Analysis of spatio-temporal fungal growth dynamics under different environmental conditions

Traditionally, fungal growth dynamics were assessed manually, limiting the research to a few environmental conditions and/or fungal species. Fortunately, more automated ways of measurement are gaining momentum due to the availability of cheap imaging and processing equipment and the development of dedicated image analysis algorithms. In this paper, we use image analysis to assess the impact of environmental conditions on the growth dynamics of two economically important fungal species, Coniophora puteana and Rhizoctonia solani. Sixteen environmental conditions combining four temperatures (15, 20, 25 and 30°C) and four relative humidity (RH) conditions (65, 70, 75 and 80% RH) were tested. Fungal growth characteristics were extracted from images of the growing fungi, taken at regular points in time. Advanced time series analysis was applied to quantitatively compare the effect of the environmental conditions on these growth characteristics. The evolution of the mycelial area and the number of tips over time resulted in typical sigmoidal growth curves. Other growth characteristics such as the mean hyphal segment length did not vary significantly over time. Temperature and RH usually had a combined effect on the growth dynamics of the mycelial area and the number of tips. When defining optimal growth conditions for a fungus, it is therefore of primordial importance that the effect of temperature and RH is assessed simultaneously. At the most extreme conditions we tested, the mycelium most probably experienced water stress when developing over the inert Petri dish surface. An RH of 65% (independent of temperature) for C. puteana and a temperature of 30 °C (independent of RH) for both C. puteana and R. solani therefore always resulted in limited fungal growth, while the optimal growing conditions were at 20 °C and 75% RH and at 25°C and 80% RH for R. solani and at 20°C and 75% RH for C. puteana. The method applied in this study offers an updated and broader alternative to classical and narrowly focused studies on fungal growth dynamics, and is well suited to efficiently assess the effect of environmental conditions on fungal growth

Factors affecting growth of the arbuscular mycorrhizal fungal mycelium

Arbuscular mycorrhizal relationsliips link plant root systems and zygomycetous fungi from the family Glomales in a symbiotic association which provides a carbon source to the fungus and benefits to the plant in terms of nutrient and water acquisition, and disease resistance. Root system colonisation is characterised by the formation of intracellular hyphae and internal fungal structures, the arbuscles and vesicles. Colonisation occurs after root penetration by fimgal hyphae originating from either spores or extra-radical hyphae. Tire extra-radical hyphae, which form the fungal mycelium in the soil and have a primary role in host plant nutrition, are a largely neglected feature of the symbiosis due to the difficulties involved in their study.In the present work, methodologies were developed to allow in vitro studies of extra-radical hyphae and thus circumvent problems associated with the inaccessibility and opacity of the soil environment. Root pieces colonised by the arbuscular mycorrhizal (AM) fungus Glomus etunicatum Becker & Gerdemann (S329, INVAM, USA) were inoculated onto dialysis membrane overlying transparent high purity agarose gel. This restricted the hyphae to a two-dimensional growth form which facilitated observation using microscopy and image analysis techniques, and enabled morphological measurements to be taken without the added complexity of three dimensional growth.The two major influences 011 AM fimgal growth are those of soil and of plant origin. The present work studied the effects of plant related factors on AM fungal hyphae. Host and non-host plant factors, and derivatives of these, were found to influence hyphal growth as characterised both by length measurements and morphological parameters. Host root exudates reduced hyphal growth, apparently as a result of changes in the overall distribution of hyphae within the mycelium, as measured by fractal dimension (FD). Non-host exudates and plant flavonoid compounds also decreased hyphal growth, but this result could not be attributed to changes in either branching or mycelial organisation. Root extracts of host and non­host plants did not significantly affect hyphal length, but did affect mycelial morphology. Although host root extracts appeared to have no effect 011 branching, they did alter hyphal distribution within the mycelium, as evidenced by an increase in FD. Non-host root extracts increased both branching and FD. Colonised host plant root extracts increased both hyp ha I length and branching, but had 110 significant effect on hyphal distribution within the mycelium.Results are discussed in the context of previous work earned out on germ tube hyphae of AM fungi from spore inoculum. Observations of extra-radical hyphae showed differential responses when compared with germ tube hyphae more commonly studied by previous authors. This indicates that different phases of the fungal life cycle respond in different ways to similar environmental influences

