Internal Transcribed Spacer Amplicons Research Articles

The comparative plastisphere microbial community profile at Kung Wiman beach unveils potential plastic-specific degrading microorganisms.

Plastic waste is a global environmental issue that impacts the well-being of humans, animals, plants, and microorganisms. Microplastic contamination has been previously reported at Kung Wiman Beach, located in Chanthaburi province along with the Eastern Gulf of Thailand. Our research aimed to study the microbial population of the sand and plastisphere and isolate microorganisms with potential plastic degradation activity. Plastic and sand samples were collected from Kung Wiman Beach for microbial isolation on agar plates. The plastic samples were identified by Fourier-transform infrared spectroscopy. Plastic degradation properties were evaluated by observing the halo zone on mineral salts medium (MSM) supplemented with emulsified plastics, including polystyrene (PS), polylactic acid (PLA), polyvinyl chloride (PVC), and bis (2-hydroxyethyl) terephthalate (BHET). Bacteria and fungi were identified by analyzing nucleotide sequence analysis of the 16S rRNA and internal transcribed spacer (ITS) regions, respectively. 16S and ITS microbiomes analysis was conducted on the total DNA extracted from each sample to assess the microbial communities. Of 16 plastic samples, five were identified as polypropylene (PP), four as polystyrene (PS), four as polyethylene terephthalate (PET), two as high-density polyethylene (HDPE), and one sample remained unidentified. Only 27 bacterial and 38 fungal isolates were found to have the ability to degrade PLA or BHET on MSM agar. However, none showed degradation capabilities for PS or PVC on MSM agar. Notably, Planococcus sp. PP5 showed the highest hydrolysis capacity of 1.64 ± 0.12. The 16S rRNA analysis revealed 13 bacterial genera, with seven showing plastic degradation abilities: Salipiger, Planococcus, Psychrobacter, Shewanella, Jonesia, Bacillus, and Kocuria. This study reports, for the first time of the BHET-degrading properties of the genera Planococcus and Jonesia. Additionally, The ITS analysis identified nine fungal genera, five of which demonstrated plastic degradation abilities: Aspergillus, Penicillium, Peacilomyces, Absidia, and Cochliobolus. Microbial community composition analysis and linear discriminant analysis effect size revealed certain dominant microbial groups in the plastic and sand samples that were absent under culture-dependent conditions. Furthermore, 16S and ITS amplicon microbiome analysis revealed microbial groups were significantly different in the plastic and sand samples collected. We reported on the microbial communities found on the plastisphere at Kung Wiman Beach and isolated and identified microbes with the capacity to degrade PLA and BHET.

Host- and Fusarium-Adapted Bacterial Consortia Alter Microbial Community Structures in Arabidopsis Roots and Suppress Fusarium oxysporum

The plant-associated microbiota confers beneficial traits to the plant host by promoting growth and preventing disease. However, it is not fully understood how the host and its associated microbiota interact with pathogens. In this work, we studied how the host plant modulates its associated microbiome to suppress disease. For this, we used two Arabidopsis thaliana lines with different host responses to Fusarium oxysporum f. sp. mathioli (FOM). We isolated bacterial consortia (BCs) from FOM-infected or healthy host plants of the two lines of Arabidopsis and studied their effect on the root-associated microbiota and FOM progression in the following generations of Arabidopsis plants. Root bacterial and fungal communities were profiled using 16S ribosomal RNA and internal transcribed spacer amplicon sequencing, respectively, while quantitative PCR was used for assessment of FOM quantities in shoots of Arabidopsis. Host- or pathogen-adapted BCs significantly reduced FOM quantities in shoots of both the resistant Col-0 and the susceptible Ler-0 Arabidopsis lines. Several bacterial taxa, including Chthoniobacter, Bacillus, Chryseobacterium and Actinoplanes negatively correlated with FOM, suggestive of an antagonistic effect. Furthermore, both host- and pathogen-adapted BCs significantly affected community composition with distinct differentially abundant taxa and co-occurrence network structures. Taken together, our findings suggest that using a subcommunity selection approach is a potential route for exploiting plant-associated rhizosphere microbiomes for engineering disease resilient microbiomes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

White-nose syndrome restructures bat skin microbiomes.

