Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insect

Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insect

Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism

BACKGROUND: Inorganic polyphosphate (poly P) occurs universally in all organisms from bacteria to man. It functions, for example, as a phosphate and energy store, and is involved in the activation and regulation of proteins. Despite its ubiquitous occurrence and important functions, it is unclear how poly P is synthesized or how poly P metabolism is regulated in higher eukaryotes. This work describes a systematic analysis of poly P levels in yeast knockout strains mutated in almost every non-essential gene. RESULTS: After three consecutive screens, 255 genes (almost 4% of the yeast genome) were found to be involved in the maintenance of normal poly P content. Many of these genes encoded proteins functioning in the cytoplasm, the vacuole or in transport and transcription. Besides reduced poly P content, many strains also exhibited reduced total phosphate content, showed altered ATP and glycogen levels and were disturbed in the secretion of acid phosphatase. CONCLUSION: Cellular energy and phosphate homeostasis is suggested to result from the equilibrium between poly P, ATP and free phosphate within the cell. Poly P serves as a buffer for both ATP and free phosphate levels and is, therefore, the least essential and consequently most variable component in this network. However, strains with reduced poly P levels are not only affected in their ATP and phosphate content, but also in other components that depend on ATP or free phosphate content, such as glycogen or secreted phosphatase activity

The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity

Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2

Clinical Aureobasidium Isolates Are More Fungicide Sensitive than Many Agricultural Isolates.

Fungicide applications in agriculture and medicine can promote the evolution of resistant, pathogenic fungi, which is a growing problem for disease management in both settings. Nonpathogenic mycobiota are also exposed to fungicides, may become tolerant, and could turn into agricultural or medical problems, for example, due to climate change or in immunocompromised individuals. However, quantitative data about fungicide sensitivity of environmental fungi is mostly lacking. Aureobasidium species are widely distributed and frequently isolated yeast-like fungi. One species, A. pullulans, is used as a biocontrol agent, but is also encountered in clinical samples, regularly. Here, we compared 16 clinical and 30 agricultural Aureobasidium isolates based on whole-genome data and by sensitivity testing with the 3 fungicides captan, cyprodinil, and difenoconazole. Our phylogenetic analyses determined that 7 of the 16 clinical isolates did not belong to the species A. pullulans. These isolates clustered with other Aureobasidium species, including A. melanogenum, a recently separated species that expresses virulence traits that are mostly lacking in A. pullulans. Interestingly, the clinical Aureobasidium isolates were significantly more fungicide sensitive than many isolates from agricultural samples, which implies selection for fungicide tolerance of non-target fungi in agricultural ecosystems. IMPORTANCE Environmental microbiota are regularly found in clinical samples and can cause disease, in particular, in immunocompromised individuals. Organisms of the genus Aureobasidium belonging to this group are highly abundant, and some species are even described as pathogens. Many A. pullulans isolates from agricultural samples are tolerant to different fungicides, and it seems inevitable that such strains will eventually appear in the clinics. Selection for fungicide tolerance would be particularly worrisome for species A. melanogenum, which is also found in the environment and exhibits virulence traits. Based on our observation and the strains tested here, clinical Aureobasidium isolates are still fungicide sensitive. We, therefore, suggest monitoring fungicide sensitivity in species, such as A. pullulans and A. melanogenum, and to consider the development of fungicide tolerance in the evaluation process of fungicides

Harnessing the Microbiomes of Suppressive Composts for Plant Protection: From Metagenomes to Beneficial Microorganisms and Reliable Diagnostics

Soil-borne diseases cause significant yield losses worldwide, are difficult to treat and often only limited options for disease management are available. It has long been known that compost amendments, which are routinely applied in organic and integrated farming as a part of good agricultural practice to close nutrient cycles, can convey a protective effect. Yet, the targeted use of composts against soil-borne diseases is hampered by the unpredictability of the efficacy. Several studies have identified and/or isolated beneficial microorganisms (i.e., bacteria, oomycetes, and fungi) from disease suppressive composts capable of suppressing pathogens (e.g., Pythium and Fusarium) in various crops (e.g., tomato, lettuce, and cucumber), and some of them have been developed into commercial products. Yet, there is growing evidence that synthetic or complex microbial consortia can be more effective in controlling diseases than single strains, but the underlying molecular mechanisms are poorly understood. Currently, a major bottleneck concerns the lack of functional assays to identify the most potent beneficial microorganisms and/or key microbial consortia from complex soil and compost microbiomes, which can harbor tens of thousands of species. This focused review describes microorganisms, which have been isolated from, amended to or found to be abundant in disease-suppressive composts and for which a beneficial effect has been documented. We point out opportunities to increasingly harness compost microbiomes for plant protection through an integrated systems approach that combines the power of functional assays to isolate biocontrol and plant growth promoting strains and further prioritize them, with functional genomics approaches that have been successfully applied in other fields of microbiome research. These include detailed metagenomics studies (i.e., amplicon and shotgun sequencing) to achieve a better understanding of the complex system compost and to identify members of taxa enriched in suppressive composts. Whole-genome sequencing and complete assembly of key isolates and their subsequent functional profiling can elucidate the mechanisms of action of biocontrol strains. Integrating the benefits of these approaches will bring the long-term goals of employing microorganisms for a sustainable control of plant pathogens and developing reliable diagnostic assays to assess the suppressiveness of composts within reach

MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

Abstract Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance. Graphical abstract MALDI biotyping is the method of choice for the repeated, rapid identification of a defined number of species

The MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for Colorimetric Determination of Fungal Cell Densities

The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting

Inorganic polyphosphate occurs in the cell wall of <it>Chlamydomonas reinhardtii </it>and accumulates during cytokinesis

<p>Abstract</p> <p>Background</p> <p>Inorganic polyphosphate (poly P), linear chains of phosphate residues linked by energy rich phosphoanhydride bonds, is found in every cell and organelle and is abundant in algae. Depending on its localization and concentration, poly P is involved in various biological functions. It serves, for example, as a phosphate store and buffer against alkali, is involved in energy metabolism and regulates the activity of enzymes. Bacteria defective in poly P synthesis are impaired in biofilm development, motility and pathogenicity. PolyP has also been found in fungal cell walls and bacterial envelopes, but has so far not been measured directly or stained specifically in the cell wall of any plant or alga.</p> <p>Results</p> <p>Here, we demonstrate the presence of poly P in the cell wall of <it>Chlamydomonas reinhardtii </it>by staining with specific poly P binding proteins. The specificity of the poly P signal was verified by various competition experiments, by staining with different poly P binding proteins and by correlation with biochemical quantification. Microscopical investigation at different time-points during growth revealed fluctuations of the poly P signal synchronous with the cell cycle: The poly P staining peaked during late cytokinesis and was independent of the high intracellular poly P content, which fluctuated only slightly during the cell cycle.</p> <p>Conclusion</p> <p>The presented staining method provides a specific and sensitive tool for the study of poly P in the extracellular matrices of algae and could be used to describe the dynamic behaviour of cell wall poly P during the cell cycle. We assume that cell wall poly P and intracellular poly P are regulated by distinct mechanisms and it is suggested that cell wall bound poly P might have important protective functions against toxic compounds or pathogens during cytokinesis, when cells are more vulnerable.</p