscopic observations revealed that Si induced the accumulation of silicified cells in leaves, reduced spore accumulation, decreased stomatal size, and protected organelles from B. setariae damage. In addition, Si increased the activity of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), reduced reactive oxygen species production (malondialdehyde and hydrogen peroxide) and modulated the expression of genes associated with hormone signalling (PR1, TGA, AOS, AOC, LOX, PYL8, and SnRK2), leading to the accumulation of abscisic acid and jasmonic acid and inhibiting SA synthesis. Si also activated the activity of metabolism-related enzymes (polyphenol oxidase and phenylalanine ammonia lyase) and the gene expression of PAL-dependent genes (PAL, C4H, and 4CL), regulating the accumulation of metabolites, such as chlorogenic acid and lignin. The antifungal test showed that chlorogenic acid (15ug µL-1) had a significant inhibitory effect on the growth of B. setariae. This study is the first to demonstrate the inhibitory effect of Si on B. setariae in sugarcane, highlighting Si as a promising and environmentally friendly strategy for managing brown stripe disease.

Fusarium sacchari hypovirus 1, a Member of Hypoviridae with Virulence Attenuation Capacity in Phytopathogenic Fusarium Species.

In a survey of mycoviruses in Fusarium species that cause sugarcane Pokkah boeng disease, twelve Fusarium strains from three Fusarium species (F. sacchari, F. andiyazi, and F. solani) were found to contain Fusarium sacchari hypovirus 1 (FsHV1), which we reported previously. The genomes of these variants range from 13,966 to 13,983 nucleotides, with 98.6% to 99.9% nucleotide sequence identity and 98.70% to 99.9% protein sequence similarity. Phylogenetic analysis placed these FsHV1 variants within the Alphahypovirus cluster of Hypoviridae. Intriguingly, no clear correlation was found between the geographic origin and host specificity of these viral variants. Additionally, six out of the twelve variants displayed segmental deletions of 1.5 to 1.8 kilobases, suggesting the existence of defective viral dsRNA. The presence of defective viral dsRNA led to a two-thirds reduction in the dsRNA of the wild-type viral genome, yet a tenfold increase in the total viral dsRNA content. To standardize virulence across natural strains, all FsHV1 strains were transferred into a single, virus-free Fusarium recipient strain, FZ06-VF, via mycelial fusion. Strains of Fusarium carrying FsHV1 exhibited suppressed pigment synthesis, diminished microspore production, and a marked decrease in virulence. Inoculation tests revealed varying capacities among different FsHV1 variants to modulate fungal virulence, with the strain harboring the FsHV1-FSA1 showing the lowest virulence, with a disease severity index (DSI) of 3.33, and the FsHV1-FS1 the highest (DSI = 17.66). The identification of highly virulent FsHV1 variants holds promise for the development of biocontrol agents for Pokkah boeng management.

Ratoon Stunting Disease of Sugarcane: A Review Emphasizing Detection Strategies and Challenges.

Sugarcane (Saccharum hybrid) is an important cash crop grown in tropical and subtropical countries. Ratoon stunting disease (RSD), caused by a xylem-inhabiting bacterium, Leifsonia xyli subsp. xyli (Lxx) is one of the most economically significant diseases globally. RSD results in severe yield losses because its highly contagious nature and lack of visually identifiable symptoms make it harder to devise an effective management strategy. The efficacy of current management practices is hindered by implementation difficulties caused by lack of resources, high cost, and difficulties in monitoring. Rapid detection of the causal pathogen in vegetative planting material is crucial for sugarcane growers to manage this disease. Several microscopic, serological, and molecular-based methods have been developed and used for detecting the RSD pathogen. Although these methods have been used across the sugarcane industry worldwide to diagnose Lxx, some lack reliability or specificity, are expensive and time-consuming to apply, and most of all, are not suitable for on-farm diagnosis. In recent decades, there has been significant progress in the development of integrated isothermal amplification-based microdevices for accurate human and plant pathogen detection. There is a significant opportunity to develop a novel diagnostic method that integrates nanobiosensing with isothermal amplification within a microdevice format for accurate Lxx detection. In this review, we summarize (i) the historical background and current knowledge of sugarcane ratoon stunting disease, including some aspects related to transmission, pathosystem, and management practices; and (ii) the drawbacks of current diagnostic methods and the potential for application of advanced diagnostics to improve disease management.

