Beneficial Proapoptotic Effect of Heterobasidion Annosum Extract in Colorectal Cancer Xenograft Mouse Model.

Fungal extracts possess potential anticancer activity against many malignant neoplastic diseases. In this research, we focused on the evaluation of Heterobasidion annosum (HA) extract in colorectal cancer in an in vivo model. The mice with implanted DLD-1 human cancer cells were given HA extract, the referential drug-5-fluorouracil (5FU), or were treated with its combination. Thereafter, tumor volume was measured and apoptotic proteins such as caspase-8, caspase-3, p53, Bcl-2, and survivin were analyzed in mice serum with an ELISA assay. The Ki-67 protein was assessed in tumor cells by immunohistochemical examination. The biggest volumes of tumors were confirmed in the DLD-1 group, while the lowest were observed in the population treated with 5FU and/or HA extract. The assessment of apoptosis showed increased concentrations of caspase 8 and p53 protein after the combined administration of 5FU and HA extract. The levels of survivin and Bcl-2 were decreased in all tested groups compared to the DLD-1 group. Moreover, we observed a positive reaction for Ki-67 protein in all tested groups. Our findings confirm the apoptotic effect of extract given alone or with 5FU. The obtained results are innovative and provide a basis for further research concerning the antitumor activity of the HA extract, especially in the range of its interaction with an anticancer chemotherapeutic agent.

Transcript Dynamics in Wounded and Inoculated Scots Pine.

Comparative transcriptome analysis provides a useful tool for the exploration of plant-pathogen interaction by allowing in-depth comparison of gene expression between unaffected, inoculated and wounded organisms. Here we present the results of comparative transcriptome analysis in genetically identical one-year-old Scots pine ramets after wounding and inoculation with Heterobasidion annosum. We identified 230 genes that were more than 2-fold upregulated in inoculated samples (compared to controls) and 116 downregulated genes. Comparison of inoculated samp les with wounded samples identified 32 differentially expressed genes (30 were upregulated after inoculation). Several of the genes upregulated after inoculation are involved in protection from oxidative stress, while genes involved in photosynthesis, water transport and drought stress tolerance were downregulated. An NRT3 family protein was the most upregulated transcript in response to both inoculation and wounding, while a U-box domain-containing protein gene was the most upregulated gene comparing inoculation to wounding. The observed transcriptome dynamics suggest involvement of auxin, ethylene, jasmonate, gibberellin and reactive oxygen species pathways and cell wall modification regulation in response to H. annosum infection. The results are compared to methyl jasmonate induced transcriptome dynamics.

Heterobasidion annosum Induces Apoptosis in DLD-1 Cells and Decreases Colon Cancer Growth in In Vivo Model.

Application of substances from medicinal mushrooms is one of the interesting approaches to improve cancer therapy. In this study, we commenced a new attempt in the field of Heterobasidion annosum (Fr.) Bref. sensu lato to further extend our knowledge on this basidiomycete fungus. For this purpose, analysis of the active substances of Heterobasidion annosum methanolic extract and also its influence on colorectal cancer in terms of in vitro and in vivo experiments were performed. In vivo studies on mice were conducted to verify its acute toxicity and to further affirm its anticancer potential. Results indicated that all the most common substances of best known medicinal mushrooms that are also responsible for their biological activity are present in tested extracts. In vitro tests showed a high hemocompatibility and a significant decrease in viability and proliferation of DLD-1 cells in a concentration-dependent manner of Heterobasidion annosum extract. The studies performed on xenograft model of mice showed lower tendency of tumor growth in the group of mice receiving Heterobasidion annosum extract as well as mild or moderate toxicity. Obtained results suggest beneficial potential of Heterobasidion annosum against colon cancer as cytotoxic agent or as adjuvant anticancer therapy.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Diversity and impact of single-stranded RNA viruses in Czech Heterobasidion populations.

