A Plant Stress-Responsive Bioreporter Coupled With Transcriptomic Analysis Allows Rapid Screening for Biocontrols of Necrotrophic Fungal Pathogens.

Streptomyces are soil-borne Actinobacteria known to produce a wide range of enzymes, phytohormones, and metabolites including antifungal compounds, making these microbes fitting for use as biocontrol agents in agriculture. In this study, a plant reporter gene construct comprising the biotic stress-responsive glutathione S-transferase promoter GSTF7 linked to a luciferase output (GSTF7:luc) was used to screen a collection of Actinobacteria candidates for manipulation of plant biotic stress responses and their potential as biocontrol agents. We identified a Streptomyces isolate (KB001) as a strong candidate and demonstrated successful protection against two necrotrophic fungal pathogens, Sclerotinia sclerotiorum and Rhizoctonia solani, but not against a bacterial pathogen (Pseudomonas syringe). Treatment of Arabidopsis plants with either KB001 microbial culture or its secreted compounds induced a range of stress and defense response-related genes like pathogenesis-related (PR) and hormone signaling pathways. Global transcriptomic analysis showed that both treatments shared highly induced expression of reactive oxygen species and auxin signaling pathways at 6 and 24 h posttreatment, while some other responses were treatment specific. This study demonstrates that GSTF7 is a suitable marker for the rapid and preliminary screening of beneficial bacteria and selection of candidates with potential for application as biocontrols in agriculture, including the Streptomyces KB001 that was characterized here, and could provide protection against necrotrophic fungal pathogens.

Tackling Control of a Cosmopolitan Phytopathogen: Sclerotinia.

Phytopathogenic members of the Sclerotinia genus cause widespread disease across a broad range of economically important crops. In particular, Sclerotinia sclerotiorum is considered one of the most destructive and cosmopolitan of plant pathogens. Here, were review the epidemiology of the pathogen, its economic impact on agricultural production, and measures employed toward control of disease. We review the broad approaches required to tackle Sclerotinia diseases and include cultural practices, crop genetic resistance, chemical fungicides, and biological controls. We highlight the benefits and drawbacks of each approach along with recent advances within these controls and future strategies.

Developing Actinobacterial Endophytes as Biocontrol Products for Fusarium pseudograminearum in Wheat.

Crown rot of wheat, caused by Fusarium pseudograminearum, results in millions of dollars of yield losses globally each year. Management strategies to control crown rot are limited and there are concerns about development of fungicide resistance so novel treatment strategies are desirable. A collection of endophytic Actinobacteria was screened for their ability to suppress the growth of F. pseudograminearum and the development of crown rot symptoms in wheat with the aim of identifying candidates that can be developed into biocontrol products. The ability of the Actinobacteria isolates to suppress the growth of three different F. pseudograminearum strains in vitro was assessed using agar-plate competition assays. Soil-free seedling assays were used to screen for suppression of development of early disease symptoms in the susceptible wheat (Triticum aestivum) cv. Tamaroi. Four of the isolates were tested in a glasshouse pot experiment to assess their ability to decrease disease symptoms and prevent yield losses in wheat cv. Tamaroi grown to maturity in an unsterilized soil. The screening of 53 isolates identified two Streptomyces isolates, MH71 and MH243, with very strong antifungal activity against F. pseudograminearum strains in agar-plate competition and seedling assays. In the glasshouse pot trial, plants treated with seed coatings of either MH71 or MH243 had > 24% lower disease severity than control plants infected with F. pseudograminearum. These two cultures show potential for development as biocontrol products because they are easy to culture, grow on relatively inexpensive media, produce highly durable spores and can be delivered to plants as a seed coat.

Draft Genome Sequences of Streptomyces sp. Strains MH60 and 111WW2.

We report here the draft genome sequences, annotations, and predictions of secondary metabolite gene clusters of two endophytic Streptomyces species isolated from wheat plants growing in the Western Australian wheat belt. These strains, Streptomyces sp. strains MH60 and 111WW2, possess antifungal and/or plant growth-promoting activities.

Draft Genome Sequence of Rhodococcus sp. Strain 66b.

We report here the draft genome sequence and annotation of Rhodococcus sp. strain 66b isolated from the soil of southwest Western Australia. This strain exhibits a range of bioactivities, including plant growth promotion, biosurfactant production, and wax degradation. Whole-genome sequencing was conducted to uncover the underlying mechanisms.

