The first ITS1 profiling of honey samples from the Southeast Asian region Lombok, Bali and Banggi Island.

Southern Asian flowers offer honeybees a diversity of nectar. Based on its geographical origin, honey quality varies. Traditional methods are less authentic than DNA-based identification. The origin of honey is determined by pollen, polyphenolic, and macro-microorganisms. In this study, amplicon sequencing targets macro-microorganisms in eDNA using the ITS1 region to explore honey's geographical location and authentication. The variety of honey samples was investigated using ITS1 with Illumina sequencing. For all four honey samples, raw sequence reads showed 979,380 raw ITS1 amplicon reads and 375 ASVs up to the phylum level. The highest total number of 202 ASVs up to phylum level identified Bali honey with 211,189 reads, followed by Banggi honey with 309,207 a total number of 111 ASVs, and Lombok represents only 63 ASVs up to phylum level with several read 458,984. Based on Shannon and Chao1, honey samples from Bali (B2) and (B3) exhibited higher diversity than honey from Lombok (B1) and green honey from Sabah (B4), while the Simpson index showed that Banggi honey (B4) had higher diversity. Honey samples had significant variance in mycobiome taxonomic composition and abundance. Zygosaccharomyces and Aspergillus were the main genera found in Lombok honey, with percentages of 68.81% and 29.76% respectively. Bali honey samples (B2 and B3) were identified as having a significant amount of the genus Aureobasidium, accounting for 40.81% and 25% of the readings, respectively. The microbiome composition of Banggi honey (B4) showed a high presence of Zygosaccharomyces 45.17% and Aureobasidium 35.24%. The ITS1 analysis effectively distinguishes between honey samples of different origins and its potential as a discriminatory tool for honey origin and authentication purposes.

Fecal fungal microbiota alterations associated with clinical phenotypes in Crohn's disease in southwest China.

Although previous studies reported that gut fungal microbiota was associated with Crohn's disease (CD), only a few studies have focused on the correlation between gut fungi and clinical phenotypes of CD. Here, we aimed to analyze the association between intestinal fungi and the occurrence of CD, disease activity, biological behaviors, and perianal lesions. Stool samples from subjects meeting the inclusion and exclusion criteria were collected for running internal transcribed spacer 2 (ITS2) high-throughput sequencing. Then, correlation analysis was conducted between intestinal fungi and different clinical groups. There were 45 patients with CD and 17 healthy controls (HCs) enrolled. Results showed that two phyla, Rozellomycota and Mortierellomycota, were not present in patients with CD compared to HCs. At the same time, there was a higher abundance of fungal genera and species belonging to the phylum Ascomycota in patients with CD. SparCC network analysis showed fewer interactions among the fungal communities in patients with CD compared to HCs. Exophiala dermatitidis was positively associated with the clinical active stage and platelet count. The genus Candida was with significantly higher abundance in the non-B1 CD group based on the Montreal classification. Clonostachys, Humicola, and Lophiostoma were significantly enriched in patients with CD with perianal lesions. Our results demonstrated that the composition of the intestinal fungal microbiota in patients with CD and HCs was markedly different, some of which might play a pathogenic role in the occurrence of CD and perianal lesions. Exophiala dermatitidis and genus Candida might be associated with active disease stage and type non-B1 CD (CD with intestinal stenosis or penetrating lesions, or both), respectively.

Host genotype controls ecological change in the leaf fungal microbiome.

Leaf fungal microbiomes can be fundamental drivers of host plant success, as they contain pathogens that devastate crop plants and taxa that enhance nutrient uptake, discourage herbivory, and antagonize pathogens. We measured leaf fungal diversity with amplicon sequencing across an entire growing season in a diversity panel of switchgrass (Panicum virgatum). We also sampled a replicated subset of genotypes across 3 additional sites to compare the importance of time, space, ecology, and genetics. We found a strong successional pattern in the microbiome shaped both by host genetics and environmental factors. Further, we used genome-wide association (GWA) mapping and RNA sequencing to show that 3 cysteine-rich receptor-like kinases (crRLKs) were linked to a genetic locus associated with microbiome structure. We confirmed GWAS results in an independent set of genotypes for both the internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA markers. Fungal pathogens were central to microbial covariance networks, and genotypes susceptible to pathogens differed in their expression of the 3 crRLKs, suggesting that host immune genes are a principal means of controlling the entire leaf microbiome.

