A novel and complete gene cluster involved in the degradation of aniline by Delftia sp. AN3

A recombinant strain, Escherichia coli JM109-AN1, was obtained by constructing of a genomic library of the total DNA of Delftia sp. AN3 in E. coli JM109 and screening for catechol 2,3-dioxygenase activity. This recombinant strain could grow on aniline as sole carbon, nitrogen and energy source. Enzymatic assays revealed that the exogenous genes including aniline dioxygenase （AD） and catechol 2,3-dioxygenase （C230） genes could well express in the recombinant strain with the activities of AD and C230 up to 0.31 U/mg wet cell and 1.92 U/mg crude proteins, respectively. The AD or C23O of strain AN3 could only catalyze aniline or catechol but not any other substituted substrates. This recombinant strain contained a recombinant plasmid, pKC505-AN1, in which a 29.7-kb DNA fragment from Delftia sp. AN3 was inserted. Sequencing and open reading frame （orfs） analysis of this 29.7 kb fragment revealed that it contained at least 27 orfs, among them a gene cluster （consisting of at least 16 genes, named danQTA1A2BRDCEFG1HIJKG2） was responsible for the complete metabolism of aniline to TCA-cycle intermediates. This gene cluster could be divided into two main parts, the upper sequences consisted of 7 genes （danQTA1A2BRD） were predicted to encode a multi-component aniline dioxygenase and a LysR-type regulator, and the central genes （danCEFG1HIJKG2） were expected to encode meta-cleavage pathway enzymes for catechol degradation to TCA-cycle intermediates. Unlike clusters tad from Delftia tsuruhatensis AD9 and tdn from Pseudomonas putida UCC22, in this gene cluster, all the genes were in the same transcriptional direction. There was only one set of C230 gene （danC） and ferredoxin-like protein gene （danD）. The presence of only one set of these two genes and specificity of AD and C230 might be the reason for strain AN3 could only degrade aniline. The products of danQTA1A2BRDC showed 99%-100% identity to those from Delftia acidovorans 7N, and 50%-85% identity to those of tad cluster from D. tsuruhatensis AD9 in amino acid residues. Besides this dan cluster, the 29.7 kb fragment also contained genes encoding the trans-membrane transporter and transposases which might be needed for transposition of the gene cluster. Pulsed-field gel electrophoresis （PFGE） and plasmid curing experiments suggested that the dan cluster might be encoded on the chromosome of strain AN3. The GenBank accession number for the dan cluster of Delftia sp. AN3 is DQ661649.

Marine Bacterial Biofilms in Bioremediation of Polycyclic Aromatic Hydrocarbons(PAHs) Under Terrestrial Condition in a Soil Microcosm

Polycyclic aromatic hydrocarbons （PAHs） in soil retain for a quite long period due to their hydrophobicity and aggregation properties. Biofilm-forming marine bacterial consortium （named as NCPR）, composed of Stenotrophomonas acidaminiphila NCW702, Alcaligenes faecalis NCW402, Pseudomonas mendocina NR802, Pseudornonas aeruginosa N6P6, and Pseudomonas pseudoalcaligenes NP103, was used for the bioremediation of PAHs in a soil microcosm. Phenanthrene and pyrene were used as reference PAHs. Parameters that can affect PAH degradation, such as chemotaxis, solubility of PAHs in extracellular polymeric substances （EPS）, and catechol 2,3-dioxygenase （C230） activity, were evaluated. P. aeruginosa N6P6 and P. pseudoalcaligenes NP103 showed chemotactic movement towards both the reference PAHs. The solubility of both the PAHs was increased with an increase in EPS concentration （extracted from all the 5 selected isolates）. Significantly （P 〈 0.001） high phenanthrene （70.29%） and pyrene （55.54%） degradation was observed in the bioaugmented soil microcosm. The C230 enzyme activity was significantly （P 〈 0.05） higher in the bioaugmented soil mi- crocosm with phenanthrene added at 173.26 ± 2.06 nmol rain-1 mg-1 protein than with pyrene added at 61.80 ± 2.20 nmol min-1 mg-1 protein. The C230 activity and gas chromatography-mass spectrometer analyses indicated catechol pathway of phenanthrene metabolism. However, the metabolites obtained from the soil microcosm added with pyrene revealed both the catechol and phthalate pathways for pyrene degradation.

