Interkingdom and intrakingdom interactions in the microbiome of Heterobasidion fruiting body and associated decayed woody tissues.

IMPORTANCE: We applied macro- (forest stand and forest management) and micro-scale (bacterial and fungal community) analyses for a better understanding of the Heterobasidion pathosystem and associated wood decay process. The core microbiome, as defined by hierarchy analysis and a consistent model, and environmental factors correlation with the community assembly were found to be novel.

Thermal Tolerance Data and Molecular Identification Are Useful for the Diagnosis, Control and Modeling of Diseases Caused by Thielaviopsis paradoxa.

Several economically important diseases of forest trees and agricultural crops in many parts of the world have been linked to the ascomycete fungal pathogen Thielaviopsis paradoxa. This study compared the growth rate of 41 isolates of T. paradoxa sourced from different hosts and two countries (Nigeria and Papua New Guinea (PNG)) under six temperature levels (22 °C, 25 °C, 30 °C, 32 °C, 34 °C and 35 °C). Phylogenetic relationships were obtained from the analysis of their nuclear ribosomal DNA internal transcribed sequence (ITS) data. While all the isolates from PNG and few from Nigeria grew optimally between 22 °C and 32 °C, the majority had their highest growth rate (2.9 cm/day) between 25 °C and 32 °C. Growth performances were generally low between 34 °C and 35 °C; no isolate from the sugar cane grew at these high temperatures. The oil palm isolate DA029 was the most resilient, with the highest growth rate (0.97 cm/day) at 35 °C. Phylogenetic analysis delineated five clusters: a very large clade which accommodates the majority (30 Nigerian and 3 PNG oil palm isolates) and four small clades containing two members each. To a large extent, the clustering pattern failed to address the temperature-isolate relationship observed. However, only the four small clades represent isolates with similar temperature tolerances. It is most likely that wider and robust analyses with more diverse isolates and genetic markers will provide better insight on thermal resilience of T. paradoxa. Additionally, future research to establish relationships between vegetative growth at different temperatures and of different pathogenicity and disease epidemiology merits being explored. The results might provide useful information for the formulation of effective management and control strategies against the pathogen, especially in this era of climate change.

Analysis of Transcriptome and Terpene Constituents of Scots Pine Genotypes Inherently Resistant or Susceptible to Heterobasidion annosum.

Root and stem rot caused by Heterobasidion annosum is a severe problem in boreal Scots pine. Dissecting the features of disease resistance is generally an essential step in resistance breeding in plants and forest trees. In this study, we explored inherent resistance factors of Scots pine against H. annosum. A total of 236 families consisting of 85 full-sib (FS), 35 half-sib population mix (HSpm), and 116 half-sib (HS) families of Scots pine seedlings were inoculated with a H. annosum isolate. We sampled needle tissues before inoculation for terpene measurements and RNA sequencing. Based on the lesion area, the extremes of 12 resistant and 12 susceptible families were selected for further analyses. Necrotic lesions resulting from fungal infection were in a weak to moderate relationship with the plant height. Monoterpenes were the principal terpene compounds observed in Scots pine seedlings. Concentrations of 3-carene were significantly higher in pine genotypes inherently resistant compared with susceptible seedlings. By contrast, susceptible genotypes had significantly higher proportions of α-pinene. Gene ontology analysis of differential expressed transcripts (DETs) revealed that response to biotic factors was enriched in resistant seedlings. Functional characterization of individual DETs revealed that higher expression of transcripts involved in response to abiotic stress was common in susceptible genotypes. This observation was supported by the annotation of hub genes in a key module that was significantly correlated with the lesion trait through weighted gene co-expression network analysis (WGCNA) of 16 HS and HSpm samples. These findings contribute to our understanding of constitutive resistance factors of Scots pine against Heterobasidion root and stem rot diseases.

The dark septate endophyte Phialocephala sphaeroides suppresses conifer pathogen transcripts and promotes root growth of Norway spruce.