The skin microbiome is an essential line of host defense against pathogens, yet our understanding of microbial communities and how they change when hosts become infected is limited. We investigated skin microbial composition in three North American bat species (Myotis lucifugus, Eptesicus fuscus, and Perimyotis subflavus) that have been impacted by the infectious disease, white-nose syndrome, caused by an invasive fungal pathogen, Pseudogymnoascus destructans. We compared bacterial and fungal composition from 154 skin swab samples and 70 environmental samples using a targeted 16S rRNA and internal transcribed spacer amplicon approach. We found that for M. lucifugus, a species that experiences high mortality from white-nose syndrome, bacterial microbiome diversity was dramatically lower when P. destructans was present. Key bacterial families-including those potentially involved in pathogen defense-significantly differed in abundance in bats infected with P. destructans compared to uninfected bats. However, skin bacterial diversity was not lower in E. fuscus or P. subflavus when P. destructans was present despite populations of the latter species declining sharply from white-nose syndrome. The fungal species present on bats substantially overlapped with the fungal taxa present in the environment at the site where the bat was sampled, but fungal community composition was unaffected by the presence of P. destructans for any of the three bat species. This species-specific alteration in bat skin bacterial microbiomes after pathogen invasion may suggest a mechanism for the severity of white-nose syndrome in M. lucifugus but not for other bat species impacted by the disease.IMPORTANCEInherent complexities in the composition of microbiomes can often preclude investigations of microbe-associated diseases. Instead of single organisms being associated with disease, community characteristics may be more relevant. Longitudinal microbiome studies of the same individual bats as pathogens arrive and infect a population are the ideal experiment but remain logistically challenging; therefore, investigations like our approach that are able to correlate invasive pathogens to alterations within a microbiome may be the next best alternative. The results of this study potentially suggest that microbiome-host interactions may determine the likelihood of infection. However, the contrasting relationship between Pd and the bacterial microbiomes of Myotis lucifugus and Perimyotis subflavus indicate that we are just beginning to understand how the bat microbiome interacts with a fungal invader such as Pd.

Fungal and Bacterial Microbiome in Sinus Mucosa of Patients with and without Chronic Rhinosinusitis.

Dysbiosis of the sinonasal microbiome has been implicated in the pathogenesis of chronic rhinosinusitis (CRS). However, the mycobiome remains largely understudied, and microbial alterations associated with specific CRS subtypes have yet to be delineated. The objective of this study is to investigate the fungal and bacterial microbiome of sinus mucosa in CRS patients with and without nasal polyposis (CRSwNP and CRSsNP) versus healthy controls. Sinus mucosa was obtained from 92 patients (31 CRSsNP, 31 CRSwNP, and 30 controls) undergoing endoscopic sinus/skull base surgery. Data regarding demographics, Lund-MacKay scores, and histopathology were collected. Fungal and bacterial microbiome analysis was performed utilizing internal transcribed spacer amplicon and 16S rRNA sequencing. Beta diversity of the sinonasal mycobiome differed significantly between CRS and controls (p = 0.001) and between CRSwNP and controls (p = 0.049), but not between CRSwNP and CRSsNP (p = 0.32) nor between CRSsNP and controls (p = 0.06). With respect to the bacterial microbiome, significantly lower alpha diversity was observed between CRS and controls (p < 0.001), CRSwNP versus controls (p < 0.001), and CRSsNP versus controls (p < 0.001). Beta diversity was also significantly different at the genus level between CRSwNP and CRSsNP (p = 0.019), CRSwNP and controls (p = 0.002)), and CRSsNP and controls (p < 0.001). However, alpha and beta diversity did not differ significantly between CRS patients with/without eosinophils or correlate with Lund-MacKay scores. Differences in mycobiota diversity in CRS patients in comparison with controls suggest that alterations in the mycobiome may contribute to disease pathogenesis. Our findings also confirmed that diminished diversity among bacterial communities is associated with CRS and that significant differences are present in microbial composition between CRSwNP and CRSsNP. 3 Laryngoscope, 134:1054-1062, 2024.

