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Drosophila border cell clusters model collective cell migration. Airyscan super-resolution microscopy enables fine-scale description of cluster shape and texture. Here we describe how to convert Airyscan images of border cell clusters into 3D models of the surface and detect regions of convex and concave curvature. We use spectral decomposition analysis to compare surface textures across genotypes to determine how genes of interest impact cluster surface geometry. This protocol applies to border cells and could generalize to additional cell types. For complete details on the use and execution of this protocol, please refer to Gabbert et al.1.

BACKGROUND: The colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi in Glomeromycota and fine root endophytes in Endogonales (Mucoromycota), as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid. RESULTS: In this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to a clade composed of Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Both plant symbiotic lineages, Glomeromycota and Endogonales, lack numerous thiamine metabolism genes but the lack of fatty acid synthesis genes is specific to AM fungi. Many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the analyzed AM fungal lineages, e.g. the high affinity phosphorus transporter Pho89. CONCLUSION: Based on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that among these lineages, two independent evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus so far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, and 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces.

BackgroundCost-effective production of biofuels from lignocellulose requires the fermentation of D-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.ResultsWe screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.ConclusionCollectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

Abstract Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.

Abstract Understanding the typology of smallholder maize farmers is crucial to elucidate the diversity in local farming systems, and therefore, devise effective interventions and policies that maximize the impact of agricultural innovations in South-Kivu province, eastern Democratic Republic of Congo (DRC). This study aimed at characterizing maize farmers in South-Kivu province and clustering them based on various socioeconomic and agronomic factors to identify specific production constraints and devise more effective interventions. A household survey among 300 South-Kivu maize farmers allowed to identify four distinct clusters based on farm size, land use, income, and agricultural technology adoption. The clusters ranged from resource-poor smallholder (Types 1 and 2) to medium-resource semi-innovative producers (Type 3) and highly equipped innovative farmers (Type 4). Key discriminative factors included total farm size, maize farm size, farming experience, access to agricultural credit, income, and innovation adoption rate. This study revealed specific constraints for each cluster, including low soil fertility, poor weed management, low market access, and labor scarcity. This study’s findings demonstrate the value of farm typology in guiding targeted interventions to address specific constraints and improve maize productivity, and suggest specific strategic directions for research and development projects in rural areas of the South-Kivu province.

Diverse members of early-diverging Mucoromycota, including mycorrhizal taxa and soil-associated Mortierellaceae, are known to harbor Mollicutes-related endobacteria (MRE). It has been hypothesized that MRE were acquired by a common ancestor and transmitted vertically. Alternatively, MRE endosymbionts could have invaded after the divergence of Mucoromycota lineages and subsequently spread to new hosts horizontally. To better understand the evolutionary history of MRE symbionts, we generated and analyzed four complete MRE genomes from two Mortierellaceae genera: Linnemannia (MRE-L) and Benniella (MRE-B). These genomes include the smallest known of fungal endosymbionts and showed signals of a tight relationship with hosts including a reduced functional capacity and genes transferred from fungal hosts to MRE. Phylogenetic reconstruction including nine MRE from mycorrhizal fungi revealed that MRE-B genomes are more closely related to MRE from Glomeromycotina than MRE-L from the same host family. We posit that reductions in genome size, GC content, pseudogene content, and repeat content in MRE-L may reflect a longer-term relationship with their fungal hosts. These data indicate Linnemannia and Benniella MRE were likely acquired independently after their fungal hosts diverged from a common ancestor. This work expands upon foundational knowledge on minimal genomes and provides insights into the evolution of bacterial endosymbionts.

Abstract DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

Abstract This study focused on quantifying and valorizing domestic waste in Bukavu, a rapidly growing city in eastern Democratic Republic of Congo (DRC). With increasing anthropogenic pressure, waste management has become a pressing issue, yet documentation in this area is still limited. This study aimed to fill this gap by providing comprehensive data on domestic waste generation, composition, and disposal practices in Bukavu city. Through field surveys and analysis, waste quantities and types were documented, revealing significant challenges in waste management infrastructure and practices. Additionally, the study explores opportunities for waste valorization, particularly through composting, given the region's agricultural potential and growing urban food demand. The findings revealed that ~ 5% of the domestic waste is biodegradable, with the majority consisting of metals and plastics. These wastes are predominantly used as livestock feed (10%), incinerated (66%), or buried (17%), with ~ 5% undergoing composting. Waste management is primarily handled by children (82%), with very few non-governmental organizations (NGOs) involved in such activities. Among the three composting methods evaluated, outputs from vermicomposting demonstrated superior qualities in terms of promoting crop growth, increasing yield, and achieving a high germination index. Overall, the application of composts improved plant growth and yield parameters of the two major legumes (common bean and soybean). Future interventions should explore the implementation of large-scale composting units at the household or citywide level, while also considering additional strategies to enhance the quality of the compost products. Such efforts are crucial in mitigating the environmental and health impacts of urban household waste and promoting sustainable practices in urban agriculture.

