Investigations on the occurrence of fungal root endophytes and an associated mycovirus in context with apple replant disease

Apple replant disease (ARD) negatively affects the production in nurseries and orchards worldwide. A biotic cause of the disease is most likely since soil disinfection can restore plant growth. Fungi have appeared to contribute to the complex of biotic factors, but up to now the actual cause of the disease remains unknown. Further, environmentally friendly and practically applicable mitigation strategies are missing. Fungal root endophytes were isolated in two central experiments of the ORDIAmur consortium. Dark septate endophytes (Leptodontidium spp.) were frequently isolated from apple roots. An abundant occurrence of Nectriaceae fungi (Dactylonectria torresensis and Ilyonectria robusta) was found in ARD roots. Reference sites displayed a different characteristic fungal community. In roots grown in irradiated soil, a reduction of the number of isolated fungi and a changed composition of the fungal community was found. To investigate the effect of fungal endophytes on apple plants a quick and soil-free bio test in Petri dishes was developed using perlite. Inoculated fungi isolated from ARD roots induced neutral (Plectosphaerella, Pleotrichocladium, and Zalerion) to negative (Cadophora, Calonectria, Dactylonectria, Ilyonectria, and Leptosphaeria) plant reactions. After re-isolation, most of the Nectriaceae isolates were confirmed as pathogens. Microscopic analyses of ARD-affected roots revealed necroses caused by an unknown fungus that forms cauliflower-like (CF) structures in diseased cortex cells. Two extraction methods, Harris Uni-Core punch and laser microdissection, were applied to further identify the fungus by PCR. Different Nectriaceae species were identified which form intracellular CF structures during the infection process. Both extraction methods can be used to identify also yet unculturable fungi from selected root areas of interest and help to avoid time-consuming isolations. Mycoviruses can influence their fungal hosts in several ways and may alter virulence (hyper- or hypovirulence) or toxin production. A hypovirulence-associated mycovirus has the potential to act as a sustainable control of fungal plant pathogens. Here, the sequence of a novel dsRNA virus originating from Dactylonectria torresensis is described, named Dactylonectria torresensis alternavirus 1 (DtAV1), which is a putative member of “Alternaviridae”. In this work, Nectriaceae were demonstrated to be involved in ARD. Further investigations of microorganism and plant interactions are needed to clarify the cause of the disease, which will then help to develop targeted control strategies.Bundes Ministerium für Bildung und Forschung/BonaRes (Modul A): ORDIAmur/031B0025/E

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease

First report of a chrysovirus infecting a member of the fungal genus Ilyonectria

The fungus Ilyonectria pseudodestructans belongs to the family Nectriaceae and was found to be part of the endophytic microbiome of apple trees (Malus x domestica, Borkh.) with apple replant disease (ARD). After dsRNA extraction, a mycoviral infection became evident. Here, we report the identification of a new virus, tentatively named “Ilyonectria pseudodestructans chrysovirus 1” (IpCV1), as the first member of the proposed new species “Alphachrysovirus ilyonectriae” within the genus Alphachrysovirus. This is the first report of a chrysovirus infecting a member of the fungal genus Ilyonectria. IpCV1 has a tripartite dsRNA genome with a total length of 8944 bp. The segments are 3439 bp, 2850 bp, and 2655 bp in length, and each dsRNA carries a single ORF. The encoded viral proteins are a 125.92-kDa RNA-dependent RNA polymerase, a 100.75-kDa coat protein, and one protein of unknown function with a predicted molecular mass of 93.04 kDa. The 5´ and 3´ UTRs are comparatively short and are 79 to 91 bp and 62 to 148 bp in length, respectively. This study provides the basis for further investigations of the impact of IpCV1 on its host and the etiology of ARD

Molecular characterization of two new alternaviruses identified in members of the fungal family Nectriaceae

Since the first report in 2009, at least ten additional viruses have been identified and assigned to the proposed virus family Alternaviridae. Here we report two new mycoviruses tentatively assigned to this family, both identified as members of the fungal family Nectriaceae, which were isolated from surface-disinfected apple roots (Malus x domestica, Borkh.) affected by apple replant disease (ARD). ARD is a highly complex, worldwide-occurring disease resulting from plant reactions to a disturbed (micro)-biome and leads to high economic losses every year. The first alternavirus characterized in this study was identified in a Dactylonectria torresensis isolate. The virus was tentatively named dactylonectria torresensis alternavirus 1 (DtAV1) as the first member of the proposed new species Alternavirus dactylonectriae. The second virus was identified in an isolate of Ilyonectria robusta and was tentatively named ilyonectria robusta alternavirus 1 (IrAV1) as the first member of the proposed new species Alternavirus ilyonectriae. Full genomic sequences of the viruses were determined and are presented. Further, we found hints for putative components of a methyl transferase machinery using in silico approaches. This putative protein domain is encoded by segment 2. However, this result only establishes the basis for subsequent studies in which the function must be confirmed experimentally in vitro. Thus, this is the first study where a function is predicted to all three genomic segments within the group of the alternaviruses. These findings provide further insights into the virome of ARD-associated fungi and are therefore another brick in the wall of understanding the complexity of the disease

Exploring the principles of self-healing polymers based on halogen bond interactions