Soil fungal communities affect the chemical quality of flue-cured tobacco leaves in Bijie, Southwest China.

Soil microorganisms could affect the quality of tobacco leaves, however, little is known about the association of tobacco chemical components and soil fungal communities. In the present study, the relationship between soil fungi and tobacco quality based on chemical components in Bijie was investigated. The results showed that the total harmony scores (THS) of the analyzed tobacco leaves ranged from 46.55 ± 3.5 to 91.55 ± 2.25. Analyses of chemical components revealed that high contents of nicotine (≥ 1.06%) and sugar (total sugar: ≥ 22.96%, reducing sugar: ≥ 19.62%), as well as low potassium level (≤ 2.68%) were the main factors limiting the quality of flue-cured tobacco leaves. Pearson correlation analysis indicated that soil nitrate, available potassium/phosphorous, and organic matter significantly correlated with tobacco nicotine, potassium, and chloride levels (p < 0.05). Besides, the analysis of alpha- and beta-diversity of soil fungal communities implied that fungal structure rather than the richness affected the chemical quality of tobacco. In detail, the relative abundance of Humicola olivacea species in soils was positively correlated with the THS of tobaccos (r = 0.52, p < 0.05). Moreover, the species including Mortierella alpina, Mortierella hyalina, Tausonia pullulan, and Humicola olivacea were negatively correlated with tobacco sugar (r ≤ - 0.45, p < 0.05) while, Codinaea acaciae and Saitozyma podzolica species were negatively correlated with tobacco nicotine (r ≤ - 0.51, p < 0.05). The present study provides a preliminary basis for utilizing fungal species in soils to improve the chemical quality of tobacco in the studied area.

Profiling the Human Oral Mycobiome in Tissue and Saliva Using ITS2 DNA Metabarcoding Compared to a Fungal-Specific Database.

The advent of high-throughput sequencing has caused a paradigm shift from the one-pathogen one-disease model to the significance of dysbiosis of the oral microbiome, including the oral mycobiome. The oral mycobiome can be profiled by a method modified from that used to profile the bacteriome with 16S rRNA gene primers. The first modification is to include an initial fungus lysis step that ensures representative yields of fungal DNA. The second step is to use a reliable target, the ITS1 and/or ITS2 regions of the 23S rRNA, to define the oral fungal population, and modifications of library preparation required to deal with the variable sized amplicons generated. In this chapter, a proven microbiomic approach to identify fungal populations in oral tissue samples associated with cancer is described. This approach is also applicable to the study of the salivary mycobiome in both healthy and diseased individuals.

Effect of HIV/HAART and Other Clinical Variables on the Oral Mycobiome Using Multivariate Analyses.

