Germination of Penicillium paneum Conidia Is Regulated by 1-Octen-3-ol, a Volatile Self-Inhibitor

Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25degreesC and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development

Germination inhibitors of fungal spores: identification and mode of action

Fungi can be found in a wide variety of environments, such as seeds, plants, soil, water, insects, and food products. Fungi and their toxic metabolites cause losses of food products, and diseases in plants and animals, and may have adverse effects on human health. A crucial step in fungalcolonisationand infection is the germination process, subsequently resulting inmycelialgrowth. The aim of this thesis was to study fungal germination and the effects of antifungal compounds on this process usingPenicilliumpaneum conidia andFusariumculmorummacroconidiaas model organisms.An antifungal compound produced by Bacillussubtilis YM 10-20 was identified and characterized as aniturin-like compound belonging to theiturinfamily. This compound inhibited fungal germination and growth ofPenicilliumpaneum conidia. Fluorescence probes in combination with flowcytometryand scanning electron microscopy were applied to assess the action of the antifungal compound against spores. Destruction and morphological changes of conidia in the presence of the inhibitor were observed. Theiturin-like compoundpermeabilisedfungal spore membranes and blocked germination. Another compound was identified and characterized fromdense conidia suspensions ofPenicilliumpaneum.A volatile compound(s) were produced under high-density conditions by the conidia themselves. It inhibitedmycelialgrowth of different species of fungi belonging to a variety of genera, suggesting a broad action range.1-Octen-3-ol was identified as a volatile self-inhibitor of spore germination.This compoundinfluenced different developmental processes during the P.paneum life cycle namelymicrocycleconidiationand spore germination.The compound henceforth acts as a fungal hormone in the life cycle of the fungus. The mode of action of 1-octen-3-ol was investigated. It targets the membrane affecting intracellular pH, respiration, and protein synthesis.The sequence of events in the germination process ofmulticompartmentFusariumculmorummacroconidiaand the effects of antifungal compounds on this process was investigated by measuring the intracellular pH (pH in) employing fluorescence ratio imaging microscopy (FRIM). During germ tube formation, clear differentiation between the compartments of themacroconidiumwas observed. Germ tubes develop preferably from apical cells and with low frequency from middle compartments. ThepH in varied among different compartments and during different stages of germination.Addition ofantifungals,nystatinandnonanoicacid during germination, affectspH inof the conidia and their differentiation.The work described in this thesis highlights the sequence of events in the germination process of fungal spores, notably conidia from Penicilliumpaneum andmulticompartmentmacroconidia fromFusariumculmorum and the effect of antifungal compounds. Detailed insight in the fungal germination process and action of antifungal compounds may contribute to a more efficient control of fungal infection of plants and animals, food spoilage and toxin production

1-Octen-3-ol inhibits conidia germination of Penicillium paneum despite of mild effects on membrane permeability, respiration, intracellular pH, and changes the protein composition

1-Octen-3-ol is a volatile germination self-inhibitor produced by Penicillium paneum that blocks the germination process. The size of conidia treated with 1-octen-3-ol was similar to that of freshly harvested conidia. Exposure to 1-octen-3-ol resulted in staining of 10¿20% of the conidia with PI and TOTO, fluorescent DNA probes that cannot enter cells with an intact membrane, whereas only 3¿5% of non-treated conidia were stained. Addition of 1-octen-3-ol to germinating conidia resulted in transient dissipation of the pH gradient. From this, we conclude that slight permeabilisation of the fungal membrane occurs in the presence of the inhibitor. Two-dimensional gel electrophoresis analysis of protein patterns revealed striking differences between non-germinated conidia, germinated conidia and 1-octen-3-ol-treated conidia. In conclusion, 1-octen-3-ol has mild effects on the plasma membrane, but interferes with essential metabolic processes, such as swelling and germination of the conidia, but in a reversible manne

Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination

Multicelled conidia are formed by many fungal species, but germination of these spores is scarcely studied. Here, the germination and the effects of antimicrobials on multicompartment macroconidia of Fusarium culmorum were investigated. Germ-tube formation was mostly from apical compartments. The intracellular pH (pHin) of the different individual cells of the macroconidia was monitored during germination. The pHin varied among different compartments and during different stages of germination. The internal pH was lowest in ungerminated cells and rose during germ-tube formation and was highest in new germ tubes. Antifungal compounds affect the pHin and differentiation of the conidia. The pHin inside the macroconidial compartments was lowered very fast in the presence of nystatin (1 and 4 ¿g/ml). At sublethal doses (0.3 ¿g/ml), the apical compartments were preferentially targeted showing lower pHin values. The reduced germination capacity of apical compartments under these conditions was compensated by an increased germination capacity of middle compartments

An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores

Aims: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti . Methods and Results: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. Conclusions: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. Significance and Impact of the Study: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination