The nature of saprophytic and mycoparasitic hyphal growth of Trichoderma spp. has been studied extensively, yet its initiation via conidial germination in this genus is less well understood. Using near-synchronous germinating cultures of Trichoderma asperelloides, we followed the morphological progression from dormant conidia to initial polar growth to germling formation and to evidence for first branching. We found that the stage-specific transcriptional profile of T. asperelloides is one of the most dynamic described to date: transcript abundance of over 5000 genes—comprising approximately half of the annotated genome—was unremittingly reduced in the transition from dormancy to polar growth. Conversely, after the onset of germination, the transcript abundance of approximately a quarter of the genome was unremittingly elevated during the transition from elongation to initial branching. These changes are a testimony to the substantial developmental events that accompany germination. Bayesian network analysis identified several chitinase- and glucanase-encoding genes as active transcriptional hubs during germination. Furthermore, the expression of specific members of the chitin synthase and glucan elongase families was significantly increased during germination in the presence of Rhizoctonia solani—a known host of the mycoparasite—indicating that host recognition can occur during the early stages of mycoparasite development.

Secondary metabolites (SMs) are low-molecular-weight compounds that often mediate interactions between fungi and their environments. Fungi enriched with SMs are of significant research interest to agriculture and medicine, especially from the aspects of pathogen ecology and environmental epidemiology. Secondary metabolite clusters (SMCs) encode the machinery for fungal toxin production. However, understanding their function and analyzing their products requires investigation of the developmental and environmental conditions in which they are expressed. Gene expression is often restricted to specific and unexamined stages of the life cycle. Therefore, we applied comparative genomics analyses to identify SMCs in Neurospora crassa and analyzed extensive transcriptomic data spanning nine independent experiments from diverse developmental and environmental conditions to reveal their life cycle-specific gene expression patterns. We reported 20 SMCs comprising 177 genes—a manageable set for investigation of the roles of SMCs across the life cycle of the fungal model N. crassa—as well as gene sets coordinately expressed in 18 predicted SMCs during asexual and sexual growth under three nutritional and two temperature conditions. Divergent activity of SMCs between asexual and sexual development was reported. Of 126 SMC genes that we examined for knockout phenotypes, al-2 and al-3 exhibited phenotypes in asexual growth and conidiation, whereas os-5, poi-2, and pmd-1 exhibited phenotypes in sexual development. SMCs with annotated function in mating and crossing were actively regulated during the switch between asexual and sexual growth. Our discoveries call for attention to roles that SMCs may play in the regulatory switches controlling mode of development, as well as the ecological associations of those developmental stages that may influence expression of SMCs. IMPORTANCE Secondary metabolites (SMs) are low-molecular-weight compounds that often mediate interactions between fungi and their environments. Fungi enriched with SMs are of significant research interest to agriculture and medicine, especially from the aspects of pathogen ecology and environmental epidemiology. However, SM clusters (SMCs) that have been predicted by comparative genomics alone have typically been poorly defined and insufficiently functionally annotated. Therefore, we have investigated coordinate expression in SMCs in the model system N. crassa, and our results suggest that SMCs respond to environmental signals and to stress that are associated with development. This study examined SMC regulation at the level of RNA to integrate observations and knowledge of these genes in various growth and development conditions, supporting combining comparative genomics and inclusive transcriptomics to improve computational annotation of SMCs. Our findings call for detailed study of the function of SMCs during the asexual-sexual switch, a key, often-overlooked developmental stage.