Plant-associated microbes including dark septate endophytes (DSEs) of forest trees play diverse functional roles in host fitness including growth promotion and increased defence. However, little is known about the impact on the fungal transcriptome and metabolites during tripartite interaction involving plant host, endophyte and pathogen. To understand the transcriptional regulation of endophyte and pathogen during co-infection, Norway spruce (Picea abies) seedlings were infected with DSE Phialocephala sphaeroides, or conifer root-rot pathogen Heterobasidion parviporum, or both. Phialocephala sphaeroides showed low but stable transcripts abundance (a decrease of 40%) during interaction with Norway spruce and conifer pathogen. By contrast, H. parviporum transcripts were significantly reduced (92%) during co-infection. With RNA sequencing analysis, P. sphaeroides experienced a shift from cell growth to anti-stress and antagonistic responses, while it repressed the ability of H. parviporum to access carbohydrate nutrients by suppressing its carbohydrate/polysaccharide-degrading enzyme machinery. The pathogen on the other hand secreted cysteine peptidase to restrict free growth of P. sphaeroides. The expression of both DSE P. sphaeroides and pathogen H. parviporum genes encoding plant growth promotion products were equally detected in both dual and tripartite interaction systems. This was further supported by the presence of tryptophan-dependent indolic compound in liquid culture of P. sphaeroides. Norway spruce and Arabidopsis seedlings treated with P. sphaeroides culture filtrate exhibited auxin-like phenotypes, such as enhanced root hairs, and primary root elongation at low concentration but shortened primary root at high concentration. The results suggested that the presence of the endophyte had strong repressive or suppressive effect on H. parviporum transcripts encoding genes involved in nutrient acquisition.

Bacteria Community Inhabiting Heterobasidion Fruiting Body and Associated Wood of Different Decay Classes.

The microbiome of Heterobasidion-induced wood decay of living trees has been previously studied; however, less is known about the bacteria biota of its perennial fruiting body and the adhering wood tissue. In this study, we investigated the bacteria biota of the Heterobasidion fruiting body and its adhering deadwood. Out of 7,462 operational taxonomic units (OTUs), about 5,918 OTUs were obtained from the fruiting body and 5,469 OTUs were obtained from the associated dead wood. Interestingly, an average of 52.6% of bacteria biota in the fruiting body was shared with the associated dead wood. The overall and unique OTUs had trends of decreasing from decay classes 1 to 3 but increasing in decay class 4. The fruiting body had the highest overall and unique OTUs number in the fourth decay class, whereas wood had the highest OTU in decay class 1. Sphingomonas spp. was significantly higher in the fruiting body, and phylum Firmicutes was more dominant in wood tissue. The FAPROTAX functional structure analysis revealed nutrition, energy, degradation, and plant-pathogen-related functions of the communities. Our results also showed that bacteria communities in both substrates experienced a process of a new community reconstruction through the various decay stages. The process was not synchronic in the two substrates, but the community structures and functions were well-differentiated in the final decay class. The bacteria community was highly dynamic; the microbiota activeness, community stability, and functions changed with the decay process. The third decay class was an important turning point for community restructuring. Host properties, environmental factors, and microbial interactions jointly influenced the final community structure. Bacteria community in the fruiting body attached to the living standing tree was suppressed compared with those associated with dead wood. Bacteria appear to spread from wood tissue of the standing living tree to the fruiting body, but after the tree is killed, bacteria moved from fruiting body to wood. It is most likely that some of the resident endophytic bacteria within the fruiting body are either parasitic, depending on it for their nutrition, or are mutualistic symbionts.

Molecular and Chemical Screening for Inherent Disease Resistance Factors of Norway Spruce (Picea abies) Clones Against Conifer Stem Rot Pathogen Heterobasidion parviporum.

