A note on the incidence of reverse complementary fungal ITS sequences in the public sequence databases and a software tool for their detection and reorientation

Reverse complementary DNA sequences––sequences that are inadvertently cast backward and in which all purines and pyrimidines are transposed––are not uncommon in sequence databases, where they may introduce noise into sequence-based research. We show that about 1% of the public fungal ITS sequences, the most commonly sequenced genetic marker in mycology, are reverse complementary, and we introduce an open source software solution to automate their detection and reorientation. The MacOSX/Linux/UNIX software operates on public or private datasets of any size, although some 50 base pairs of the 5.8S gene of the ITS region are needed for the analysis.     

A study on reproductive performance and related factors of zebu cows in pastoral herds in a semi-arid area of Tanzania

A prospective longitudinal study was carried out from September 2001 to June 2004 in three adjacent villages in a semi-arid area of Tanzania. The objectives of this study were to measure the intervals between calving and either resumption of cyclical activity or confirmation of pregnancy, to estimate calving intervals, and to investigate the effect of factors assumed to be related to postpartum reproductive performance. A total of 275 lactation periods from 177 Tanzanian Shorthorn Zebu cows managed in a traditional pastoral system in 46 households were initially included. Animals were initially screened for brucelosis and thereafter examined by palpation per rectum at 2-week intervals. Body condition score (scale 1 to 5) was assessed and girth measurement (cm) taken. Occurrence of other reproductive events such as calving, abortion, death of calf, culling and reason for culling were recorded. In a subset of 98 lactation periods from 91 cows milk samples for progesterone (P4) determination were collected twice per week from day 7 after calving to the time of confirmed pregnancy or until milk production ceased before pregnancy. The data were analysed both univariately and in multivariable Cox proportional hazard (frailty) models. The mean (+/-S.E.M.) calving interval was 500+/-13.6 days. Positive reactors in the brucellosis test were 15.6% of the tested animals. Milk P4 analysis showed the rate of abortion/late embryo loss to be 14.3%. Calf mortality rates varied between 14.6 and 17.4%. A positive relationship was found between the outcome variables likelihood of cyclical activity and likelihood of pregnancy in the Cox model, and the explanatory variables: parity and body condition score (BCS) at calving. A negative relationship was found between the outcome variables, and the explanatory variables: maximum BCS loss and calf survival/mortality. Calving in the rainy season was associated with an increased likelihood of pregnancy.     

UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open-access database. UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present UNITE contains 758 ITS sequences from 455 species and 67 genera of ECM fungi. UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment either to pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ECM fungi. The UNITE database is accessible through the URL http://unite.zbi.ee     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

Resistance and resilience of the forest soil microbiome to logging-associated compaction

Soil compaction is a major disturbance associated with logging, but we lack a fundamental understanding of how this affects the soil microbiome. We assessed the structural resistance and resilience of the microbiome using a high-throughput pyrosequencing approach in differently compacted soils at two forest sites and correlated these findings with changes in soil physical properties and functions. Alterations in soil porosity after compaction strongly limited the air and water conductivity. Compaction significantly reduced abundance, increased diversity, and persistently altered the structure of the microbiota. Fungi were less resistant and resilient than bacteria; clayey soils were less resistant and resilient than sandy soils. The strongest effects were observed in soils with unfavorable moisture conditions, where air and water conductivities dropped well below 10% of their initial value. Maximum impact was observed around 6–12 months after compaction, and microbial communities showed resilience in lightly but not in severely compacted soils 4 years post disturbance. Bacteria capable of anaerobic respiration, including sulfate, sulfur, and metal reducers of the Proteobacteria and Firmicutes, were significantly associated with compacted soils. Compaction detrimentally affected ectomycorrhizal species, whereas saprobic and parasitic fungi proportionally increased in compacted soils. Structural shifts in the microbiota were accompanied by significant changes in soil processes, resulting in reduced carbon dioxide, and increased methane and nitrous oxide emissions from compacted soils. This study demonstrates that physical soil disturbance during logging induces profound and long-lasting changes in the soil microbiome and associated soil functions, raising awareness regarding sustainable management of economically driven logging operations.