High‑throughput sequencing view on the magnitude of global fungal diversity

High-throughput DNA sequencing has dramatically transformed several areas of biodiversity research including mycol-ogy.Despite limitations,high-throughput sequencing is nowadays a predominant method to characterize the alpha and beta diversity of fungal communities.Across the papers utilizing high-throughput sequencing approaches to study natural habitats in terrestrial ecosystems worldwide,>200 studies published until 2019 have generated over 250 million sequences of the primary mycological metabarcoding marker,the nuclear ribosomal internal transcribed spacer 2(ITS2).Here we show that at a 97%sequence similarity threshold,the total richness of non-singleton fungal taxa across the studies published so far is 1.08 million,mostly Ascomycota(56.8%of the taxa)and Basidiomycota(36.7%of the taxa).The Chao-1 estimate of the total extant fungal diversity based on this dataset is 6.28 million taxa,representing a conservative estimate of global fungal species richness.Soil and litter represent the habitats with the highest alpha diversity of fungi followed by air,plant shoots,plant roots and deadwood with Chao-1 predictions,for samples containing 5000 sequences,of 1219,569,392,228,215 and 140 molecular species,respectively.Based on the high-throughput sequencing data,the highest proportion of unknown fungal species is associated with samples of lichen and plant tissues.When considering the use of high-throughput sequenc-ing for the estimation of global fungal diversity,the limitations of the method have to be taken into account,some of which are sequencing platform-specific while others are inherent to the metabarcoding approaches of species representation.In this respect,high-throughput sequencing data can complement fungal diversity predictions based on methods of traditional mycology and increase our understanding of fungal biodiversity.

Diversity of foliar endophytes in wind-fallen Picea abies trees

The unmanaged Norway spruce montane forests in the Bohemian Forest National Park(Czech Republic)suffered from repeated large-scale bark beetle outbreaks in last decade.In this study,the diversity of culturable foliar endophyte microfungi in needles originating from eleven recently wind-fallen trees in this area was surveyed.Our aims were to describe their diversity and to determine the relative host and organ specificities of isolated endophyte species to estimate the species pool and abundance of foliar endophytes before the forest degradation.Microfungi were isolated from surface-sterilized needles,and the outgrowing strains were identified based on morphological and molecular characteristics(analyses of ITS1,ITS2 and partial 28S rDNA).Fungal communities in the needles were diverse,with ascomycetes(mostly anamorphs of Helotiales)dominating basidiomycetes.The most frequent species(Phacidiopycnis sp.,Cistella acuum,Sirococcus sp.and two species of Chalara)did not correspond with those recorded in previous studies of Picea spp.For example,the widely distributed Lophodermium piceae was rarely recorded in this study.This pattern may be caused by different methods of sterilization and cultivation or by physiological characteristics of the needles,or it may reflect the species distribution in the studied area.Members of the Helotiales,along with sequences from GenBank,showed substantial overlap in host affinities,most prominently between Pinus and Picea,and also among species from distant plant lineages.

Microhabitat heterogeneity associated with Vanilla spp.and its influences on the microbial community of leaf litter and soil

The impact of forest microhabitats on physiochemical properties of the soil and that of microbial communities on tropical soils remain poorly understood.To elucidate the effect of tropical forest stand on leaf litter and soil microbial communities,we studied enzyme activities,microbial biomass,and diversity in three distinct microhabitats in terms of plant richness,diameter at breast height(DBH),and physiochemical properties of soil and litter,each associated with a different Vanilla sp.In the soil,positive correlations were found between electrical conductivity(EC)and total organic carbon(TOC)with phosphatase activity,and between nitrogen(N)and water-soluble carbon(WSC)content with urease activity(UA).In the litter,the water content was positively correlated with bacterial and fungal biomass,and N and WSC contents were positively correlated with fungal biomass.Positive correlations were found between plant richness and UA in the soil,plant richness and fungal biomass in the soil and litter,and DBH and fungal biomass in the litter.Amplicon sequencing revealed differences between microhabitats in the relative abundance of some fungal and bacterial taxa and in the bacterial community composition of both litter and soil.Bacterial richness and diversity were different between microhabitats,and,in litter samples,they were negatively correlated with DBH and plant richness,respectively.By contrast,none of the soil and litter physiochemical properties were significantly correlated with microbial diversity.Our results show that significant shifts in enzyme activity,microbial biomass,and diversity in the microhabitats were driven by key abiotic and biotic factors depending on the soil or litter sample type.