Root and stem rot of conifer trees caused by Heterobasidion annosum species complex leads to huge economic losses in Europe, yet not much is known about the molecular and chemical basis for host resistance. To identify inherent chemical or molecular markers in clones found to be either resistant or susceptible, we sampled needle tissues of all the clones before pathogen inoculation. We conducted a short-term resistance screening by using the pathogen H. parviporum to inoculate 70 Norway spruce clones. Based on lesion size, subsets of highly susceptible and resistant clones were further analyzed. Terpene detection and RNA sequencing were performed to explore inherent variations in genotypes differing in resistance to pathogenic challenge at chemical and transcriptional levels. A negative correlation emerged between resistance and growth. Terpene profiles of resistant clones showed higher content of monoterpenes and sesquiterpenes, with concomitant increased transcript abundance of genes involved in the terpenoid pathway. A set of upregulated genes relevant to flavonoid biosynthesis was observed in resistant genotypes, whereas higher transcripts of lignin biosynthetic genes were prevalent in susceptible clones. Genes involved in flavonoid and lignin biosynthesis as well as terpene content may have a role in facilitating resistance of Norway spruce against H. parviporum. Our results provide strong support on the feasibility of sampling needle tissues before pathogen inoculation, and the approach could be of value for large-scale screening of novel biomarkers for durable resistance. The additional insights could form a basis for further research on resistance screening in this pathosystem.

The dark septate endophyte Phialocephala sphaeroides confers growth fitness benefits and mitigates pathogenic effects of Heterobasidion on Norway spruce.

Forest trees frequently interact with a diverse range of microorganisms including dark septate endophytes (DSEs) and fungal pathogens. Plant defense responses to either individual pathogens or endophytes have been widely studied, but very little is known on the effect of coinfection on host defenses. To study the impact of coinfection or tripartite interaction on plant growth and host defenses, Norway spruce (Picea abies (L.) Karst) seedlings were inoculated with a DSE Phialocephala sphaeroides or with a root pathogen Heterobasidion parviporum Niemela & Korhonen or coinfected with both fungi. The results showed that the DSE promoted the root growth of spruce seedlings. Control seedlings without any inoculum were subjected to sequencing and used as a baseline for identification of differentially expressed genes (DEGs). RNA-seq analysis of seedlings inoculated with P. sphaeroides, infected with H. parviporum or coinfected with both fungi resulted in a total of 5269 DEGs. The majority of DEGs were found in P. sphaeroides-inoculated seedlings. Lignin biosynthesis pathways were generally activated during fungal infections. The pattern was distinct with endophyte inoculation. The majority of the genes in the flavonoid biosynthesis pathway were generally suppressed during fungal infections. A specific transcriptional response to P. sphaeroides inoculation was the increased transcripts of genes involved in jasmonic acid biosynthesis, mitogen-activated protein kinases signaling pathway, plant hormone signal transduction and calcium-mediated signaling. This may have potentially contributed to promoting the root growth of seedlings. Although the coinfection suppressed the induction of numerous genes, no negative effect on the growth of the spruce seedlings occurred. We conclude that the subsequent H. parviporum infection triggered reprogramming of host metabolism. Conversely, the endophyte (P. sphaeroides), on the other hand, counteracted the negative effects of H. parviporum on the growth of the spruce seedlings.

Comparative Genomics Reveals Potential Mechanisms of Plant Beneficial Effects of a Novel Bamboo-Endophytic Bacterial Isolate Paraburkholderia sacchari Suichang626.

Plant-beneficial microbes have drawn wide attention due to their potential application as bio-control agents and bio-fertilizers. Moso bamboo, which is among the monocots with the highest growth rate, lives perennially with abundant microbes that may benefit annually growing crops. Genome information of moso bamboo associated bacteria remains underexplored. We isolated and identified a novel Paraburkholderia strain Suichang626 from moso bamboo roots. Growth promoting effects of Suichang626 on both moso bamboo and seedlings of the model dicot Arabidopsis thaliana were documented in laboratory conditions. To gain insight into the genetic basis of this growth promotion effect, we sequenced the genome of Suichang626. Evidenced by genome-wide phylogeny data, we propose that Suichang626 is a novel strain of Paraburkholderia sacchari. Gene homologs encoding biosynthesis of the plant growth-promoting chemicals, acetoin and 2,3-butanediol, were identified in the genome of Suichang626. Comparative genomics was further performed with plant-beneficial and plant/animal pathogenic species of Paraburkholderia and Burkholderia. Genes related to volatile organic compounds, nitrogen fixation, and auxin biosynthesis were discovered specifically in the plant growth-promoting species of both genera.