The oral microbiome is considered an important factor in health and disease. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial communities in a large cohort of HIV-positive and -negative individuals. The purpose of the present study was to similarly analyze the oral mycobiome in the same cohort. To identify fungi, the internal transcribed spacer 2 (ITS2) of the fungal rRNA genes was sequenced using oral rinse samples from 149 HIV-positive and 88 HIV-negative subjects that had previously undergone bacterial amplicon sequencing. Quantitative PCR was performed for total fungal content and total bacterial content. Interestingly, samples often showed predominance of a single fungal species with four major clusters predominated by Candida albicans, Candida dubliniensis, Malassezia restricta, or Saccharomyces cerevisiae Quantitative PCR analysis showed the Candida-dominated sample clusters had significantly higher total fungal abundance than the Malassezia or Saccharomyces species. Of the 25 clinical variables evaluated for potential influences on the oral mycobiome, significant effects were associated with caries status, geographical site of sampling, sex, HIV under highly active antiretroviral therapy (HAART), and missing teeth, in rank order of statistical significance. Investigating specific interactions between fungi and bacteria in the samples often showed Candida species positively correlated with Firmicutes or Actinobacteria and negatively correlated with Fusobacteria, Proteobacteria, and Bacteroidetes Our data suggest that the oral mycobiome, while diverse, is often dominated by a limited number of species per individual; is affected by several clinical variables, including HIV positivity and HAART; and shows genera-specific associations with bacterial groups.IMPORTANCE The oral microbiome is likely a key element of homeostasis in the oral cavity. With >600 bacterial species and >160 fungal species comprising the oral microbiome, influences on its composition can have an impact on both local and systemic health. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial community in a large cohort of HIV-positive and -negative subjects. We describe here a comprehensive analysis of the oral mycobiome in the same cohort. Similar to the bacterial community, HIV under highly active antiretroviral therapy (HAART) had a significant impact on the mycobiome composition, but with less impact compared to other clinical variables. Additionally, unlike the oral bacterial microbiome, the oral mycobiome is often dominated by a single species with 4 major clusters of fungal communities. Together, these results suggest the oral mycobiome has distinct properties compared with the oral bacterial community, although both are equally impacted by HIV.

Candida albicans disorder is associated with gastric carcinogenesis.

Background: Bacterial infection is associated with gastric carcinogenesis. However, the relationship between nonbacterial components and gastric cancer (GC) has not been fully explored. We aimed to characterize the fungal microbiome in GC. Methods: We performed ITS rDNA gene analysis in cancer lesions and adjacent noncancerous tissues of 45 GC cases from Shenyang, China. Obtaining the OTUs and combining effective grouping, we carried out species identifications, alpha and beta diversity analyses, and FUNGuild functional annotation. Moreover, differences were compared and tested between groups to better investigate the composition and ecology of fungi associated with GC and find fungal indicators. Results: We observed significant gastric fungal imbalance in GC. Principal component analysis revealed separate clusters for the GC and control groups, and Venn diagram analysis indicated that the GC group showed a lower OTU abundance than the control. At the genus level, the abundances of 15 fungal biomarkers distinguished the GC group from the control, of which Candida (p = 0.000246) and Alternaria (p = 0.00341) were enriched in GC, while Saitozyma (p = 0.002324) and Thermomyces (p = 0.009158) were decreased. Combining the results of Welch's t test and Wilcoxon rank sum test, Candida albicans (C. albicans) was significantly elevated in GC. The species richness Krona pie chart further revealed that C. albicans occupied 22% and classified GC from the control with an area under the receiver operating curve (AUC) of 0.743. Random forest analysis also confirmed that C. albicans could serve as a biomarker with a certain degree of accuracy. Moreover, compared with that of the control, the alpha diversity index was significantly reduced in the GC group. The Jaccard distance index and the Bray abundance index of the PCoA clarified separate clusters between the GC and control groups at the species level (p = 0.00051). Adonis (PERMANOVA) analysis and ANOVA showed that there were significant differences in fungal structure among groups (p = 0.001). Finally, FUNGuild functional classification predicted that saprotrophs were the most abundant taxa in the GC group. Conclusions: This study revealed GC-associated mycobiome imbalance characterized by an altered fungal composition and ecology and demonstrated that C. albicans can be a fungal biomarker for GC. With the significant increase of C. albicans in GC, the abundance of Fusicolla acetilerea, Arcopilus aureus, Fusicolla aquaeductuum were increased, while Candida glabrata, Aspergillus montevidensis, Saitozyma podzolica and Penicillium arenicola were obviously decreased. In addition, C. albicans may mediate GC by reducing the diversity and richness of fungi in the stomach, contributing to the pathogenesis of GC.

Gut mycobiome: A promising target for colorectal cancer.

The human gut is mainly habited by a staggering amount and abundance of bacteria as well as fungi. Gut dysbiosis is believed as a pivotal factor in colorectal cancer (CRC) development. Lately increasing evidence from animal or clinical studies suggested that fungal disturbance also contributed to CRC development. This review summarized the current status of fungal dysbiosis in CRC and highlighted the potential tumorigenic mechanisms of fungi. Then the fungal markers and some therapeutic strategies for CRC were discussed. It would provide a better understanding of the correlation of mycobiota and CRC, and modulating fungal community would be a promising target against CRC.