Heterobasidion annosum sensu lato comprises some of the most devastating pathogens of conifers. Exploring virocontrol as a potential strategy to mitigate economic losses caused by these fungi holds promise for the future. In this study, we conducted a comprehensive screening for viruses in 98 H. annosum s.l. specimens from different regions of Czechia aiming to identify viruses inducing hypovirulence. Initial examination for dsRNA presence was followed by RNA-seq analyses using pooled RNA libraries constructed from H. annosum and Heterobasidion parviporum, with diverse bioinformatic pipelines employed for virus discovery. Our study uncovered 25 distinct ssRNA viruses, including two ourmia-like viruses, one mitovirus, one fusarivirus, one tobamo-like virus, one cogu-like virus, one bisegmented narna-like virus and one segment of another narna-like virus, and 17 ambi-like viruses, for which hairpin and hammerhead ribozymes were detected. Coinfections of up to 10 viruses were observed in six Heterobasidion isolates, whereas another six harbored a single virus. Seventy-three percent of the isolates analyzed by RNA-seq were virus-free. These findings show that the virome of Heterobasidion populations in Czechia is highly diverse and differs from that in the boreal region. We further investigated the host effects of certain identified viruses through comparisons of the mycelial growth rate and proteomic analyses and found that certain tested viruses caused growth reductions of up to 22% and significant alterations in the host proteome profile. Their intraspecific transmission rates ranged from 0% to 33%. Further studies are needed to fully understand the biocontrol potential of these viruses in planta.IMPORTANCEHeterobasidion annosum sensu lato is a major pathogen causing significant damage to conifer forests, resulting in substantial economic losses. This study is significant as it explores the potential of using viruses (virocontrol) to combat these fungal pathogens. By identifying and characterizing a diverse array of viruses in H. annosum populations from Czechia, the research opens new avenues for biocontrol strategies. The discovery of 25 distinct ssRNA viruses, some of which reduce fungal growth and alter proteome profiles, suggests that these viruses could be harnessed to mitigate the impact of Heterobasidion. Understanding the interactions between these viruses and their fungal hosts is crucial for developing effective, environmentally friendly methods to protect conifer forests and maintain ecosystem health. This study lays the groundwork for future research on the application of mycoviruses in forest disease management.

Rapid detection of Heterobasidion annosum using a loop-mediated isothermal amplification assay.

Heterobasidion annosum is one of the most aggressive pathogens of Pinus forests in Europe, causing considerable economic losses. To detect H. annosum for disease diagnosis and control, we developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA sequences of H. annosum. In our study, this LAMP assay was found to be capable of efficiently amplifying the target gene within 60 min at 63°C. In specificity tests, H. annosum was positively detected, and other species were negative. The detection limit of this assay was found to be 100 pg·µL-1, and the assay was also successfully tested for use with basidiospore suspensions and wood samples. This study provides a rapid method for diagnosing root and butt rot caused by H. annosum, which will be of use in port surveillance of logs imported from Europe.

An Experiment in Denmark to Infect Wounded Sitka Spruce with the Rotstop Isolate of Phlebiopsis gigantea, and Its Implications for the Control of Heterobasidion annosum in Britain.

The formulation of a Finnish isolate of the saprotrophic wood-rotting fungus Phlebiopsis gigantea into the biocontrol agent (BCA) Rotstop, which is used to prevent infection of Norway spruce stumps by aerial basidiospores of H. annosum, has led to its application to more than 200,000 ha of forest in Scandinavia and North Europe. The success of this treatment opens the possibility of introducing the Rotstop strain into Britain for use on Sitka spruce stumps, which at present (2022) are lacking adequate prophylactic treatment. However, Rotstop is probably non-native to Britain and to North America (the ancestral home of this spruce), and we do not know if this xylem-decaying BCA can invade standing trees. In this paper, we describe a trial into this issue conducted for the U.K. Forestry Commission in Denmark, in a country where both Rotstop and Sitka spruce have been naturalised. It was preliminary to further stump treatment trials, and is relevant to long-term issues surrounding stump treatment in Britain. Inoculations into 44-year-old standing Sitka spruce with 20 mm wooden Scots pine plugs pre-colonised with Rotstop resulted in decay of the S1, S2, S3 and middle lamellae of sapwood above and below the wounds after 11-18 months. In contrast, infection of sapwood occurred in only one of 39 wounds on the same trees challenged with oidial spore inoculants adpressed to undamaged xylem sapwood during the same period. While the results suggest that release of Rotstop into the productive stands of Sitka spruce in Britain would be unlikely to cause long-term commercial losses to wounded trees, the work highlights issues relating to the assessment of risk associated with the introduction of non-native BCAs within the forest environment.