Intracellular polyamines regulate redox homeostasis with cAMP-PKA signalling during sexual mating/filamentation and pathogenicity of Sporisorium scitamineum.

Sugarcane smut caused by Sporisorium scitamineum seriously impairs sugarcane production and quality. Sexual mating/filamentation is a critical step of S. scitamineum pathogenesis, yet the regulatory mechanisms are not fully understood. In this study, we identified the SsAGA, SsODC, and SsSAMDC genes, which are involved in polyamine biosynthesis in S. scitamineum. Deletion of SsODC led to complete loss of filamentous growth after sexual mating, and deletion of SsAGA or SsSAMDC caused reduced filamentation. Double deletion of SsODC and SsSAMDC resulted in auxotrophy for putrescine (PUT) and spermidine (SPD) when grown on minimal medium (MM), indicating that these two genes encode enzymes that are critical for PUT and SPD biosynthesis. We further showed that low PUT concentrations promoted S. scitamineum filamentation, while high PUT concentrations suppressed filamentation. Disrupted fungal polyamine biosynthesis also resulted in a loss of pathogenicity and reduced fungal biomass within infected plants at the early infection stage. SPD formed a gradient from the diseased part to nonsymptom parts of the cane stem, suggesting that SPD is probably favourable for fungal virulence. Mutants of the cAMP-PKA (SsGPA3-SsUAC1-SsADR1) signalling pathway displayed up-regulation of the SsODC gene and elevated intracellular levels of PUT. SsODC directly interacted with SsGPA3, and sporidia of the ss1uac1ΔodcΔ mutant displayed abundant pseudohyphae. Furthermore, we found that elevated PUT levels caused accumulation of intracellular reactive oxygen species (ROS), probably by suppressing transcription of ROS-scavenging enzymes, while SPD played the opposite role. Overall, our work proves that polyamines play important roles in the pathogenic development of sugarcane smut fungus, probably by collaboratively regulating intracellular redox homeostasis with the cAMP-PKA signalling pathway.

Transmission of Xanthomonas albilineans by the spittlebug, Mahanarva fimbriolata (Hemiptera: Cercopidae), in Brazil: first report of an insect vector for the causal agent of sugarcane leaf scald.

Leaf scald is a destructive sugarcane disease caused by the bacterium Xanthomonas albilineans (Ashby) Dowson. This pathogen presents the gene cluster SPI-1 T3SS, a conserved feature in pathogens vectored by animals. In this study, the competence of Mahanarva fimbriolata (Stål), a spittlebug commonly found in sugarcane fields in Brazil, was evaluated for the transmission of X. albilineans. Artificial probing assays were conducted to investigate the ability of M. fimbriolata adults to acquire X. albilineans from artificial diets containing the pathogen with subsequent inoculation of X. albilineans into pathogen-free diets. Plant probing assays with M. fimbriolata adults were conducted to evaluate the acquisition of X. albilineans from diseased source plants and subsequent inoculation of healthy recipient sugarcane plants. The presence of X. albilineans DNA in saliva/diet mixtures of the artificial probing assays and both insects and plants of the plant probing assays were checked using TaqMan assays. The artificial probing assays showed that M. fimbriolata adults were able to acquire and inoculate X. albilineans in diets. Plant probing assays confirmed the competence of M. fimbriolata to transmit X. albilineans to sugarcane. Over the entire experiment, 42% of the insects had acquired the pathogen and successful inoculation of the pathogen occurred in 18% of the recipient-susceptible sugarcane plants at 72 or 96 h of inoculation access period. Assays evidenced the vector competence of M. fimbriolata for transmission of X. albilineans, opening new pathways for investigating the biology and the economic impacts of the interaction between X. albilineans and M. fimbriolata.

Effects of Pesticides on Red Rot of Planted Sugarcane.

