Response of soil health indicators to dung, urine and mineral fertilizer application in temperate pastures.

Healthy soils are key to sustainability and food security. In temperate grasslands, not many studies have focused on soil health comparisons between contrasting pasture systems under different management strategies and treatment applications (e.g. manures and inorganic fertilisers). The aim of this study was to assess the responses of soil health indicators to dung, urine and inorganic N fertiliser in three temperate swards: permanent pasture not ploughed for at least 20 years (PP), high sugar ryegrass with white clover targeted at 30% coverage reseeded in 2013 (WC), and high sugar ryegrass reseeded in 2014 (HG). This study was conducted on the North Wyke Farm Platform (UK) from April 2017 to October 2017. Soil health indicators including soil organic carbon (SOC, measured by loss of ignition and elemental analyser), dissolved organic carbon (DOC), total nitrogen (TN), C:N ratio, soil C and N bulk isotopes, pH, bulk density (BD), aggregate stability, ergosterol concentration (as a proxy for fungi biomass), and earthworms (abundance, mass and density) were measured and analysed before and after application of dung and N fertilizer, urine and N fertiliser, and only N fertiliser. The highest SOC, TN, DOC, ergosterol concentration and earthworms as well as the lowest BD were found in PP, likely due to the lack of ploughing. Differences among treatments were observed due to the application of dung, resulting in an improvement in chemical indicators of soil health after 50 days of its application. Ergosterol concentration was significantly higher before treatment applications than at the end of the experiment. No changes were detected in BD and aggregate stability after treatment applications. We conclude that not enough time had passed for the soil to recover after the ploughing and reseeding of the permanent pasture, independently of the sward composition (HG or WC). Our results highlight the strong influence of the soil management legacy in temperate pasture and the positive effects of dung application on soil health over the short term. In addition, we point out the relevance of using standardised methods to report soil health indicators and some methodological limitations.

The stable oxygen isotope ratio of resin extractable phosphate derived from fresh cattle faeces.

RATIONALE: Phosphorus losses from agriculture pose an environmental threat to watercourses. A new approach using the stable oxygen isotope ratio of oxygen in phosphate (δ18 OPO4 value) may help elucidate some phosphorus sources and cycling. Accurately determined and isotopically distinct source values are essential for this process. The δ18 OPO4 values of animal wastes have, up to now, received little attention. METHODS: Phosphate (PO4 ) was extracted from cattle faeces using anion resins and the contribution of microbial PO4 was assessed. The δ18 OPO4 value of the extracted PO4 was measured by precipitating silver phosphate and subsequent analysis on a thermal conversion elemental analyser at 1400°C, with the resultant carbon monoxide being mixed with a helium carrier gas passed through a gas chromatography (GC) column into a mass spectrometer. Faecal water oxygen isotope ratios (δ18 OH2O values) were determined on a dual-inlet mass spectrometer through a process of headspace carbon dioxide equilibration with water samples. RESULTS: Microbiological results indicated that much of the extracted PO4 was not derived directly from the gut fauna lysed during the extraction of PO4 from the faeces. Assuming that the faecal δ18 OH2O values represented cattle body water, the predicted pyrophosphatase equilibrium δ18 OPO4 (Eδ18 OPO4 ) values ranged between +17.9 and +19.9‰, while using groundwater δ18 OH2O values gave a range of +13.1 to +14.0‰. The faecal δ18 OPO4 values ranged between +13.2 and +15.3‰. CONCLUSIONS: The fresh faecal δ18 OPO4 values were equivalent to those reported elsewhere for agricultural animal slurry. However, they were different from the Eδ18 OPO4 value calculated from the faecal δ18 OH2O value. Our results indicate that slurry PO4 is, in the main, derived from animal faeces although an explanation for the observed value range could not be determined.

Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds.

Application of monoclonal antibodies in quantifying fungal growth dynamics during aerobic spoilage of silage.

Proliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony-forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on-farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post-aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on-farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health.

Exploitation of endophytes for sustainable agricultural intensification.

Intensive agriculture, which depends on unsustainable levels of agrochemical inputs, is environmentally harmful, and the expansion of these practices to meet future needs is not economically feasible. Other options should be considered to meet the global food security challenge. The plant microbiome has been linked to improved plant productivity and, in this microreview, we consider the endosphere - a subdivision of the plant microbiome. We suggest a new definition of microbial endophyte status, the need for synergy between fungal and bacterial endophyte research efforts, as well as potential strategies for endophyte application to agricultural systems.

Transcriptional reprogramming underpins enhanced plant growth promotion by the biocontrol fungus Trichoderma hamatum GD12 during antagonistic interactions with Sclerotinia sclerotiorum in soil.

The free-living soil fungus Trichoderma hamatum strain GD12 is notable amongst Trichoderma strains in both controlling plant diseases and stimulating plant growth, a property enhanced during its antagonistic interactions with pathogens in soil. These attributes, alongside its markedly expanded genome and proteome compared with other biocontrol and plant growth-promoting Trichoderma strains, imply a rich potential for sustainable alternatives to synthetic pesticides and fertilizers for the control of plant disease and for increasing yields. The purpose of this study was to investigate the transcriptional responses of GD12 underpinning its biocontrol and plant growth promotion capabilities during antagonistic interactions with the pathogen Sclerotinia sclerotiorum in soil. Using an extensive mRNA-seq study capturing different time points during the pathogen-antagonist interaction in soil, we show that dynamic and biphasic signatures in the GD12 transcriptome underpin its biocontrol and plant (lettuce) growth-promoting activities. Functional predictions of differentially expressed genes demonstrate the enrichment of transcripts encoding proteins involved in transportation and oxidation-reduction reactions during both processes and an over-representation of siderophores. We identify a biphasic response during biocontrol characterized by a significant induction of transcripts encoding small-secreted cysteine-rich proteins, secondary metabolite-producing gene clusters and genes unique to GD12. These data support the hypothesis that Sclerotinia biocontrol is mediated by the synthesis and secretion of antifungal compounds and that GD12's unique reservoir of uncharacterized genes is actively recruited during the effective biological control of a plurivorous plant pathogen.

Investigating the beneficial traits of Trichoderma hamatum GD12 for sustainable agriculture-insights from genomics.

Trichoderma hamatum strain GD12 is unique in that it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens and can induce systemic resistance to foliar pathogens. This study extends previous work in lettuce to demonstrate that GD12 can confer beneficial agronomic traits to other plants, providing examples of plant growth promotion in the model dicot, Arabidopsis thaliana and induced foliar resistance to Magnaporthe oryzae in the model monocot rice. We further characterize the lettuce-T. hamatum interaction to show that bran extracts from GD12 and an N-acetyl-ß-D-glucosamindase-deficient mutant differentially promote growth in a concentration dependent manner, and these differences correlate with differences in the small molecule secretome. We show that GD12 mycoparasitises a range of isolates of the pre-emergence soil pathogen Sclerotinia sclerotiorum and that this interaction induces a further increase in plant growth promotion above that conferred by GD12. To understand the genetic potential encoded by T. hamatum GD12 and to facilitate its use as a model beneficial organism to study plant growth promotion, induced systemic resistance and mycoparasitism we present de novo genome sequence data. We compare GD12 with other published Trichoderma genomes and show that T. hamatum GD12 contains unique genomic regions with the potential to encode novel bioactive metabolites that may contribute to GD12's agrochemically important traits.