The Salivary Mycobiome Contains 2 Ecologically Distinct Mycotypes.

A broad range of fungi has been detected in molecular surveys of the oral mycobiome. However, knowledge is still lacking on interindividual variability of these communities and the ecologic and clinical significance of oral fungal commensals. In this cross-sectional study, we use internal transcribed spacer 1 amplicon sequencing to evaluate the salivary mycobiome in 59 subjects, 36 of whom were scheduled to receive cancer chemotherapy. Analysis of the broad population structure of fungal communities in the whole cohort identified 2 well-demarcated genus-level community types (mycotypes), with Candida and Malassezia as the main taxa driving cluster partitioning. The Candida mycotype had lower diversity than the Malassezia mycotype and was positively correlated with cancer and steroid use in these subjects, smoking, caries, utilizing a removable prosthesis, and plaque index. Mycotypes were also associated with metabolically distinct bacteria indicative of divergent oral environments, with aciduric species enriched in the Candida mycotype and inflammophilic bacteria increased in the Malassezia mycotype. Similar to their fungal counterparts, coexisting bacterial communities associated with the Candida mycotype showed lower diversity than those associated with the Malassezia mycotype, suggesting that common environmental pressures affected bacteria and fungi. Mycotypes were also seen in an independent cohort of 24 subjects, in which cultivation revealed Malassezia as viable oral mycobiome members, although the low-abundance Malassezia sympodialis was the only Malassezia species recovered. There was a high degree of concordance between the molecular detection and cultivability of Candida, while cultivation showed low sensitivity for detection of the Malassezia mycotype. Overall, our work provides insights into the oral mycobiome landscape, revealing 2 community classes with apparently distinct ecologic constraints and specific associations with coexisting bacteria and clinical parameters. The utility of mycotypes as biomarkers for oral diseases warrants further study.

A European ECMM-ESCMID survey on goals and practices for mycobiota characterisation using next-generation sequencing.

Although substantial efforts have been made to investigate about the composition of the microbiota, fungi that constitute the mycobiota play a pivotal role in maintaining microbial communities and physiological processes in the body. Here, we conducted an international survey focusing on laboratory's current procedures regarding their goals and practices of mycobiota characterisation using NGS. A questionnaire was proposed to laboratories affiliated to working groups from ECMM (NGS study group) and ESCMID (ESGHAMI and EFISG study groups). Twenty-six questionnaires from 18 countries were received. The use of NGS to characterise the mycobiota was not in routine for most of the laboratories (N = 23, 82%), and the main reason of using NGS was primary to understand the pathophysiology of a dysbiosis (N = 20), to contribute to a diagnosis (N = 16) or to implement a therapeutic strategy (N = 12). Other reported reasons were to evaluate the exposome (environmental studies) (N = 10) or to investigate epidemics (N = 8). Sputum is the main sample studied, and cystic fibrosis represents a major disease studied via the analysis of pulmonary microbiota. No consensus has emerged for the choice of the targets with 18S, ITS1 and ITS2 used alternatively among the laboratories. Other answers are detailed in the manuscript. We report a photography of mycobiota analysis that may become a major tool in the near future. We can draw some conclusions on the diversity of approaches within the answers of the 27 laboratories and underline the need for standardisation.

Species composition of arbuscular mycorrhizal communities changes with elevation in the Andes of South Ecuador.