Novel species of fungi described in this study include those from various countries as follows: Australia, Agaricus albofoetidus, Agaricus aureoelephanti and Agaricus parviumbrus on soil, Fusarium ramsdenii from stem cankers of Araucaria cunninghamii, Keissleriella sporoboli from stem of Sporobolus natalensis, Leptosphaerulina queenslandica and Pestalotiopsis chiaroscuro from leaves of Sporobolus natalensis, Serendipita petricolae as endophyte from roots of Eriochilus petricola, Stagonospora tauntonensis from stem of Sporobolus natalensis, Teratosphaeria carnegiei from leaves of Eucalyptus grandis × E. camaldulensis and Wongia ficherai from roots of Eragrostis curvula. Canada, Lulworthia fundyensis from intertidal wood and Newbrunswickomyces abietophilus (incl. Newbrunswickomyces gen. nov.)on buds of Abies balsamea. Czech Republic, Geosmithia funiculosa from a bark beetle gallery on Ulmus minor and Neoherpotrichiella juglandicola (incl. Neoherpotrichiella gen. nov.)from wood of Juglans regia. France, Aspergillus rouenensis and Neoacrodontium gallica (incl. Neoacrodontium gen. nov.)from bore dust of Xestobium rufovillosum feeding on Quercus wood, Endoradiciella communis (incl. Endoradiciella gen. nov.)endophyticin roots of Microthlaspi perfoliatum and Entoloma simulans on soil. India, Amanita konajensis on soil and Keithomyces indicus from soil. Israel, Microascus rothbergiorum from Stylophora pistillata. Italy, Calonarius ligusticus on soil. Netherlands , Appendopyricularia juncicola (incl. Appendopyricularia gen. nov.), Eriospora juncicola and Tetraploa juncicola on dead culms of Juncus effusus, Gonatophragmium physciae on Physcia caesia and Paracosmospora physciae (incl. Paracosmospora gen. nov.)on Physcia tenella, Myrmecridium phragmitigenum on dead culm of Phragmites australis, Neochalara lolae on stems of Pteridium aquilinum, Niesslia nieuwwulvenica on dead culm of undetermined Poaceae, Nothodevriesia narthecii (incl. Nothodevriesia gen. nov.) on dead leaves of Narthecium ossifragum and Parastenospora pini (incl. Parastenospora gen. nov.)on dead twigs of Pinus sylvestris. Norway, Verticillium bjoernoeyanum from sand grains attached to a piece of driftwood on a sandy beach. Portugal, Collybiopsis cimrmanii on the base of living Quercus ilex and amongst dead leaves of Laurus and herbs. South Africa , Paraproliferophorum hyphaenes (incl. Paraproliferophorum gen. nov.) on living leaves of Hyphaene sp. and Saccothecium widdringtoniae on twigs of Widdringtonia wallichii. Spain, Cortinarius dryosalor on soil, Cyphellophora endoradicis endophytic in roots of Microthlaspi perfoliatum, Geoglossum laurisilvae on soil, Leptographium gemmatum from fluvial sediments, Physalacria auricularioides from a dead twig of Castanea sativa , Terfezia bertae and Tuber davidlopezii in soil. Sweden, Alpova larskersii, Inocybe alpestris and Inocybe boreogodeyi on soil. Thailand, Russula banwatchanensis, Russula purpureoviridis and Russula lilacina on soil. Ukraine, Nectriella adonidis on over wintered stems of Adonis vernalis. USA, Microcyclus jacquiniae from living leaves of Jacquinia keyensis and Penicillium neoherquei from a minute mushroom sporocarp. Morphological and culture characteristics are supported by DNA barcodes.

The filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed. The filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed.

Fruit skin reticulation is accompanied by the formation of a wound-periderm tissue made of suberized cells. The regulatory networks overseeing skin reticulation during fruit development were extensively studied, yet how reticulation affects post-harvest traits remains unknown. We addressed this notion using the common Cucumis sativus and the skin-cracked Sikkim (Cucumis sativus var. sikkimensis) cucumbers. Light and electron microscopy in consort with gas chromatography-mass spectrometry revealed that sativus fruit skin is made of the typical cutin polymer, while the skin of sikkimensis fruit comprised of the aromatic suberin polymer. Comparative post-harvest experiments with different storage temperatures revealed that sikkimensis fruit are more resilient to chilling injuries arise during cold storage, exhibiting lower rates of weight losses, ethylene and CO2, electrolyte leakage and lipid peroxidation. We further demonstrate that different storage temperatures affect the contents of skin polymers cutin and suberin in a differential manner.

Conjugal plasmids constitute a major engine for horizontal gene transfer in bacteria, and are key drivers of the spread of antibiotic resistance, virulence, and metabolic functions. Bacteria in terrestrial habitats often inhabit surfaces that are not constantly water-saturated, where microscopic surface wetness (MSW), comprised of thin liquid films and microdroplets, permanently or intermittently occurs. How physical properties of microdroplets, and of the surfaces they reside on, affect plasmid transfer rates is not well understood. Here, building on microscopy-based microdroplet experiments, we examined the relation between droplet properties (size and spread) and plasmid transfer rates at single-cell and individual droplet resolution, using Pseudomonas putida as a model species. We show that transfer rates increase with droplet size, due to higher densities of cells on the surface in larger droplets, resulting from lower ratio between the area of the liquid-solid interface and droplet volumes. We further show that surface hydrophobicity promotes transfer rates via the same mechanism. Our results provide new insights into how physical properties of surfaces and MSW affect plasmid transfer rates, and more generally, microbial interactions mediated by cell-to-cell contact, with important implications for our understanding of the ecology and evolution of bacteria in unsaturated environments.

