Antibody-guided in vivo imaging of Aspergillus fumigatus lung infections during anti-fungal azole treatment

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: Due to their large size, the raw imaging data that support the findings of this study are directly available from the corresponding authors upon reasonable request. Derived data have been compiled in the Source Data file provided with this paper. Any remaining data supporting the findings from this study are available from the corresponding author upon reasonable request.Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA, and its response to azole treatment, is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo and 3D light sheet fluorescence microscopy ex vivo to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.Ministry of Culture and Science of North Rhine-WestphaliaGoverning Mayor of Berlin including Science and ResearchFederal Ministry of Education and ResearchEuropean Union FP7Werner Siemens Foundatio

Improving Management and Understanding of Major Diseases of Sugar Beet

Sugar beet is a sugar-yielding crop, that contributes 25% of the global sucrose production. Economic production of sugar beet is hampered by Cercospora leaf spot (CLS) (Cercospora beticola), Rhizoctonia crown and root rot (RCRR) (Rhizoctonia solani), Sclerotinia root rot (SRR) (Sclerotinia sclerotiorum), and Rhizopus root rot (Rhizopus arrhizus) diseases. These diseases can reduce yield by 15 to 40%. On CLS, buildup of fungicide-resistance strains is a major issue due to poor implementation of fungicides and understanding of disease development at early stages. The identification of germplasm resistant to RCRR disease is hindered by the lack of effective inoculation methods. Identification of SRR and RRR pathogens is crucial for their proper management. The objectives of this research were 1. to evaluate the role of adjuvants in improving the efficacy of fungicides on CLS, 2. to characterize the infection process during early stages of infection by C. beticola, 3. to identify an effective inoculation method for RCRR, and 4. to identify and characterize the causal organisms of SSR and RRR. The value of adjuvants was evaluated in greenhouse and field conditions. Application of fungicides with or without adjuvants before disease onset reduced disease severity of CLS in greenhouse condition. In field conditions, additions of adjuvants did not improve the effectiveness of fungicides and few of them negatively impacted root yield. The initial stage of infection on CLS susceptible and resistant sugar beet variety were compared using confocal microscopy. C. beticola biomass accumulation, percent leaf cell death and disease severity were all significantly greater in the susceptible variety compared to the resistant variety (P<0.05). R. solani inoculated on the crown and roots were compared in a replicated trial in greenhouse conditions. The root inoculation method provided a more consistent disease rating of the sugar beet variety in the greenhouse for screening of RCRR cultivars in a resistance breeding program. Based on morphological and molecular techniques, causal organisms of SRR and RRR were characterized and was found to be pathogenic to sugar beet varieties tested in-vitro and in the greenhouse conditions

Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes

<p>Abstract</p> <p>Background</p> <p>The hemibiotrophic fungus <it>Moniliophthora perniciosa </it>is the causal agent of Witches' broom, a disease of <it>Theobroma cacao</it>. The pathogen life cycle ends with the production of basidiocarps in dead tissues of the infected host. This structure generates millions of basidiospores that reinfect young tissues of the same or other plants. A deeper understanding of the mechanisms underlying the sexual phase of this fungus may help develop chemical, biological or genetic strategies to control the disease.</p> <p>Results</p> <p>Mycelium was morphologically analyzed prior to emergence of basidiomata by stereomicroscopy, light microscopy and scanning electron microscopy. The morphological changes in the mycelium before fructification show a pattern similar to other members of the order <it>Agaricales</it>. Changes and appearance of hyphae forming a surface layer by fusion were correlated with primordia emergence. The stages of hyphal nodules, aggregation, initial primordium and differentiated primordium were detected. The morphological analysis also allowed conclusions on morphogenetic aspects. To analyze the genes involved in basidiomata development, the expression of some selected EST genes from a non-normalized cDNA library, representative of the fruiting stage <it>of M. perniciosa</it>, was evaluated. A macroarray analysis was performed with 192 selected clones and hybridized with two distinct RNA pools extracted from mycelium in different phases of basidiomata formation. This analysis showed two groups of up and down-regulated genes in primordial phases of mycelia. Hydrophobin coding, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase genes were grouped among the up-regulated. In the down-regulated group relevant genes clustered coding calmodulin, lanosterol 14 alpha demethylase and PIM1. In addition, 12 genes with more detailed expression profiles were analyzed by RT-qPCR. One aegerolysin gene had a peak of expression in mycelium with primordia and a second in basidiomata, confirming their distinctiveness. The number of transcripts of the gene for plerototolysin B increased in reddish-pink mycelium and indicated an activation of the initial basidiomata production even at this culturing stage. Expression of the glucose transporter gene increased in mycelium after the stress, coinciding with a decrease of adenylate cyclase gene transcription. This indicated that nutrient uptake can be an important signal to trigger fruiting in this fungus.</p> <p>Conclusion</p> <p>The identification of genes with increased expression in this phase of the life cycle of <it>M. perniciosa </it>opens up new possibilities of controlling fungus spread as well as of genetic studies of biological processes that lead to basidiomycete fruiting. This is the first comparative morphologic study of the early development both <it>in vivo </it>and <it>in vitro </it>of <it>M. perniciosa </it>basidiomata and the first description of genes expressed at this stage of the fungal life cycle.</p