Arbuscular mycorrhizal fungi (AMF) are the most prominent mycobionts of plants in the tropics, yet little is known about their diversity, species compositions and factors driving AMF distribution patterns. To investigate whether elevation and associated vegetation type affect species composition, we sampled 646 mycorrhizal samples in locations between 1000 and 4000 m above sea level (masl) in the South of Ecuador. We estimated diversity, distribution and species compositions of AMF by cloning and Sanger sequencing the 18S rDNA (the section between AML1 and AML2) and subsequent derivation of fungal OTUs based on 99% sequence similarity. In addition, we analyzed the phylogenetic structure of the sites by computing the mean pairwise distance (MPD) and the mean nearest taxon difference (MNTD) for each elevation level. It revealed that AMF species compositions at 1000 and 2000 masl differ from 3000 and 4000 masl. Lower elevations (1000 and 2000 masl) were dominated by members of Glomeraceae, whereas Acaulosporaceae were more abundant in higher elevations (3000 and 4000 masl). Ordination of OTUs with respect to study sites revealed a correlation to elevation with a continuous turnover of species from lower to higher elevations. Most of the abundant OTUs are not endemic to South Ecuador. We also found a high proportion of rare OTUs at all elevations: 79-85% of OTUs occurred in less than 5% of the samples. Phylogenetic community analysis indicated clustering and evenness for most elevation levels indicating that both, stochastic processes and habitat filtering are driving factors of AMF community compositions.

Responses of Soil Bacterial and Fungal Communities to Organic and Conventional Farming Systems in East China.

Organic farming is considered an effective form of sustainable agricultural management. However, understanding of soil microbial diversity and composition under long-term organic and conventional farming is still limited and controversial. In this study, the Illumina MiSeq platform was applied to investigate the responses of soil bacterial and fungal diversity and compositions to organic farming (OF) and improved conventional farming (CF, applied straw retention) in the rice-wheat rotation system. The results highlighted that the alpha diversity of microbial communities did not differ significantly, except for higher bacterial diversity under OF. However, there were significant differences in the compositions of the soil bacterial and fungal communities between organic and conventional farming. Under our experimental conditions, through the ecological functional analysis of significant different or unique bacterial and fungal taxonomic members at the phyla and genus level, OF enhanced nitrogen, sulfur, phosphorus and carbon dynamic cycling in soil with the presence of Nodosilinea, Nitrospira, LCP-6, HB118, Lyngbya, GOUTA19, Mesorhizobium, Sandaracinobacter, Syntrophobacter and Sphingosinicella, and has the potential to strengthen soil metabolic ability with Novosphingobium. On the other hand, CF increased the intensity of nitrogen cycling with Ardenscatena, KD1-23, Iamia, Nitrosovibrio and Devosia, but enriched several pathogen fungal members, including Coniochaeta, Corallomycetella, Cyclaneusma, Cystostereum, Fistulina, Curvularia and Dissoconium.

Dysbiosis in the oral bacterial and fungal microbiome of HIV-infected subjects is associated with clinical and immunologic variables of HIV infection.

BACKGROUND: The effect of smoking on microbial dysbiosis and the potential consequence of such shift on markers of HIV disease is unknown. Here we assessed the relationship of microbial dysbiosis with smoking and markers of HIV disease. METHODS: Oral wash was collected from: (1) HIV-infected smokers (HIV-SM, n = 48), (2) HIV-infected non-smokers (HIV-NS, n = 24), or (3) HIV-uninfected smokers (UI-SM, n = 24). Microbial DNA was extracted and their bacterial and fungal microbiota (bacteriome and mycobiome, respectively) were characterized using Ion-Torrent sequencing platform. Sequencing data were compared using clustering, diversity, abundance and inter-kingdom correlations analyses. RESULTS: Bacteriome was more widely dispersed than mycobiome, there was no noticeable difference in clustering between groups. Richness of oral bacteriome in HIV-SM was significantly lower than that of UI-SM (P ≤ .03). Diversity of HIV-NS was significantly lower than that of HIV-SM or UI-SM at phylum level (P ≤ .02). Abundance of Phylum Firmicutes was significantly decreased in HIV-NS compared to HIV-SM and UI-SM (P = .007 and .027, respectively), while abundance of Proteobacteria was significantly increased in HIV-NS compared to HIV-SM and UI-SM (P = .0005 and .011, respectively). Fungal phyla did not differ significantly between the three cohorts. Cumulative smoking was positively correlated with Facklamia but negatively with Enhydrobacter, and current alcohol use was negatively correlated with Geniculata. Bacteria Facklamia exhibited weakly positive correlation with longer PI duration (r = 0.094, P = 0.012), and a negative correlation with nadir CD4 count (r = -0.345; P = 0.004), while Granulicatella was negatively correlated with nadir CD4 count (r = -0.329; P = 0.007). Fungus Stemphylium correlated negatively with nadir CD4 (r = -0.323; P = 0.008). CONCLUSIONS: Dysbiosis of the oral microbiota is associated with clinical and immunologic variables in HIV-infected patients.

