An Integrative View of the Phyllosphere Mycobiome of Native Rubber Trees in the Brazilian Amazon.

The rubber tree, Hevea brasiliensis, is a neotropical Amazonian species. Despite its high economic value and fungi associated with native individuals, in its original area in Brazil, it has been scarcely investigated and only using culture-dependent methods. Herein, we integrated in silico approaches with novel field/experimental approaches and a case study of shotgun metagenomics and small RNA metatranscriptomics of an adult individual. Scientific literature, host fungus, and DNA databases are biased to fungal taxa, and are mainly related to rubber tree diseases and in non-native ecosystems. Metabarcoding retrieved specific phyllospheric core fungal communities of all individuals, adults, plantlets, and leaves of the same plant, unravelling hierarchical structured core mycobiomes. Basidiomycotan yeast-like fungi that display the potential to produce antifungal compounds and a complex of non-invasive ectophytic parasites (Sooty Blotch and Flyspeck fungi) co-occurred in all samples, encompassing the strictest core mycobiome. The case study of the same adult tree (previously studied using culture-dependent approach) analyzed by amplicon, shotgun metagenomics, and small RNA transcriptomics revealed a high relative abundance of insect parasite-pathogens, anaerobic fungi and a high expression of Trichoderma (a fungal genus long reported as dominant in healthy wild rubber trees), respectively. Altogether, our study unravels new and intriguing information/hypotheses of the foliar mycobiome of native H. brasiliensis, which may also occur in other native Amazonian trees.

Comparative genome analyses suggest a hemibiotrophic lifestyle and virulence differences for the beech bark disease fungal pathogens Neonectria faginata and Neonectria coccinea.

Neonectria faginata and Neonectria coccinea are the causal agents of the insect-fungus disease complex known as beech bark disease (BBD), known to cause mortality in beech forest stands in North America and Europe. These fungal species have been the focus of extensive ecological and disease management studies, yet less progress has been made toward generating genomic resources for both micro- and macro-evolutionary studies. Here, we report a 42.1 and 42.7 mb highly contiguous genome assemblies of N. faginata and N. coccinea, respectively, obtained using Illumina technology. These species share similar gene number counts (12,941 and 12,991) and percentages of predicted genes with assigned functional categories (64 and 65%). Approximately 32% of the predicted proteomes of both species are homologous to proteins involved in pathogenicity, yet N. coccinea shows a higher number of predicted mitogen-activated protein kinase genes, virulence determinants possibly contributing to differences in disease severity between N. faginata and N. coccinea. A wide range of genes encoding for carbohydrate-active enzymes capable of degradation of complex plant polysaccharides and a small number of predicted secretory effector proteins, secondary metabolite biosynthesis clusters and cytochrome oxidase P450 genes were also found. This arsenal of enzymes and effectors correlates with, and reflects, the hemibiotrophic lifestyle of these two fungal pathogens. Phylogenomic analysis and timetree estimations indicated that the N. faginata and N. coccinea species divergence may have occurred at ∼4.1 million years ago. Differences were also observed in the annotated mitochondrial genomes as they were found to be 81.7 kb (N. faginata) and 43.2 kb (N. coccinea) in size. The mitochondrial DNA expansion observed in N. faginata is attributed to the invasion of introns into diverse intra- and intergenic locations. These first draft genomes of N. faginata and N. coccinea serve as valuable tools to increase our understanding of basic genetics, evolutionary mechanisms and molecular physiology of these two nectriaceous plant pathogenic species.

Diaporthe Seed Decay of Soybean [Glycine max (L.) Merr.] Is Endemic in the United States, But New Fungi Are Involved.

Diaporthe seed decay can compromise seed quality in soybean [Glycine max (L.) Merr.] in the warm and humid production areas of the United States during crop maturation. In the current study, 45 isolates of Diaporthe were recovered from seed sampled from soybean fields affected by Diaporthe-associated diseases in eight U.S. states in 2017. The isolates obtained belonged to 10 species of Diaporthe based on morphology and phylogenetic analyses of the internal transcribed spacer, partial translation elongation factor 1-α, and ß-tubulin gene sequences. The associated species included D. aspalathi, D. caulivora, D. kongii, D. longicolla, D. sojae, D. ueckerae, D. unshiuensis, and three novel fungi, D. bacilloides, D. flavescens, and D. insulistroma. One isolate each of the 10 species was examined for pathogenicity on seed of cultivar Sava under controlled conditions. Seven days postinoculation, significant differences in the percentages of decayed seeds and seedling necrosis were observed among the isolates and the noninoculated control (P < 0.0001). While the isolates of D. bacilloides, D. longicolla, and D. ueckerae caused a significantly greater percentage of decayed seeds (P < 0.0001), the isolate of D. aspalathi caused the greatest seedling necrosis (P < 0.0001). The observation of new fungi causing Diaporthe seed decay suggests the need for a more comprehensive survey in U.S. soybean producing areas since members of the genus Diaporthe appear to form a complex that causes seed decay.

Genome Resources for Thelonectria rubi, the Causal Agent of Nectria Canker of Caneberry.