Dimethylarsenate (DMA) exposure influences germination rates, arsenic uptake and arsenic species formation in wheat.

The contamination of cereals with arsenic (As) is a global health and agronomic concern. This study compared the physiological response, As uptake and As speciation in the grains and above ground tissues of 20 wheat cultivars exposed to 5 mg As kg-1 soil as either arsenate (AsV) or dimethylarsenate (DMA) under glasshouse conditions. Germination rates for the majority of cultivars exceeded 80% for the majority of cultivars when exposed to AsV, but fell significantly to 20-40% when exposed to DMA. For a number of cultivars, grain yields were 20-50% lower when plants were exposed to DMA compared to AsV. Grain As concentrations were between 0.6 and 1.6 µg As g-1 grain across the twenty cultivars when exposed to AsV, whereas grain As concentrations were much higher (2.2-4.6 µg As g-1 grain) when exposed to DMA. When plants were exposed to AsV, 100% of the As present in the grain was found as inorganic As while in plants exposed to DMA, 70-90% of As was present as DMA with the remainder found as inorganic As. DMA is believed to be incorporated by plants via silica (Si) acid channels and assessment of grain Si concentrations demonstrated that up to 40% less Si was accumulated in grains when plants were exposed to DMA. The decreased germination rates and grain yields in the presence of DMA is similar to the symptoms described for straight head disease in rice, which has been linked to DMA exposure. The results presented here indicate some analogous processes occur in wheat to those described in rice. We hypothesise that exposure to DMA may have inhibited Si-metabolism and translocation which resulted in both developmental impairment and possibly an increased susceptibility to soil pathogens.

Selenium speciation in wheat grain varies in the presence of nitrogen and sulphur fertilisers.

This study investigated whether selenium species in wheat grains could be altered by exposure to different combinations of nitrogen (N) and sulphur (S) fertilisers in an agronomic biofortification experiment. Four Australian wheat cultivars (Mace, Janz, Emu Rock and Magenta) were grown in a glasshouse experiment and exposed to 3 mg Se kg-1 soil as selenate (SeVI). Plants were also exposed to 60 mg N kg-1 soil as urea and 20 mg S kg-1 soil as gypsum in a factorial design (N + S + Se; N + Se; S + Se; Se only). Plants were grown to maturity with grain analysed for total Se concentrations via ICP-MS and Se species determined via HPLC-ICP-MS. Grain Se concentrations ranged from 22 to 70 µg Se g-1 grain (dry mass). Selenomethionine (SeMet), Se-methylselenocystine (MeSeCys), selenohomolanthionine (SeHLan), plus a large concentration of uncharacterised Se species were found in the extracts from grains. SeMet was the major Se species identified accounting for between 9 and 24 µg Se g-1 grain. Exposure to different N and S fertiliser combinations altered the SeMet content of Mace, Janz and Emu Rock grain, but not that of Magenta. MeSeCys and SeHLan were found in far lower concentrations (<4 µg Se g-1 grain). A large component of the total grain Se was uncharacterisable (>30 % of total grain Se) in all samples. When N fertiliser was applied (with or without S), the proportion of uncharacterisable Se increased between 60 and 70 % of the total grain Se. The data presented here indicate that it is possible to alter the content of individual Se species in wheat grains via biofortification combined with manipulation of N and S fertiliser regimes. This has potential significance in alleviating or combating both Se deficiency and Se toxicity effects in humans.

A composite guanyl thiourea (GTU), dicyandiamide (DCD) inhibitor improves the efficacy of nitrification inhibition in soil.