Alphapartitiviruses of Heterobasidion Wood Decay Fungi Affect Each Other's Transmission and Host Growth.

Heterobasidion spp. root rot fungi are highly destructive forest pathogens of the northern boreal forests, and are known to host a diverse community of partitiviruses. The transmission of these mycoviruses occurs horizontally among host strains via mycelial anastomoses. We revealed using dual cultures that virus transmission rates are affected by pre-existing virus infections among two strains of H. annosum. The transmission efficacy of mycovirus HetPV15-pa1 to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90% to a partitivirus-free recipient. On contrary, pre-existing virus infections of two closely related strains of HetPV11 hindered each other's transmission, but had unexpectedly dissimilar effects on the transmission of more distantly related viruses. The co-infection of HetPV13-an1 and HetPV15-pa1 significantly reduced host growth, whereas double infections including HetPV11 strains had variable effects. Moreover, the results showed that RdRp transcripts are generally more abundant than capsid protein (CP) transcripts and the four different virus strains express unique transcripts ratios of RdRp and CP. Taken together, the results show that the interplay between co-infecting viruses and their host is extremely complex and highly unpredictable.

Retrotransposon expression in response to in vitro inoculation with two fungal pathogens of Scots pine (Pinus sylvestris L.).

OBJECTIVE: Conifer genomes show high genetic diversity in intergenic regions that contain diverse sets of transposable elements with dominating long terminal repeat (LTR) retrotransposons (RE). Transcription of RE in response to environmental stimuli could produce various types of regulatory non-coding RNAs, but global genomic methylation changes could result in a coincidental expression of normally silent genomic regions. Expression of several RE families was evaluated in Scots pine seedlings after controlled inoculations with two fungal species that exhibit different modes of pathogenicity (necrotrophic and likely biotrophic); data compared to the overall RE distribution in genome. Recognition of regulatory non-coding RNA involved in host-pathogen interplay could be valuable in understanding defence mechanisms of perennial plants. RESULTS: In the case of necrotrophic fungi Heterobasidion annosum (HA), short activation followed by restriction of RE expression was revealed after inoculation and during the spread of the pathogen. After inoculation with Lophodermium seditiosum (LS), an early increase in RE expression was revealed with the spread of the pathogen and subsequent transcription rise in all seedlings. Our observations indicate that in the complex plant genome multiple RE families constitutively express in response to pathogen invasion and these sequences could undergo regulation related to host response or pathogen influence.

CYPome of the conifer pathogen Heterobasidion irregulare: Inventory, phylogeny, and transcriptional analysis of the response to biocontrol.

The molecular mechanisms underlying the interaction of the pathogen, Heterobasidion annosum s.l., the conifer tree and the biocontrol fungus, Phlebiopsis gigantea have not been fully elucidated. Members of the cytochrome P450 (CYP) protein family may contribute to the detoxification of components of chemical defence of conifer trees by H. annosum during infection. Additionally, they may also be involved in the interaction between H. annosum and P. gigantea. A genome-wide analysis of CYPs in Heterobasidion irregulare was carried out alongside gene expression studies. According to the Standardized CYP Nomenclature criteria, the H. irregulare genome has 121 CYP genes and 17 CYP pseudogenes classified into 11 clans, 35 families, and 64 subfamilies. Tandem CYP arrays originating from gene duplications and belonging to the same family and subfamily were found. Phylogenetic analysis showed that all the families of H. irregulare CYPs were monophyletic groups except for the family CYP5144. Microarray analysis revealed the transcriptional pattern for 130 transcripts of CYP-encoding genes during growth on culture filtrate produced by P. gigantea. The high level of P450 gene diversity identified in this study could result from extensive gene duplications presumably caused by the high metabolic demands of H. irregulare in its ecological niches.