Red rot, caused by Colletotrichum falcatum, is an important constraint to sugarcane production. In Louisiana, red rot primarily affects planted seed-cane and is more severe when billets (stalk sections) are planted rather than whole stalks. At planting, application of seed-treatment pesticides, particularly a combination of a fungicide and the insecticide thiamethoxam, has improved stand establishment and increased yields in billet plantings in Louisiana. However, information on the effect of chemicals on disease development is lacking. Greenhouse experiments were conducted to evaluate stalk rot symptom severity and initial plant growth for billets dip-treated with a combination of the fungicides azoxystrobin and propiconazole, thiamethoxam, a combination of both fungicides and the insecticide, and, as a control, untreated billets. Reductions in disease severity recorded for different treatments were similar for billets inoculated with the fungus or exposed to natural inoculum. Disease severity was consistently reduced by the combination treatment, while reductions resulting from treatment with fungicides and insecticide alone were variable. Reductions occurred for both internode and node rot severity. The effects of pesticide treatments on plant growth after 6 weeks were minor; however, there was evidence of disease adversely affecting germination, particularly for nontreated billets exposed to natural inoculum, where germination was reduced by one third. The treatments that reduced disease severity prevented this reduction. The results provide evidence that reduction in disease severity is an important contributor to the stand establishment and yield improvements observed for treated billets in field experiments.

Designing and validation of specific primers for the quantitative detection of bacteria in sugarcane inoculant.

Endophytic diazotrophic plant growth-promoting bacteria Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Paraburkholderia tropica (Ppe8T), Gluconacetobacter diazotrophicus (Pal5T), and Nitrospirillum amazonense (CBAmC) have been used as inoculants for sugarcane. The genome sequences of these strains were used to design a set of specific primers for the real-time PCR (qPCR) assay. Primer specificity was confirmed by conventional PCR using the genomic DNAs of 25 related bacterial species and the five target strains. The qPCR assays were conducted using root and shoot samples from two sugarcane varieties (RB867515 and RB92579). These samples were collected both with and without inoculation, using the target strains specified in this study. The sugarcane plants were grown in a greenhouse, utilizing a substrate composed of sterile sand and vermiculite in a 2:1 ratio, for a duration of 55 days. The primers designed for this study successfully amplified target DNA fragments from each of the bacterial species, enabling their differentiation at the species level. The total bacterial population present in the sugarcane quantified using qPCR was on average 105.2 cells g-1 of fresh tissue. Across both evaluated varieties, it was observed that the population of inoculated bacteria tended to decrease over time and became more concentrated in the sugarcane roots compared to the aerial parts. The qPCR results suggest that both the host and the microbes influence the endophytic population and the bacterial number decreases with plant age.

Enzymatic potential of endophytic fungi: xylanase production by Colletotrichum boninense from sugarcane biomass.

Endophytic fungi constitute a major part of the still unexplored fungal diversity and have gained interest as new biological sources of natural active compounds, including enzymes. Endophytic fungi were isolated from soybean leaves and initially screened on agar plates for the production of CMCase (carboxymethylcellulase), xylanase, amylase and protease. The highest Enzymatic Indexes (IE) were verified for xylanase (2.14 and 1.31) with the fungi M6-A6P5F2 and M12-A5P3F1.2 and CMCase (1.92 and 1.62) with the fungi M13-A9P2F1 and M12-A5P3F1.2, respectively. The production of xylanase and CMCase by the selected fungi was evaluated in submerged cultivation using beechwood xylan and carboxymethylcellulose (CMC), as well as sugarcane straw and bagasse in different ratios as carbon sources. Both types of lignocellulosic biomass proved to be good inducers of enzymatic activity. The best xylanase producer among the isolates was identified as Colletotrichum boninense. With this fungus, the highest xylanase activity was obtained with a sugarcane straw-bagasse mixture in a 50:50 ratio (383.63 U mL-1), a result superior to that obtained with the use of beechwood xylan (296.65 U mL-1). Regardingthe kinetic behavior of the crude xylanase, there was found optimal pH of 5.0 and optimal temperatures of 50°C and 60°C. At 40°C and 50°C, xylanase retained 87% and 76% of its initial catalytic activity, respectively. These results bring new perspectives on bioprospecting endophytic fungi for the production of enzymes, mainly xylanase, as well as the exploitation of agro-industrial by-products, such as sugarcane straw and bagasse.

Survival of five diazotrophic bacterial strains in different substrates used in sugarcane seedling propagation.

