Search

Background: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). Results: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. Conclusion: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

Ridomil Gold SL (45.3% a.i. mefenoxam) is a widely used chemical fungicide for the control of oomycetes. However, its impact on fungal communities remains unexplored. Therefore, the goal of this study was to examine the effects of mefenoxam on the temporal dynamics of fungal taxonomic and functional diversities during carrot cultivation under four treatment groups: mefenoxam application with and without Pythium inoculation, and untreated control groups with and without Pythium inoculation. Our in vitro sensitivity assay showed that the maximum recommended concentration of mefenoxam, 0.24 ppm, did not suppress the mycelial growth of P. irregulare. At 100 ppm, mycelial growth was only reduced by 11.4%, indicating that the isolate was resistant to mefenoxam. MiSeq sequencing data revealed transient taxonomic variations among treatments 2 weeks post-treatment. Mortierella dominated the fungal community in the mefenoxam-Pythium combination treatment, as confirmed through PCR using our newly designed Mortierella-specific primers. Conversely, mefenoxam-Pythium combination had adverse effects on Penicillium, Trichoderma, and Fusarium, and decrease the overall alpha diversity. However, these compositional changes gradually reverted to those observed in the control by the 12th week. The predicted ecological functions of fungal communities in all Pythium and mefenoxam treatments shifted, leading to a decrease in symbiotrophs and plant pathogen functional groups. Moreover, the community-level physiological profiling approach, utilizing 96-well Biolog FF microplates, showed discernible variations in the utilization of 95 diverse carbon sources among the treatments. Notably, arbutin, L-arabinose, Tween 80, and succinamic acid demonstrated a strong positive association with Mortierella. Our findings demonstrate that a single application of mefenoxam at its recommended rate triggers substantial taxonomic and functional shifts in the soil fungal community. Considering this

Soil desertification poses a critical ecological challenge in arid and semiarid climates worldwide, leading to decreased soil productivity due to the disruption of essential microbial community processes. Fungi, as one of the most important soil microbial communities, play a crucial role in enhancing nutrient and water uptake by plants through mycorrhizal associations. However, the impact of overgrazing-induced desertification on fungal community structure, particularly in the Caatinga biome of semiarid regions, remains unclear. In this study, we assessed the changes in both the total fungal community and the arbuscular mycorrhizal fungal community (AMF) across 1. Natural vegetation (native), 2. Grazing exclusion (20 years) (restored), and 3. affected by overgrazing-induced degradation (degraded) scenarios. Our assessment, conducted during both the dry and rainy seasons in Irauçuba, Ceará, utilized Internal Transcribed Spacer (ITS) gene sequencing via Illumina® platform. Our findings highlighted the significant roles of the AMF families Glomeraceae (∼71% of the total sequences) and Acaulosporaceae (∼14% of the total sequences) as potential key taxa in mitigating climate change within dryland areas. Moreover, we identified the orders Pleosporales (∼35% of the total sequences) and Capnodiales (∼21% of the total sequences) as the most abundant soil fungal communities in the Caatinga biome. The structure of the total fungal community differed when comparing native and restored areas to degraded areas. Total fungal communities from native and restored areas clustered together, suggesting that grazing exclusion has the potential to improve soil properties and recover fungal community structure amid global climate change challenges.

Reptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel

While the intestinal microbiome seems a major driver of persistent immune defects in people with HIV (PWH), little is known about its fungal component, the mycobiome. We assessed the inter-kingdom mycobiome–bacteriome interactions, the impact of diet, and the association with the innate and adaptive immunity in PWH on antiretroviral therapy. We included 24 PWH individuals and 12 healthy controls. We sequenced the Internal Transcribed Spacer 2 amplicons, determined amplicon sequence variants, measured biomarkers of the innate and adaptive immunity in blood and relations with diet. Compared to healthy controls, PWH subjects exhibited a distinct and richer mycobiome and an enrichment for Debaryomyces hansenii, Candida albicans, and Candida parapsilosis. In PWH, Candida and Pichia species were strongly correlated with several bacterial genera, including Faecalibacterium genus. Regarding the links between the mycobiome and systemic immunology, we found a positive correlation between Candida species and the levels of proinflammatory cytokines (sTNF-R2 and IL-17), interleukin 22 (a cytokine implicated in the regulation of mucosal immunity), and CD8+ T cell counts. This suggests an important role of the yeasts in systemic innate and adaptive immune responses. Finally, we identified inter-kingdom interactions implicated in fiber degradation, short-chain fatty acid production, and lipid metabolism, and an effect of vegetable and fiber intake on the mycobiome. Therefore, despite the great differences in abundance and diversity between the bacterial and fungal communities of the gut, we defined the changes associated with HIV, determined several different inter-kingdom associations, and found links between the mycobiome, nutrient metabolism, and systemic immunity, European Development Regional Found "A way to achieve Europe" (ERDF), Instituto de Salud Carlos III, Depto. de Nutrición y Ciencia de los Alimentos, Fac. de Farmacia, TRUE, pub

The world is facing severe consequences of chemical fertilizers and synthetic pesticides. As a safer and eco-friendly option for crop nutrition and pest control, endophytic fungi (EFs) are now hotpots in sustainable crop production research. This comprehensive review elucidated the potentials of EF in sustainable crop production amid various biotic and abiotic stress conditions as well as their extraordinary role in phyto- and bioremediation. A critical investigation on how climatic factors such as temperature, rainfall, nutrient level, pH and storage condition affect the survival and performance of EFs are also presented. On-going research majorly focused on the application of endophytic fungi as crop growth promoters, biocontrol agents, and stress mediators. However, a lack of in-depth understanding of the underlying mechanism is predominant. Nevertheless, challenges of successful isolation and identification of EFs hinder their commercialization. Modern genomic technologies and nanotechnology-based applications of EFs can help in overcome the barriers. This review will provide direction for future research where EFs will lead next-generation sustainable strategies to cope with climate change.