Mathematical and Experimental Investigation of Yeast Colony Development – A Model System for the Growth of Filamentous Fungi in Heterogeneous Environments

In the presented study, dimorphic yeasts were applied as model organisms to study the growth of fungal mycelia. When environmental conditions are chosen appropriately, yeast colonies are built up of well separated individual cells. Thus, in contrast to fungal mycelia the translocation of nutrients and information within the colony can be neglected. The study focuses on the question of how the growth behaviour of a population of single cells is regulated, and which differences can be expected when nutrient translocation actually occurs. To answer this question, at first, an effective method for the highly resolved estimation of biomass distributions inside the colonies was developed. This method facilitates a dynamic non-invasive monitoring of colony development. Furthermore, mathematical models were established which describe the development of the colonies based on the behaviour of discrete individual cells. Growth simulations allow a quantitative prediction, and, thereby, an in silico testing of hypothetic regulatory mechanisms. The growth behaviour of yeast colonies was investigated applying the model organisms Candida boidinii and Yarrowia lipolytica. The yeasts were cultivated on solid agar substrates at various degrees of carbon and nitrogen limitation, respectively. The highest gain of understanding was achieved for the growth of both yeasts on glucose as the limiting carbon source: Investigations showed that mycelial yeast colonies adapt to declining nutrient concentrations by decreasing the cell density in their mycelium while the growth rate of the colony diameter remains constant. Under glucose limitation, the yeast C. boidinii grows diffusion-limited, i.e., the growth of the population is controlled by the amount of nutrient that diffuses towards the colony. The cessation of growth coincides with the depletion of the primary nutrient source glucose from the growth substrate. In contrast to these findings, it was shown that Y. lipolytica colonies continue to extend even after the complete consumption of glucose. In the absence of the primary nutrient source, the yeast assimilates biomass from the inner colony regions to facilitate the growth of the population. The suggested mechanism of coupled extension and decay processes was verified by a number of experiments. However, the mechanism which facilitates the transport of decay products to the growing colony boundary, i.e., the actual nature of the decay process, remains unclear. Mathematical simulations show that a continuous colony extension on the decay products of dying cells cannot be explained by the assumption that colonies are built up of uncoordinatedly growing single cells. Therefore, a hypothesis for the growth of Y. lipolytica colonies was derived which suggests that these populations are built up of tube-like hyphal cells. Accordingly, the measured drop of biomass density in the inner colony areas is the consequence of a cytoplasm transport towards the growing edge of the mycelium where it is assimilated as a secondary nutrient resource in the absence of glucose. It has to be emphasized that this hypothesis also provides a mechanistic explanation for the vacuolisation of hyphae in mycelia of higher fungi.In der vorgestellten Arbeit wurden dimorphe Hefen als Modellorganismen für die Untersuchung des Wachstums von