‘Symptomless’ infection by Botrytis cinerea

The study was carried out to clarify the nature of symptomless infection by Botrytis cinerea and to what extent it differs from aggressive necrotic infection in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless plants were produced by dry spore inoculation in plants growing in controlled environmental conditions or in glasshouses. Plating out of surface-disinfected and non-surface-disinfected samples of inoculated, apparently healthy, plants on selective medium revealed that the fungus was spreading from the initial inoculation site to newly developing plant organs both internally and externally. Similar findings were obtained in microscope experiments in which host plants were inoculated with GFP labelled B. cinerea and symptomless spreading was monitored under confocal laser scanning microscope. Spore germination on leaf surface was followed by development of sub-cuticular vesicles and plant cell damage in the infected epidermal cell and a few nearby cells. Sparsely branched long hyphae arose from the vesicles and spread on the leaf surface; spread was mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells. In the late symptomless phase, mycelium arising from single vesicles formed several mycelial networks on leaves. Experiments were carried out to compare the extent of gene expression in symptomless and necrotic infections, using RT-qPCR. Expression of selected genes was quantified in tissue samples based on the amount of mRNA of the respective genes found. In both host species, the mRNA concentration of signalling genes bcg1, bmp1 and calcineurin, and the pathogenicity genes bcsod1 and bcpg1 were similar to or slightly greater in symptomless samples than in necrotic samples. The mRNA of the signalling gene bac and pathogenicity genes bcbot1 and bcnep1, were not detected or detected in lower abundance than in necrosis. In lettuce, the leaves developing distant from the site of inoculation showed similar results to A. thaliana, but in healthy leaves close to the site of inoculation mRNA concentrations of bac and bcnep1 were similar to necrotic samples. Thus, in both host species, the fungus grew along with the plant and moved to newly growing plant parts without producing symptoms; during this growth some pathogenicity genes were less expressed than in necrotic infection

Epidemiology of Fusarium oxysporum f. sp. cucumerinum in greenhouse cucumbers

Fusarium Bosporus f. sp. cucumerinum is the fungal pathogen responsible for Fusarium vascular wilt of cucumber. The options for managing Fusarium wilt in greenhouse cucumbers are limited by our poor understanding of the modes of survival and dissemination of the pathogen. Aerial dissemination of the pathogen was investigated following the development of a highly specific and sensitive quantitative real-time PCR assay that reliably detected as few as 100 Fusarium oxysporum f. sp. cucumerinum genome copies in environmental matrices. Numbers of both macroconidia and microconidia were variable in greenhouse air samples at different times of day. A potential relationship between fluctuation in relative humidity and spore number was found. While this shows that the pathogen can be aerially disseminated, airborne spores were unable to infect wound stem sites. These results suggest that aerial inoculum propagates and disseminates the pathogen, but that infection is primarily through the root after aerial spores are deposited on the soil surface. Aerial dissemination was also found to occur through insect vectors. Sciarid and shore flies could carry between 1 × 102 and 1 × 106 pathogen genome copies/individual. Experimentally, sciarid and shore flies acquired F. oxysporum f. sp. cucumerinum following exposures to agar cultures of the pathogen of up to 94 hours and were found to transfer the pathogen, resulting in disease expression in a glasshouse transmission trial