Thelonectria rubi is the causal agent of Nectria canker of Rubus spp. Here, we report a high-quality draft genome sequence for this pathogen, which also represents the first genome sequence for a Thelonectria species. The genome assembly was 44.6 Mb in size, assembled into 669 scaffolds and consisting of 12,973 predicted protein-coding genes. The availability of genome data for T. rubi provides a critical additional resource for an important plant pathogen and will be useful for fungal biology, comparative genomic, taxonomic and population studies of this and related species.

Exploration of stem endophytic communities revealed developmental stage as one of the drivers of fungal endophytic community assemblages in two Amazonian hardwood genera.

Many aspects of the dynamics of tropical fungal endophyte communities are poorly known, including the influence of host taxonomy, host life stage, host defence, and host geographical distance on community assembly and composition. Recent fungal endophyte research has focused on Hevea brasiliensis due to its global importance as the main source of natural rubber. However, almost no data exist on the fungal community harboured within other Hevea species or its sister genus Micrandra. In this study, we expanded sampling to include four additional Hevea spp. and two Micrandra spp., as well as two host developmental stages. Through culture-dependent and -independent (metagenomic) approaches, a total of 381 seedlings and 144 adults distributed across three remote areas within the Peruvian Amazon were sampled. Results from both sampling methodologies indicate that host developmental stage had a greater influence in community assemblage than host taxonomy or locality. Based on FunGuild ecological guild assignments, saprotrophic and mycotrophic endophytes were more frequent in adults, while plant pathogens were dominant in seedlings. Trichoderma was the most abundant genus recovered from adult trees while Diaporthe prevailed in seedlings. Potential explanations for that disparity of abundance are discussed in relation to plant physiological traits and community ecology hypotheses.

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.

Unexpected diversity of basidiomycetous endophytes in sapwood and leaves of Hevea.

Research on fungal endophytes has expanded dramatically in recent years, but little is known about the diversity and ecological roles of endophytic basidiomycetes. Here we report the analysis of 310 basidiomycetous endophytes isolated from wild and planted populations of the rubber tree genus, Hevea. Species accumulation curves were nonasymptotic, as in the majority of endophyte surveys, indicating that more sampling is needed to recover the true diversity of the community. One hundred eighteen OTUs were delimited, representing nine orders of Basidiomycota (Agaricales, Atheliales, Auriculariales, Cantharellales, Hymenochaetales, Polyporales, Russulales, Septobasidiales, Tremellales). The diversity of basidiomycetous endophytes found inhabiting wild populations of Hevea was comparable to that present in plantations. However, when samples were segregated by tissue type, sapwood of wild populations was found to contain a higher number of species than sapwood of planted trees. Seventy-five percent of isolates were members of the Polyporales, the majority in the phlebioid clade. Most of the species belong to clades known to cause a white-rot type of wood decay. Two species in the insect-associated genus Septobasidium were isolated. The most frequently isolated genera included Bjerkandera, Ceriporia, Phanerochaete, Phlebia, Rigidoporus, Tinctoporellus, Trametes (Polyporales), Peniophora, Stereum (Russulales) and Coprinellus (Agaricales), all of which have been reported as endophytes from a variety of hosts, across wide geographic locations. Literature records on the geographic distribution and host association of these genera revealed that their distribution and substrate affinity could be extended if the endophytic niche was investigated as part of fungal biodiversity surveys.

Novel endophytic lineages of Tolypocladium provide new insights into the ecology and evolution of Cordyceps-like fungi.

The objective of this study was to identify a group of unknown endophytic fungal isolates from the living sapwood of wild and planted Hevea (rubber tree) populations. Three novel lineages of Tolypocladium are described based on molecular and morphological data. Findings from this study open a window for novel hypotheses regarding the ecology and role of endophytes within plant communities as well as trait evolution and potential forces driving diversification of Cordyceps-like fungi. This study stresses the importance of integrating asexual and sexual fungal states for a more complete understanding of the natural history of this diverse group. In addition, it highlights the study of fungi in the sapwood of tropical trees as habitat for the discovery of novel fungal lineages and substrate associations.

Impact of organic and conventional management on the phyllosphere microbial ecology of an apple crop.

Bacterial communities associated with the phyllosphere of apple trees (Malus domestica cv. Enterprise) grown under organic and conventional management were assessed to determine if increased biological food safety risks might be linked with the bacterial communities associated with either treatment. Libraries of 16S rRNA genes were generated from phyllosphere DNA extracted from a wash made from the surfaces of leaves and apples from replicated organic and conventional treatments. 16S rRNA gene libraries were analyzed with software designed to identify statistically significant differences between bacterial communities as well as shared and unique phylotypes. The identified diversity spanned eight bacterial phyla and 14 classes in the pooled organic and conventional libraries. Significant differences between organic and conventional communities were observed at four of six time points (P < 0.05). Despite the identification of significantly diverse microfloras associated with organic and conventional treatments, no detectable differences in the presence of potential enteric pathogens could be associated with either organic or conventional management. Neither of the bacterial genera most commonly associated with produce-related illness outbreaks (Salmonella and Escherichia) was observed in any of the libraries. The impressive bacterial diversity that was documented in this study provides a valuable contribution to our developing understanding of the total microbial ecology associated with the preharvest phyllospheres of food crops. The fact that organic and conventional phyllosphere bacterial communities were significantly different at numerous time points suggests that crop management methods may influence the bacterial consortia associated with the surfaces of fruits and vegetables.