MinION sequencing of fungi in Sub-Saharan African air, and a novel LAMP assay for rapid detection of the tropical phytopathogenic genus Lasiodiplodia

To date, there have been no DNA-based metabarcoding studies into airborne fungi in tropical Sub-Saharan Africa. In this initial study, 10 air samples were collected onto Vaseline-coated acrylic rods mounted on drones flown at heights of 15-50 metres above ground for 10-15 minutes at three sites in Ghana. Purified DNA was extracted from air samples, the internal transcribed spacer (ITS) region was amplified using fungal-specific primers, and minION third-generation amplicon sequencing was undertaken with downstream bioinformatics analyses utilizing GAIA cloud-based software (at genus taxonomic level). Principal Co-Ordinate analyses based on Bray-Curtis beta diversity dissimilarity values found no clear evidence for structuring of fungal air communities based on geographic location (east vs. central Ghana), underlying vegetation type (cocoa vs. non-cocoa) or height above ground level (15-23 m vs. 25-50 m). In Ghanaian air samples, despite the very short flight times, ~90 operational taxonomic units (OTUs) were identified in each sample, with no statistical differences in alpha diversity between air samples from different locations, vegetation types or height above ground. In Ghanaian air, fungal assemblages were skewed at the phylum taxonomic level towards the ascomycetes (53.7%) as opposed to basidiomycetes (24.6%); at the class level, the Dothideomyectes were predominant (29.8%) followed by the Agaricomycetes (21.8%). The most common fungal genus in Ghanaian air was cosmopolitan and globally ubiquitous Cladosporium (9.9% of reads). Interestingly, many fungal genera containing economically important phytopathogens of tropical crops were also identified in Ghanaian air, including Corynespora, Fusarium and Lasiodiplodia. Consequently, a novel loop-mediated isothermal amplification (LAMP) assay, based on translation elongation factor-1α sequences, was developed and tested for rapid, sensitive and specific detection of the fungal phytopathogenic genus Lasiodiplodia. The potential applications for improved tropical disease management are considered

Evolution of decreased sensitivity to azole fungicides in western European populations of Plenodomus lingam (cause of Phoma leaf spot / stem canker on oilseed rape)

Plenodomus lingam (Leptosphaeria maculans) and P. biglobosus (L. biglobosa) are related fungal pathogens causing Phoma leaf spot and stem canker, an internationally damaging disease of oilseed rape (Brassica napus) and other brassicas. In Europe, fungicides used for disease management are mainly sterol 14α-demethylase (CYP51) inhibitors (DMIs/azoles); quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs) are also used. Decreased DMI sensitivity has emerged in Australian and eastern European P. lingam populations. Decreased sensitivity is mediated by promoter inserts in CYP51 resulting in target site overexpression. In the present study, based on in vitro sensitivity testing, we report decreased DMI (prothioconazole-desthio and mefentrifluconazole) sensitivity in modern western European isolates of P. lingam (collected 2022-23) compared to baseline historical (1992-2005) isolates. Around 85% of the modern western European P. lingam isolates collected, for which the CYP51 promoter region was sequenced, carried a promoter insert but target site alterations were not detected. Six different CYP51 promoter inserts were identified, with a 237 bp fragment of the Sahana transposable element most frequently detected. Inserts were typically associated with a 3 to 10 fold decrease in sensitivity to the DMIs tested. In contrast to P. lingam, PCR screening revealed that CYP51 promoter inserts were absent in modern western European P. biglobosus isolates (collected 2021-23). The combined data indicate P. lingam isolates lacking an insert were similarly (or slightly more) sensitive to the DMIs tested for P. biglobosus, whereas those carrying an insert were slightly less sensitive than P. biglobosus. No clear evidence for substantive sensitivity shifts to the QoI (pyraclostrobin) or SDHI (boscalid) fungicides tested was obtained for either Plenodomus species

An agrogeophysical modelling framework for the detection of soil compaction spatial variability due to grazing using field-scale electromagnetic induction data.

Soil compaction is a regarded as a major environmental and economical hazard, degrading soils across the world. Changes in soil properties due to compaction are known to lead to decrease in biomass and increase in greenhouse gas emissions, nutrient leaching and soil erosion. Quantifying adverse impacts of soil compaction and developing strategies for amelioration relies on an understanding of soil compaction extent and temporal variability. The main indicators of soil compaction (i.e., reduction of pore space, increase in bulk density and decrease in soil transport properties) are relatively easy to quantify in laboratory conditions but such traditional point-based methods offer little information on soil compaction extent at the field scale. Recently, geophysical methods have been proposed to provide non-invasive information about soil compaction. In this work, we developed an agrogeophysical modelling framework to help address the challenges of characterizing soil compaction across grazing paddocks using electromagnetic induction (EMI) data. By integrative modelling of grazing, soil compaction, soil processes and EMI resistivity anomalies, we demonstrate how spatial patterns of EMI observations can be linked to management leading to soil compaction and concurrent modifications of soil functions. The model was tested in a dairy farm in the midlands of Ireland that has been grazed for decades and shows clear signatures of grazing-induced compaction. EMI data were collected in the summer of 2021 and autumn of 2022 under dry and wet soil moisture conditions, respectively. For both years, we observed decreases of apparent electrical resistivity at locations that with visible signatures of compaction such as decreased vegetation and water ponding (e.g., near the water troughs and gates). A machine learning algorithm was used to cluster EMI data with three unique cluster signatures assumed to be representative of heavy, moderately, and non-compacted field zones. We conducted 1D process-based simulations corresponding to non-compacted and compacted soils. The modelled EMI signatures agree qualitatively and quantitatively with the measured EMI data, linking decreased electrical resistivities to zones that were visibly compacted. By providing a theoretical framework based on mechanistic modelling of soil management and compaction, our work may provide a strategy for utilizing EMI data for detection of soil degradation due to compaction