Comparative analysis of genome sequences of the conifer tree pathogen, Heterobasidion annosum s.s.

The causal agent of root and butt rot of conifer trees, Heterobasidion annosum, is widespread in boreal forests and economically responsible for annual loss of approximately 50 million euros to forest industries in Finland alone and much more at European level. In order to further understand the pathobiology of this fungus at the genome level, a Finnish isolate of H. annosum sensu stricto (isolate 03012) was sequenced and analyzed with the genome sequences of 23 white-rot and 13 brown-rot fungi. The draft genome assembly of H. annosum has a size of 31.01 Mb, containing 11,453 predicted genes. Whole genome alignment showed that 84.38% of H. annosum genome sequences were aligned with those of previously sequenced H. irregulare TC 32-1 counterparts. The result is further supported by the protein sequence clustering analysis which revealed that the two genomes share 6719 out of 8647 clusters. When sequencing reads of H. annosum were aligned against the genome sequences of H. irregulare, six single nucleotide polymorphisms were found in every 1 kb, on average. In addition, 98.68% of SNPs were found to be homo-variants, suggesting that the two species have long evolved from different niches. Gene family analysis revealed that most of the white-rot fungi investigated had more gene families involved in lignin degradation or modification, including laccases and peroxidase. Comparative analysis of the two Heterobasidion spp. as well as white-/brown-rot fungi would provide new insights for understanding the pathobiology of the conifer tree pathogen.

Heterobasidion wood decay fungi host diverse and globally distributed viruses related to Helicobasidium mompa partitivirus V70.

Viruses of the Partitiviridae family occur at a relatively low frequency in the fungal genus Heterobasidion, but show high genetic diversity. Here, we describe four novel partitivirus species that infect three Heterobasidion species that are pathogens of conifers: H. annosum, H. parviporum and H. irregulare. We show that these viruses, designated Heterobasidion partitivirus 12 (HetPV12), HetPV13, HetPV14 and HetPV15, form a phylogenetically distinct clade together with the previously described Heterobasidion partitivirus 3 (HetPV3) found in the H. insulare species complex and Helicobasidium mompa partitivirus V70, both members of the genus Alphapartitivirus. Closely related strains of HetPV13 (over 97% polymerase identity at the nucleotide level) occur in H. annosum and H. parviporum, suggesting recent transmission of this virus species between the two fungal host species. Moreover, the occurrence of nearly identical HetPV13 strains in Finland and Poland (ca 1400km apart) indicates that the dispersal capacity of Heterobasidion partitiviruses is high. Viruses related to HetPV3 have a global distribution but only ca 2.7% overall prevalence among isolates of Heterobasidion. In three cases, these HetPV3-related viruses co-infected their hosts with distantly related partitiviruses or Heterobasidion RNA virus 6.

Tansley Review No. 19 Environmental and endogenous controls of developmental pathways: variation and its significance in the forest pathogen, Heterobasidion annosum.

Cultural studies of the forest root pathogen Heterobasidion annosum (Fr.) Bref. have shown that the mycelium of the fungus possesses a high degree of developmental versatility, being able to grow in a variety of distinctive functional modes. Consequently the fungus can alternate between conidiogenous and meiotic reproductive pathways, rapid, sparsely branched and slow, densely branched mycelial extension, nutritive and non-nutritive modes, and'juvenile'and 'senescent'phases. This ability is probably of considerable importance in natural infection cycles and provides an interesting model for examining interrelationships between developmental regulation and host-pathogen interactions generally. It also raises important questions about the interface between environmental and endogenous controls of developmental pathways. CONTENTS Summary 245 I. Introduction 245 II. Spore germination and mycelial extension 246 III. Production of non-nutritive mycelium 249 IV. Sporophore production, senescence, somatic incompatibility and extracellular enzyme production 251 V. Discussion 254 Acknowledgements 255 References 255.

Diffuse competition for heterogeneous substrate in soil among six species of wood-decomposing basidiomycetes.