Introduction: Colonization by pathogenic microorganisms in medical devices used in the intensive care unit is a risk factor for the increase in infections associated with health care that escalates the morbidity and mortality of intubated patients. In Colombia, fungal colonization of endotracheal tubes has not been described, which could allow inferring new therapeutic options for the benefit of patients.Objective: To describe the fungi that colonize the endotracheal tubes of patients in the intensive care unit, along with their profile of sensitivity to antifungals. Materials and methods: A descriptive observational study was carried out in 2 clinics for 12 months. Endotracheal tubes were collected from patients in intensive care units. These were processed for culture and identification of fungi, and their sensitivity profile to antifungals.Results: 121 endotracheal tubes obtained from 113 patients were analyzed, of which 41.32% were colonized by the fungi Candida albicans (64.62%), Candida non-albicans (30.77%), Cryptococcus (3.08%) and molds (1.54%). All the fungi evaluated presented a high sensitivity to antifungals, on average 91%.Conclusion: Fungal colonization was found in the endotracheal tubes of patients under invasive mechanical ventilation; the sensitivity profile in these patients is favorable. A clinical study is required to find possible correlations between the colonizing microorganisms and their probability of generating infection., Introducción. La colonización por microorganismos patógenos en los dispositivos médicos de uso en la unidad de cuidado intensivo, es un factor de riesgo para el aumento de infecciones asociadas a la atención en salud que aumenta la morbilidad y mortalidad de los pacientes intubados. En Colombia no se ha descrito la colonización por hongos de tubos endotraqueales, lo que podría permitir inferir nuevas opciones terapéuticas en beneficio de los pacientes.Objetivo. Describir los hongos que colonizan los tubos endotraqueales de pacientes en unidad de cuidado intensivo, junto con su perfil de sensibilidad a los antifúngicos.Materiales y métodos. Se realizó un estudio observacional descriptivo, en 2 clínicas, durante 12 meses. Se recolectaron tubos endotraqueales de pacientes de las unidades de cuidado intensivo. Estos fueron procesados para cultivo e identificación de hongos, y su perfil de sensibilidad a antifúngicos.Resultados. Se analizaron 121 tubos endotraqueales obtenidos de 113 pacientes, de los cuales el 41,32% fueron colonizados por los hongos Cándida albicans (64,61%), Cándida no albicans (30,77%), Cryptococcus (3,08%) y mohos (1,54%). Todos los hongos evaluados presentaronuna alta sensibilidad a los antifúngicos, en promedio del 91,00%.Conclusión. Se encontró colonización fúngica en los tubos endotraqueales de pacientes bajo ventilación mecánica invasiva. El perfil de sensibilidad en estos pacientes es favorable. Se requiere un estudio clínico donde se realice la correlación entre los microorganismos colonizadores y su probabilidad de generar infección.

Bee-fungus associations are common, and while most studies focus on entomopathogens, emerging evidence suggests that bees associate with a variety of symbiotic fungi that can influence bee behavior and health. Here, we review nonpathogenic fungal taxa associated with different bee species and bee-related habitats. We synthesize results of studies examining fungal effects on bee behavior, development, survival, and fitness. We find that fungal communities differ across habitats, with some groups restricted mostly to flowers (Metschnikowia), while others are present almost exclusively in stored provisions (Zygosaccharomyces). Starmerella yeasts are found in multiple habitats in association with many bee species. Bee species differ widely in the abundance and identity of fungi hosted. Functional studies suggest that yeasts affect bee foraging, development, and pathogen interactions, though few bee and fungal taxa have been examined in this context. Rarely, fungi are obligately beneficial symbionts of bees, whereas most are facultative bee associates with unknown or ecologically contextual effects. Fungicides can reduce fungal abundance and alter fungal communities associated with bees, potentially disrupting bee-fungi associations. We recommend that future study focus on fungi associated with non-honeybee species and examine multiple bee life stages to document fungal composition, abundance, and mechanistic effects on bees.

Coccidioidomycosis is a typically respiratory fungal disease that, in the United States, occurs primarily in Arizona and California. In California, most coccidioidomycosis cases occur in the San Joaquin Valley, a primarily agricultural region where the disease poses a risk for outdoor workers. We collected 710 soil samples and 265 settled dust samples from nine sites in the San Joaquin Valley and examined how Coccidioides detection varied by month, site, and the presence and abundance of other fungal species. We detected Coccidioides in 89 of 238 (37.4%) rodent burrow soil samples at five undeveloped sites and were unable to detect Coccidioides in any of 472 surface and subsurface soil samples at four agricultural sites. In what is the largest sampling effort undertaken on agricultural land, our results provide no evidence that agricultural soils in the San Joaquin Valley harbor Coccidioides. We found no clear association between Coccidioides and the greater soil fungal community, but we identified 19 fungal indicator species that were significantly associated with Coccidioides detection in burrows. We also did not find a seasonal pattern in Coccidioides detection in the rodent burrow soils we sampled. These findings suggest both the presence of a spore bank and that coccidioidomycosis incidence may be more strongly associated with Coccidioides dispersal than Coccidioides growth. Finally, we were able to detect Coccidioides in only five of our 265 near-surface settled dust samples, one from agricultural land, where Coccidioides was undetected in soils, and four from undeveloped land, where Coccidioides was common in the rodent burrow soils we sampled. Our ability to detect Coccidioides in few settled dust samples indicates that improved methods are likely needed moving forward, though raises questions regarding aerial dispersal in Coccidioides, whose key transmission event likely occurs over short distances in rodent burrows from soil to naïve rodent lungs.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

Bacterial and fungal root endophytes can impact the fitness of their host plants, but the relative importance of drivers for root endophyte communities is not well known. Host plant species, the composition and density of the surrounding plants, space, and abiotic drivers could significantly affect bacterial and fungal root endophyte communities. We investigated their influence in endophyte communities of alpine plants across a harsh high mountain landscape using high-throughput sequencing. There was less compositional overlap between fungal than bacterial root endophyte communities, with four 'cosmopolitan' bacterial OTUs found in every root sampled, but no fungal OTUs found across all samples. We found that host plant species, which included nine species from three families, explained the greatest variation in root endophyte composition for both bacterial and fungal communities. We detected similar levels of variation explained by plant neighborhood, space, and abiotic drivers on both communities, but the plant neighborhood explained less variation in fungal endophytes than expected. Overall, these findings suggest a more cosmopolitan distribution of bacterial OTUs compared to fungal OTUs, a structuring role of the plant host species for both communities, and largely similar effects of the plant neighborhood, abiotic drivers, and space on both communities.

