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A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola
(2019)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
A genetic toolbox for exploring the life style of the rock-inhabiting black fungus Knufia petricola
(2020)
After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish subaerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of microcolonial black fungi such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis (Nai et al. 2013, Fungal Genet Biol). We developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence of the strain A95 is paving the way for studying interactions of K. petricola with environmental stressors, material surfaces, soil matrices and phototrophic symbionts.
Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements.
In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern.
Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria.
At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy.
The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined.
The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion.
Despite the recognized excellence of virology and bioinformatics, these two communities have interacted surprisingly sporadically, aside from some pioneering work on HIV-1 and influenza. Bringing together the Expertise of bioinformaticians and virologists is crucial, since very specific but fundamental computational approaches are required for virus research, particularly in an era of big data. Collaboration between virologists and bioinformaticians is necessary to improve existing analytical tools, cloud-based systems, computational resources, data sharing approaches, new diagnostic tools, and bioinformatic training. Here, we highlight current progress and discuss potential avenues for future developments in this promising era of virus bioinformatics. We end by presenting an overview of current technologies, and by outlining some of the Major challenges and Advantages that bioinformatics will bring to the field of virology.
RNA virus populations are composed of highly diverse individuals that form a cloud of related sequences commonly referred to as a ‘quasispecies’1–3. This diversity arises as a consequence of low-fidelity genome replication4,5. By contrast, DNA Virus populations contain more uniform individuals with similar fitness6.
Genome diversity is often correlated with increased Fitness in RNA viruses, while DNA viruses are thought to require more faithful genome replication. During DNA replication, erroneously incorporated bases are removed by a 3′-5′ exonuclease, a highly conserved enzymatic function of replicative DNA but not RNA polymerases. This proofreading process enhances replication fidelity and ensures the genome integrity of DNA organisms, including large DNA viruses7. Here, we show that a herpesvirus can tolerate impaired exonucleolytic proofreading, resulting in DNA virus populations, which, as in RNA viruses8, are composed of highly diverse genotypes of variable individual fitness. This indicates that herpesvirus mutant diversity may compensate for individual Fitness loss. Notably, in vivo infection with diverse virus populations results in a marked increase in virulence compared to genetically homogenous parental virus. While we cannot exclude that the increase in virulence is caused by selection of and/or interactions between individual genotypes, our findings are consistent with quasispecies dynamics. Our results contrast with traditional views of DNA virus replication and evolution, and indicate that a substantial increase in population diversity can lead to higher virulence.
Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, which are hindered by slow growth, lack of sexual cycles and transformation difficulties, CRISPR/Cas9-based genetic tools were implemented (Erdmann et al. 2022, Front Fungal Biol). Now efficient targeted mutagenesis of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Still more progress on assigning functions to yet unknown genes can be expected if a forward genetics approach is available. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis of K. petricola. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase (AcTPase) and by this the regulatable transposition of the resistance cassette-containing Ds transposon is desired. However, endogenous promoters for nitrate assimilation and galactose catabolism - often used in fungi for regulatable gene expression - are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in oligotrophic fungi. Therefore, the metabolism-independent Tet-on system was combined with the AcTPase coding sequence and subsequently transformed into Ds-carrying K. petricola strains. In total, four auxotrophic Ac|Ds starter strains containing the Ds transposon at different position of ade2 or ura3 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA-lacking media resulted in prototrophic colonies (revertants) for some but not all Ac|Ds strains. Currently, the transposition events in the obtained revertants are studied to validate the procedure. First amplicon sequencing of excision sites revealed footprint patterns, proving the transposon jumped.
A similar secretome disturbance as a hallmark of non-pathogenic Botrytis cinerea ATMT-mutants?
(2019)
The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect
hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciensmediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted Proteins in the infection process. In the twelve non-pathogenic mutants, the Differentiation of infection cushions was also impaired, suggesting a link between the Penetration structures and the secretion of proteins involved in the virulence of the pathogen.
Traceable and reliable chemical element analysis of aerosols by X-ray spectrometry was investigated using aerosol samples from field campaigns which have been measured in the GIXRF-beamline at BESSY.
The reference-free XRF approach allows for a traceable analysis of the mass deposition.
Traceable quantification by means of XRF can be transfered to benchtop instrumentation used in the laboratory Chemical and dimensional analysis of deposited aerosol allows for a comprehensive analysis of aerosols, e.g. for toxicity assessment and determination of the source The folowing elements could be identified and quantified in the field samples: Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, W, Pb.
Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple
genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.
Free-living and (ecto)-mycorrhizal fungi enhance rock weathering. In their roles as mineral weathering agents and mutualistic partners of phototrophs, fungi supply primary producers like plants and phototrophic microorganisms with mineral-derived nutrients. The exact mechanisms behind fungus-induced mineral weathering processes are however not well understood. Progress can be achieved here by reproducible experimental simulations of the natural processes, using well-characterised model organisms and minerals.
In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes.
Three experimental systems, which generate long-term microbiological stability, were developed to study the impact of the WT and ΔKppks on olivine weathering: (1) batch and (2) mixed flow dissolution experiments, and (3) biofilm cultivation experiments. In addition, state-of-the-art analytical techniques were used to monitor changes in the growth medium, as well as of the mineral surface and biofilm-mineral interface.
Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis of the Mg, Si and Fe concentrations in the reacted growth medium was used to quantify olivine dissolution. In abiotic controls, Mg and Si dissolved congruently, while Fe precipitated. The measured olivine dissolution rates at pH 6 were two orders of magnitude lower than previously reported, but similar at acidic pH. X-ray photoelectron spectroscopy (XPS) analyses of the olivine surface confirmed the presence of Fe (oxyhydr)oxide precipitates. Transmission electron microscopy (TEM) imaging of an abiotically reacted polished olivine section from the long-term cultivation experiment showed the presence of an amorphous layer enriched in Fe. All these observations indicate that the precipitation of Fe (oxyhydr)oxides on the olivine surface inhibits olivine dissolution.
Both tested rock-inhabiting fungal strains affect Fe precipitation as well as olivine dissolution. Evaluation of the WT and ΔKppks revealed that the WT formed less biomass but could take up higher amounts of metals (e.g. Fe) and was more efficient in its attachment to olivine. The WT and ΔKppks enhanced olivine dissolution as demonstrated by higher Mg and Si concentration in the reacted growth medium. They furthermore prevented Fe precipitation by binding Fe and retaining it in solution, thereby allowing olivine dissolution to proceed. The WT cells that were attached to the olivine surface were particularly efficient at inhibiting Fe precipitation. By binding Fe directly at the olivine surface, the WT cells removed the inhibition of olivine dissolution almost completely. TEM analysis of polished olivine sections, colonised by a fungal biofilm for seven months, supported this hypothesis. After long-term fungus-olivine interaction, the Fe-enriched, amorphous layer did not develop, and the olivine surface was stronger etched compared to the abiotic control.
To study the effect of mutualism on mineral weathering, K. petricola was grown with the cyanobacterium, Nostoc punctiforme ATCC 29133. Both partners showed an enhanced growth and formed a stratified biofilm which attached more strongly to olivine. Nevertheless, the olivine dissolution rate of the fungus-cyanobacterium consortium was moderate.
Rock weathering simulation systems developed here are promising research instruments. The experimental conditions allow for the alteration of the studied mineral surface, while the clear definition of these conditions delivers a stable growth of microorganisms. The latter makes these systems universally applicable, especially in combination with integrative multidisciplinary analytics. Processes underlying environmental and biological effects on rock weathering, metal corrosion, plastic degradation, or the deterioration of any other substrate can be studied reproducibly and over a long period of time. The chemical and biological complexity of these simulation systems mimics natural rock weathering processes. The mineral dissolution rates generated in this study are therefore relevant to natural ecosystems.
The development of a test to evaluate the degradation of semi-volatile fuels as diesel by microorganisms is presented. This method is based on the principles described in the CEC-L-103 Standard procedure that is exclusively meant for testing the biodegradability of non-volatile lubricants. Therefore, significant modifications involve aseptic conditions for testing specific microorganisms and conducting the test in closed vessels avoiding evaporation losses, while fuel quantification using gas chromatography-flame ionization detection (GC-FID) is retained. It is suggested that the modified procedure should enable routine application for semi-volatile hydrocarbon-based fuels. GC-FID provides additionally valuable information on the alteration of fuel component patterns during biodegradation. The procedure was successfully tested using two bacteria (Pseudomonas aeruginosa and Sphingomonas sp.) and two yeasts (Moesziomyces sp. and Candida sp.) isolated from real diesel contamination cases. All tested microorganisms caused a significant degradation of diesel fuel achieving hydrocarbon degradation percentages ranging from 23% to 35%. Specific aspects on the test modification and prospects for further modification regarding targeted investigations in the field of fuel contamination by microorganisms are briefly discussed.