Enriched soil amendments influenced soil fertility, herbage yield and bioactive principle of medicinal plant (Cassia angustifolia Vahl.) grown in two different soils

High cost of chemical fertilizers and poor nutrient content in conventional organic sources (manure, compost, charcoal etc.) can be addressed through development of enriched organic amendments. However, there is a need to evaluate enriched organic amendments as a potential alternative of chemical fertilizers. Therefore, an effort was made to prepare enriched organic amendments through blending distillation waste of aromatic plant biomass (DWB) with naturally available low-grade rock phosphate (RP) and waste mica (WM). Enrich compost (ENC) was produced from DWB in a natural composting process, blended with mineral powder, whereas biochar fortified mineral (BFM) was prepared by blending biochar, derived from DWB through hydrothermal reaction, with mineral powder. The main aims of the present study were to investigate the impacts of ENC and BFM applications on soil properties, and herbage yield and quality of a medicinal herb Senna (Cassia angustifolia Vahl.). The performances of ENC and BFM at two different rates (2.5 and 5 t ha−1) were compared with the application of conventional farmyard manure (FYM, 5 t ha−1) and chemical fertilizers (CF, NPK 60-40-20 kg ha−1) in two different soils in a pot experiment. Both, ENC and EBC improved soil quality and fertility by increasing soil organic carbon, available nutrients, microbial biomass and enzyme activity. The ENC and BFM increased total herbage yields by 21 and 16.3 % compared to FYM. In both soils, the CF treatment produced the maximum dry herbage yields (32.7–37.4 g pot−1), which however were comparable to ENC (31.9–33.7 g pot−1) and BFM (30.7–35.1 g pot−1) treatments. Bioactive compound (sennoside) production in senna was significantly improved by ENC and BFM compared to CF. The present study indicates that ENC and BFM could not only help to overcome the limitation of conventional FYM, but also have the potentials to substitute costly chemical fertilizers, particularly in medicinal plant cultivation

Unravelling the impact of soil types on zinc, iron, and selenium concentrations in grains and straw of wheat/Amblyopyrum muticum and wheat/Triticum urartu doubled haploid lines

The concentration of mineral nutrients in plants is associated with bioavailabilities of soil mineral nutrients, which are regulated by various soil physio-chemical properties. A pot experiment was conducted to investigate the effects of soil type on grain and straw zinc (Zn), iron (Fe) and selenium (Se) concentrations of wheat/Amblyopyrum muticum and wheat/Triticum urartu doubled haploid lines. A set of 42 treatments in a factorial combination with 21 genotypes and two soil types collected from Ngabu and Chitedze Research Stations in Malawi was laid in a randomised complete block design (RCBD) in three replicates. Pre-experiment soil Zn and Fe were extracted using DTPA extraction method followed by analysis with inductively coupled plasma-mass spectrometry (ICP-MS). Aqua-regia hotplate acid digestion was used to extract soil Se and analysis was done using ICPM-MS. Grain and straw samples were digested using nitric acid digestion (HNO3) and analysed using ICP-MS. Soil analysis results showed that the two soils had the same textural class (Sandy clay loam), but different mineral concentrations, pH levels and percentage organic matter. Analysis of variance revealed a ~two-fold higher Zn concentration in grains grown in low pH, high Zn soils (Chitedze soils) compared to grains grown in high pH, low Zn soils (Ngabu soils). Variation in grain Zn concentration was associated with the genotypes (p = 0002), soil type (p = <0.0001), and their interaction (p = 0.035). Grain Fe was 1.3-fold higher in low pH than in high pH soils, and it was influenced by genotypes (p = < 0.0001) and soil type (p = <0.0001). Grain Se was highly associated with soil type (p = <0.0001), and it was 30-fold higher in high pH than in low pH soils. Straw Zn was generally higher in plants grown in Chitedze soils than Ngabu soils, whilst straw Se was higher in plants grown in Ngabu soils than Chitedze soils. The findings demonstrate the significance of soil physio-chemical properties for mineral accumulation and distribution to plant parts, thus informing future breeding programs on importantconsiderations on crop genetic biofortification with the three mineral element

Identification and functional characterisation of a locus for target site integration in Fusarium graminearum

Background Fusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration. Results Here, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1. Conclusion The TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation

The Functional Diversity of the High-Affinity Nitrate Transporter Gene Family in Hexaploid Wheat: Insights from Distinct Expression Profiles