High resolution ITS amplicon melting analysis as a tool to analyse microbial communities of household biofilms in ex-situ models

Biofilms are the most common growth types of microorganisms. These complex communities usually consist of different species and are embedded in an extracellular matrix containing polymers, proteins and DNA. This matrix offers protection against different (a)biotic environmental factors and generally increases resistances. Higher resistances against antibiotics are one of the main reasons why biofilms are often associated with healthcare settings. Nevertheless, they are also found in domestic settings, mostly in humid places with abundant nutrients like dishwashers or washing machines. Biofilms in these areas show individual compositions and are influenced for example by temperature, frequency of use or the age of the device.In this study, we introduce a model for the ex-situ cultivation of domestic biofilms from household appliances. Furthermore, we tested the ability of high resolution melting analysis (HRMA) as a tool for analysing these biofilms. Our goal was to maintain a high amount of complexity in the ex-situ biofilms that is characterized by the melting behavior of the contained DNA. Dishwasher and washing machine biofilms were sampled in private households and cultivated for 10 d. After DNA extraction, 16S rDNA was sequenced and melting behavior of the bacterial Internal Transcribed Spacer (ITS) region was analysed. Additionally, testing for independence of continuous new sampling, storage of cultivated biofilms in glycerol stocks and following recultivation of them was done up to three times.Our results show that a high level of complexity could be maintained in the ex-situ biofilms after 10 d of cultivation, although in general the bacterial diversity slightly decreased compared to the original biofilm in most cases. Recultivation of a similar biofilm from glycerol stocks was possible as well with some impact by various factors. Differences in the bacterial composition of biofilms could clearly made visible by HRMA although it was not possible to match peaks to a specific phylogenetic group. Still, HRMA proved to be a less costly and time consuming alternative to sequencing for the characterization of biofilms.

Features of combined gut bacteria and fungi from a Chinese cohort of colorectal cancer, colorectal adenoma, and post-operative patients.

Colorectal cancer (CRC) accounts for the third highest morbidity burden among malignant tumors worldwide. Previous studies investigated gut microbiome changes that occur during colorectal adenomas (CRA) progression to overt CRC, thus highlighting the importance of the gut microbiome in carcinogenesis. However, few studies have examined gut microbiome characteristics across the entire spectrum, from CRC development to treatment. The study used 16S ribosomal ribonucleic acid and internal transcribed spacer amplicon sequencing to compare the composition of gut bacteria and fungi in a Chinese cohort of healthy controls (HC), CRC patients, CRA patients, and CRC postoperative patients (PP). Our analysis showed that beta diversity was significantly different among the four groups based on the gut bacterial and fungal data. A total of 51 species of bacteria and 8 species of fungi were identified in the HC, CRA, CRC, and PP groups. Correlation networks for both the gut bacteria and fungi in HC vs. CRA, HC vs. CRC, and HC vs. PP indicated some hub bacterial and fungal genera in each model, and the correlation between bacterial and fungal data indicated that a highly significant negative correlation exists among groups. Quantitative polymerase chain reaction (qPCR) analysis in a large cohort of HC, CRC, CRA, and PP patients demonstrated a significantly increasing trend of Fusobacterium nucleatum, Bifidobacterium bifidum, Candida albicans, and Saccharomyces cerevisiae in the feces of CRC patients than that of HC patients (p < 0.01). However, the abundance levels of CRA and PP were significantly lower in HC patients than those in CRC patients. Further studies are required to identify the functional consequences of the altered bacterial/fungal composition on metabolism and CRC tumorigenesis in the host.

Cadmium inhibits powdery mildew colonization and reconstructs microbial community in leaves of the hyperaccumulator plant Sedum alfredii

Understanding the influence of the heavy metal cadmium (Cd) on the phyllosphere microbiome of hyperaccumulator plants is crucial for enhancing phytoremediation. The characteristics of the phyllosphere of Sedum alfredii Hance, a hyperaccumulator plant, were investigated using 16S rRNA and internal transcribed spacer amplicon sequencing of powdery mildew-infected leaves treated or untreated with Cd. The results showed that the colonization of powdery mildew caused severe chlorosis and necrosis in S. alfredii leaves, and the relative abundance of Leotiomycetes in infected leaves increased dramatically and significantly decreased phyllosphere microbiome diversity. However, S. alfredii preferentially accumulated higher concentrations of Cd in the leaves of infected plants than in uninfected plants by powdery mildew, which in turn significantly inhibited powdery mildew colonization in leaves; the relative abundance of the fungal class Leotiomycetes in infected leaves decreased, and alpha and beta diversities of the phyllosphere microbiome significantly increased with Cd treatment in the infected plants. In addition, the inter-kingdom networks in the microbiota of the infected leaves treated with Cd presented many nodes and edges, and the highest inter-kingdom modularity compared to the untreated infected leaves, indicating a highly connected microbial community. These results suggest that Cd significantly inhibits powdery mildew colonization by altering the composition of the phyllosphere microbiome in S. alfredii leaves, paving the way for efficient heavy metal phytoremediation and providing a new perspective on defense strategies against heavy metals.

Sour Orange Microbiome Is Affected by Infections of Plenodomus tracheiphilus Causal Agent of Citrus Mal Secco Disease

Mal secco is a severe vascular citrus disease (MSD) caused by the mitosporic fungus Plenodomus tracheiphilus (Pt). The pathogen enters through wounds on the above- and below-ground parts of the tree. The susceptible species sour orange (Citrus aurantium) is the most commonly used rootstock for lemon trees in Italy. In this study, sour orange seedlings were wound-inoculated with P. tracheiphilus in leaves or roots. Six months post-inoculation, the microbial communities of rhizosphere, endorhizosphere, and xylem endosphere samples from inoculated and healthy plants were analyzed by 16S rRNA and ITS (internal transcribed spacer) amplicon sequencing. The DNA of Pt was quantified by real-time PCR in the three compartments. According to our results, the endorhizosphere of root-inoculated plants showed the highest concentration of the pathogen DNA. Bacterial populations of potentially beneficial taxa (e.g., Pseudomonas and Burkholderia) were depleted in the rhizosphere of the inoculated plants. Infection through leaves and roots also produced a network-wide rewiring of microbial associations in sour orange roots. Overall, our findings revealed community-level changes induced by Pt infection in the sour orange root and xylem microbiome, providing further insights into the beneficial multispecies interactions in Citrus-associated microbial communities.

Soil Chemistry and Soil History Significantly Structure Oomycete Communities in Brassicaceae Crop Rotations.

Oomycetes are critically important in soil microbial communities, especially for agriculture, where they are responsible for major declines in yields. Unfortunately, oomycetes are vastly understudied compared to bacteria and fungi. As such, our understanding of how oomycete biodiversity and community structure vary through time in the soil remains poor. Soil history established by previous crops is one factor known to structure other soil microbes, but this has not been investigated for its influence on oomycetes. In this study, we established three different soil histories in field trials; the following year, these plots were planted with five different Brassicaceae crops. We hypothesized that the previously established soil histories would structure different oomycete communities, regardless of their current Brassicaceae crop host, in both the roots and rhizosphere. We used a nested internal transcribed spacer amplicon strategy incorporated with MiSeq metabarcoding, where the sequencing data was used to infer amplicon sequence variants of the oomycetes present in each sample. This allowed us to determine the impact of different soil histories on the structure and biodiversity of the oomycete root and rhizosphere communities from the five different Brassicaceae crops. We found that each soil history structured distinct oomycete rhizosphere communities, regardless of different Brassicaceae crop hosts, while soil chemistry structured the oomycete communities more during a dry year. Interestingly, soil history appeared specific to oomycetes but was less influential for bacterial communities previously identified from the same samples. These results advance our understanding of how different agricultural practices and inputs can alter edaphic factors to impact future oomycete communities. Examining how different soil histories endure and impact oomycete biodiversity will help clarify how these important communities may be assembled in agricultural soils. IMPORTANCE Oomycetes cause global plant diseases that result in substantial losses, yet they are highly understudied compared to other microbes, like fungi and bacteria. We wanted to investigate how past soil events, like changing crops in rotation, would impact subsequent oomycete communities. We planted different oilseed crops in three different soil histories and found that each soil history structured a distinct oomycete community regardless of which new oilseed crop was planted, e.g., oomycete communities from last year's lentil plots were still detected the following year regardless of which new oilseed crops we planted. This study demonstrated how different agricultural practices can impact future microbial communities differently. Our results also highlight the need for continued monitoring of oomycete biodiversity and quantification.

Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

Fungal species participate in vast numbers of processes in the landscape around us. However, their cryptic mycelial growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enables identification and relative quantification of fungal community members across well-replicated experimental settings.Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, i.e., the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon pool to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

Legacy effects of fumigation on soil bacterial and fungal communities and their response to metam sodium application

BackgroundSoil microorganisms are integral to maintaining soil health and crop productivity, but fumigation used to suppress soilborne diseases may affect soil microbiota. Currently, little is known about the legacy effects of soil fumigation on soil microbial communities and their response to fumigation at the production scale. Here, 16S rRNA gene and internal transcribed spacer amplicon sequencing was used to characterize the bacterial and fungal communities in soils from intensively managed crop fields with and without previous exposure to metam sodium (MS) fumigation. The effect of fumigation history, soil series, and rotation crop diversity on microbial community variation was estimated and the response of the soil microbiome to MS application in an open microcosm system was documented.ResultsWe found that previous MS fumigation reduced soil bacterial diversity but did not affect microbial richness and fungal diversity. Fumigation history, soil series, and rotation crop diversity were the main contributors to the variation in microbial β-diversity. Between fumigated and non-fumigated soils, predominant bacterial and fungal taxa were similar; however, their relative abundance varied with fumigation history. In particular, the abundance of Basidiomycete yeasts was decreased in fumigated soils. MS fumigation also altered soil bacterial and fungal co-occurrence network structure and associations. In microcosms, application of MS reduced soil microbial richness and bacterial diversity. Soil microbial β-diversity was also affected but microbial communities of the microcosm soils were always similar to that of the field soils used to establish the microcosms. MS application also induced changes in relative abundance of several predominant bacterial and fungal genera based on a soil’s previous fumigation exposure.ConclusionsThe legacy effects of MS fumigation are more pronounced on soil bacterial diversity, β-diversity and networks. Repeated fumigant applications shift soil microbial compositions and may contribute to differential MS sensitivity among soil microorganisms. Following MS application, microbial richness and bacterial diversity decreases, but microbial β-diversity was similar to that of the field soils used to establish the microcosms in the short-term (< 6 weeks). The responses of soil microbiome to MS fumigation are context dependent and rely on abiotic, biotic, and agricultural management practices.

Evaluation of the Ecological Environment Affected by Cry1Ah1 in Poplar.

