Molecular detection of Paracoccidioides spp. in environmental samples from the Southwestern Amazon.

Paracoccidioidomycosis is an infection with the potential for environmental dissemination, especially in regions of hot and humid climate, where human cases have been recorded in the Southwestern Amazon of Brazil, specifically in the state of Acre. Despite studies providing information about the presence of these fungi in soil and animal samples, such as armadillos, further investigations are still needed to determine the epidemiological distribution of the genus Paracoccidioides. The aim of this study was to detect the occurrence of Paracoccidioides fungi in the Southwestern Amazon. To achieve this, 60 soil samples were collected from armadillo burrows on rural properties in the in the municipalities of Acrelândia, Bujari, Plácido de Castro, Rio Branco, Sena Madureira, and Senador Guiomard, located in the state of Acre, Brazil. Fungal DNA was extracted from these samples using the DNEASY® PowerSoil kit-Quiagen, followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-R as internal primers. DNA amplification products of about 380 bp compatible with Paracoccidioides spp. were detected in six samples (10%), being sequenced and identified as P. brasiliensis. These findings indicate that the soils of the Acre state could be considered a potential source for Paracoccidioides spp., suggesting that local infections are likely.

Metabolome Dynamics of Smutted Sugarcane Reveals Mechanisms Involved in Disease Progression and Whip Emission.

Sugarcane smut disease, caused by the biotrophic fungus Sporisorium scitamineum, is characterized by the development of a whip-like structure from the plant meristem. The disease causes negative effects on sucrose accumulation, fiber content and juice quality. The aim of this study was to exam whether the transcriptomic changes already described during the infection of sugarcane by S. scitamineum result in changes at the metabolomic level. To address this question, an analysis was conducted during the initial stage of the interaction and through disease progression in a susceptible sugarcane genotype. GC-TOF-MS allowed the identification of 73 primary metabolites. A set of these compounds was quantitatively altered at each analyzed point as compared with healthy plants. The results revealed that energetic pathways and amino acid pools were affected throughout the interaction. Raffinose levels increased shortly after infection but decreased remarkably after whip emission. Changes related to cell wall biosynthesis were characteristic of disease progression and suggested a loosening of its structure to allow whip growth. Lignin biosynthesis related to whip formation may rely on Tyr metabolism through the overexpression of a bifunctional PTAL. The altered levels of Met residues along with overexpression of SAM synthetase and ACC synthase genes suggested a role for ethylene in whip emission. Moreover, unique secondary metabolites antifungal-related were identified using LC-ESI-MS approach, which may have potential biomarker applications. Lastly, a putative toxin was the most important fungal metabolite identified whose role during infection remains to be established.

Functional analysis of oxidative burst in sugarcane smut-resistant and -susceptible genotypes.

MAIN CONCLUSION: Smut pathogen induced an early modulation of the production and scavenging of reactive oxygen species during defence responses in resistant sugarcane that coincided with the developmental stages of fungal growth. Sporisorium scitamineum is the causal agent of sugarcane smut disease. In this study, we characterized sugarcane reactive oxygen species (ROS) metabolism in response to the pathogen in smut-resistant and -susceptible genotypes. Sporisorium scitamineum teliospore germination and appressorium formation coincided with H2O2 accumulation in resistant plants. The superoxide dismutase (SOD) activity was not responsive in any of the genotypes; however, a higher number of isoenzymes were detected in resistant plants. In addition, related to resistance were lipid peroxidation, a decrease in catalase (CAT), and an increase in glutathione S-transferase (GST) activities and an earlier transcript accumulation of ROS marker genes (CAT3, CATA, CATB, GST31, GSTt3, and peroxidase 5-like). Furthermore, based on proteomic data, we suggested that the source of the increased hydrogen peroxide (H2O2) may be due to a protein of the class III peroxidase, which was inhibited in the susceptible genotype. H2O2 is sensed and probably transduced through overlapping systems related to ascorbate-glutathione and thioredoxin to influence signalling pathways, as revealed by the presence of thioredoxin h-type, ascorbate peroxidase, and guanine nucleotide-binding proteins in the infected resistant plants. Altogether, our data depicted the balance of the oxidative burst and antioxidant enzyme activity in the outcome of this interaction.

RNAseq Transcriptional Profiling following Whip Development in Sugarcane Smut Disease.

Sugarcane smut disease is caused by the biotrophic fungus Sporisorium scitamineum. The disease is characterized by the development of a whip-like structure from the primary meristems, where billions of teliospores are produced. Sugarcane smut also causes tillering and low sucrose and high fiber contents, reducing cane productivity. We investigated the biological events contributing to disease symptoms in a smut intermediate-resistant sugarcane genotype by examining the transcriptional profiles (RNAseq) shortly after inoculating the plants and immediately after whip emission. The overall picture of disease progression suggests that premature transcriptional reprogramming of the shoot meristem functions continues until the emergence of the whip. The guidance of this altered pattern is potentially primarily related to auxin mobilization in addition to the involvement of other hormonal imbalances. The consequences associated with whip emission are the modulation of typical meristematic functions toward reproductive organ differentiation, requiring strong changes in carbon partitioning and energy production. These changes include the overexpression of genes coding for invertases and trehalose-6P synthase, as well as other enzymes from key metabolic pathways, such as from lignin biosynthesis. This is the first report describing changes in the transcriptional profiles following whip development, providing a hypothetical model and candidate genes to further study sugarcane smut disease progression.

Complete Genome Sequence of Sporisorium scitamineum and Biotrophic Interaction Transcriptome with Sugarcane.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

Biochemical and histological characterization of tomato mutants.

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H(2)O(2)) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.