High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies

Mehlich 3 as an indicator of grain nutrient concentration for five crops in sub-Saharan Africa

Soil testing for available nutrients is an important tool to determine fertilizer rates, however many standard methods test the availability of a single nutrient only. In contrast, Mehlich 3 (M3) is a multi-element test for predicting crop yield responses to the addition of macro and micro-nutrients. However, the M3 test has rarely been validated against crop nutrient concentrations, which limits its application for dietary improvement studies in sub-Saharan Africa. This study used 1,096 paired soil and crop samples of five crop types: maize, rice, sorghum, teff and wheat, covering a broad range of soil types and soil properties in Ethiopia and Malawi (e.g., pH 4.5 - 8.8; Olsen P <1 - 280 ppm). The samples were selected based on “high” or “low” grain nutrient concentrations, and the respective soil available nutrients were measured with M3 and other extraction tests: CaCl2 (P, K, Mg, Mn), Ca(NO3)2 (K and Mg), Olsen P, sequential extraction (S), and DTPA (Mn, Fe and Zn). The primary objective was to test how well the M3 nutrient concentrations corresponded to grain nutrient concentrations. The M3 concentrations followed the trend of the “high” and “low” grain concentrations in nearly all nutrients and crops, and this was statistically significant in teff and wheat for all nutrients. The results were best for macronutrients, and slightly less good for micronutrients, probably partly because the concentration of micronutrients in the selected soil samples was generally quite low. Compared to the other multi-element extractant (CaCl2), the M3 test corresponded better to the grain concentrations of K and Mg, and equally well to Olsen P, sequential extraction (S), and DTPA predictions of P, S, Zn and Fe, respectively. M3 extracted much greater concentrations than the other tests, and this was more pronounced in alkaline soils. Given that the M3 test corresponded well to grain nutrient concentrations across a range of soils and crops in SSA, we conclude it could be regarded as a suitable universal test for many nutrients. We also defined thresholds for M3 values, defining below optimum, optimum and above optimum soil fertility status. These results validate the use of the M3 extractant to assess soil fertility and develop fertilizer recommendations for improved produce quality to enhance diets

How much organic carbon could the soil store? The carbon sequestration potential of Australian soil

Soil is a huge carbon (C) reservoir, but where and how much extra C can be stored is unknown. Current methods to estimate the maximum amount of mineral-associated organic carbon (MAOC) stabilized in the fine fraction (clay + silt, <20μm) fit through the MAOC versus clay + silt relationship, not their maxima, making their estimates more uncertain and unreliable. We need a function that ‘envelopes’ that relationship. Here, using 5089 observations, we estimated that the uppermost 30 cm of Australian soil holds 13 Gt (10–18 Gt) of MAOC. We then fitted frontier lines, by soil type, to the relationship between MAOC and the percentage of clay + silt to estimate the maximum amounts of MAOC that Australian soils could store in their current environments, and calculated the MAOC deficit, or C sequestration potential. We propagated the uncertainties from the frontier line fitting and mapped the estimates of these values over Australia using machine learning and kriging with external drift. The maps show regions where the soil is more in MAOC deficit and has greater sequestration potential. The modelling shows that the variation over the whole continent is determined mainly by climate, linked to vegetation and soil mineralogy. We find that the MAOC deficit in Australian soil is 40 Gt (25–60 Gt). The deficit in the vast rangelands is 20.84 Gt (13.97–29.70 Gt) and the deficit in cropping soil is 1.63 Gt (1.12–2.32 Gt). Management could increase C sequestration in these regions if the climate allowed it. Our findings provide new information on the C sequestration potential of Australian soils and highlight priority regions for soil management. Australia could benefit environmentally, socially and economically by unlocking even a tiny portion of its soil's C sequestration potential

Mapping the ratio of agricultural inputs to yields reveals areas with potentially less sustainable farming.

Fertilisers and pesticides are major sources of the environmental harm that results from farming, yet it remains difficult to target reductions in their impacts without compromising food production. We suggest that calculating the ratio of agrochemical inputs to yield can provide an indication of the potential sustainability of farmland, with those areas that have high input relative to yield being considered as less sustainable. Here we design an approach to characterise such Input to Yield Ratios (IYR) for four inputs that can be plausibly linked to environmental impacts: the cumulative risk resulting from pesticide exposure for honeybees and for earthworms, and the amount of nitrogen or phosphorus fertiliser applied per unit area. We capitalise on novel national-scale data to assess IYR for wheat farming across all of England. High-resolution spatial patterns of IYR differed among the four inputs, but hotspots, where all four IYRs were high, were in key agricultural regions not usually characterised as having low suitability for cropping. By scaling the magnitude of each input against crop yield, the IYR does not penalise areas of high yield with higher inputs (important for food production), or areas with low yields but which are achieved with low inputs (important as low impact areas). Instead, the IYR provides a globally applicable framework for evaluating the broad patterns of trade-offs between production and environmental risk, as an indicator of the potential for harm, over large scales. Its use can thus inform targeting to improve agricultural sustainability, or where one might switch to other land uses such as ecosystem restoration