Pilzmyzelien eingesetzt. Bei geeigneter Wahl der Umgebungsbedingungen sind Hefekolonien aus Einzelzellen aufgebaut, wodurch im Gegensatz zu Myzelien höherer Pilze der Transport von Nährstoffen und Informationen innerhalb der Kolonie vernachlässigt werden kann. Im Mittelpunkt der Untersuchungen stand die Frage, wie das Wachstumsverhalten einer Population individueller Zellen reguliert ist, bzw. welche Unterschiede sich ergeben, wenn ein Nährstofftransport tatsächlich stattfindet. Um diese Fragestellungen bearbeiten zu können, wurde zunächst eine effektive Methode zur hoch ortsaufgelösten Bestimmung der Biomasseverteilung innerhalb der Kolonien entwickelt. Diese Methode ermöglicht ein dynamisches nichtinvasives Monitoring der Entwicklung einer Kolonie. Weiterhin wurden mathematische Modelle entwickelt, die das Wachstumsverhalteeiner Population auf der Grundlage des Verhaltens von diskreten Einzelzellen beschreibt. Die Wachstumssimulationen erlauben quantitative Vorhersagen und damit ein in silico Testen der Auswirkungen von hypothetischen Regulationsmechanismen. Das Wachstumsverhalten von Hefekolonien wurde anhand der Modellorganismen Candida boidinii und Yarrowia lipolytica untersucht. Die Hefen wurden auf festen Agar-Nährböden bei verschieden starker Kohlenstoff- und Stickstofflimitation kultviert. Der größte Erkenntnisgewinn wurde dabei für das Wachstum beider Hefen auf Glukose als limitierender Kohlenstoffquelle erzielt: Die Untersuchungen ergaben, dass myzelartig wachsende Hefekolonien bei sinkenden Nährstoffkonzentrationen eine geringere Zelldichte aber einen konstante Wachstumsgeschwindigkeit des Koloniedurchmessers aufweisen. Die Hefe C. boidinii wächst unter Glukoselimitation diffusionslimitiert, d.h. das Wachstum der Population wird durch die Menge der zur Kolonie diffundierenden Nährstoffe bestimmt. Der Abbruch des Koloniewachstums fällt mit dem Verbrauch der primären Nähstoffquelle Glukose zusammen. Im Gegensatz dazu konnte für das Wachstum von Y. lipolytica gezeigt werden, dass sich die Kolonien auch nach dem vollständigen Verbrauch von Glukose weiter ausdehnen. Im Abwesenheit der primären Nährstoffquelle nutzt die Hefe Zerfallsprodukte eigener Zellmasse aus dem Inneren der Kolonie als Nährstoff, um das weitere Wachstum der Population zu gewährleisten. Während der vorgeschlagene gekoppelte Ausdehnungs- und Zerfallprozess durch eine Reihe von Versuchen experimentell abgesichert wurde, bleibt der Mechanismus des Transports der Zerfallsprodukte zum Kolonierand, bzw. die eigentliche Natur des Zerfallsprozesses unklar. Simulationsrechnungen ergaben, dass eine kontinuierliche Ausdehnung der Kolonie auf Zellzerfallsprodukten sterbender Zellen nicht durch die Annahme erklärt werden kann, dass die Kolonien aus unkoordiniert wachsenden Einzelzellen aufgebaut sind. Aus diesem Grunde wurde für das Wachstum von Y. lipolytica die Hypothese abgeleitet, dass das Myzelium dieser Hefe aus schlauchartigen Hyphenzellen aufgebaut ist. Der gemessene Abfall der Biomassekonzentration im Kolonieinneren ist demnach die Konsequenz des Transports von Zytoplasma hin zum wachsenden Kolonierand, wo es in Abwesenheit von Glukose als sekundäre interne Nährstoffquelle assimiliert wird. Es ist zu beachten, dass diese Hypothese auch eine mechanistische Erklärung für die Ursachen der Vakuolisierung in Myzelien höherer filamentöser Pilze gibt

Use of the fungal pathogen Hirsutella cryptosclerotium sp. nov. for the biocontrol of Rastrococcus invadens (Pseudococcidae)