Competition among six wood decay fungi was studied using 15×15 mm wood blocks placed in 250×250 mm plastic trays filled with unsterilized sand or clay. The wood blocks were preinoculated with Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm., Phanerochaete sanguinea (Fr.) Hjortstam, Coniophora sp. DC. ex Me"rat, Armillaria borealis Marxmuller and Korhonen and Hypholoma capnoides (Fr.) Kummer before they were combined in all possible combinations in the trays. Two methods were used, one with all wood blocks inoculated, and one with sterilized non-inoculated wood blocks distributed between the inoculated ones. Wood blocks preinoculated with the six species were also used in a pairwise competition test. Following incubation for 9 months in darkness at 21°C, mycelia were reisolated and identified. R. bicolor was most successful at invading through the soil and replacing other species in the wood blocks. P. sanguinea, Coniophora sp. and H. capnoides also had some success.

Population structure and responses to disturbance of the basidiomycete Resinicium bicolor.

Resinicium bicolor (Alb. & Schw. ex Fr.) Parm. [=Odontia bicolor (Alb. & Schw. ex Fr.) Bres.] is an outcrossing resupinate basidiomycete associated with root and butt rots of trees, but is itself only very weakly pathogenic. The distribution of genets among every spruce stump in a 70-year-old 1250 m2 spruce stand was analysed using somatic incompatibility testing. R. bicolor was present on 40% of 8-to 10-year-old stumps. Nineteen genets were found occupying 32 stumps; yielding probabilities of colonisation following establishment by basidiospores of 0.20-0.24 and by mycelial extension or dispersal of 0.16-0.20. The probability of colonisation decreased with increasing distance from a point of establishment. R. bicolor responded to both enrichment and destructive disturbances by the formation of an extensive cord system which enabled it to colonise discontinuously distributed resources and to overgrow fungi adjacent to it in a single resource unit, including Heterobasidion annosum.

Partitiviruses of a fungal forest pathogen have species-specific quantities of genome segments and transcripts.

Heterobasidion partitiviruses infect forest pathogenic fungi of the genus Heterobasidion. We have studied the amounts of genomes and transcripts of four partitiviruses isolated from four different Heterobasidion strains infecting different host trees in Greece, Poland, Finland, and China. Heterobasidion partitiviruses have bisegmented genomes encoding coat protein and RNA-dependent RNA polymerase. Our results show that the coat protein genome segment is generally more abundant in infected mycelia than the RNA-dependent RNA polymerase segment and that this bias persists also at transcript levels. The different virus species all have unique ratios of the genome segments and the ratio is generally stable over different temperatures and hosts. The amounts of transcripts of each virus respond to host growth temperatures in a distinctive and consistent manner. The Heterobasidion partitiviruses studied here affect only rarely the growth of their natural hosts but do influence the growth of a new host more frequently.

Secondary metabolite comparison of the species within the Heterobasidion annosum s.l. complex.

The metabolite production of the five members of the fungal species complex Heterobasidion annosum s.l., i.e. H. annosum s.s., H. abietinum, H. parviporum, H. occidentale and H. irregulare, was analyzed by LC-HRMS. The five members are described to have differences in host preferences: H. annosum s.s. and H. irregulare are pine infecting species, and H. parviporum, H. occidentale and H. abietinum are non-pine infecting. Principal component analysis (PCA) of the LC-HRMS data showed that samples from the five species could be separated into five groups and in accordance with the differences in host preferences. Twenty-three compounds, important to the observed PCA grouping, were isolated and identified. The main contributor to the separation of the pine infecting species from the non-pine infecting species in PC1 was the benzohydrofuran fomannoxin, which was only detected in the pine infecting species H. annosum s.s. and H. irregulare. These two species were further separated in PC3, and one major contributor here was the sesquiterpene deoxyfomannosin A. The three non-pine infecting species were separated in PC2, by epoxydrimenol that was detected in only two of the species and further in PC4, where a few fomannoxin related compounds were important for the grouping. During the work, three unknown compounds were isolated and described: 3-hydroxy-2-(1,3-dihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde, 3-hydroxy-2-(1,2,3-trihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde and 3-hydroxy-2,3-dihydrobenzofuran-5-carboxylic acid.