An inoculant containing selected bacterial strains can be easily applied during the nursery please process, but in this case, substrate composition can affect its survival and, in consequence, bacterial colonization. The aim of this study was to evaluate the survival of five diazotrophic bacterial strains/species applied individually on 13 different substrates used in sugarcane seedling nurseries considering an active population higher than 105 cells g-1. In addition, one experiment was performed using two commercial substrates, coconut fiber, and Multiplant™, inoculated or not with a mixture of five bacterial strains to evaluate plant growth after 30 days. These strains are combined inoculants selected for sugarcane acting as plant growth promoters. Bacterial counts were determined every seven days using the Most Probable Number technique with four different semi-solid N-free media specific for each strain tested over 35 days. The survival order, independent of the substrate tested, was: Paraburkholderia tropica Pt-PPe8T > Nitrospirillum amazonense Na-CBAMc > Herbaspirillum seropedicae Hs-HRC54 = H. rubrisubalbicans Hr-HCC103 > Gluconacetobacter diazotrophicus Gd-PAL5T. All tested substrates influenced the bacterial survival, especially after 21 days of incubation. The population size can be partially controlled by the substrate pH and stimulated by the addition of slow-release fertilizer. Besides the differences in the bacterial population present in the two commercial substrates, plant growth was found to be stimulated by the inoculated bacteria, depending on the substrate and its sugarcane cultivar tested. The selection of a substrate used to produce new plantlets of sugarcane can contribute to bacterial survival and improve bacterial colonization.

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification.

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Genome Organization of Four Brazilian Xanthomonas albilineans Strains Does Not Correlate with Aggressiveness.

An integrative approach combining genomics, transcriptomics, and cell biology is presented to address leaf scald disease, a major problem for the sugarcane industry. To gain insight into the biology of the causal agent, the complete genome sequences of four Brazilian Xanthomonas albilineans strains with differing virulence capabilities are presented and compared to the GPEPC73 reference strain and FJ1. Based on the aggressiveness index, different strains were compared: Xa04 and Xa11 are highly aggressive, Xa26 is intermediate, and Xa21 is the least, while, based on genome structure, Xa04 shares most of its genomic features with Xa26, and Xa11 share most of its genomic features with Xa21. In addition to presenting more clustered regularly interspaced short palindromic repeats (CRISPR) clusters, four more novel prophage insertions are present than the previously sequenced GPEPC73 and FJ1 strains. Incorporating the aggressiveness index and in vitro cell biology into these genome features indicates that disease establishment is not a result of a single determinant factor, as in most other Xanthomonas species. The Brazilian strains lack the previously described plasmids but present more prophage regions. In pairs, the most virulent and the least virulent share unique prophages. In vitro transcriptomics shed light on the 54 most highly expressed genes among the 4 strains compared to ribosomal proteins (RPs), of these, 3 outer membrane proteins. Finally, comparative albicidin inhibition rings and in vitro growth curves of the four strains also do not correlate with pathogenicity. In conclusion, the results disclose that leaf scald disease is not associated with a single shared characteristic between the most or the least pathogenic strains. IMPORTANCE An integrative approach is presented which combines genomics, transcriptomics, and cell biology to address leaf scald disease. The results presented here disclose that the disease is not associated with a single shared characteristic between the most pathogenic strains or a unique genomic pattern. Sequence data from four Brazilian strains are presented that differ in pathogenicity index: Xa04 and Xa11 are highly virulent, Xa26 is intermediate, and Xa21 is the least pathogenic strain, while, based on genome structure, Xa04 shares with Xa26, and Xa11 shares with X21 most of the genome features. Other than presenting more CRISPR clusters and prophages than the previously sequenced strains, the integration of aggressiveness and cell biology points out that disease establishment is not a result of a single determinant factor as in other xanthomonads.

Engineered Sucrose Metabolism Improves the Smut Disease Suppression Potency of Pseudomonas sp. ST4.

Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.

Assessment of the rhizosphere fungi and bacteria recruited by sugarcane during smut invasion.

Whip smut is one of the most serious and widely spread sugarcane diseases. Plant-associated microbes play various roles in conferring advantages to the host plant. Understanding the microbes associated with sugarcane roots will help develop strategies for the biocontrol of smut. Therefore, the present study explored microbe-mediated sugarcane response to smut invasion via 16S rRNA and ITS metabarcoding survey of the rhizosphere soils of resistant and susceptible sugarcane varieties. The bacterial and fungal diversity in the rhizosphere soils differed between the resistant and susceptible varieties. The bacterial genera Sphingomonas, Microcoleus_Es-Yyy1400, Marmoricola, Reyranella, Promicromonospora, Iamia, Phenylobacterium, Aridibacter, Actinophytocola, and Edaphobacter and one fungal genus Cyphellophora were found associated with smut resistance in sugarcane. Detailed analysis revealed that the majority of bacteria were beneficial, including the actinomycete Marmoricola and Iamia and Reyranella with denitrification activity. Analysis of bacterial network interaction showed that three major groups interacted during smut invasion. Meanwhile, seven of these genera appeared to interact and promote each other's growth. Finally, functional annotation based on the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database predicted that the abundant bacteria are dominated by oxygenic photoautotrophy, photoautotrophy, and phototrophy functions, which may be related to smut resistance in sugarcane. The present study thus provides new insights into the dynamics of the sugarcane rhizosphere microbial community during smut invasion.