Physiochemical and molecular features of the necrotic lesion in the Heterobasidion-Norway spruce pathosystem.

In the forest of Northern Hemisphere, the fungi Heterobasidion annosum (Fr.) Bref. s.l. causes severe root and stem rot diseases, dramatically reducing the wood quality of conifer trees. The hallmark of the host response during the infection process is the formation of necrotic lesions and reaction zones. To characterize physiochemical and molecular features of the necrotic lesion, we conducted artificial inoculations on Norway spruce plants at different developmental stages: seedlings, young and mature trees. The results were further compared against data available on the formation of reaction zones. Strong necrosis browning or enlarged necrotic lesions were observed in infected tissues. This was accompanied by elevated pH. However, the increased pH, around 6.0 in necrotic lesions, was not as high as that documented in reaction zones, above 7.0 as marked by the intensity of the blue colour in response to 2,6-dichlorophenol indophenol dye. Peroxidase activity increased in infected plants and RNA-seq analysis of necrotic lesions showed marked upregulation of defence-related genes. Our findings highlight similarities and differences between the reaction zone and necrotic lesion formation in response of conifer trees to biotic stress.

Comparative Genomics Platform and Phylogenetic Analysis of Fungal Laccases and Multi-Copper Oxidases.

Laccases (EC 1.10.3.2), a group of multi-copper oxidases (MCOs), play multiple biological functions and widely exist in many species. Fungal laccases have been extensively studied for their industrial applications, however, there was no database specially focused on fungal laccases. To provide a comparative genomics platform for fungal laccases, we have developed a comparative genomics platform for laccases and MCOs (http://laccase.riceblast.snu.ac.kr/). Based on protein domain profiles of characterized sequences, 3,571 laccases were predicted from 690 genomes including 253 fungi. The number of putative laccases and their properties exhibited dynamic distribution across the taxonomy. A total of 505 laccases from 68 genomes were selected and subjected to phylogenetic analysis. As a result, four clades comprised of nine subclades were phylogenetically grouped by their putative functions and analyzed at the sequence level. Our work would provide a workbench for putative laccases mainly focused on the fungal kingdom as well as a new perspective in the identification and classification of putative laccases and MCOs.

Author Correction: Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

Bacterial Community Structure of Pinus Thunbergii Naturally Infected by the Nematode Bursaphelenchus Xylophilus.

Pine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a devastating disease in conifer forests in Eurasia. However, information on the effect of PWD on the host microbial community is limited. In this study, the bacterial community structure and potential function in the needles, roots, and soil of diseased pine were studied under field conditions using Illumina MiSeq coupled with Phylogenetic Investigation of Communities by Reconstruction of Unobserved states (PICRUSt) software. The results showed that the community and functional structure of healthy and diseased trees differed only in the roots and needles, respectively (p < 0.05). The needles, roots, and soil formed unique bacterial community and functional structures. The abundant phyla across all samples were Proteobacteria (41.9% of total sequence), Actinobacteria (29.0%), Acidobacteria (12.2%), Bacteroidetes (4.8%), and Planctomycetes (2.1%). The bacterial community in the healthy roots was dominated by Acidobacteria, Planctomycetes, and Rhizobiales, whereas in the diseased roots, Proteobacteria, Firmicutes, and Burkholderiales were dominant. Functionally, groups involved in the cell process and genetic information processing had a higher abundance in the diseased needles, which contributed to the difference in functional structure. The results indicate that PWD can only affect the host bacteria community structure and function in certain anatomical regions of the host tree.