Imperial Users onl

Fungal susceptibility of bio-based building materials

Bio-based building materials are made from renewable resources and produced with considerably less energy and associated carbon emissions than many traditional building materials. This makes them essential building elements in the much needed transition towards a more sustainable building industry. Many bio-based building materials are (to some extent) biodegradable, an excellent quality at the end of a material’s service life as it solves waste issues, but a less desirable feature during use. When an organic material is exposed to favourable moisture and temperature conditions as well as to degrading organisms, its functional and aesthetic service life can decrease. The risk of fungal decay depends on the environmental conditions and the material resistance. The purpose of this PhD thesis was to determine how different material characteristics affect the decay risk of bio-based building materials. A test method was developed to assess the importance of material chemistry as compared to material structure and moisture dynamics on durability. State-of-the-art methods (LFNMR, IR, X-ray CT) were applied to better understand the moisture dynamics of wood-based panels and bio-based insulation materials and a method was developed to assess the influence of material structure on decay progress with X-ray CT. As material moisture dynamics and structure have a (major) influence on durability and decay risk, there is a great opportunity to tailor bio-based building materials for diversified end uses and moisture conditions, ensuring an increased service life

Working with Mycorrhizas in Forestry and Agriculture

Crop Production/Industries,

Terpene arms race in the Seiridium cardinale - Cupressus sempervirens pathosystem

The canker-causing fungus Seiridium cardinale is the major threat to Cupressus sempervirens worldwide. We investigated the production of terpenes by canker-resistant and susceptible cypresses inoculated with S. cardinale, the effect of these terpenes on fungal growth, and the defensive biotransformation of the terpenes conducted by the fungus. All infected trees produced de novo terpenes and strongly induced terpenic responses, but the responses were stronger in the cankerresistant than the susceptible trees. In vitro tests for the inhibition of fungal growth indicated that the terpene concentrations of resistant trees were more inhibitory than those of susceptible trees. The highly induced and de novo terpenes exhibited substantial inhibition (more than a fungicide reference) and had a high concentration-dependent inhibition, whereas the most abundant terpenes had a low concentration-dependent inhibition. S. cardinale biotransformed three terpenes and was capable of detoxifying them even outside the fungal mycelium, in its immediate surrounding environment. Our results thus indicated that terpenes were key defences efficiently used by C. sempervirens, but also that S. cardinale is ready for the battle

Quantification of superficial growth and the pigmentation of filamentous fungi by effect of oscillating magnetic field

This work was to quantify the superficial growth and the pigmentation of filamentous fungi colonies by effect of an extremely low frequency oscillating magnetic field (OMF-ELF) depending on nutritional status of these microorganisms in two culture media. OMF-ELF of 2 mT by 60 Hz/200 V during 2 h (optimization of an experimental design) was applied at two filamentous fungi strains isolated from indoor environments: Aspergillus niger (hyaline mycelium seen under the optic microscope) and Cladosporium cladosporioides (pigmented mycelium), this latter strain was used as a negative control. The strains were inoculated in Petri dishes with Malt Extract Agar (MEA) and Czapek Dox Agar (CDA), incubated at 30ºC and photographs were taken every 4 hours for 10 days. The area (superficial growth) and color intensity (pigmentation) of these colonies were evaluated by digital images processing (DIP) using MatLab®. The stimulating effect of OMF-ELF on the cellular metabolism of both fungal strains was demonstrated, with an increase of the superficial growth and a greater variation in pigmentation (indicative of sporulation by conidiogenesis) in CDA until reaching values of spores similar to when there are enough nutrients (MEA). The predictive mathematical model obtained by optimization of the experimental design to predict the growth of Aspergillus niger colony was validated with the algorithm implemented for the DIP. It was concluded that the OMF-ELF stimulated the metabolism of the studied fungal strains independently of the nutrients in the culture media and that the DIP is a precise technique to quantify the macroscopic phenomena that occur due to magnetobiological effects at the cellular level