Roles of ß-Indole Acetic Acid (IAA) Producing Endophytic Bacteria on the Recovery of Plant Growth and Survival Ability of Sugarcane Infected White Leaf Disease (SWLD).

ß-Indole acetic acid is produced in the rhizosphere by endophytic bacteria and promotes plant growth. Effects of bacterial IAA producers (BIPs) on plant growth and recovery of sugarcane seedlings infected with phytoplasma causing white leaf disease (SWLD) were examined. Fifty-five endophytic bacteria isolated from rice roots were collected from the Mekong River Delta, Vietnam. Seven isolates showed ß-Indole acetic acid production in culture medium supplemented with tryptophan. Interestingly, two of them (BC17 and BTII2) produced the highest ß-Indole acetic acid after 4 days of culture. Based on 16S rRNA sequences and phylogenetic analysis, the BC17 and BTII2 isolates were identified as Delftia lacustris and Rahnella aquatilis, respectively. Plant growth induced by the BC17 and BTII2 isolates showed statistically significant differences in height, root length and fresh weight of rice seedlings compared with non-treatment as the control. Treatment of two bacterial isolates in SWLD infected sugarcane plants also showed differences in height of sugarcane seedlings, while gradual symptoms of exposure decreased plant mortality compared to non-treatment as the control. BIPs were shown to be efficient biofertilizer inoculants that promoted plant growth and also ameliorated damage caused by phytoplasma-associated diseases at the sugarcane seedling stage.

Synergistic Effects of Organosilicon and Cu(OH)2 in Controlling Sugarcane Leaf Scald Disease.

Sugarcane leaf scald is a systemic disease caused by Xanthomonas albilineans that limits sugarcane yield and quality. Previous research has shown that exogenous application of copper hydroxide to plants is effective in controlling this disease. However, long-term bactericide use causes serious "3R" problems: resistance, resurgence, and residue. It is therefore urgent to discover new methods for the improvement of bactericide efficiency and efficacy. In the present study, disease index values for leaf scald were measured in sugarcane seedlings over time to determine the effects of different concentrations of copper hydroxide, types of silicon additive, and treatment timing after inoculation with X. albilineans on controlling sugarcane leaf scald disease. Our results show copper hydroxide mixed with organosilicon additive could improve the bactericide efficiency and efficacy and reduce the growth of pathogenic bacteria, even at a reduced concentration in both laboratory and field conditions. This study provides an important practical model for controlling sugarcane leaf scald disease by reducing the concentration of bactericide and increasing its efficacy in sugarcane fields.

Comparative genome analysis unravels pathogenicity of Xanthomonas albilineans causing sugarcane leaf scald disease.

BACKGROUND: Xanthomonas is a genus of gram-negative bacterium containing more than 35 species. Among these pathogenic species, Xanthomonas albilineans (Xal) is of global interest, responsible for leaf scald disease in sugarcane. Another notable Xanthomonas species is Xanthomonas sachari (Xsa), a sugarcane-associated agent of chlorotic streak disease. RESULT: The virulence of 24 Xanthomonas strains was evaluated by disease index (DI) and Area Under Disease Progress Curve (AUDPC) in the susceptible inoculated plants (GT 46) and clustered into three groups of five highly potent, seven mild virulent, and twelve weak virulent strains. The highly potent strain (X. albilineans, Xal JG43) and its weak virulent related strain (X. sacchari, Xsa DD13) were sequenced, assembled, and annotated in the circular genomes. The genomic size of JG43 was smaller than that of DD13. Both strains (JG43 and DD13) lacked a Type III secretory system (T3SS) and T6SS. However, JG43 possessed Salmonella pathogenicity island-1 (SPI-1). More pathogen-host interaction (PHI) genes and virulent factors in 17 genomic islands (GIs) were detected in JG43, among which six were related to pathogenicity. Albicidin and a two-component system associated with virulence were also detected in JG43. Furthermore, 23 Xanthomonas strains were sequenced and classified into three categories based on Single Nucleotide Polymorphism (SNP) mutation loci and pathogenicity, using JG43 as a reference genome. Transitions were dominant SNP mutations, while structural variation (SV) is frequent intrachromosomal rearrangement (ITX). Two essential genes (rpfC/rpfG) of the two-component system and another gene related to SNP were mutated to understand their virulence effect. The mutation of rpfG resulted in a decrease in pathogenicity. CONCLUSION: These findings revealed virulence of 24 Xanthomonas strains and variations by 23 Xanthomonas strains. We sequenced, assembled, and annotated the circular genomes of Xal JG43 and Xsa DD13, identifying diversity detected by pathogenic factors and systems. Furthermore, complete genomic sequences and sequenced data will provide a theoretical basis for identifying pathogenic factors responsible for sugarcane leaf scald disease.