Predicting interspecies interactions is a key challenge in microbial ecology, as such interactions shape the composition and functioning of microbial communities. However, predicting microbial interactions is challenging since they can vary considerably depending on species' metabolic capabilities and environmental conditions. Here, we employ machine learning models to predict pairwise interactions between culturable bacteria based on their phylogeny, monoculture growth capabilities, and interactions with other species. We trained our models on one of the largest available pairwise interactions dataset containing over 7500 interactions between 20 species from 2 taxonomic groups that were cocultured in 40 different carbon environments. Our models accurately predicted both the sign (accuracy of 88%) and the strength of effects (R2 of 0.87) species had on each other's growth. Encouragingly, predictions with comparable accuracy could be made even when not relying on information about interactions with other species, which are often hard to measure. However, species' monoculture growth was essential to the model, as predictions based solely on species' phylogeny and inferred metabolic capabilities were significantly less accurate. These results bring us a step closer to a predictive understanding of microbial communities, which is essential for engineering beneficial microbial consortia.Competing Interest StatementThe authors have declared no competing interest.

The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 “core” mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway—evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1. We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.

Interspecies interactions shape the structure and function of microbial communities. In particular, positive, growth-promoting interactions can substantially affect the diversity and productivity of natural and engineered communities. However, the prevalence of positive interactions and the conditions in which they occur are not well understood. To address this knowledge gap, we used kChip, an ultrahigh-throughput coculture platform, to measure 180,408 interactions among 20 soil bacteria across 40 carbon environments. We find that positive interactions, often described to be rare, occur commonly and primarily as parasitisms between strains that differ in their carbon consumption profiles. Notably, nongrowing strains are almost always promoted by strongly growing strains (85%), suggesting a simple positive interaction-mediated approach for cultivation, microbiome engineering, and microbial consortium design.

Studying the role of predator-prey interactions in food-web stability and species coexistence in the environment is arduous. Here, Cohen et al. use a combination of community and single-cell analyses to show that bacterial predators can regulate prey populations in the species-rich environments of wastewater treatment plants. A fundamental question in community ecology is the role of predator-prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.

Wastewater treatment plants (WWTPs) are continuously exposed to sub-inhibitory concentrations of antibiotics that are thought to contribute to the spreading of antibiotic resistant bacteria and antibiotic resistance genes, which are eventually released to downstream environments through effluents. In order to understand the effects of sub-inhibitory concentrations of antibiotics on sludge microbiome and resistome, we spiked a conventional activated sludge (CAS) model system with ciprofloxacin, a common fluoroquinolone antibiotic, from 0.0001 mg/L (about twice the typical ciprofloxacin concentration observed in municipal wastewater) up to 0.1 mg/L (one order of magnitude below the clinical MIC for Enterobacteriaceae) for 151 days. The abundance of ciprofloxacin resistant bacteria and qnrS, a plasmid-associated gene that confers resistance to quinolones, in activated sludge and in effluents of control and spiked CAS reactors, showed no measurable effect of the antibiotic amendment. This was also true for the bacterial community structure and for indicators of WW treatment such as N removal efficiency. Surprisingly, temporal fluctuations in both reactors could explain the observed internal variability of these antibiotic resistance determinants better than the hypothesized antibiotic-driven selective pressure. Overall, this work shows that the core sludge microbiome in CAS systems is resilient to sub-inhibitory concentrations of ciprofloxacin at a functional, structural, and antibiotic resistance levels.

Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring.-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.

