Austropuccinia psidii uses tetrapolar mating and produces meiotic spores in older infections on Eucalyptus grandis.

Austropuccinia psidii is the causal agent of myrtle rust, a fungal disease that infects over 480 species in the Myrtaceae. A. psidii is a biotrophic pathogen that reproduces sexually and asexually. Sexual reproduction has been previously shown on Syzygium jambos and little is known about its reproductive biology on other hosts or whether populations that were formerly structured by host range can outcross on universally susceptible hosts. We investigated if mating genes in three genomes of A. psidii were under selection as a proxy for whether different strains can reproduce sexually on a shared host. We examined three homologs of the STE3.2 gene, sequences of which were near-identical in the three genomes, and the homeodomain locus, which contained two alleles of two homeodomain genes in each genome. A. psidii likely uses tetrapolar mating. Pheromone/receptor loci were distal to homeodomain loci, and based on haplotypes of a phased assembly, mate compatibility is regulated by multiallelic HD genes and biallelic STE3.2 genes; the third homolog of STE3.2 (STE3.2-1) was present in both haplotypes, and our study supports hypotheses this gene does not regulate mate recognition. Populations of A. psidii formerly structured by host range could potentially outcross on universal hosts based on their related mating genes, however this hypothesis should remain theoretical given the implications for biosecurity. Additionally, we searched for core meiotic genes in genomes of A. psidii, four species of Puccinia, and Sphaerophragmium acaciae through comparative genomics based on 136 meiosis-related orthologous genes modeled from Mycosarcoma maydis. Meiotic genes are conserved in rust fungi at family rank. We analyzed the expression of two meiotic and four mitotic genes of A. psidii on E. grandis over a 28-day time course to validate that identified meiotic genes were upregulated in teliospores. Three mitotic genes were significantly downregulated in samples collected 28 days after inoculation (DAI) compared to 14 DAI. Expression of meiotic genes was significantly up-regulated in samples collected 28 DAI compared to 14 DAI, indicating a temporal switch from production of uredinia (mitotic stage) to telia in the life cycle, which we hypothesize may be in response to leaf ageing.

Specific plant induced biofilm formation in Methylobacterium species.

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.

Characterization of the contaminant bacterial communities in sugarcane first-generation industrial ethanol production.

The industrial ethanolic fermentation process is operated in distilleries, either in fed-batch or continuous mode. A consequence of the large industrial ethanol production is bacterial contamination in the fermentation tanks, which is responsible for significant economic losses. To investigate this community, we accessed the profile of bacterial contaminant from two distilleries in Brazil, each operating a different fermentation mode, throughout sugarcane harvest of 2013-2014. Bacterial communities were accessed through Illumina culture-independent 16S rDNA gene sequencing, and qPCR was used to quantify total bacteria abundance. Both ethanol production modes showed similar bacterial abundance, around 105 gene copies/mL. 16S rDNA sequencing showed that 92%-99% of the sequences affiliated to Lactobacillus genus. Operational taxonomic units differently represented belonged mainly to Lactobacillus, but also to Weissella, Pediococcus, Acetobacter and Anaeosporobacter, although in lower abundance. Alpha-diversity only showed a correlation through the fermentation tanks in continuous mode, where it was always higher in the second and third tanks. Beta-diversity clearly separated the two distilleries and metagenome prediction reinforces clusterization within distilleries. Despite certain variations between bacterial community in the distilleries throughout harvest season, Lactobacillus were the main genera reported in both distilleries and bacterial community seemed to persist along time, suggesting bacterial reinfestation.

Molecular variability and genetic relationship among Brazilian strains of the sugarcane smut fungus.

Sporisorium scitamineum is the fungus that causes sugarcane smut disease. Despite of the importance of sugarcane for Brazilian agribusiness and the persistence of the pathogen in most cropping areas, genetic variation studies are still missing for Brazilian isolates. In this study, sets of isolates were analyzed using two molecular markers (AFLP and telRFLP) and ITS sequencing. Twenty-two whips were collected from symptomatic plants in cultivated sugarcane fields of Brazil. A total of 41 haploid strains of compatible mating types were selected from individual teliospores and used for molecular genetic analyses. telRFLP and ITS analyses were expanded to six Argentine isolates, where the sugarcane smut was first recorded in America. Genetic relationship among strains suggests the human-mediated dispersal of S. scitamineum within the Brazilian territory and between the two neighboring countries. Two genetically distinct groups were defined by the combined analysis of AFLP and telRFLP. The opposite mating-type strains derived from single teliospores were clustered together into these main groups, but had not always identical haplotypes. telRFLP markers analyzed over two generations of selfing and controlled outcrossing confirmed the potential for emergence of new variants and occurrence of recombination, which are relevant events for evolution of virulence and environmental adaptation.

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.

Specific plant induced biofilm formation in Methylobacterium species

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.

Molecular characterization of a beta-1,4-endoglucanase from an endophytic Bacillus pumilus strain.

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a beta-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5-8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6-8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50-60 degrees C are required over long incubation periods.