Populus is a genus of globally significant plantation trees used widely in industrial and agricultural production. Poplars are easily damaged by Micromelalopha troglodyta and Hyphantria cunea, resulting in decreasing quality. Bt toxin-encoded by the Cry gene has been widely adopted in poplar breeding because of its strong insect resistance. There is still no comprehensive and sufficient information about the effects of Cry1Ah1-modified (CM) poplars on the ecological environment. Here, we sampled the rhizosphere soils of field-grown CM and non-transgenic (NT) poplars and applied 16S rRNA and internal transcribed spacer amplicon Illumina MiSeq sequencing to determine the bacterial community associated with the CM and NT poplars. Based on the high-throughput sequencing of samples, we found that the predominant taxa included Proteobacteria (about 40% of the total bacteria), Acidobacteria (about 20% of the total bacteria), and Actinobacteria (about 20% of the total bacteria) collected from the natural rhizosphere of NT and CM poplars. In addition, studies on the microbial diversity of poplar showed that Cry1Ah1 expression has no significant influence on rhizosphere soil alkaline nitrogen, but significantly affects soil phosphorus, soil microbial biomass nitrogen, and carbon. The results exhibited a similar bacterial community structure between CM varieties affected by the expression of Cry1Ah1 and non-transgenic poplars. In addition, Cry1Ah1 expression revealed no significant influence on the composition of rhizosphere microbiomes. These results broadly reflect the effect of the Bt toxin-encoded by Cry1Ah1 on the ecology and environment and provide a clear path for researchers to continue research in this field in the future.

Profiling the Fungal Microbiome after Fecal Microbiota Transplantation for Graft-versus-Host Disease: Insights from a Phase 1 Interventional Study

Disruption of the intestinal bacterial microbiota is frequently observed in the context of allogeneic hematopoietic cell transplantation (HCT) and is particularly pronounced in patients who develop graft-versus-host disease (GVHD). Donor fecal microbiota transplantation (FMT) restores gut microbial diversity and reduces GVHD in HCT recipients. The composition of the intestinal fungal community in patients with GVHD, and whether fungal taxa are transferred during FMT are currently unknown. We performed a secondary analysis of our clinical trial of FMT in patients with steroid-refractory GVHD with a focus on the mycobiota. We characterized the fecal mycobiota of 17 patients and healthy FMT donors using internal transcribed spacer amplicon sequencing. The donor who provided the majority of FMT material in our study represents an n-of-one study of the intestinal flora over time. In this donor, mycobiota composition fluctuated over time while the bacterial microbiota remained stable over 16 months. Fungal DNA was detected more frequently in baseline stool samples from patients with steroid-refractory GVHD than in patients with steroid-dependent GVHD. We could detect fungal taxa in the majority of samples but did not see evidence of mycobiota transfer from donor to recipient. Our study demonstrates the feasibility of profiling the mycobiota alongside the more traditional bacterial microbiota, establishes the methodology, and provides a first insight into the mycobiota composition of patients with GVHD.

Molecular Identification of Aspergillus flavus isolated from some maize (Zea mays L.) varieties across Abuja, Nigeria

This study aims at isolating and molecularly characterizing Aspergillus flavus from maize (Zea mays L.) varieties (Samaz 15,20, 37, 52, Oba super 6, 9, 39, 98, and DK 777) obtained across Abuja, Nigeria. Maize varieties were obtained across the district areas of Abuja namely; Kuje, Kwali, Gwagalada, AMAC, Abaji, Bwari and UniAbuja Experimental field. The potato dextrose agar as a medium was used in growing the fungi from the maize varieties seeds for 7 days at 25oC and then sub-cultured to obtain an isolate into potato dextrose broth. The mycelial mass produced was processed to extract its DNA using genomic DNA extraction of Zymo, D6005 for fungi and bacteria. Molecular Identification of the Internal transcribed spacer (ITS) region for each A. flavus isolate was carried out with ITS1 and ITS4 primers. anf18S rRNA gene region was also amplified with ITS5 and ITS4 primers. 2.0% agarose gels were electrophoresed for 30 minutes in 0.5× buffer, Dye and 60 mM EDTA at 80 V. A gel documentation system was used in determining PCR product sizes by evaluating with internal Mol. Wt. Standards of Thermo Scientific GeneRuler 100 bp DNA Ladder. Fifteen (15) of the twenty (20) fungal isolates were found to be positive with PCR amplification of ITS regions and amplicon sizes of 600 base pairs (bp) for A. flavus were obtained. All the twenty isolates have the presence of 18S rRNA 300-400bp. Thirteen of the fungal growth from the fourteen maize varieties obtained across Abuja were identified as A. flavus.