An emerging crop, intermediate wheatgrass (IWG) is resistant to the natural occurrence of most important cereal diseases and is currently under domestication to improve its yield. An IWG population consisting of two parental lines and 154 individual F1 progenies were inoculated with F. graminearum spores in a field setting to check their performance against Fusarium head blight (FHB) over two consecutive years. The higher humidity in 2022 lead to higher FHB severity and incidence, and higher DON (deoxynivalenol) mycotoxin accumulation than in 2021. DON was mostly restricted to the chaff in 2021, whereas in 2022, DON was found in the grain as well. Considering all the FHB aspects checked in this experiment, four IWG progeny individuals (plants: P-17, P-43, P-107, and P-139) were found to consistently perform better than the parental lines and the other progeny individuals during both years. The second part of this thesis analyzed the influence of F. graminearum on the mycobiome of IWG spikelet and flag leaf samples from two different locations, Winnipeg and Carman, MB. A total 372 IWG samples (half of which were inoculated with F. graminearum [Winnipeg] and the other half, not [Carman]) were used for fungal community profiling via ITS amplicon sequencing using PacBio technology. There were significant differences in the fungal community among the IWG spike samples from Winnipeg and Carman. As expected, F. graminearum dominated IWG spike mycobiomes at the inoculated Winnipeg site, whereas in Carman, Alternaria dominated. Also, the presence of F. graminearum seemed to influence the broader spike mycobiome and the presence of beneficial fungi was less where F. graminearum dominated. The mycobiome data also revealed that the pathogenic fungi Alternaria, Nigrospora, Septoriella, Stemphylium, Claviceps, Bipolaris, and Drechslera can become potential threats to IWG in the future. The last part of this thesis sheds light on the interaction between F. graminearum and another

An emerging crop, intermediate wheatgrass (IWG) is resistant to the natural occurrence of most important cereal diseases and is currently under domestication to improve its yield. An IWG population consisting of two parental lines and 154 individual F1 progenies were inoculated with F. graminearum spores in a field setting to check their performance against Fusarium head blight (FHB) over two consecutive years. The higher humidity in 2022 lead to higher FHB severity and incidence, and higher DON (deoxynivalenol) mycotoxin accumulation than in 2021. DON was mostly restricted to the chaff in 2021, whereas in 2022, DON was found in the grain as well. Considering all the FHB aspects checked in this experiment, four IWG progeny individuals (plants: P-17, P-43, P-107, and P-139) were found to consistently perform better than the parental lines and the other progeny individuals during both years. The second part of this thesis analyzed the influence of F. graminearum on the mycobiome of IWG spikelet and flag leaf samples from two different locations, Winnipeg and Carman, MB. A total 372 IWG samples (half of which were inoculated with F. graminearum [Winnipeg] and the other half, not [Carman]) were used for fungal community profiling via ITS amplicon sequencing using PacBio technology. There were significant differences in the fungal community among the IWG spike samples from Winnipeg and Carman. As expected, F. graminearum dominated IWG spike mycobiomes at the inoculated Winnipeg site, whereas in Carman, Alternaria dominated. Also, the presence of F. graminearum seemed to influence the broader spike mycobiome and the presence of beneficial fungi was less where F. graminearum dominated. The mycobiome data also revealed that the pathogenic fungi Alternaria, Nigrospora, Septoriella, Stemphylium, Claviceps, Bipolaris, and Drechslera can become potential threats to IWG in the future. The last part of this thesis sheds light on the interaction between F. graminearum and another

Both exposure to antibiotics at birth and delivery via Caesarean section influence the gut bacteriome’s development in infants. Using 16S rRNA and internal transcribed spacer sequencing on the Ion Torrent platform, we employed network analysis to investigate the bacterial and fungal interkingdom relationships in the gut microbiome from birth to age 18 months in a prospective cohort study of 140 infants. The gut microbiome at ages six and 18 months revealed distinctive microbial interactions, including both positive and negative associations between bacterial and fungal genera in the gut ecosystem. Perinatal factors, delivery mode and intrapartum antibiotic exposure affected the associations between bacterial and fungal species. In infants exposed and unexposed to perinatal antibiotics, the gut microbiome formed distinct networks for the bacteriome and mycobiome. The fungi Saccharomyces, Trichosporon, Pezoloma, Cystofilobasidium, Rigidoporus and Fomitopsis were strongly associated with exposure to antibiotics at birth. Hyaloscypha, Trichosporon, Fomitopsis and Vishniacozyma were strongly associated with the control group that was not exposed to antibiotics. Five distinct networks were formed according to delivery mode. The present study confirms that bacteria and fungi clearly interact in the infant gut ecosystem. Furthermore, perinatal factors appear to influence the relationships between bacteria and fungi in the developing gut microbiome.

The human gut microbiota is comprised of a variety of microbes, such as bacteria, fungi, viruses, and archaea. Furthermore, the gut contains bacterial extracellular vesicles (bEVs), small particles secreted by bacteria. The health effects of the gut microbiota have been explored but it is yet unclear when the gut microbiota starts developing. The womb was earlier assumed to be sterile, but recent studies imply that the fetus may already be exposed to microbes in the womb. Colonization of the gut is usually assessed from the first-pass meconium, the first stool passed after birth, which is assumed to serve as a proxy for the fetal gut microbiota. This cohort study of 513 mother-infant pairs was undertaken to elucidate the timing of gut microbiota development and factors affecting it. The first-pass meconium and later stool samples were collected from the infants and placenta and amniotic fluid from the mothers. 16S rRNA gene sequencing was performed to characterize the bacteriome and bEVs in the samples and internal transcribed spacer (ITS) gene sequencing to characterize the mycobiome. Questionnaires were used to collect information from the families on environmental and lifestyle conditions during pregnancy in order to assess the effect of prenatal factors on the microbial composition of the initial gut microbiota. A detectable meconium bacteriome and mycobiome were characterized, but no detectable placenta or amniotic fluid microbiota could be identified. The meconium microbiota was mainly affected by birth factors, including the mode of delivery and use of intrapartum antibiotics. Prenatal factors had little effect on the composition of the meconium bacteriome. The bEVs characterized in the meconium were found to be unaffected by the birth factors. Birth factors affected gut mycobiome development in the infants at least until the age of 18 months. It is concluded that the neonatal gut is probably colonized during birth, and that the birth factors influen, Tiivistelmä Ihmisen suoliston mikrobiomi koostuu monenlaisista mikrobeista, kuten bakteereista, sienistä, viruksista ja arkeista. Lisäksi suolisto sisältää bakteeriperäisiä ekstrasellulaarisia vesikkeleitä (bEV), jotka ovat pieniä partikkeleita, joita bakteerit erittävät. Suoliston mikrobiomin terveysvaikutuksia on tutkittu jo laajasti, mutta suoliston mikrobiomin alkuperää ei vielä tunneta tarkkaan. Aikaisemmin kohtua on pidetty sikiölle steriilinä kasvuympäristönä, mutta viimeaikainen tutkimus osoittaa, että sikiö saattaa altistua mikrobeille kohdussa. Suoliston kolonisointia on tutkittu lapsenpihkan eli mekoniumin, lapsen elämän ensimmäisen ulosteen avulla, jonka uskotaan kuvaavan sikiön suoliston mikrobiomia. Tutkimus koostui 513 äiti-lapsiparin kohortista, ja tutkimuksen tarkoituksena oli havainnollistaa suoliston mikrobiomin kolonisointiaikaa sekä sen kehitykseen vaikuttavia tekijöitä. Keräsimme mekonium- ja ulostenäytteitä lapsilta sekä istukka- ja lapsivesinäytteitä äideiltä, ja suoritimme 16S rRNA -geenisekvensoinnin tutkimaan bakteriomia ja bEV:itä ja ITS-geenisekvensoinnin tutkimaan mykobiomia näytteissä. Lisäksi suoritimme kyselytutkimuksen perheille heidän elinympäristöstään ja elintavoistaan raskauden aikana tutkiaksemme prenataalisia ja synnytyksenaikaisia tekijöitä, jotka ovat voineet vaikuttaa varhaisen suoliston mikrobiomin kehitykseen. Mekoniumista löytyi havaittava bakteriomi ja mykobiomi, mutta istukasta ja lapsivedestä havaittavaa mikrobiomia ei löytynyt. Mekoniumin mikrobiomiin vaikuttivat eritoten synnytyksenaikaiset tekijät, mukaan lukien synnytystapa ja synnytyksenaikaiset antibiootit. Prenataalisilla tekijöillä oli pieni vaikutus mekonium bakteriomiin. Löysimme myös mekoniumista bEV:itä, joihin synnytyksenaikaiset tekijät eivät vaikuttaneet. Lisäksi totesimme, että synnytyksenaikaiset tekijät vaikuttivat suoliston mykobiomin kehitykseen lapsilla vähintään 18 kk ikäisiksi asti. Tämä tutkimus osoitti, että varhainen suoliston mikrobiomi tode