Abstract Background The colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi in Glomeromycota and fine root endophytes in Endogonales (Mucoromycota), as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid. Results In this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to a clade composed of Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Both plant symbiotic lineages, Glomeromycota and Endogonales, lack numerous thiamine metabolism genes but the lack of fatty acid synthesis genes is specific to AM fungi. Many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the analyzed AM fungal lineages, e.g. the high affinity phosphorus transporter Pho89. Conclusion Based on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that among these lineages, two independent evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

Abstract The preservation of cultural heritage, including ancient photographic materials, is of paramount importance in the field of conservation science. In this context, the microbial diversity of ‘Skull and Crossbones’, a 1983 photograph by Robert Mapplethorpe printed on silver gelatine, was assessed. We employed both culture-dependent and culture-independent methods to characterize microbial communities inhabiting this artwork. Vibrational Raman micro spectroscopy and FT-IR spectroscopy were utilized to assess the chemical degradation condition and characterize the chemical components of the silver gelatin print. The combination of molecular sequencing methods (Sanger and HTS approach) and non-invasive vibrational spectroscopy yielded valuable insights into the microbial communities thriving on photographic material and the chemical degradation of the print. Isolated fungal strains were added to the Fungal Collection at the University of Naples Federico II, and their deteriorative potential was investigated by adding substrates, commonly used in canvas photographs to the culture media. These results establish a link between microbial communities colonizing ancient photographic materials, paper decomposition, and the enzymatic patterns of the retrieved microorganisms. This information is invaluable for understanding and addressing biodeterioration progression on valuable works of art, such as historical photographs, which remain understudied.

Summary: Drosophila border cell clusters model collective cell migration. Airyscan super-resolution microscopy enables fine-scale description of cluster shape and texture. Here we describe how to convert Airyscan images of border cell clusters into 3D models of the surface and detect regions of convex and concave curvature. We use spectral decomposition analysis to compare surface textures across genotypes to determine how genes of interest impact cluster surface geometry. This protocol applies to border cells and could generalize to additional cell types.For complete details on the use and execution of this protocol, please refer to Gabbert et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis. Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Abstract Soil depletion constitutes a major challenge for agriculture and food security in highlands of eastern Democratic Republic of Congo (DRC). This study aimed to assess the effectiveness of the split-application of nitrogen fertilizer on grain yield and profitability of maize on Nitisols in eastern DRC. The urea fertilizer (100 kg ha−1) was applied in single, two, and three split-applications on three maize varieties for two cropping seasons. Results showed that maize growth and yield parameters varied significantly with N splitting strategy, varieties, and cropping season (p

ABSTRACTQuality seed is one of the most important farm inputs. The Ituri province, in northeastern Democratic Republic of Congo (DRC), faces enormous challenges in implementing a coherent formal seed sector, an essential gage for seed quality. This study aimed at diagnosing the Ituri seed value chain to propose improvements at all levels. Data collection was conducted using focus group discussions and direct interviews with key players at all levels, household surveys across the province, and a review of seed intervention programme reports, farm visit reports and other literature. Results showed that the seed system in the northeastern DRC is mainly informal due to several socioeconomic and technical factors. Less than a third of crops in the region had a formal seed delivery system. The cash flow in the seed sector was high owing to high seed demands from humanitarian organizations and farmer support structures, estimated at ∼1.5 million US$ in 2022 for the top three commercial seed commodities (common beans, maize and peanuts). However, the province seed production capacity through formal pathway is low (only ∼35 and 69% of the demands for common bean and maize, respectively, are covered by local productions), meaning that the seed demand is met either through importation or fraudulent conversion of farmer-saved seed. Due to lack of technical capacities, Ituri seed multipliers mainly produce open pollinated and synthetic varieties, while hybrids are imported from neighbouring countries. The lack of functional breeders in the provincial seed production chain, disproportionately low seed multipliers-to-distributors ratio, the weakness of the seed certification and regulation services, the border porosity, poor coordination among actors, and low awareness of farmers on seed qualities and advantages of using certified quality seed are the main structural and functional factors hindering the well-functioning of northeastern DRC seed system. This study provided an insight on the characteristics, stakeholders and challenges facing the seed sector in northeastern DRC and proposed an action plan for its improvement.