Culturable Endophytes Associated with Soybean Seeds and Their Potential for Suppressing Seed-Borne Pathogens.

Seed-borne pathogens in crops reduce the seed germination rate and hamper seedling growth, leading to significant yield loss. Due to the growing concerns about environmental damage and the development of resistance to agrochemicals among pathogen populations, there is a strong demand for eco-friendly alternatives to synthetic chemicals in agriculture. It has been well established during the last few decades that plant seeds harbor diverse microbes, some of which are vertically transmitted and important for plant health and productivity. In this study, we isolated culturable endophytic bacteria and fungi from soybean seeds and evaluated their antagonistic activities against common bacterial and fungal seed-borne pathogens of soybean. A total of 87 bacterial isolates and 66 fungal isolates were obtained. Sequencing of 16S rDNA and internal transcribed spacer amplicon showed that these isolates correspond to 30 and 15 different species of bacteria and fungi, respectively. Our antibacterial and antifungal activity assay showed that four fungal species and nine bacterial species have the potential to suppress the growth of at least one seed-borne pathogen tested in the study. Among them, Pseudomonas koreensis appears to have strong antagonistic activities across all the pathogens. Our collection of soybean seed endophytes would be a valuable resource not only for studying biology and ecology of seed endophytes but also for practical deployment of seed endophytes toward crop protection.

Landscape scale ecology of Tetracladium spp. fungal root endophytes

BackgroundThe genus Tetracladium De Wild. (Ascomycota) has been traditionally regarded as a group of Ingoldian fungi or aquatic hyphomycetes—a polyphyletic group of phylogenetically diverse fungi which grow on decaying leaves and plant litter in streams. Recent sequencing evidence has shown that Tetracladium spp. may also exist as root endophytes in terrestrial environments, and furthermore may have beneficial effects on the health and growth of their host. However, the diversity of Tetracladium spp. communities in terrestrial systems and the factors which shape their distribution are largely unknown.ResultsUsing a fungal community internal transcribed spacer amplicon dataset from 37 UK Brassica napus fields we found that soils contained diverse Tetracladium spp., most of which represent previously uncharacterised clades. The two most abundant operational taxonomic units (OTUs), related to previously described aquatic T. furcatum and T. maxilliforme, were enriched in roots relative to bulk and rhizosphere soil. For both taxa, relative abundance in roots, but not rhizosphere or bulk soil was correlated with B. napus yield. The relative abundance of T. furcatum and T. maxilliforme OTUs across compartments showed very similar responses with respect to agricultural management practices and soil characteristics. The factors shaping the relative abundance of OTUs homologous to T. furcatum and T. maxilliforme OTUs in roots were assessed using linear regression and structural equation modelling. Relative abundance of T. maxilliforme and T. furcatum in roots increased with pH, concentrations of phosphorus, and increased rotation frequency of oilseed rape. It decreased with increased soil water content, concentrations of extractable phosphorus, chromium, and iron.ConclusionsThe genus Tetracladium as a root colonising endophyte is a diverse and widely distributed part of the oilseed rape microbiome that positively correlates to crop yield. The main drivers of its community composition are crop management practices and soil nutrients.

Stem traits, compartments and tree species affect fungal communities on decaying wood.

SummaryDead wood quantity and quality is important for forest biodiversity, by determining wood‐inhabiting fungal assemblages. We therefore evaluated how fungal communities were regulated by stem traits and compartments (i.e. bark, outer‐ and inner wood) of 14 common temperate tree species. Fresh logs were incubated in a common garden experiment in a forest site in the Netherlands. After 1 and 4 years of decay, the fungal composition of different compartments was assessed using Internal Transcribed Spacer amplicon sequencing. We found that fungal alpha diversity differed significantly across tree species and stem compartments, with bark showing significantly higher fungal diversity than wood. Gymnosperms and Angiosperms hold different fungal communities, and distinct fungi were found between inner wood and other compartments. Stem traits showed significant afterlife effects on fungal communities; traits associated with accessibility (e.g. conduit diameter), stem chemistry (e.g. C, N, lignin) and physical defence (e.g. density) were important factors shaping fungal community structure in decaying stems. Overall, stem traits vary substantially across stem compartments and tree species, thus regulating fungal communities and the long‐term carbon dynamics of dead trees.

Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, β-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum, and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 μg OTA kg−1 dry mycelium.

Focused Microbiome Shifts in Reconstructed Wetlands Correlated with Elevated Copper Concentrations Originating from Micronized Copper Azole-Treated Wood.

Micronized copper (Cu) azole (MCA) wood preservative formulations include Cu in nano form, and relatively little is known about longer term effects of Cu leached from MCA into wetland ecosystems. We tested the hypothesis that changes in soil microbiomes within reconstructed freshwater wetlands will be associated with exposure to elevated Cu concentrations originating from immersed MCA-treated wood stakes. Eight replicate communities were assembled with Willamette Valley (OR, USA) flood plain soil and clonally propagated wetland plants within mesocosms. Inundated communities were equilibrated for 5 months before installation of MCA or control southern yellow pine stakes (n = 4 communities/experimental group). Soil samples were collected for 16S and internal transcribed spacer amplicon sequencing to quantify responses in prokaryotes and eukaryotes, respectively, at 15 time points, spanning two simulated seasonal dry downs, for up to 678 days. Physiochemical properties of water and soil were monitored at 20 and 12 time points respectively, over the same period. For both taxonomic groups of organisms, phylogenetic diversity increased and was positively correlated with elapsed days. Furthermore, there was significant divergence among eukaryotes during the second year based on experimental group. Although the composition of taxa underwent succession over time, there was significantly reduced relative abundance of sequence variants from Gomphonema diatoms and Scutellinia fungi in communities where MCA wood stakes were present compared with the controls. These focused microbiome shifts were positively correlated with surface water Cu and soil Cu concentrations, which were significantly elevated in treated communities. The reconstructed communities were effective systems for assessing potential impacts to wetland microbiomes after exposure to released copper. The results further inform postcommercialization risk assessments on MCA-treated wood. Environ Toxicol Chem 2021;40:3351-3368.Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Detection of Pathogenic and Beneficial Microbes for Roselle Wilt Disease.

Wilt disease of roselle (Hibiscus sabdariffa L.) is common in Taiwan; however, the causative agent remains unknown. The stems of wilted roselle are browned, slightly constricted, and covered by white aerial hyphae, suggesting that potential pathogens may originate from soil. To identify the potential pathogens, we conducted a rhizosphere microbiota survey in phenotypically healthy and diseased plants through fungal internal transcribed spacer (ITS) and bacterial 16S rRNA amplicon sequencing for uncovering the microbial compositions in the roselle rhizosphere. The fungal family Nectriaceae exhibited significantly higher abundance in diseased rhizospheres than in healthy rhizospheres, and this bacterial community was more specific to geography (i.e., plot-dependent) than to rhizosphere disease status. However, a few bacterial groups such as Bacilli were associated with the healthy rhizosphere. Fusarium species were the most dominant species of Nectriaceae in the survey and became the main target for potential pathogen isolation. We successfully isolated 119 strains from diseased plants in roselle fields. Koch’s postulates were used to evaluate the pathogenicity of these strains; our results indicated that Fusarium solani K1 (FsK1) can cause wilting and a rotted pith in roselles, which was consistent with observations in the fields. This is the first demonstration that F. solani can cause roselle wilt in Taiwan. Furthermore, these newly isolated strains are the most dominant operational taxonomic units detected in ITS amplicon sequencing in diseased rhizospheres, which serves as further evidence that F. solani is the main pathogen causing the roselle wilt disease. Administration of Bacillus velezensis SOI-3374, a strain isolated from a healthy roselle rhizosphere, caused considerable anti-FsK1 activity, and it can serve as a potential biocontrol agent against roselle wilt disease.