Fungal diversity in oil palm leaves showing symptoms of Fatal Yellowing disease.

Oil palm (Elaeis guineensis Jacq.) is an excellent source of vegetable oil for biodiesel production; however, there are still some limitations for its cultivation in Brazil such as Fatal Yellowing (FY) disease. FY has been studied for many years, but its causal agent has never been determined. In Colombia and nearby countries, it was reported that the causal agent of Fatal Yellowing (Pudrición del Cogollo) is the oomycete Phytophthora palmivora, however, several authors claim that Fatal Yellowing and Pudrición del Cogollo (PC) are different diseases. The major aims of this work were to test, using molecular biology tools, Brazilian oil palm trees for the co-occurrence of the oomycete Phytophthora and FY symptoms, and to characterize the fungal diversity in FY diseased and healthy leaves by next generation sequencing. Investigation with specific primers for the genus Phytophthora showed amplification in only one of the samples. Analysis of the fungal ITS region demonstrated that, at the genus level, different groups predominated in all symptomatic samples, while Pyrenochaetopsis and unclassified fungi predominated in all asymptomatic samples. Our results show that fungal communities were not the same between samples at the same stage of the disease or among all the symptomatic samples. This is the first study that describes the evolution of the microbial community in the course of plant disease and also the first work to use high throughput next generation sequencing to evaluate the fungal community associated with leaves of oil palm trees with and without symptoms of FY.

The Mycobiome: Impact on Health and Disease States.

The term "microbiome" refers to microorganisms (microbiota) and their genomes (metagenome) coexisting with their hosts. Some researchers coined the term "second genome" to underscore the importance of the microbiota and its collective metagenome on their host's health and/or disease. It is now undeniable that the commensal fungal microorganisms, alongside the other components of the microbiota, play a central role in association with the human host. In recognition, projects were launched nationally and internationally to unify efforts to characterize the microbiome and elucidate the functional role of the microbiota and the mechanism(s) by which these organisms and their metabolites (metabolome) may affect health and disease states. In this article, we will highlight the role of the fungal community as an indispensable component of the microbiome.

Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation.

INTRODUCTION AND PURPOSE: Propidium monoazide (PMA)-pretreatment has increasingly been applied to remove the bias from dead or damaged cell artefacts, which could impact the microbiota analysis by high-throughput sequencing. Our study aimed to determine whether a PMA-pretreatment coupled with high-throughput sequencing analysis provides a different picture of the airway mycobiome and bacteriome. RESULTS AND DISCUSSION: We compared deep-sequencing data of mycobiota and microbiota of 15 sputum samples from 5 cystic fibrosis (CF) patients with and without prior PMA-treatment of the DNA-extracts. PMA-pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic units (OTUs) with a relative abundance greater than or equal to 1%), but caused a significant difference in the intermediate community (less than 1%) when analyzing the alpha biodiversity Simpson index (p = 0.03). Regarding PMA impact on the airway mycobiota evaluated for the first time here; no significant differences in alpha diversity indexes between PMA-treated and untreated samples were observed. Regarding beta diversity analysis, the intermediate communities also differed more dramatically than the total and abundant ones when studying both mycobiome and bacteriome. Our results showed that only the intermediate (or low abundance) population diversity is impacted by PMA-treatment, and therefore that abundant taxa are mostly viable during acute exacerbation in CF. Given such a cumbersome protocol (PMA-pretreatment coupled with high-throughput sequencing), we discuss its potential interest within the follow-up of CF patients. Further studies using PMA-pretreatment are warranted to improve our "omic" knowledge of the CF airways.