The Conifer Root and Stem Rot Pathogen (Heterobasidion parviporum): Effectome Analysis and Roles in Interspecific Fungal Interactions.

Heterobasidion parviporum Niemelä & Korhonen is an economically important basidiomycete, causing root and stem rot disease of Norway spruce (Picea abies (L.) Karst) in Northern Europe. The H. parviporum genome encodes numerous small secreted proteins, which might be of importance for interacting with mycorrhiza symbionts, endophytes, and other saprotrophs. We hypothesized that small secreted proteins from H. parviporum (HpSSPs) are involved in interspecific fungal interaction. To identify HpSSP-coding genes potentially involved, we screened the H. parviporum effectome and compared their transcriptomic profiles during fungal development and in planta tree infection. We further conducted phylogenetic analysis, and identified a subset of hypothetical proteins with nonpredicted domain or unknown function as HpSSPs candidates for further characterization. The HpSSPs candidates were selected based on high-quality sequence, cysteine residue frequency, protein size, and in planta expression. We subsequently explored their roles during in vitro interaction in paired cultures of H. parviporum with ectomycorrhizal Cortinarius gentilis, endophytic Phialocephala sphaeroides, saprotrophs (Mycena sp., Phlebiopsis gigantea, and Phanerochaete chrysosporium), respectively. The transcriptomic profile revealed that a large proportion of effector candidates was either barely expressed or highly expressed under all growth conditions. In vitro dual-culture test showed that P. sphaeroides and C. gentilis were overgrown by H. parviporum. The barrage zone formation or no physical contact observed in paired cultures with the saprotrophs suggest they had either combative interaction or antibiosis effect with H. parviporum. Several HpSSPs individuals were up- or downregulated during the nonself interactions. The results of HpSSPs gene expression patterns provide additional insights into the diverse roles of SSPs in tree infection and interspecific fungal interactions.

Fourier-transform infrared (FT-IR) spectroscopy analysis discriminates asymptomatic and symptomatic Norway spruce trees.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 - 912 cm-1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 - 994 cm-1 and 1104 - 994 cm-1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Evaluation of potential genetic and chemical markers for Scots pine tolerance against Heterobasidion annosum infection.

MAIN CONCLUSION: Two terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum. Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted α-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, ß-caryophyllene and α-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

Genome description of Phlebia radiata 79 with comparative genomics analysis on lignocellulose decomposition machinery of phlebioid fungi.

BACKGROUND: The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. RESULTS: The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. CONCLUSIONS: The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.

Chlorophyll fluorescence imaging for monitoring effects of Heterobasidion parviporum small secreted protein induced cell death and in planta defense gene expression.

Heterobasidion parviporum Niemelä & Korhonen is a necrotrophic fungal pathogen of Norway spruce (Picea abies). The H. parviporum genome encodes numerous necrotrophic small secreted proteins (SSP) which might be important for promoting and sustaining the disease development. However, their transcriptional dynamics and plant defense response during infection are largely unknown. In this study, we identified a necrotrophic SSP named HpSSP35.8 and its coding gene was highly expressed in the pre-symptomatic phase of the host (Norway spruce) infection. We explored the impact of HpSSP35.8 on non-host Nicotiana benthamiana using Agrobacterium-mediated transient expression system under visible spectrum RGB imaging and chlorophyll fluorescence imaging. The results showed that HpSSP35.8 triggered a form of SSP-associated programmed cell death, accompanied by a decrease in the plant photosynthetic activity. Defense-related genes including WRKY12, ethylene response factor (ERF1α) and a chitinase gene PR4 were up-regulated in both HpSSP35.8-N. benthamiana interaction and H. parviporum-Norway spruce pathosystem. This study also highlighted the potential to use the chlorophyll fluorescence imaging approach to monitor both the indirect effects of SSP and also for the selection of other potential effector-like protein candidates.