Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system

In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation

An Assessment of the Lolium perenne (Perennial Ryegrass) Seedborne Microbiome across Cultivars, Time, and Biogeography: Implications for Microbiome Breeding

Research into the bacterial component of the seed microbiome has been intensifying, with the aim of understanding its structure and potential for exploitation. We previously studied the intergenerational seed microbiome of one cultivar of perennial ryegrass with and without one strain of the commercially deployed fungal endophyte Epichloë festucae var. lolii. The work described here expands on our previous study by exploring the bacterial seed microbiome of different commercial cultivar/Epichloë festucae var. lolii combinations in collections of single seeds from the harvest year 2016. In this dataset, a cultivar effect could be seen between the seed microbiomes from cultivars Alto and Trojan. The bacterial component of the seed microbiome from pooled seeds from a single cultivar/E. festucae var. lolii combination harvested from 13 seed production farms around Canterbury in the year 2018 was also studied. This dataset allows the effect of different production locations on the bacterial seed microbiome to be examined. By comparing the two sets of data, bacteria from the genera Pantoea, Pseudomonas, Duganella, Massilia, and an unknown Enterobacteriaceae were observed to be in common. This core bacterial microbiome was stable over time but could be affected by supplemental taxa derived from the growth environment of the parental plant; differing microbiomes were seen between different seed production farms. By comparison to a collection of bacterial isolates, we demonstrated that many of the members of the core microbiome were culturable. This allows for the possibility of exploiting these microbes in the future

Metarhizium spp. isolates effective against Queensland fruit fly juvenile life stages in soil.

Queensland fruit fly, Bactrocera tryoni, Froggatt (Diptera: Tephritidae) is Australia's primary fruit fly pest species. Integrated Pest Management (IPM) has been adopted to sustainably manage this polyphagous species with a reduced reliance on chemical pesticides. At present, control measures are aimed at the adult stages of the fly, with no IPM tools available to target larvae once they exit the fruit and pupate in the soil. The use of entomopathogenic fungi may provide a biologically-based control method for these soil-dwelling life stages. The effectiveness of fungal isolates of Metarhizium and Beauveria species were screened under laboratory conditions against Queensland fruit fly. In bioassays, 16 isolates were screened for pathogenicity following exposure of third-instar larvae to inoculum-treated vermiculite used as a pupation substrate. The best performing Metarhizium sp. isolate achieved an average percentage mortality of 93%, whereas the best performing Beauveria isolate was less efficient, with an average mortality of 36%. Susceptibility to infection during different development stages was investigated using selected fungal isolates, with the aim of assessing all soil-dwelling life stages from third-instar larvae to final pupal stages and emerging adults. Overall, the third larval instar was the most susceptible stage, with average mortalities between 51-98% depending on the isolate tested. Moreover, adult mortality was significantly higher when exposed to inoculum during pupal eclosion, with mortalities between 56-76% observed within the first nine days post-emergence. The effect of temperature and inoculum concentration on insect mortality were assessed independently with candidate isolates to determine the optimum temperature range for fungal biological control activity and the rate required for application in field conditions. Metarhizium spp. are highly efficacious at killing Queensland fruit fly and have potential for use as biopesticides to target soil-dwelling and other life stages of B. tryoni

Enhanced Arbovirus Surveillance with High-Throughput Metatranscriptomic Processing of Field-Collected Mosquitoes

Surveillance programs are essential for the prevention and control of mosquito-borne arboviruses that cause serious human and animal diseases. Viral metatranscriptomic sequencing can enhance surveillance by enabling untargeted, high-throughput arbovirus detection. We used metatranscriptomic sequencing to screen field-collected mosquitoes for arboviruses to better understand how metatranscriptomics can be utilised in routine surveillance. Following a significant flood event in 2016, more than 56,000 mosquitoes were collected over seven weeks from field traps set up in Victoria, Australia. The traps were split into samples of 1000 mosquitoes or less and sequenced on the Illumina HiSeq. Five arboviruses relevant to public health (Ross River virus, Sindbis virus, Trubanaman virus, Umatilla virus, and Wongorr virus) were detected a total of 33 times in the metatranscriptomic data, with 94% confirmed using reverse transcription quantitative PCR (RT-qPCR). Analysis of metatranscriptomic cytochrome oxidase I (COI) sequences enabled the detection of 12 mosquito and two biting midge species. Screening of the same traps by an established public health arbovirus surveillance program corroborated the metatranscriptomic arbovirus and mosquito species detections. Assembly of genome sequences from the metatranscriptomic data also led to the detection of 51 insect-specific viruses, both known and previously undescribed, and allowed phylogenetic comparison to past strains. We have demonstrated how metatranscriptomics can enhance surveillance by enabling untargeted arbovirus detection, providing genomic epidemiological data, and simultaneously identifying vector species from large, unsorted mosquito traps

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane- 1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species. © 2005 Elsevier Ireland Ltd. All rights reserved

Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales).

The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose

Transcriptome analysis of Neotyphodium and Epichloë grass endophytes

Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloe festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource, They permit the interrogation of 3806 Neotyphodium genes (Nchip (TM) rnicroarray), and 4195 Neotyphodium and 920 Epichloe genes (EndoChip (TM) microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass-symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloe was performed. (c) 2006 Elsevie

Mean values for growth attributes and virulence of the three <i>M</i>. <i>anisopliae</i> s.l. isolates MQ1, MM2, and MGV1 at different temperatures.

Data represents means of replicates ± standard error. Treatments followed by different letters indicate significant differences (P < 0.05) based on non-parametric Kruskal-Wallis (KW)of percent data followed a Bonferroni post hoc analysis.</p

Fig 6 -

(a) Mean percentage pupal mortality of the isolate MQ1 towards third-instar larvae of Queensland fruit fly in non-sterile orchard soil. Data represents means of replicates ± standard error. Treatments followed by different letters indicate significant difference (p .05) through generalised linear mixed model analysis of proportional data followed a Tukey HSD test. (b) Linear regression for proportion juvenile mortality with 95% confidence interval indicated in grey (9.127−02+(1.223−08×viable rate applied)+0.084).</p

Fig 3 -

Proportion of germinated conidia over 24 hours for MQ1, MM2, and MGV1 at different temperatures: (a) 21°C, (b) 27°C, and (c) 33°C.</p