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 fungal<span class=SpellE>colonisation</span>and infection is the germination process, subsequently resulting in<span class=SpellE>mycelial</span>growth. The aim of this thesis was to study fungal germination and the effects of antifungal compounds on this process using<span class=SpellE>Penicillium</span><span class=SpellE>paneum</span> conidia and<span class=SpellE>Fusarium</span><span class=SpellE>culmorum</span><span class=SpellE>macroconidia</span>as model organisms.<o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%'><span lang=EN-US style='font-size:12.0pt;mso-bidi-font-size:10.0pt;line-height:130%; mso-ansi-language:EN-US'>An antifungal compound produced by Bacillus<span class=SpellE>subtilis</span> YM 10-20 was identified and characterized as an<span class=SpellE>iturin</span>-like compound belonging to the<span class=SpellE>iturin</span>family. This compound inhibited fungal germination and growth of<span class=SpellE>Penicillium</span><span class=SpellE>paneum</span> conidia. Fluorescence probes in combination with flow<span class=SpellE>cytometry</span>and 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. The<span class=SpellE>iturin</span>-like compound<span class=SpellE>permeabilised</span>fungal spore membranes and blocked germination. Another compound was identified and characterized from<span style='mso-bidi-font-style:italic'>dense conidia suspensions of</span><span class=SpellE>Penicillium</span><span class=SpellE>paneum</span>.<span style='mso-bidi-font-style:italic'></span>A volatile compound(s) were produced under high-density conditions by the conidia themselves. It inhibited<span class=SpellE>mycelial</span>growth 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.<span class=GramE>This compound<span style='font-size:11.0pt;mso-bidi-font-size:10.0pt; line-height:130%'></span>influenced different developmental processes during the P.<span class=SpellE>paneum</span> life cycle namely<span class=SpellE>microcycle</span><span class=SpellE>conidiation</span>and spore germination.</span>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.<span style='mso-bidi-font-style:italic'><o:p></o:p></span></span><p class=MsoNormal style='text-align:justify;line-height:130%'><span lang=EN-US style='font-size:12.0pt;mso-bidi-font-size:10.0pt;line-height:130%; mso-ansi-language:EN-US'>The sequence of events in the germination process of<span class=SpellE>multicompartment</span><span class=SpellE>Fusarium</span><span class=SpellE>culmorum</span><span class=SpellE>macroconidia</span>and the effects of antifungal compounds on this process was investigated by measuring the intracellular pH (<span class=SpellE>pH _in</span>) employing fluorescence ratio imaging microscopy (FRIM). During germ tube formation, clear differentiation between the compartments of the<span class=SpellE>macroconidium</span>was observed. Germ tubes develop preferably from apical cells and with low frequency from middle compartments. The<span class=SpellE>pH _in</span> varied among different compartments and during different stages of germination.</span><span lang=EN-US style='mso-ansi-language:EN-US'></span><span class=GramE><span lang=EN-US style='font-size:12.0pt;mso-bidi-font-size:10.0pt; line-height:130%;mso-ansi-language:EN-US'>Addition of<span class=SpellE>antifungals</span>,<span class=SpellE>nystatin</span>and<span class=SpellE>nonanoic</span>acid during germination, affects<span class=SpellE>pH _in</span>of the conidia and their differentiation.</span></span><span lang=EN-US style='font-size:12.0pt;mso-bidi-font-size:10.0pt;line-height:130%;mso-ansi-language: EN-US'><o:p></o:p></span><p class=MsoNormal style='text-align:justify;line-height:130%'><span lang=EN-US style='font-size:12.0pt;mso-bidi-font-size:10.0pt;line-height:130%; mso-ansi-language:EN-US'>The work described in this thesis highlights the sequence of events in the germination process of fungal spores, notably conidia from <span class=SpellE>Penicillium</span><span class=SpellE>paneum</span> and<span class=SpellE>multicompartment</span><span class=SpellE>macroconidia</span> from<span class=SpellE>Fusarium</span><span class=SpellE>culmorum</span> 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