Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.

BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

Comparative mitogenomics of Agaricomycetes: Diversity, abundance, impact and coding potential of putative open-reading frames.

The mitochondrion is an organelle found in eukaryote organisms, and it is vital for different cellular pathways. The mitochondrion has its own DNA molecule and, because its genetic content is relatively conserved, despite the variation of size and structure, mitogenome sequences have been widely used as a promising molecular biomarker for taxonomy and evolution in fungi. In this study, the mitogenomes of two fungal species of Agaricomycetes class, Phellinotus piptadeniae and Trametes villosa, were assembled and annotated for the first time. We used these newly sequenced mitogenomes for comparative analyses with other 55 mitogenomes of Agaricomycetes available in public databases. Mitochondrial DNA (mtDNA) size and content are highly variable and non-coding and intronic regions, homing endonucleases (HEGs), and unidentified ORFs (uORFs) significantly contribute to the total size of the mitogenome. Furthermore, accessory genes (most of them as HEGs) are shared between distantly related species, most likely as a consequence of horizontal gene transfer events. Conversely, uORFs are only shared between taxonomically related species, most probably as a result of vertical evolutionary inheritance. Additionally, codon usage varies among mitogenomes and the GC content of mitochondrial features may be used to distinguish coding from non-coding sequences. Our results also indicated that transposition events of mitochondrial genes to the nuclear genome are not common. Despite the variation of size and content of the mitogenomes, mitochondrial genes seemed to be reliable molecular markers in our time-divergence analysis, even though the nucleotide substitution rates of mitochondrial and nuclear genomes of fungi are quite different. We also showed that many events of mitochondrial gene shuffling probably happened amongst the Agaricomycetes during evolution, which created differences in the gene order among species, even those of the same genus. Altogether, our study revealed new information regarding evolutionary dynamics in Agaricomycetes.

A multiscale study of fungal endophyte communities of the foliar endosphere of native rubber trees in Eastern Amazon.

Hevea brasiliensis is a native hyperdiverse tree species in the Amazon basin with great economic importance since it produces the highest quality natural rubber. H. brasiliensis, in its natural habitat, may harbor fungal endophytes that help defend against phytopathogenic fungi. In this work, we investigated the fungal endophytic communities in two pristine areas in Eastern Amazon (Anavilhanas National Park - ANP and Caxiuanã National Forest - CNF) at different spatial scales: regional, local, individual (tree), and intra-individual (leaflet). Using a culture-based approach, 210 fungal endophytes were isolated from 240 sampling units and assigned to 46 distinct MOTUs based on sequencing of the nrITS DNA. The community compositions of the endophytomes are different at both regional and local scales, dominated by very few taxa and highly skewed toward rare taxa, with many endophytes infrequently isolated across hosts in sampled space. Colletotrichum sp. 1, a probably latent pathogen, was the most abundant endophytic putative species and was obtained from all individual host trees in both study areas. Although the second most abundant putative species differed between the two collection sites, Clonostachys sp. 1 and Trichoderma sp. 1, they are phylogenetically related (Hypocreales) mycoparasites. Thus, they probably exhibit the same ecological function in the foliar endosphere of rubber tree as antagonists of its fungal pathogens.