Positive interactions, including intraspecies cooperation and interspecies mutualisms, play crucial roles in shaping the structure and function of many ecosystems, ranging from plant communities to the human microbiome. While the evolutionary forces that form and maintain positive interactions have been investigated extensively, the influence of positive interactions on the ability of species to adapt to new environments is still poorly understood. Here, we use numerical simulations and theoretical analyses to study how positive interactions impact the likelihood that populations survive after an environment deteriorates, such that survival in the new environment requires quick adaptation via the rise of new mutants-a scenario known as evolutionary rescue. We find that the probability of evolutionary rescue in populations engaged in positive interactions is reduced significantly. In cooperating populations, this reduction is largely due to the fact that survival may require at least a minimal number of individuals, meaning that adapted mutants must arise and spread before the population declines below this threshold. In mutualistic populations, the rescue probability is decreased further due to two additional effects-the need for both mutualistic partners to adapt to the new environment, and competition between the two species. Finally, we show that the presence of cheaters reduces the likelihood of evolutionary rescue even further, making it extremely unlikely. These results indicate that while positive interactions may be beneficial in stable environments, they can hinder adaptation to changing environments and thereby elevate the risk of population collapse. Furthermore, these results may hint at the selective pressures that drove co-dependent unicellular species to form more adaptable organisms able to differentiate into multiple phenotypes, including multicellular life. Author summary Many ecosystems are exposed to rapidly changing environmental conditions, from global warming to overuse of antibiotics in medicine and agriculture. Therefore, there is great interest in elucidating the factors that affect the ability of ecosystems to adapt to these changes. While many such factors have been recently investigated, the effect of interactions within a community on its ability to adapt remain largely unexplored. In this work, we focus on the effect of positive interactions, in the form of cooperation between individual or different species, on the ability of communities to adapt to new environments. Using simulations and theoretical analysis, we find that positive interactions significantly reduce the probability of survival of cooperative communities in changing environments, elevating the risk of populations' extinction. Furthermore, we suggest that the need for an adaptable solution of cooperation could have played a part in the circumstances leading to the transition between unicellular and multicellular life.

There is growing evidence that symbiotic microbes can influence multiple nutrition-related behaviors of their hosts, including locomotion, feeding, and foraging. However, how the microbiome affects nutrition-related behavior is largely unknown. Here, we demonstrate clear sexual dimorphism in how the microbiome affects foraging behavior of a frugivorous fruit fly, Drosophila suzukii. Female flies deprived of their microbiome (axenic) were consistently less active in foraging on fruits than their conventional counterparts, even though they were more susceptible to starvation and starvation-induced locomotion was notably more elevated in axenic than conventional females. Such behavioral change was not observed in male flies. The lag of axenic female flies but not male flies to forage on fruits is associated with lower oviposition by axenic flies, and mirrored by reduced food seeking observed in virgin females when compared to mated, gravid females. In contrast to foraging intensity being highly dependent on the microbiome, conventional and axenic flies of both sexes showed relatively consistent and similar fruit preferences in foraging and oviposition, with raspberries being preferred among the fruits tested. Collectively, this work highlights a clear sex-specific effect of the microbiome on foraging and locomotion behaviors in flies, an important first step toward identifying specific mechanisms that may drive the modulation of insect behavior by interactions between the host, the microbiome, and food.

The small size of bacterial cells necessitates rapid adaption to sudden environmental changes. InBdellovibrio bacteriovorus, an obligate predator of bacteria common in oligotrophic environments, the non-replicative, highly motile attack phase (AP) cell must invade a prey to ensure replication. AP cells swim fast and respire at high rates, rapidly consuming their own contents. How the predator survives in the absence of prey is unknown. We show that starvation for prey significantly alters swimming patterns and causes exponential decay in prey-searching cells over hours, until population-wide swim-arrest. Swim-arrest is accompanied by changes in energy metabolism, enabling rapid swim-reactivation upon introduction of prey or nutrients, and a sweeping change in gene expression and gene regulation that largely differs from those of the paradigmatic stationary phase. Swim-arrest is costly as it imposes a fitness penalty in the form of delayed growth. We track the control of the swim arrest-reactivation process to cyclic-di-GMP (CdG) effectors, including two motility brakes. CRISPRi transcriptional inactivation, and in situ localization of the brakes to the cell pole, demonstrated their essential role for effective survival under prey-induced starvation. Thus, obligate predators evolved a unique CdG-controlled survival strategy, enabling them to sustain their uncommon lifestyle under fluctuating prey supply.

Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria commonly encountered in the environment. In dual predator-prey cultures, prey accessibility ensures optimal feeding and replication and rapid BALO population growth. However, the environmental prey landscape is complex, as it also incorporates non-prey cells and other particles. These may act as decoys, generating unproductive encounters which in turn may affect both predator and prey population dynamics. In this study, we hypothesized that increasing decoy:prey ratios would bring about increasing costs on the predator's reproductive fitness. We also tested the hypothesis that different BALOs and decoys would have different effects. To this end, we constructed prey landscapes including petiplasmic or epibiotic predators including two types of decoy under a large range of initial decoy:prey ratio, and mixed cultures containing multiple predators and prey. We show that as decoy:prey ratios increase, the maximal predator population sizes is reduced and the time to reach it significantly increases. We found that BALOs spent less time handling non-prey (including superinfection-immune invaded prey) than prey cells, and did not differentiate between efficient and less efficient prey. This may explain why in multiple predator and prey cultures, less preferred prey appear to act as decoy.

Elevated CO2 stimulates plant growth and affects quantity and composition of root exudates, followed by response of its microbiome. Three scenarios representing nitrate fertilization regimes: limited (30 ppm), moderate (70 ppm) and excess nitrate (100 ppm) were compared under ambient and elevated CO2 (eCO(2), 850 ppm) to elucidate their combined effects on root-surface-associated bacterial community abundance, structure and function. Wheat root-surface-associated microbiome structure and function, as well as soil and plant properties, were highly influenced by interactions between CO2 and nitrate levels. Relative abundance of total bacteria per plant increased at eCO(2) under excess nitrate. Elevated CO2 significantly influenced the abundance of genes encoding enzymes, transporters and secretion systems. Proteobacteria, the largest taxonomic group in wheat roots (similar to 75%), is the most influenced group by eCO(2) under all nitrate levels. Rhizobiales, Burkholderiales and Pseudomonadales are responsible for most of these functional changes. A correlation was observed among the five gene-groups whose abundance was significantly changed (secretion systems, particularly type VI secretion system, biofilm formation, pyruvate, fructose and mannose metabolism). These changes in bacterial abundance and gene functions may be the result of alteration in root exudation at eCO(2), leading to changes in bacteria colonization patterns and influencing their fitness and proliferation.

Aims The Mediterranean fruit fly (the medfly) causes major losses of agricultural fruits. Its microbiome is mainly composed of various Enterobacteriaceae that contribute to nutrient acquisition and are associated with the fly's development. Moreover, the performance of males produced by the sterile insect technique is improved by providing mass-reared insects with specific gut bacteria. Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria that efficiently preys upon diverse Enterobacteriaceae, making it a potential disruptor of the fly's microbiome. We hypothesized that the fly's microbiome can be targeted to control the insect. Methods and Results Inoculation of B. bacteriovorus as free-swimming or encapsulated cells into gut extracts significantly reduced gut bacterial abundance, sustaining predator survival. Similar treatments applied to adult flies showed that the predators also survived in the gut environment. While addition of the predators did not affect total gut bacterial abundance and end-point fly mortality, a shift in the gut community structure, measured by high-throughput community sequencing was observed. Conclusions The bacterial predator of bacteria B. bacteriovorus can prey and survive in vivo in the medfly gut. Significance and Impact of the Study This study establishes the potential of BALOs to affect the microbiome of insect hosts.

Bdellovibrio and like organisms (BALOs) prey on Gram-negative bacteria in the planktonic phase as well as in biofilms, with the ability to reduce prey populations by orders of magnitude. During the last few years, evidence has mounted for a significant ecological role for BALOs, with important implications for our understanding of microbial community dynamics as well as for applications against pathogens, including drug-resistant pathogens, in medicine, agriculture and aquaculture, and in industrial settings for various uses. However, our understanding of biofilm predation by BALOs is still very fragmentary, including gaps in their effect on biofilm structure, on prey resistance, and on evolutionary outcomes of both predators and prey. Furthermore, their impact on biofilms has been shown to reach beyond predation, as they are reported to reduce biofilm structures of non-prey cells (including Gram-positive bacteria). Here, we review the available literature on BALOs in biofilms, extending known aspects to potential mechanisms employed by the predators to grow in biofilms. Within that context, we discuss the potential ecological significance and potential future utilization of the predatory and enzymatic possibilities offered by BALOs in medical, agricultural and environmental applications.