Quantitative chemical analysis of airborne particulate matter (PM) is vital for the understanding of health effects in indoor and outdoor environments, as well as for enforcing air quality regulations. Typically, airborne particles are sampled over long time periods on filters, followed by lab-based analysis, e.g., with inductively coupled plasma mass spectrometry (ICP-MS). Within the EURAMET EMPIR AEROMET project, cascade impactor aerosol sampling was combined for the first time with on-site total reflection X-ray fluorescence (TXRF) spectroscopy to develop a tool for quantifying particle element compositions within short time intervals and even on-site. This makes variations of aerosol chemistry observable with time resolution of only a few hours and with good size resolution in the PM10 range. A proof of principles of this methodological approach and the comparison to standard methods within the scope of a field campaign will be presented. Secondly, aerosol sampling and TXRF analysis seems suitable for the quantification of elements in indoor aerosols as well and may provide an important enhancement of existing methods for the analysis of organic species in aerosols (such as sampling and TD-GC/MS). As an example, the TXRF analysis of particles emitted from laser printers under controlled conditions in an environmental test chamber will be presented.
With the advent of widely accessible and cost-effective next-generation sequencing technologies, it has become increasingly feasible to study insect immunity on a deep genomic or transcriptomic level. Here we introduce a protocol that is aimed at exploiting transcriptomic data to study immunity in non-model insect organisms. We provide instructions for an entire workflow, starting with successfiil extraction of insect RNA through to bioinformatic guidelines for the effective analysis of mRNA sequencing data. The RNA extraction procedure is based on TRIzol Reagent and a spin-column clean-up Step. The bioinformatic pipeline is intended to help users identify immune genes from de novo transcriptome data and includes guidelines for conducting differential gene expression analyses on transcriptomic data. The immune gene prediction method is based on inferring protein homologs with HMMERand Blastp and talces Advantage ofthe ImmunoDB database, which is a valuable resource for research on insect immune-related genes and gene families. The differential gene expression analysis procedure utilizes the DESeq2 package as implemented in R. We hope this protocol will serve as a usefi.il resource for researchers aiming to study immunity in non-model insect species.
The application of fracture mechanics to the determination of the fatigue behavior of weldments is discussed with the focus on classic fatigue, i.e., the overall fatigue life and the fatigue strength in terms of an S-N curve and the endurance limit. The following issues are addressed: specific features of short fatigue crack propagation, an adequate initial crack size, multiple crack propagation and its statistical treatment as well as welding residual stresses. As an example, an approach of the authors is applied to the determination of FAT classes for a butt weld with varying weld toe geometry.
Algal biofilm façades are an alternative to traditional green façades which can help to improve biodiversity and air quality within cities. They present a low maintenance approach in which subaerial algae are grown directly on concrete substrates. The intrinsic bioreceptivity of the substrate is a critical factor in successful facade colonisation. Existing research has identified several environmental and material properties which influence concrete bioreceptivity, however a consensus has yet to be made on which properties are most influential and how the interaction between properties may promote algal biofilm growth under specific conditions.
Recent studies have shown that host-microbiota interactions can lead to dramatic changes in host phenotype especially behaviour. We investigate the causal drivers of microbe-associated shifts in host phenotype by examining how feeding behaviour is modulated by a bacterial infection in an omnivorous cockroach: Blatta orientalis. We conducted food-choice experiments after challenging hosts with the common entomopathogenic soil bacterium Pseudomonas entomophila to understand the impact of bacterial pathogens on host macronutrient preference. We find that immune challenge by this bacterium drives a sharp decline in carbohydrate intake and results in a relative increase in the ratio of protein to carbohydrate consumed. Additionally, infected cockroaches reduce their overall nutrient intake. We show for the first time that cockroach feeding behaviour is dynamically modulated by a pathogen. In contrast to studies on Spodoptera moths, this modulation does not impact any of the immune parameters we measured: it does not affect the abundance of immune related proteins in the hemolymph, hemolymph antimicrobial activity, or survival. This leads to the possibility that as long-lived omnivorous species B. orientalis may be better adapted to unpredictable variation in food availability and quality. An illness-induced anorexia-like response which is thought to assist hosts in limiting nutritional resources available to pathogens is therefore the most likely cause of the observed phenotype. Reduction of carbohydrate intake by sick individuals would be consistent with such an explanation.