Executioner-caspase activation has been considered a point-of-no-return in apoptosis. However, numerous studies report survival from caspase activation after treatment with drugs or radiation. An open question is whether cells can recover from direct caspase activation without pro-survival stress responses induced by drugs. To address this question, we engineered a HeLa cell line to express caspase-3 inducibly and combined it with a quantitative caspase activity reporter. While high caspase activity levels killed all cells and very low levels allowed all cells to live, doses of caspase activity sufficient to kill 15 to 30% of cells nevertheless allowed 70 to 85% to survive. At these doses, neither the rate, nor the peak level, nor the total amount of caspase activity could accurately predict cell death versus survival. Thus, cells can survive direct executioner-caspase activation, and variations in cellular state modify the outcome of potentially lethal caspase activity. Such heterogeneities may underlie incomplete tumor cell killing in response to apoptosis-inducing cancer treatments.

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.

Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration in vivo.

Sri Lankan literature in English occupies an important place in postcolonial literature but can also position itself in the arc of ecocriticism. The aim of this paper is to explore Shehan Karunatilaka’s 2022 Booker Prize winning novel The Seven Moons of Maali Almeida by tracing a transition from despair to hope. Thematising the civil war in Sri Lanka, the novel depicts the Afterlife from the perspective of a recently dead photographer called Maali, who tries to solve the mystery of his death and retrieve some of his photographs. In the plot, despair and hope are of paramount importance and take on interesting traits. While the portrayal of despair strongly resembles its Elizabethan conception and can pave the way for intertextual interactions with Spenser’s The Faerie Queene, hope takes on ecocritical traits through a positive form of fragmentation, which bridges human/nonhuman boundaries, and a decolonial reading of the Sri Lankan landscape. By analysing specific excerpts from the novel, this paper will underline the ecocritical themes in the novel and highlight compelling connections with Spenser’s text. Thus, Sri Lankan ecocritical literature will be explored and interesting aspects of Karunatilaka’s novel, such as the transition from despair to hope, will be brought to the foreground.

Although in postcolonial ecocritical literature the agency of animals finds full recognition, representations of insects are so rare that curiosity arises about their untapped ecocritical meaning. Interestingly, in Namwali Serpell’s The Old Drift, a chorus of mosquitoes takes centre stage. In the first section of this paper, I argue that Serpell’s novel recontextualises classical references, placing them between adaptation to the conventions of epic poems and innovation. In the second section, the ecocritical meanings of the chorus of mosquitoes are analysed and framed in relation to the concepts of ‘simulacrum’ and post nature.

In developing countries, studies on sustainable preservation of forests (including non-timber forest products NTFPs) have been poorly carried out for promoting alternative income generating activities (IGA) to alleviate pressure on praised resources by local communities living close by protected areas. This study aimed at (1) inventorying NTFPs exploited by local communities around the Kahuzi Biega National Park (KBNP), (2) assessing their therapeutic values and nutritional contributions, and (3) analyzing their rural and urban market values to determine their potential as alternative income sources as well as the major constraints in their exploitation. Individual interviews were conducted with 790 rural and urban households in Kabare, Walungu, Kalehe, and Bukavu City, eastern Democratic Republic of Congo (DRC) coupled with focus group discussions with key stakeholders around KBNP. Street vendors, restaurants, hotels, and market owners were also interviewed on uses, awareness of the nutritional and therapeutic values, and factors hindering NTFPs wide use in Bukavu urban environments. Physicochemical analyses were also conducted on main NTFPs to determine their nutritional values. Results showed that local populations around KBNP exploit at least 55 NTFPs, mainly for self-consumption as food, feed, and medicine. NTFPs represent the second most important income source around KBNP, accounting for 23.3 % of total households’ income after agriculture that generates 25.7 % of total households’ income. Income from NTFP trade was primarily used for food supply (29 %), investment in small businesses (19.6 %), children's education (17.7 %), and healthcare expenses (5.1 %). These NTFPs had varying nutritional values (in terms of proximate composition, essential minerals, and bioactive compounds). Solanum nigrum (24.9 %), Termitomyces robustus (18.0 %), Amaranthus viridis (17.6 %), and Piper nigrum (16.9 %) are valuable sources of proteins while Zingiber officinale (25.5 %), T. robustus (15.4 %) and P. nigrum (14.3 %) are rich in dietary fibres. Piper nigrum (37.0 and 128.3 mg/kg) and Basella alba (35.1 and 108.7 mg/kg) had the highest zinc and iron contents, respectively. Some of the most nutritious NTFPs, such as S. nigrum, Dioscorea spp., and P. nigrum had high contents in anti-nutritional elements and should be processed properly to maximize bioavailability. Besides, NTFPs created employment opportunities for collectors, vendors, supermarkets, hotels, and restaurants both in rural and urban areas. Though varying with rural communities, the main challenges in the NTFP value chain were low market values (48.6 %), short shelf life (22.5 %), and excessive taxation (19.1 %) while in urban areas, seasonality (36.9 %), scarcity (17.7 %), and prohibiting high prices (17.7 %) were the major limiting factors. This study highlights the significant role played by NTFPs in providing healthy and nutritious food and income for households surrounding the KBNP, and thus emphasizes the necessity for their promotion as alternative income sources and healthy diets to alleviate poverty and hidden hunger around the KBNP.