Biological control of Botrytis spp. by Ulocladium atrum : an ecological analysis

Plant pathogenic fungi from the genus Botrytis cause economically important diseases in a wide range of crops during the production phase as well as post harvest phase. Control is based on the frequent use of fungicides. Alternative approaches for control are studied because of the development of fungicide resistance in the pathogen and environmental concerns.The fungal saprophytic antagonist Ulocladium atrum is an effective biological control agent of B. cinerea in cyclamen. U. atrum may also be effective against B. cinerea in other crops and against other Botrytis spp. The biocontrol effect of U. atrum is based on a competitive interaction with B. cinerea in plant tissue. An immuno-histological technique was developed to visualize the interaction between the mycelia of Botrytis spp. and U. atrum in plant tissue and quantify colonization levels. Nutrient competition was found to be the dominant antagonistic mechanism between the species and pre-emptive colonization of plant tissue by U. atrum was identified as the most promising biocontrol strategy. Necrotic plant tissue is the primary target for U. atrum applications, since this is the (only) niche available to both Botrytis spp. and U. atrum .Pre-emptive colonization of naturally necrotic tissue by U. atrum was highly effective against B. cinerea in cyclamen, whereas U. atrum applications against B. elliptica in lily were ineffective. A comparison of both pathosystems suggests three criteria that govern the potential success of U. atrum as a biocontrol agent. 1) There must be a niche in which U. atrum competes with the pathogen; 2) The competitive ability of U. atrum in this niche has to be sufficient to allow for competitive exclusion of the pathogen. 3) The niche from which the pathogen can be excluded has to be essential for disease development. Colonies of U. atrum have lower radial growth rates than those of the Botrytis spp. studied. In practical biocontrol, this competitive disadvantage of the antagonist can be overcome by applying a high density of its conidia in a uniform distribution on the target tissue.</p

Development of Novel Image Analysis Methods for the Morphological Quantification of Filamentous Fungi

Mycelial morphology is a critically important process property in fermentations of lamentous micro-organisms, as particular phenotypes are associated with maximum productivity. The design of systems capable of rapidly and accurately characterising morphology within a given process represents a signi cant challenge to biotechnologists, as the complex phenotypes that are manifested are often not easily quanti ed. A system has been developed for high-resolution characterisation of lamentous fungal growth, using membrane immobilization and fully-automatic imageprocessing software. The system has been used to quantify the early-stage hyphal di erentiation of Aspergillus oryzae on solid substrates, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 16 h, the number of tips was found to increase exponentially. The speci c growth rate, maximum hyphal tip extension rate and speci c branching frequency of a population of hyphae were calculated as approximately 0.27 h-1, 27 um tip-1 h-1 and 2:3x 10- 3 tips um-1 h-1 respectively. The robustness of the image-analysis system was veri ed by testing the e ect of small variations in the input parameters. Subsequent experimentation focussed on investigating the morphological development of A. oryzae in submerged culture and the associated in uence on -amylase production. The temporal variation in pellet structure and -amylase production over time was quanti ed and the potential for the use of membrane-immobilisation in submerged culture was examined. Variation in carbon source type had little morphological impact, although increasing starch concentration caused a shift from a pelleted form to dispersed, `pulp-like\u27 growth. Increasing inoculum concentration was found to result in a decrease in mean pellet diameter and an increase in -amylase production. The supplementation of fermentation media with non-ionic detergents caused a signi cant increase in -amylase production (up to 149%), but this increase did not seem to be related to observed morphological variation. Recently, fractal geometry has been employed in the study of lamentous microbes, but a clear link between fractal dimension and branching behaviour has not been demonstrated. This thesis presents an alternative means of enumerating the fractal dimension of fungal mycelial structures, by generating a `fractal signal\u27 from an object boundary. In the analysis of a population of A. oryzae mycelia, both fractal dimension and hyphal growth unit were found to increase together over time, while cultivating populations of Penicillium chrysogenum and A. oryzae mycelia under a variety of di erent conditions revealed a strong correlation between fractal dimension and hyphal growth unit. The technique has the potential to be adapted and applied to any morphological form that may be encountered in a fermentation process, providing a universally-applicable parameter for more complete data acquisition