Endophytic fungal communities living in leaves of trees can have a significant impact on their hosts,showing both beneficial and detrimental effects. Some mutualistic fungal species protect the host against pathogens or herbivores, while pathogenic species can increase the vulnerability of the host against biotic and abiotic stresses. The Swedish forestry model consists of planting nursery-produced seedlings after final felling. In the nurseries, the seedlings are grown densely in containers under greenhouse conditions and for disease prevention the Integrated Pest Management strategy is followed, where fungicides are applied to protect the plants. It is known that the intensive nursery production system affect the fungal communities present in the roots of seedlings by making them less diverse, but the effect in the foliar fungi is still unknown. The main objective of this study was to investigate the diversity of foliar fungal communities in Scots pine (Pinus sylvestris) seedlings produced in the nursery after their transplantation to the forest clear-cuts and to compare them with naturally regenerated seedlings. Needle samples of both types of seedlings were collected from three different forest clear-cut sites in Uppland, Sweden. The time scale and the exposure time of the needle were taken into account by sampling at 8-time points of the seasonal period and taking one-year old needles and current-year needles. Fungal communities were characterised by high- throughput sequencing of the ITS2 rRNA region of healthy-looking needles. Sequences were filtered and grouped into operational taxonomic units (OTU). The taxonomic classification of each OTU was determined using the PROTAX-fungi and massBLASTer databases and fungal lifestyle was assigned using the FungalTraits database. Results show that exposure time and needle age are the main factors affecting fungal diversity in needles, irrespective of whether seedlings are from nurseries or natural regeneration. Overall

Background: Chestnut forests are severely threatened by chestnut blight caused by the fungal pathogen Cryphonectria parasitica and the infected trees exhibit bark canker in the later stage of the disease. European chestnut (Castanea sativa) is further infected by Gnomoniopsis smithogilvyi, another canker-causing fungal pathogen. We explored whether and how chestnut blight is reflected in bark-inhabiting fungal communities of European chestnut and also assessed the co-occurrence of C. parasitica and G. smithogilvyi. Materials and methods: We initially investigated the fungal communities of European chestnut bark tissues and further monitored changes in these fungal communities with regard to disease progression from infection to canker formation by analyzing bark samples from asymptomatic trees, asymptomatic trees with latent C. parasitica infection, and infected trees with canker tissues, using amplicon sequencing of the ITS2 region of rDNA. Results: The results showed that fungal community composition and diversity differed between the sample types. The fungal community composition was substantially reshaped by canker formation, whereas latent C. parasitica infection and more specifically pre-canker infection period per se had a weak effect. Fungal communities of canker samples was less diverse and more dissimilar to those of other sample types. C. parasitica dominated the mycobiome of canker samples, whereas G. smithogilvyi was found in only 9% of canker samples at very low abundances. However, G. smithogilvyi was a dominant fungus in the bark of healthy plants. Conclusion: This study highlights that canker formation is the principal driver of decreasing diversity and altered composition of the mycobiome in bark tissues of European chestnut infected by C. parasitica infection. It additionally emphasizes the scarce co-occurrence of C. parasitica and G. smithogilvyi on European chestnut.

Oral candidiasis is an opportunistic infection of the oral mucosa sustained by fungi of the genus Candida. Various Candida species, with a predominance of C. albicans, normally a saprophyte of the oral cavity, may become virulent and infect the oral mucosa with variegated clinical presentation, in case of imbalance of the oral microbiota, the presence of local predisposing factors and systemic conditions that weaken the immune system. Conventionally, oral candidiasis eradication is done with the help of antifungal drugs. However, the growing phenomena of drug resistance and the increase in infections sustained by non-albicans species being less responsive to common antifungals have orientied researches towards the experimentation of alternative therapies. The present review considered the most promising alternative therapeutic proposals. The use of plant derivatives with phytotherapy is a promising option, such as probiotics, to rebalance the oral microbiota in case of dysbiosis. Finally, antimicrobial photodynamic therapy (aPDT), with highly selective fungicidal activity and free of side effects, is also being studied as a powerful alternative to drug administration. All these therapies are alternatives or supportive to the conventional treatment of recurrent and non-drug-responsive forms of oral candidiasis. However, further studies are needed to define the most active compounds, the efficacy of the therapies compared with the conventional ones, and the planning of regulated and standardized protocols.

Liver and biliary diseases affect more than a billion people worldwide, with high associated morbidity and mortality. The impact of the intestinal bacterial microbiome on liver diseases has been well established. However, the fungal microbiome, or mycobiome, has been overlooked for a long time. Recently, several studies have shed light on the role of the mycobiome in the development and progression of hepatobiliary diseases. In particular, the fungal genus Candida has been found to be involved in the pathogenesis of multiple hepatobiliary conditions. Herein, we compare colonisation and infection, describe mycobiome findings in the healthy state and across the various hepatobiliary conditions, and point toward communalities. We detail how quantitation of immune responses to fungal antigens can be employed to predict disease severity, e.g. using antibodies to Saccharomyces cerevisiae or specific anti-Candida albicans antibodies. We also show how fungal products (e.g. beta-glucans, candidalysin) activate the host's immune system to exacerbate liver and biliary diseases. Finally, we describe how the gut mycobiome can be modulated to ameliorate hepatobiliary conditions.