In another cockroach species Blatella germanica we also investigate whether in addition to pathogens, host microbiota especially gut commensals are causally responsible for shifts in host behavioural phenotype and immune competence. To address this, we conduct food-choice and immunechallenge experiments in germ-free cockroaches and naïve cockroaches.
Bioinformatics meets virology: The European virus bioinformatics center's second annual meeting
(2018)
The Second Annual Meeting of the European Virus Bioinformatics Center (EVBC), held in Utrecht, Netherlands, focused on computational approaches in virology, with topics including (but not limited to) virus discovery, diagnostics, (meta-)genomics, modeling, epidemiology, molecular structure, evolution, and viral ecology. The goals of the Second Annual Meeting were threefold: (i) to bring together virologists and bioinformaticians from across the academic, industrial, professional, and training sectors to share best practice; (ii) to provide a meaningful and interactive scientific environment to promote discussion and collaboration between students, postdoctoral fellows, and both new and established investigators; (iii) to inspire and suggest new research directions and questions. Approximately 120 researchers from around the world attended the Second Annual Meeting of the EVBC this year, including 15 renowned international speakers. This report presents an overview of new developments and novel research findings that emerged during the meeting.
Materials that support natural biodiversity on their surfaces can compensate for human activities that have a negative impact on nature and thus contribute to a carbon-neutral and nature-positive world. Specifically designing bioreceptive materials which favor the growth of biofilms on their surface is an approach complementing conventional, macroscopic green façades. But what exactly characterizes a bioreceptive substrate and how do biofilm and substrate interact? How and why does a spontaneous colonization and the formation of biofilms take place? What are biofilms and how can they be established in a laboratory setting? How can this existing knowledge be transferred to the artificial stone concrete so that this material can be tuned to increase (or decrease) its bioreceptivity?
This review paper aims at summarizing the existing state of knowledge on bioreceptive concrete and pointing out inconsistencies and contradictions which can only be removed by more interdisciplinary research in the field.
Human-made systems, also called “build environment” or “technosphere”, sustain human comfort as well as our industrial activities. These systems have become particularly widespread since the Industrial Revolution, i.e., since the 17th century. At the same time, these technical systems – buildings, monuments, energy production, transformation and transmission, water purification and supply systems - serve as new habitats for living organisms.
Life is ubiquitously present on our planet since a very long time: the Earth is 4.54 billion years old and microbial communities have played a key role on our planet for 3.7 billion years. Once human-made system appeared, microorganisms became an integral part of all types of technosphere infrastructure as well.
Here we will illustrate biosphere-technosphere interactions using a specific example of the black fungi and their impact on the efficiency of solar (photovoltaic) panels. This expanding renewable infrastructure for electricity generation is growing on all continents - and create a specific, arid habitat for stress-tolerant black fungi.
Black fungi were once discovered in hot and cold natural deserts – and now belong to the persistent colonisers of human-made deserts of solar parks. This new niche is evolving an impressive biodiversity. So far more than 60 isolates of black fungi belonging to Arthoniomycetes, Eurotiomycetes and Dothideomycetes were obtained from solar panels in Europe and Americas. Here we will present the analysis of this emerging anthropogenic biodiversity.
Opportunities for future research in the field include quantification of the microbial load on technosphere surfaces – along with characterisation of the corresponding microbial diversity. The strategy of precise measurement and characterisation will enable us to reliably determine the beneficial and harmful functions that living microorganisms play in the functioning of energy-generating systems – and technosphere in general.
There are viral, fungal, bacterial and trypanosomal pathogens that negatively impact the individual and superorganismal health of the western honey bee. One fungal pathogen, Ascosphaera apis , affects larvae and causes the disease chalkbrood. A previous genome analysis of As. apis revealed that its genome encodes for RNA interference genes, similar to other fungi and eukaryotes. Here, we examined whether As. apis -targeting double-stranded RNA species could disrupt the germination of As. apis.
We observed that when spores were co-incubated with As. apis -targeting dsRNA, fewer spores were activated for germination, suggesting an uptake of exogenous genetic material at the very onset of germination and consequent damage to essential transcripts needed for germination. Overall, these results indicate that the causative agent of chalkbrood disease, As. apis , can be successfully targeted using an RNAi-based strategy.