Towards defining the core Saccharum microbiome: input from five genotypes.

BACKGROUND: Plant microbiome and its manipulation inaugurate a new era for plant biotechnology with the potential to benefit sustainable crop production. Here, we used the large-scale 16S rDNA sequencing analysis to unravel the dynamic, structure, and composition of exophytic and endophytic microbial communities in two hybrid commercial cultivars of sugarcane (R570 and SP80-3280), two cultivated genotypes (Saccharum officinarum and Saccharum barberi) and one wild species (Saccharum spontaneum). RESULTS: Our analysis identified 1372 amplicon sequence variants (ASVs). The microbial communities' profiles are grouped by two, root and bulk soils and stem and leave when these four components are compared. However, PCoA-based data supports that endophytes and epiphytes communities form distinct groups, revealing an active host-derived mechanism to select the resident microbiota. A strong genotype-influence on the assembly of microbial communities in Saccharum ssp. is documented. A total of 220 ASVs persisted across plant cultivars and species. The ubiquitous bacteria are two potential beneficial bacteria, Acinetobacter ssp., and Serratia symbiotica. CONCLUSIONS: The results presented support the existence of common and cultivar-specific ASVs in two commercial hybrids, two cultivated canes and one species of Saccharum across tissues (leaves, stems, and roots). Also, evidence is provided that under the experimental conditions described here, each genotype bears its microbial community with little impact from the soil conditions, except in the root system. It remains to be demonstrated which aspect, genotype, environment or both, has the most significant impact on the microbial selection in sugarcane fields.

Influence of Sugarcane Variety on Rhizosphere Microbiota Under Irrigated and Water-Limiting Conditions.

Drought is one of the main problems linked to climate change that is faced by agriculture, affecting various globally important crops, including sugarcane. Environmentally sustainable strategies have been sought to mitigate the effects of climate change on crops. Among them, the use of beneficial microorganisms offers a promising approach. However, it is still necessary to understand the mechanisms that regulate plant-microorganism interactions, in normal situations and under stress. In this work, the rhizosphere metagenomes of two sugarcane varieties, one resistant and the other susceptible to drought, were compared under normal conditions and under water-limiting conditions. The results showed that for the drought-resistant sugarcane variety, bacteria belonging to the order Sphingomonadales and the family Xanthomonadaceae presented increased activities in terms of mobility, colonization, and cell growth. In contrast, the rhizosphere associated with the drought-sensitive variety exhibited increases of bacteria belonging to the family Polyangiaceae, and the genus Streptomyces, with modifications in DNA metabolism and ribosome binding proteins. The results pointed to variation in the rhizosphere microbiota that was modulated by the host plant genotype, revealing potential bacterial candidates that could be recruited to assist plants during water-limiting conditions.

Aminotransferase SsAro8 Regulates Tryptophan Metabolism Essential for Filamentous Growth of Sugarcane Smut Fungus Sporisorium scitamineum.

Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum leads to severe economic losses globally. Sexual mating/filamentation of S. scitamineum is critical for its pathogenicity, as only the dikaryotic hyphae formed after sexual mating are capable of invading the host cane. Our comparative transcriptome analysis showed that the mitogen-activated protein kinase (MAPK) pathway and the AGC kinase Agc1 (orthologous to yeast Rim15), both governing S. scitamineum mating/filamentation, were induced by elevated tryptophol level, supporting a positive regulation of S. scitamineum mating/filamentation by tryptophol. However, the biosynthesis pathway of tryptophol remains unknown in S. scitamineum. Here, we identified an aminotransferase orthologous to the established tryptophan aminotransferase Tam1/Aro8, catalyzing the first step of tryptophan-dependent indole-3-acetic acid (IAA) production as well as that of the Ehrlich pathway for tryptophol production. We designated this S. scitamineum aminotransferase as SsAro8 and found that it was essential for mating/filamentation. Comparative metabolomics analysis revealed that SsAro8 was involved in tryptophan metabolism, likely for producing important intermediate products, including tryptophol. Exogenous addition of tryptophan or tryptophol could differentially restore mating/filamentation in the ssaro8Δ mutant, indicating that in addition to tryptophol, other product(s) of tryptophan catabolism may also be involved in S. scitamineum mating/filamentation regulation. S. scitamineum could also produce IAA, partially dependent on SsAro8 function. Surprisingly, photodestruction of IAA produced the compound(s) able to suppress S. scitamineum growth/differentiation. Lastly, we found that SsAro8 was required for proper biofilm formation, oxidative stress tolerance, and full pathogenicity in S. scitamineum. Overall, our study establishes the aminotransferase SsAro8 as an essential regulator of S. scitamineum pathogenic differentiation, as well as fungus-host interaction, and therefore of great potential as a molecular target for sugarcane smut disease control. IMPORTANCE Sugarcane smut caused by the basidiomycete fungus S. scitamineum leads to massive economic losses in sugarcane plantation globally. Dikaryotic hyphae formation (filamentous growth) and biofilm formation are two important aspects in S. scitamineum pathogenesis, yet the molecular regulation of these two processes was not as extensively investigated as that in the model pathogenic fungi, e.g., Candida albicans, Ustilago maydis, or Cryptococcus neoformans. In this study, a tryptophan aminotransferase ortholog was identified in S. scitamineum, designated SsAro8. Functional characterization showed that SsAro8 positively regulates both filamentous growth and biofilm formation, respectively, via tryptophol-dependent and -independent manners. Furthermore, SsAro8 is required for full pathogenicity and, thus, is a promising molecular target for designing anti-smut strategy.

Interference of bio-control Trichoderma to enhance physical and physiological strength of sugarcane during Pokkah boeng infection.

Tremendous benefits have been derived from the use of fungicides but excessive use of chemical fungicides not only posing threat to human and animal life but also contaminates the prevailing environment. Damage by pathogenic fungi alone causes significant damage to crops like maize, rice, wheat, soybeans, and potatoes. Therefore, it becomes imperative that these diseases are checked and controlled, for which chemical pesticides are being sprayed on plants extensively. Considering the devastating damage and toxicity, the global focus has taken a drift from synthetic chemicals to nature-friendly biological control agents. The present study focuses on the use of biological control agents particularly Trichoderma in sugarcane during Pokkah boeng infection. In the present experiment, twenty promising Trichoderma strains were evaluated for plant growth promotion, lytic enzymes, and physiological and biocontrol activity. Out of the twenty, four potential Trichoderma strains were assessed in the pot experiment viz. T. harzianum strain T28, T41 and T49 and T. aureoviride strain T38. The T. harzianum (T28) showed efficient plant growth-promoting traits as it produced IAA (20.67 µg/ml), phosphorus solubilization (18.57 µg/ml), and cell wall degrading enzymes such as chitinase (24.98 µg/ml) and ß-glucanase (29.98 µg/ml). The interference of biocontrol agent T. harzianum (T28) controlled the disease by 73.55%. Apart from this, the inoculation of Trichoderma (T28) enhanced growth attributes including germination percentage (26.61%), mean tiller number (8.28 tiller/pot), individual cane length (241.5 cm), single cane weight (1.13 kg) and the number of milleable canes (6.00 cane/pot). Improvements in physiological activities at different growth stages of the sugarcane crop were observed based on the photosynthetically active radiation (PAR) on the leaf surface, transpiration rate, stomatal conductance, and photosynthetic rate. Further, improvement in juice quality parameters was also observed as it recorded the highest 0brix, sucrose, and commercial cane sugar by 21.26%, 19.28%, and 13.50%, respectively, by applying T. harzianum strain T28. Thus, results proved that T. harzianum strain T28 may be an effective eco-friendly biocontrol tool for managing Pokkah boeng disease in sugarcane. This is the first report of the biocontrol potential of Trichoderma spp. against Fusarium proliferatum causing Pokkah boeng disease in sugarcane.