The purpose of this mixed methods research study was to explore the practice of academic advising and the challenges associated with virtual advising in California higher education during the COVID-19 pandemic. Advising is a retention practice recognized as critical to student success because advisors are one of few personnel interactions students are guaranteed to have with a concerned institutional representative. Though advising is historically a face-to-face practice in a social profession, COVID-19 has forced advising to become remote due to public health concerns during the pandemic. Data was collected from 74 participants representing 43 higher education institutions across California through an online survey that analyzed how academic advising currently functions and how it has changed since COVID-19. Generally, reported challenges were largely technological, particularly regarding access to reliable technology. Advisors were mixed on working virtually but highly willing to combat technological challenges if it meant students benefited from the practice. Academic advisors and their institutions may consider testing alternative virtual advising platforms to determine best fit, implementing automated advising systems for minor advising tasks, offering optional virtual advising training to all appropriate faculty and staff, and creating a standard assessment method for all advising. Future research should explore virtual advising from the student perspective to better understand the role of virtual advising in student integration.

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Forest soil microbiomes have crucial roles in carbon storage, biogeochemical cycling and rhizosphere processes. Wildfire season length, and the frequency and size of severe fires have increased owing to climate change. Fires affect ecosystem recovery and modify soil microbiomes and microbially mediated biogeochemical processes. To study wildfire-dependent changes in soil microbiomes, we characterized functional shifts in the soil microbiota (bacteria, fungi and viruses) across burn severity gradients (low, moderate and high severity) 1 yr post fire in coniferous forests in Colorado and Wyoming, USA. We found severity-dependent increases of Actinobacteria encoding genes for heat resistance, fast growth, and pyrogenic carbon utilization that might enhance post-fire survival. We report that increased burn severity led to the loss of ectomycorrhizal fungi and less tolerant microbial taxa. Viruses remained active in post-fire soils and probably influenced carbon cycling and biogeochemistry via turnover of biomass and ecosystem-relevant auxiliary metabolic genes. Our genome-resolved analyses link post-fire soil microbial taxonomy to functions and reveal the complexity of post-fire soil microbiome activity.

Fungi survive in diverse ecological niches by secreting proteins and other molecules into the environment to acquire food and interact with various biotic and abiotic stressors. Fungal secretome content is, therefore, believed to be tightly linked to fungal ecologies. We sampled 132 genomes from the early-diverging terrestrial fungal lineage zygomycetes (Mucoromycota and Zoopagomycota) and characterized their secretome composition. Our analyses revealed that phylogeny played an important role in shaping the secretome composition of zygomycete fungi with trophic mode contributing a smaller amount. Reconstruction of the evolution of secreted digestive enzymes revealed lineage-specific expansions, indicating that Mucoromycota and Zoopagomycota followed different trajectories early in their evolutionary history. We identified the presence of multiple pathogenicity-related proteins in the lineages known as saprotrophs, suggesting that either the ecologies of these fungi are incompletely known, and/or that these pathogenicity-related proteins have important functions associated with saprotrophic ecologies, both of which invite further investigation.