An emerging crop, intermediate wheatgrass (IWG) is resistant to the natural occurrence of most important cereal diseases and is currently under domestication to improve its yield. An IWG population consisting of two parental lines and 154 individual F1 progenies were inoculated with F. graminearum spores in a field setting to check their performance against Fusarium head blight (FHB) over two consecutive years. The higher humidity in 2022 lead to higher FHB severity and incidence, and higher DON (deoxynivalenol) mycotoxin accumulation than in 2021. DON was mostly restricted to the chaff in 2021, whereas in 2022, DON was found in the grain as well. Considering all the FHB aspects checked in this experiment, four IWG progeny individuals (plants: P-17, P-43, P-107, and P-139) were found to consistently perform better than the parental lines and the other progeny individuals during both years. The second part of this thesis analyzed the influence of F. graminearum on the mycobiome of IWG spikelet and flag leaf samples from two different locations, Winnipeg and Carman, MB. A total 372 IWG samples (half of which were inoculated with F. graminearum [Winnipeg] and the other half, not [Carman]) were used for fungal community profiling via ITS amplicon sequencing using PacBio technology. There were significant differences in the fungal community among the IWG spike samples from Winnipeg and Carman. As expected, F. graminearum dominated IWG spike mycobiomes at the inoculated Winnipeg site, whereas in Carman, Alternaria dominated. Also, the presence of F. graminearum seemed to influence the broader spike mycobiome and the presence of beneficial fungi was less where F. graminearum dominated. The mycobiome data also revealed that the pathogenic fungi Alternaria, Nigrospora, Septoriella, Stemphylium, Claviceps, Bipolaris, and Drechslera can become potential threats to IWG in the future. The last part of this thesis sheds light on the interaction between F. graminearum and another

An emerging crop, intermediate wheatgrass (IWG) is resistant to the natural occurrence of most important cereal diseases and is currently under domestication to improve its yield. An IWG population consisting of two parental lines and 154 individual F1 progenies were inoculated with F. graminearum spores in a field setting to check their performance against Fusarium head blight (FHB) over two consecutive years. The higher humidity in 2022 lead to higher FHB severity and incidence, and higher DON (deoxynivalenol) mycotoxin accumulation than in 2021. DON was mostly restricted to the chaff in 2021, whereas in 2022, DON was found in the grain as well. Considering all the FHB aspects checked in this experiment, four IWG progeny individuals (plants: P-17, P-43, P-107, and P-139) were found to consistently perform better than the parental lines and the other progeny individuals during both years. The second part of this thesis analyzed the influence of F. graminearum on the mycobiome of IWG spikelet and flag leaf samples from two different locations, Winnipeg and Carman, MB. A total 372 IWG samples (half of which were inoculated with F. graminearum [Winnipeg] and the other half, not [Carman]) were used for fungal community profiling via ITS amplicon sequencing using PacBio technology. There were significant differences in the fungal community among the IWG spike samples from Winnipeg and Carman. As expected, F. graminearum dominated IWG spike mycobiomes at the inoculated Winnipeg site, whereas in Carman, Alternaria dominated. Also, the presence of F. graminearum seemed to influence the broader spike mycobiome and the presence of beneficial fungi was less where F. graminearum dominated. The mycobiome data also revealed that the pathogenic fungi Alternaria, Nigrospora, Septoriella, Stemphylium, Claviceps, Bipolaris, and Drechslera can become potential threats to IWG in the future. The last part of this thesis sheds light on the interaction between F. graminearum and another

Rock-dwelling fungi play critical ecological roles in drylands, including soil formation and nutrient cycling; however, we know very little about the identity, function and environmental preferences of these important organisms, and the mere existence of a consistent rock mycobiome across diverse arid regions of the planet remains undetermined. To address this knowledge gap, we conducted a meta-analysis of rock fungi and spatially associated soil communities, surveyed across 28 unique sites spanning four major biogeographic regions (North America, Arctic, Maritime and Continental Antarctica) including contrasting climates, from cold and hot deserts to semiarid drylands. We show that rocks support a consistent and unique mycobiome that was different from that found in surrounding soils. Lichenized fungi from class Lecanoromycetes were consistently indicative of rocks across contrasting regions, together with ascomycetous representatives of black fungi in Arthoniomycetes, Dothideomycetes and Eurotiomycetes. In addition, compared with soil, rocks had a lower proportion of saprobes and plant symbiotic fungi. The main drivers structuring rock fungi distribution were spatial distance and, to a larger extent, climatic factors regulating moisture and temperature (i.e. mean annual temperature and mean annual precipitation), suggesting that these paramount and unique communities might be particularly sensitive to increases in temperature and desertification.

Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome, Svampsamhällen har global betydelse som drivande krafter i viktiga näringskretslopp, och ingår symbioser med de flesta landväxter. I de kvävefattiga svenska boreala skogarna koloniserar ektomykorrhizasvampar majoriteten av rötter på de ekonomiskt viktiga och dominerande trädarterna gran (Picea abies) och tall (Pinus sylvestris). Detta har signifikanta implikationer för både trädnäring och nedbrytningsprocesser i marken. För att säkerställa hållbart skogsbruk på lång sikt är det nödvändigt att vi får en djupare förståelse för biotiska och abiotiska faktorer som kan påverka skogens hälsa och tillväxt. Moderna sekvenseringsmetoder som DNA amplikon-sekvensering och RNA-baserad metatranskriptomik har hittills lett till en förbättrad förståelse för svampsamhällen, men det finns fortfarande oklarheter i många av processerna de är viktiga för. I denna doktorsavhandling har jag använt dessa sekvenseringsmetoder för att fördjupa vår förståelse av svampsamhällen i boreal skog, och hur de påverkas av skogsbruk och kvävetillförsel. I avhandlingens första del har jag undersökt hur gödsling vid plantering och sådd påverkar plantornas överlevnad och tillväxt, och dessutom de associerade svampsamhällen som undersöktes med hjälp av amplikon sekvensering. I två manuskript visar jag, hur efter plantering på markberedda kalhyggen, plantorna fort blir koloniserade av olika svampar från marken, bland annat mykorrhizasvampar. Jag visar att små doser av kvävegödsel höjer överlevnad av sådda frön, och att organiskt kväve (i form av arginin) kan förbättra tidig rottillväxt vid plantering. Svampsamhällen blev inte påtagligt förändrade av denna lätta gödsling. I andra delen av denna avhandling har jag varit involverad i utvecklingen av en bioinformatisk pipeline för de novo-assemblering, såsom taxonomisk och funktionell annotering av komplex RNA sekvenseringsdata, för att göra framsteg i vår förmåga att använda metatranskriptomik (inte bara, men också) som ett alternativ till DNA amplikon sekve

Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome, Svampsamhällen har global betydelse som drivande krafter i viktiga näringskretslopp, och ingår symbioser med de flesta landväxter. I de kvävefattiga svenska boreala skogarna koloniserar ektomykorrhizasvampar majoriteten av rötter på de ekonomiskt viktiga och dominerande trädarterna gran (Picea abies) och tall (Pinus sylvestris). Detta har signifikanta implikationer för både trädnäring och nedbrytningsprocesser i marken. För att säkerställa hållbart skogsbruk på lång sikt är det nödvändigt att vi får en djupare förståelse för biotiska och abiotiska faktorer som kan påverka skogens hälsa och tillväxt. Moderna sekvenseringsmetoder som DNA amplikon-sekvensering och RNA-baserad metatranskriptomik har hittills lett till en förbättrad förståelse för svampsamhällen, men det finns fortfarande oklarheter i många av processerna de är viktiga för. I denna doktorsavhandling har jag använt dessa sekvenseringsmetoder för att fördjupa vår förståelse av svampsamhällen i boreal skog, och hur de påverkas av skogsbruk och kvävetillförsel. I avhandlingens första del har jag undersökt hur gödsling vid plantering och sådd påverkar plantornas överlevnad och tillväxt, och dessutom de associerade svampsamhällen som undersöktes med hjälp av amplikon sekvensering. I två manuskript visar jag, hur efter plantering på markberedda kalhyggen, plantorna fort blir koloniserade av olika svampar från marken, bland annat mykorrhizasvampar. Jag visar att små doser av kvävegödsel höjer överlevnad av sådda frön, och att organiskt kväve (i form av arginin) kan förbättra tidig rottillväxt vid plantering. Svampsamhällen blev inte påtagligt förändrade av denna lätta gödsling. I andra delen av denna avhandling har jag varit involverad i utvecklingen av en bioinformatisk pipeline för de novo-assemblering, såsom taxonomisk och funktionell annotering av komplex RNA sekvenseringsdata, för att göra framsteg i vår förmåga att använda metatranskriptomik (inte bara, men också) som ett alternativ till DNA amplikon sekve

Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis. We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B. The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis. Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH. Non-alcoholic fatty liver disease is one of the most co

Rock-dwelling fungi play critical ecological roles in drylands, including soil formation and nutrient cycling; however, we know very little about the identity, function and environmental preferences of these important organisms, and the mere existence of a consistent rock mycobiome across diverse arid regions of the planet remains undetermined. To address this knowledge gap, we conducted a meta-analysis of rock fungi and spatially associated soil communities, surveyed across 28 unique sites spanning four major biogeographic regions (North America, Arctic, Maritime and Continental Antarctica) including contrasting climates, from cold and hot deserts to semiarid drylands. We show that rocks support a consistent and unique mycobiome that was different from that found in surrounding soils. Lichenized fungi from class Lecanoromycetes were consistently indicative of rocks across contrasting regions, together with ascomycetous representatives of black fungi in Arthoniomycetes, Dothideomycetes and Eurotiomycetes. In addition, compared with soil, rocks had a lower proportion of saprobes and plant symbiotic fungi. The main drivers structuring rock fungi distribution were spatial distance and, to a larger extent, climatic factors regulating moisture and temperature (i.e. mean annual temperature and mean annual precipitation), suggesting that these paramount and unique communities might be particularly sensitive to increases in temperature and desertification.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

Microbes play an important role in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and asthma. While the role of bacterial pathogens has been extensively studied, the contribution of fungal species to the pathogenesis of chronic lung diseases is much less understood. The recent introduction of next generation sequencing techniques has revealed the existence of complex microbial lung communities in healthy individuals and patients with chronic respiratory disorders, with fungi being an important part of these communities’ structure (mycobiome). There is growing evidence that the components of the lung mycobiome influence the clinical course of chronic respiratory diseases, not only by direct pathogenesis but also by interacting with bacterial species and with the host’s physiology. In this article, we review the current knowledge on the role of fungi in chronic respiratory diseases, which was obtained by conventional culture and next-generation sequencing, highlighting the limitations of both techniques and exploring future research areas.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Fungal communities (mycobiome) have an important role in sustaining the resilience of complex microbial communities and maintenance of homeostasis. The mycobiome remains relatively unexplored compared to the bacteriome despite increasing evidence highlighting their contribution to host-microbiome interactions in health and disease. Despite being a small proportion of the total species, fungi constitute a large proportion of the biomass within the human microbiome and thus serve as a potential target for metabolic reprogramming in pathogenesis and disease mechanism. Metabolites produced by fungi shape host niches, induce immune tolerance and changes in their levels prelude changes associated with metabolic diseases and cancer. Given the complexity of microbial interactions, studying the metabolic interplay of the mycobiome with both host and microbiome is a demanding but crucial task. However, genome-scale modelling and synthetic biology can provide an integrative platform that allows elucidation of the multifaceted interactions between mycobiome, microbiome and host. The inferences gained from understanding mycobiome interplay with other organisms can delineate the key role of the mycobiome in pathophysiology and reveal its role in human disease., QC 20221024

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Dispersal is a key force in the assembly of fungal communities and the air is the dominant route of dispersal for most fungi. Understanding the dynamics of airborne fungi is important for determining their source and for helping to prevent fungal disease. This understanding is important in the San Joaquin Valley of California, which is home to 4.2 million people and where the airborne fungus Coccidioides is responsible for the most important fungal disease of otherwise healthy humans, coccidioidomycosis. The San Joaquin Valley is the most productive agricultural region in the United States, with the principal crops grown therein susceptible to fungal pathogens. Here, we characterize the fungal community in soil and air on undeveloped and agricultural land in the San Joaquin Valley using metabarcoding of the internal transcribed spacer 2 variable region of fungal rDNA. Using 1,002 individual samples, we report one of the most extensive studies of fungi sampled simultaneously from air and soil using modern sequencing techniques. We find that the air mycobiome in the San Joaquin Valley is distinct from the soil mycobiome, and that the assemblages of airborne fungi from sites as far apart as 160 km are far more similar to one another than to the fungal communities in nearby soils. Additionally, we present evidence that airborne fungi in the San Joaquin Valley are subject to dispersal limitation and cyclical intra-annual patterns of community composition. Our findings are broadly applicable to understanding the dispersal of airborne fungi and the taxonomic structure of airborne fungal assemblages.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Microbes play an important role in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and asthma. While the role of bacterial pathogens has been extensively studied, the contribution of fungal species to the pathogenesis of chronic lung diseases is much less understood. The recent introduction of next generation sequencing techniques has revealed the existence of complex microbial lung communities in healthy individuals and patients with chronic respiratory disorders, with fungi being an important part of these communities’ structure (mycobiome). There is growing evidence that the components of the lung mycobiome influence the clinical course of chronic respiratory diseases, not only by direct pathogenesis but also by interacting with bacterial species and with the host’s physiology. In this article, we review the current knowledge on the role of fungi in chronic respiratory diseases, which was obtained by conventional culture and next-generation sequencing, highlighting the limitations of both techniques and exploring future research areas.