In this study, novel self-healing polymers based on halogen bonds as reversible supramolecular crosslinking moieties are presented. The reversible crosslinking is facilitated by a polymer-bound bidentate halogen bond donor entity in combination with small molecule acceptor suberic acid. The binding strength of the crosslinking can be tuned via deprotonation of the diacid crosslinker. The material characteristics are investigated with several methods such as NMR and Raman spectroscopy, thermogravimetric analysis and differential scanning calorimetry as well as rheology. The tactile profile measurements have been utilized to monitor the scratch healing ability of the polymer networks revealing excellent healing efficiencies up to 99% within 2 h at a temperature of 100°C. Thus, the self-healing ability of halogen bond polymers could be quantified for the first time

Cannibalism stress response in <i>Bacillus subtilis</i>

When faced with carbon source limitation, the Gram-positive soil organism Bacillus subtilis initiates a survival strategy called sporulation, which leads to the formation ofhighly resistant endospores that allow B. subtilis to survive even long periods ofstarvation. In order to avoid commitment to this energy-demanding and irreversibleprocess, B. subtilis employs another strategy called cannibalism to delay sporulationas long as possible. Cannibalism involves the production and secretion of twocannibalism toxins, the sporulation delaying protein, SDP, and the sporulation killingfactor, SKF, which are able to lyse sensitive siblings. The lysed cells are thought tothen provide nutrients for the cannibals to slow down or even prevent them fromentering sporulation. In this study, we uncovered the role of the cell envelope stressresponse (CESR), especially the Bce-like antimicrobial peptide detoxification modules,in cannibalism stress response during stationary phase. SDP and SKF specificallyinduce Bce-like systems and some ECF σ factors in stationary phase cultures, but onlythe latter provide some degree of protection. A full Bce response is only triggered bymature toxins, but not by toxin precursors. Our study provides insights into the closerelationship between stationary phase survival and the CESR of B. subtilis.</i

The influence of inhibitory processes on affective theory of mind in young and old adults

The primary aim of this study was to examine the impact of an inhibition manipulation on the effect of age on theory of mind (ToM) in an ecologically valid, affective ToM task. Participants were 30 young and 30 old adults. The Cambridge Mindreading Face-Voice Battery was used to measure ToM; in addition, measures of fluid and crystallized intelligence were taken. Participants were subjected to three levels of inhibitory demand during ToM reasoning: emotional inhibition, non-emotional inhibition, and no inhibition. Old adults performed worse than young adults. The emotional and non-emotional inhibition conditions resulted in worse ToM performance compared to the no inhibition condition. There were no differences in the impact of the inhibition conditions on old and young adults. Regression analyses suggested that old adults’ crystallized intelligence was a significant predictor of ToM performance, whereas it did not predict young adults’ ToM performance. Results are discussed in terms of verbal ability as a possible compensatory mechanism in coping with verbal inhibitory load in ToM reasoning

Inhibition of T-antigen expression promoting glycogen synthase kinase 3 impairs merkel cell carcinoma cell growth

International audienceMerkel cell carcinoma is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). Since proliferation of MCPyV-positive MCC tumor cells strictly depends on expression of the virus-encoded T antigens (TA), these proteins theoretically represent ideal targets for different kinds of therapeutic approaches. Here we developed a cell-based assay to identify compounds which specifically inhibit growth of MCC cells by repressing TA expression. Applying this technique we screened a kinase inhibitor library and identified six compounds targeting glycogen synthase kinase 3 (GSK3) such as CHIR99021 as suppressors of TA transcription in MCC cells. Involvement of GSK3 alpha and -beta in the regulation of TA-expression was confirmed by combining GSK3A knockout with inducible GSK3B shRNA knockdown since double knockouts could not be generated. Finally, we demonstrate that CHIR99021 exhibits in vivo antitumor activity in an MCC xenograft mouse model suggesting GSK3 inhibitors as potential therapeutics for the treatment of MCC in the future

Impact of Endothelial Progenitor Cells in the Vascularization of Osteogenic Scaffolds

The microvascular endothelial network plays an important role in osteogenesis, bone regeneration and bone tissue engineering. Endothelial progenitor cells (EPCs) display a high angiogenic and vasculogenic potential. The endothelialization of scaffolds with endothelial progenitor cells supports vascularization and tissue formation. In addition, EPCs enhance the osteogenic differentiation and bone formation of mesenchymal stem cells (MSCs). This study aimed to investigate the impact of EPCs on vascularization and bone formation of a hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP)–fibrin scaffold. Three groups were designed: a scaffold-only group (A), a scaffold and EPC group (B), and a scaffold and EPC/MSC group (C). The HA/ß–TCP–fibrin scaffolds were placed in a porous titanium chamber permitting extrinsic vascularization from the surrounding tissue. Additionally, intrinsic vascularization was achieved by means of an arteriovenous loop (AV loop). After 12 weeks, the specimens were explanted and investigated by histology and CT. We were able to prove a strong scaffold vascularization in all groups. No differences regarding the vessel number and density were detected between the groups. Moreover, we were able to prove bone formation in the coimplantation group. Taken together, the AV loop is a powerful tool for vascularization which is independent from scaffold cellularization with endothelial progenitor cells’ prior implantation