This study investigated whether applying dicyandiamide (DCD) and guanyl thiourea (GTU) in conjunction with urea improves the efficacy of nitrification inhibition relative to traditional fertiliser application of urea or urea + DCD. Urea at a rate of 100 mg N kg(-1) soil was applied to soil microcosms (high nutrient tenosol and low nutrient hydrosol) which were treated with either no inhibitor (urea-only); 15 mg DCD kg(-1) soil or 15 mg DCD kg(-1) soil plus 21 mg GTU kg soil(-1). Mineral N (NH4(+) & NO3(-)) concentrations, potential nitrification rates (PNR) and abundances of ammonia oxidising bacteria (AOB) were measured over time. After 100-days incubation, ∼73 mg N kg(-1) soil was found as NH4(+) when urea + DCD + GTU were applied to the tenosol. NH4(+) concentrations were lower (11-32 mg N kg(-1) soil) when urea or urea + DCD were applied. This suggests that the application of GTU in conjunction with DCD elongated the effects of nitrification inhibition. In both soils, PNRs were faster and AOB abundances (gene copies g(-1) soil) were higher when urea was applied without nitrification inhibitors. There were, however, no differences in PNR or AOB abundances in either soil type when 'urea + DCD' or 'urea + DCD + GTU' were applied. The results indicate that the application of GTU with DCD may extend nitrification inhibition in certain soil types. This finding has the potential to improve the efficacy of commercially available and widely used inhibitors such as DCD.

Changes in glucose fermentation pathways by an enriched bacterial culture in response to regulated dissolved H2 concentrations.

It is well established that metabolic pathways in the fermentation of organic waste are primarily controlled by dissolved H2 concentrations, but there is no reported study that compares observed and predicted shifts in fermentation pathways induced by manipulating the dissolved H2 concentration. A perfusion system is presented that was developed to control dissolved H2 concentrations in the continuous fermentation of glucose by a culture highly enriched towards Thermoanaerobacterium thermosaccharolyticum (86 ± 9% relative abundance) from an originally diverse consortia in the leachate of a laboratory digester fed with municipal solid waste. Media from a 2.5 L CSTR was drawn through sintered steel membrane filters to retain biomass, allowing vigorous sparging in a separate chamber without cellular disruption. Through a combination of sparging and variations in glucose feeding rate from 0.8 to 0.2 g/L/d, a range of steady state fermentations were performed with dissolved H2 concentrations as low as an equivalent equilibrated H2 partial pressure of 3 kPa. Trends in product formation rates were simulated using a H2 regulation partitioning model. The model correctly predicted the direction of products redistribution in response to H2 concentration changes and the acetate and butyrate formation rates when H2 concentrations were less than 6 kPa. However, the model over-estimated acetate, ethanol and butanol productions at the expense of butyrate production at higher H2 concentrations. The H2 yield at the lowest dissolved H2 concentration was 2.67 ± 0.08 mol H2 /mol glucose, over 300% higher than the yield achieved in a CSTR operated without sparging.

The effect of media changes on the rate of cellulose solubilisation by rumen and digester derived microbial communities.

The aim of this study was to investigate the effect of rumen fluid and leachate-based media on the ability of rumen and anaerobic digester derived microorganisms to degrade cellulose. The results demonstrated that rumen microorganisms are not capable of solubilising cellulose, or generating biomass, at an optimal rate when grown in leachate-based media when compared to the rates achieved when grown in rumen-based media. In contrast, the rate of biomass generation and cellulose solubilisation by digester microorganisms was not strongly affected by a change in media type. Several authors have theorised that cellulose degradation rates in anaerobic digesters can be increased by inoculation with rumen-derived microorganisms. The results from this study show that this approach is unlikely to be successful, because the rumen microorganisms would likely be unable to solubilise and out-compete native solid waste microorganisms for the cellulose in a foreign (leachate based) medium.

Comparison of cellulose solubilisation rates in rumen and landfill leachate inoculated reactors.

The aim of this study was to conduct a number of controlled digestions to obtain easily comparable cellulose solubilisation rates and to compare these rates to those found in the literature to see which operational differences were significant in affecting cellulose degradation during anaerobic digestion. The results suggested that differences in volumetric cellulose solubilisation rates were not indicative of the true performance of cellulose digestion systems. When cellulose solubilisation rates were normalised by the mass of cellulose in the reactor at each time step, the comparison of the rates became more meaningful. Cellulose solubilisation was surface area limited. Therefore, changes in the loading rate of cellulose to the reactor altered the volumetric solubilisation rate without changing the mass normalised rate. Comparison of mass normalised solubilisation rates from this study and the literature demonstrated that differences in reactor configuration and operational conditions did not significantly impact on the solubilisation rate whereas the difference in composition of the microbial communities showed a marked effect. This work highlights the importance of using appropriately normalised data when making comparisons between systems with differing operational conditions.

Structure of a cellulose degrading bacterial community during anaerobic digestion.

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.