Background & aimsStudies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis.MethodsWe performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B.ResultsThe presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis.ConclusionsNon-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NAS

Humans harbour large quantities of microbes, including bacteria, fungi, viruses and archaea, in the gut. Patients with liver disease exhibit changes in the intestinal microbiota and gut barrier dysfunction. Preclinical models demonstrate the importance of the gut microbiota in the pathogenesis of various liver diseases. In this review, we discuss how manipulation of the gut microbiota can be used as a novel treatment approach for liver disease. We summarise current data on untargeted approaches, including probiotics and faecal microbiota transplantation, and precision microbiome-centered therapies, including engineered bacteria, postbiotics and phages, for the treatment of liver diseases.

Microbial communities contain a broad phylogenetic diversity of organisms; however, the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include data from the fungal internal transcribed spacer (ITS) region, five candidate DNA extraction kits were compared against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. The results are presented considering a diverse panel of host-associated and environmental sample types and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection and microbial community composition among protocols. Across all criteria, the MagMAX Microbiome kit was found to perform best. The PowerSoil Pro kit performed comparably but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food and Norgen Stool kits were included.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.

Microbes play an important role in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and asthma. While the role of bacterial pathogens has been extensively studied, the contribution of fungal species to the pathogenesis of chronic lung diseases is much less understood. The recent introduction of next generation sequencing techniques has revealed the existence of complex microbial lung communities in healthy individuals and patients with chronic respiratory disorders, with fungi being an important part of these communities’ structure (mycobiome). There is growing evidence that the components of the lung mycobiome influence the clinical course of chronic respiratory diseases, not only by direct pathogenesis but also by interacting with bacterial species and with the host’s physiology. In this article, we review the current knowledge on the role of fungi in chronic respiratory diseases, which was obtained by conventional culture and next-generation sequencing, highlighting the limitations of both techniques and exploring future research areas.

The role of gastrointestinal fungi in human health and disease is becoming more evident. The mycobiome composition and diversity of preterm infants is affected by interactions with bacteria and with clinical variables. We characterized the composition and diversity of the preterm infant mycobiome and the effect of clinical variables on it over the course of the first six postnatal weeks.

Many children spend considerable time in daycare centers and may be influenced by the indoor microorganisms there, including fungi. In this study, we investigate the indoor mycobiomes of 125 daycare centers distributed along strong environmental gradients throughout Norway. Dust samples were collected from doorframes outside and inside buildings using a community science sampling approach. Fungal communities in the dust samples were analyzed using DNA metabarcoding of the internal transcribed spacer 2 (ITS2) region. We observed a marked difference between the outdoor and indoor mycobiomes. The indoor mycobiomes included considerably more yeasts and molds than the outdoor samples, with Saccharomyces, Mucor, Malassezia, and Penicillium being among the most dominant fungal genera. Changes in the indoor fungal richness and composition correlated with numerous variables related to both outdoor and indoor conditions; there was a clear geographic structure in the indoor mycobiome composition that mirrored the outdoor climate, ranging from humid areas in western Norway to drier and colder areas in eastern Norway. Moreover, the number of children in the daycare centers, as well as various building features, influenced the indoor mycobiome composition. We conclude that the indoor mycobiomes in Norwegian daycare centers are structured by multiple factors and are dominated by yeasts and molds. This study exemplifies how community science sampling enables DNA-based analyses of a high number of samples covering wide geographic areas.

Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome, Svampsamhällen har global betydelse som drivande krafter i viktiga näringskretslopp, och ingår symbioser med de flesta landväxter. I de kvävefattiga svenska boreala skogarna koloniserar ektomykorrhizasvampar majoriteten av rötter på de ekonomiskt viktiga och dominerande trädarterna gran (Picea abies) och tall (Pinus sylvestris). Detta har signifikanta implikationer för både trädnäring och nedbrytningsprocesser i marken. För att säkerställa hållbart skogsbruk på lång sikt är det nödvändigt att vi får en djupare förståelse för biotiska och abiotiska faktorer som kan påverka skogens hälsa och tillväxt. Moderna sekvenseringsmetoder som DNA amplikon-sekvensering och RNA-baserad metatranskriptomik har hittills lett till en förbättrad förståelse för svampsamhällen, men det finns fortfarande oklarheter i många av processerna de är viktiga för. I denna doktorsavhandling har jag använt dessa sekvenseringsmetoder för att fördjupa vår förståelse av svampsamhällen i boreal skog, och hur de påverkas av skogsbruk och kvävetillförsel. I avhandlingens första del har jag undersökt hur gödsling vid plantering och sådd påverkar plantornas överlevnad och tillväxt, och dessutom de associerade svampsamhällen som undersöktes med hjälp av amplikon sekvensering. I två manuskript visar jag, hur efter plantering på markberedda kalhyggen, plantorna fort blir koloniserade av olika svampar från marken, bland annat mykorrhizasvampar. Jag visar att små doser av kvävegödsel höjer överlevnad av sådda frön, och att organiskt kväve (i form av arginin) kan förbättra tidig rottillväxt vid plantering. Svampsamhällen blev inte påtagligt förändrade av denna lätta gödsling. I andra delen av denna avhandling har jag varit involverad i utvecklingen av en bioinformatisk pipeline för de novo-assemblering, såsom taxonomisk och funktionell annotering av komplex RNA sekvenseringsdata, för att göra framsteg i vår förmåga att använda metatranskriptomik (inte bara, men också) som ett alternativ till DNA amplikon sekve

Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome, Svampsamhällen har global betydelse som drivande krafter i viktiga näringskretslopp, och ingår symbioser med de flesta landväxter. I de kvävefattiga svenska boreala skogarna koloniserar ektomykorrhizasvampar majoriteten av rötter på de ekonomiskt viktiga och dominerande trädarterna gran (Picea abies) och tall (Pinus sylvestris). Detta har signifikanta implikationer för både trädnäring och nedbrytningsprocesser i marken. För att säkerställa hållbart skogsbruk på lång sikt är det nödvändigt att vi får en djupare förståelse för biotiska och abiotiska faktorer som kan påverka skogens hälsa och tillväxt. Moderna sekvenseringsmetoder som DNA amplikon-sekvensering och RNA-baserad metatranskriptomik har hittills lett till en förbättrad förståelse för svampsamhällen, men det finns fortfarande oklarheter i många av processerna de är viktiga för. I denna doktorsavhandling har jag använt dessa sekvenseringsmetoder för att fördjupa vår förståelse av svampsamhällen i boreal skog, och hur de påverkas av skogsbruk och kvävetillförsel. I avhandlingens första del har jag undersökt hur gödsling vid plantering och sådd påverkar plantornas överlevnad och tillväxt, och dessutom de associerade svampsamhällen som undersöktes med hjälp av amplikon sekvensering. I två manuskript visar jag, hur efter plantering på markberedda kalhyggen, plantorna fort blir koloniserade av olika svampar från marken, bland annat mykorrhizasvampar. Jag visar att små doser av kvävegödsel höjer överlevnad av sådda frön, och att organiskt kväve (i form av arginin) kan förbättra tidig rottillväxt vid plantering. Svampsamhällen blev inte påtagligt förändrade av denna lätta gödsling. I andra delen av denna avhandling har jag varit involverad i utvecklingen av en bioinformatisk pipeline för de novo-assemblering, såsom taxonomisk och funktionell annotering av komplex RNA sekvenseringsdata, för att göra framsteg i vår förmåga att använda metatranskriptomik (inte bara, men också) som ett alternativ till DNA amplikon sekve

Fungal communities are major players in globally important nutrient cycling processes, and form symbioses with most terrestrial plants. In the nitrogen (N) limited Swedish boreal forest, ectomycorrhizal (EcM) fungi colonize most roots of the economically important and stand dominating conifer species, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), with significant implications for tree nutrition and decomposition processes. Long-term sustainable forestry practices require a deeper understanding of biotic and abiotic factors influencing forest health and tree growth. While high-throughput sequencing technologies such as DNA amplicon sequencing or RNA-based metatranscriptomics have furthered our understanding of fungal communities, there are still many details of EcM symbiosis and decomposition processes that we do not understand. In this thesis, I have used these sequencing methods to further our understanding of fungal communities in the boreal forest, and how they are influenced by forest management and N addition. In the first part of this thesis, I investigated how early fertilization of seeded or planted seedlings affects seedling growth and survival, and the fungal communities associated with the growing seedlings, assessed by amplicon sequencing. In two manuscripts I show that seeds or seedlings planted into scarified clearcut soil are rapidly colonized by site indigenous fungi, including many EcM species. I show that small doses of added N increase survival of sown seeds and that organic N (in the form of arginine) can increase early root growth of planted seedlings. This light fertilization did not perturb early fungal community succession. In the second part, I co-developed a workflow for de novo assembly as well as functional and taxonomic annotation of complex fungal community RNA sequencing data, in order to advance our ability to utilize metatranscriptomics (not only) as an alternative to DNA amplicon approaches. I assessed the outcome, Svampsamhällen har global betydelse som drivande krafter i viktiga näringskretslopp, och ingår symbioser med de flesta landväxter. I de kvävefattiga svenska boreala skogarna koloniserar ektomykorrhizasvampar majoriteten av rötter på de ekonomiskt viktiga och dominerande trädarterna gran (Picea abies) och tall (Pinus sylvestris). Detta har signifikanta implikationer för både trädnäring och nedbrytningsprocesser i marken. För att säkerställa hållbart skogsbruk på lång sikt är det nödvändigt att vi får en djupare förståelse för biotiska och abiotiska faktorer som kan påverka skogens hälsa och tillväxt. Moderna sekvenseringsmetoder som DNA amplikon-sekvensering och RNA-baserad metatranskriptomik har hittills lett till en förbättrad förståelse för svampsamhällen, men det finns fortfarande oklarheter i många av processerna de är viktiga för. I denna doktorsavhandling har jag använt dessa sekvenseringsmetoder för att fördjupa vår förståelse av svampsamhällen i boreal skog, och hur de påverkas av skogsbruk och kvävetillförsel. I avhandlingens första del har jag undersökt hur gödsling vid plantering och sådd påverkar plantornas överlevnad och tillväxt, och dessutom de associerade svampsamhällen som undersöktes med hjälp av amplikon sekvensering. I två manuskript visar jag, hur efter plantering på markberedda kalhyggen, plantorna fort blir koloniserade av olika svampar från marken, bland annat mykorrhizasvampar. Jag visar att små doser av kvävegödsel höjer överlevnad av sådda frön, och att organiskt kväve (i form av arginin) kan förbättra tidig rottillväxt vid plantering. Svampsamhällen blev inte påtagligt förändrade av denna lätta gödsling. I andra delen av denna avhandling har jag varit involverad i utvecklingen av en bioinformatisk pipeline för de novo-assemblering, såsom taxonomisk och funktionell annotering av komplex RNA sekvenseringsdata, för att göra framsteg i vår förmåga att använda metatranskriptomik (inte bara, men också) som ett alternativ till DNA amplikon sekve

Head and neck squamous cell carcinoma (HNSCC) tumor phenotypes and clinical outcomes are significantly influenced by etiological agents, such as HPV infection, smoking, and alcohol consumption. Accordingly, the intratumor microbiome has been increasingly implicated in cancer progression and metastasis. However, few studies characterize the intratumor microbial landscape of HNSCC with respect to these etiological agents. In this study, we aimed to investigate the bacterial and fungal landscape of HNSCC in association with HPV infection, smoking, and alcohol consumption. RNA-sequencing data were extracted from The Cancer Genome Atlas (TCGA) regarding 449 tissue samples and 44 normal samples. Pathoscope 2.0 was used to extract the microbial reads. Microbe abundance was compared to clinical variables, oncogenic signatures, and immune-associated pathways. Our results demonstrated that a similar number of dysregulated microbes was overabundant in smokers and nonsmokers, while heavy drinkers were characterized by an underabundance of dysregulated microbes. Conversely, the majority of dysregulated microbes were overabundant in HPV+ tumor samples when compared to HPV- tumor samples. Moreover, we observed that many dysregulated microbes were associated with oncogenic and metastatic pathways, suggesting their roles in influencing carcinogenesis. These microbes provide insights regarding potential mechanisms for tumor pathogenesis and progression with respect to the three etiological agents.