Chemical and Microbial Characteristics of Vermicompost Leachate and their Effect on Plant Growth

There is interest in reducing chemical fertiliser in favour of organic amendments. Vermicompost leachate is the liquid resulting from organic waste breakdown by earthworms. Little is known about effects of different organic substrates on leachate properties. Various substrates were tested for their effect on earthworms, leachate chemical and microbial composition, and benefits to plants. Effect of 18 substrates on earthworm population and mass was measured over 8 weeks. Earthworm growth and survival varied with substrate. T-RFLP analysis from 6 substrates at 4 and 8 weeks showed differences in leachate bacteria and fungi. Banana(B), green bean(G) and rockmelon(R) were used, alone and in mixes. Earthworm population, mass, life cycle stage and cocoon number were measured over 16 weeks. ARISA analysis at 10 weeks showed differences in leachate bacteria. Leachates were stored for a year at 4°C and 24°C. Most leachate chemical and microbial properties changed with storage. Leachates from substrate mixes B/R or G/R were unchanged at 4°C. Leachates were applied to soybean, serradella and wheat. Unsterile G leachate increased soybean nodulation at 8 weeks and serradella nodulation at 10 weeks, but not at 5. Plant mass increased in chemically fertilised wheat at 5 and 10 weeks, and 5 for serradella. At 10 weeks there was no difference in serradella mass. G leachate effect on nodulation was tested on serradella. G leachate was compared with substrate mix of B/G/R leachate, synthetic G leachate, and chemicals (CRS). Microbes extracted from G leachate were applied. CRS produced the highest plant height and dry weight. Inoculation with G leachate microbes increased nodule mass in plants treated with CRS. Vermicompost recycles organic waste into a product that improves legume nodulation through microbial action and may increase biologically fixed nitrogen

Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement

Molecular Evolutionary Relationships of Enteroinvasive Escherichia coli and Shigella spp.

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H− strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli

Identification and characterization of cyprinid herpesvirus-3 (cyhv-3) encoded micrornas

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA-offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3

Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA-offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3

N076_Unique_Sequence_Tags

Unique reads and read counts obtained from deep sequencing of small RNA (~17-25 nt) from Common Carp Brain cells (CCBs) infected with Cyprinid Herpesvirus-3 (CyHV-3) N076 isolate. Fully intact 3’ adapter sequences are trimmed from the 3’ end of each unique read

H361_Unique_Sequence_Tags

Unique reads and read counts obtained from deep sequencing of small RNA (~17-25 nt) from Common Carp Brain cells (CCBs) infected with Cyprinid Herpesvirus-3 (CyHV-3) H361 isolate. Fully intact 3’ adapter sequences are trimmed from the 3’ end of each unique read

Alignment of CyHV-3 pre-miRNAs to candidate homologues on the CyHV-1 and CyHV-2 genomes.

<p>CyHV-1 and CyHV-2 sequences that are underlined represent homologous sequences identified by BLASTN, sequences not underlined (i.e. remainder of input sequences) were subsequently manually inspected for sequence similarity. Nucleotides shaded in grey are conserved in all viruses in the respective alignments and those in dark grey are conserved in 2/3 viruses. <b>(A)</b> MD1111—with the exception of the first 3 nucleotides, the MD1111-miR-5p sequence is fully conserved in CyHV-2; <b>(B)</b> MD11776—the seed (bases 2–7) of MD11776-miR-3p is fully conserved and the remainder of the miRNA is also conserved in all three viruses with the exception of two mismatches; <b>(C)</b> MD11704—the MD11704-miR-5p seed region is fully conserved, the MD11704-miR-3p seed region has one mismatch to each virus.</p

Genomic locations and structures of high probability CyHV-3 pre-miRNA identified using a non-automated approach.

<p><b>(A)</b> Genomic locations of pre-miRNAs; all features on the forward strand are shown in black and those on the reverse strand are in grey. <b>(B)</b> Structures (predicted by VMir) of the high-probability CyHV-3 pre-miRNAs shown in (A). The 5′/3′ arm miRNAs and 5′/3′ arm moRNAs are highlighted in red/yellow and green/purple respectively and represent the dominant reads at these loci in the H361 infection, variants (if any) identified in the N076 infection are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125434#pone.0125434.t002" target="_blank">Table 2</a>. MiRNA* reads are indicated with an asterisk. MiRNAs partially overlapping with moRNAs are underlined.</p

Comparison of the levels of miRNAs derived from CyHV-3 pre-miRNA candidates MR5057 and MD11776.

<p>(A) Small RNA deep sequencing read counts and (B) DNA microarray miRNA probe hybridization signal intensities corrected and expressed as multiples of background (MOB). Data from both (A) and (B) were obtained using size-selected miRNA-inclusive RNA (F1 fraction; 17–25 nt) from the same <i>in vitro</i> infection (CyHV-3 H361 isolate). No corresponding hybridization signals were detected in miRNA non-inclusive RNA (F2 faction; 26–35 nt) from the same infection or in miRNA inclusive RNA (F3 fraction, 17–25 nt) from non-infected cells (Dataset S in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125434#pone.0125434.s002" target="_blank">S1 File</a>). Error bars represent the data range.</p