Bacterial communities in different locations, seasons and segments of a dairy wastewater treatment system consisting of six segments

A dairy wastewater treatment system composed of the 1st segment（no aeration） equipped with a facility for the destruction of milk fat particles, four successive aerobic treatment segments with activated sludge and a final sludge settlement segment was developed. The activated sludge is circulated through the six segments by settling sediments（activated sludge） in the 6th segment and sending the sediments beck to the 1st and 2nd segments.Microbiota was examined using samples from the non-aerated 1st and aerated 2nd segments obtained from two farms using the same system in summer or winter. Principal component analysis showed that the change in microbiota from the 1st to 2nd segments concomitant with effective wastewater treatment is affected by the concentrations of activated sludge and organic matter（biological oxygen demand [BOD]）, and dissolved oxygen（DO） content. Microbiota from five segments（1st and four successive aerobic segments） in one location was also examined. Although the activated sludge is circulating throughout all the segments, microbiota fluctuation was observed. The observed successive changes in microbiota reflected the changes in the concentrations of organic matter and other physicochemical conditions（such as DO）, suggesting that the microbiota is flexibly changeable depending on the environmental condition in the segments. The genera Dechloromonas, Zoogloea and Leptothrix are frequently observed in this wastewater treatment system throughout the analyses of microbiota in this study.

Improving ITS sequence data for identification of plant pathogenic fungi

Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours.These taxa often have complex and poorly understood life cycles,lack observable,discriminatory morphological characters,and may not be amenable to in vitro culturing.As a result,species identification is frequently difficult.Molecular(DNA sequence)data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi,with the nuclear ribosomal internal transcribed spacer(ITS)region being the most popular marker.However,international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality,making their use in the molecular identification of plant pathogenic fungi problematic.Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages.A third objective was to enrich the sequences with geographical and ecological metadata.The results-a total of 31,954 changes-are incorporated in and made available through the UNITE database for molecular identification of fungi(http://unite.ut.ee),including standalone FASTA files of sequence data for local BLAST searches,use in the next-generation sequencing analysis platforms QIIME and mothur,and related applications.The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi,and we invite all researchers with pertinent expertise to join the annotation effort.

Diverse Asgard archaea including the novel phylum Gerdarchaeota participate in organic matter degradation

Asgard is an archaeal superphylum that might hold the key to understand the origin of eukaryotes, but its diversity and ecological roles remain poorly understood. Here, we reconstructed 15 metagenomic-assembled genomes from coastal sediments covering most known Asgard archaea and a novel group, which is proposed as a new Asgard phylum named as the "Gerdarchaeota".Genomic analyses predict that Gerdarchaeota are facultative anaerobes in utilizing both organic and inorganic carbon. Unlike their closest relatives Heimdallarchaeota, Gerdarchaeota have genes encoding for cellulase and enzymes involved in the tetrahydromethanopterin-based Wood–Ljungdahl pathway. Transcriptomics showed that most of our identified Asgard archaea are capable of degrading organic matter, including peptides, amino acids and fatty acids, occupying ecological niches in different depths of layers of the sediments. Overall, this study broadens the diversity of the mysterious Asgard archaea and provides evidence for their ecological roles in coastal sediments.

Anaerobic digestion in mesophilic and room temperature conditions: Digestion performance and soil-borne pathogen survival

Tomato plant waste（TPW） was used as the feedstock of a batch anaerobic reactor to evaluate the effect of anaerobic digestion on Ralstonia solanacearum and Phytophthora capsici survival. Batch experiments were carried out for TS（total solid） concentrations of 2%, 4% and 6% respectively, at mesophilic（37 ± 1°C） and room（20–25°C） temperatures. Results showed that higher digestion performance was achieved under mesophilic digestion temperature and lower TS concentration conditions. The biogas production ranged from 71 to 416 L/kg VS（volatile solids）. The inactivation of anaerobic digestion tended to increase as digestion performance improved. The maximum log copies reduction of R. solanacearum and P. capsici detected by quantitative PCR（polymerase chain reaction） were 3.80 and 4.08 respectively in reactors with 4% TS concentration at mesophilic temperatures. However, both in mesophilic and room temperature conditions, the lowest reduction of R. solanacearum was found in the reactors with 6% TS concentration, which possessed the highest VFA（volatile fatty acid） concentration. These findings indicated that simple accumulation of VFAs failed to restrain R. solanacearum effectively, although the VFAs were considered poisonous. P. capsici was nearly completely dead under all conditions. Based on the digestion performance and the pathogen survival rate, a model was established to evaluate the digestate biosafety.