Standardised sporulation methods for Diplodia, Lasiodiplodia and Neofusicoccum

Abstract

Members of the <i>Botryosphaeriaceae</i> are widespread fungal pathogens responsible for economically important diseases in woody plants. Despite the relevance of conidia production for understanding pathogen biology, infection processes, and disease epidemiology, sporulation <i>in vitro</i> remains unpredictable and inconsistent across species. In this study, we evaluated the efficacy of Vogel's Minimal Medium (VMM) for inducing pycnidial and conidial development, which has recently been shown to be effective for this purpose in <i>Diplodia sapinea</i>, in species of <i>Diplodia</i> (n = 3), <i>Lasiodiplodia</i> (n = 2), and <i>Neofusicoccum</i> (n = 26). For this purpose, we used 123 isolates recently collected in the Canary Islands (Spain), as well as 67 <i>Neofusicoccum</i> isolates from long-term storage. All isolates were identified through multilocus phylogenetic analysis. The results showed that <i>D. africana</i>, <i>D. mutila</i>, and <i>D. seriata</i> were able to produce pycnidia, although only <i>D. seriata</i> consistently released conidia. <i>Lasiodiplodia brasiliensis</i> and <i>L. theobromae</i> successfully formed pycnidia and released conidia, while 20 of the 26 tested <i>Neofusicoccum</i> species formed pycnidia, of which 15 released conidia. Significant variation was observed in the time required for pycnidial development and conidial release, as well as in the quantity of conidia produced, both among and within species. Reduced sucrose concentration in VMM delayed pycnidia formation and conidial release and reduced sporulation yields in <i>Neofusicoccum</i> species but increased sporulation in <i>D. africana</i>, <i>D. seriata</i>, <i>L. brasiliensis</i>, and <i>L. theobromae</i>. Long-term storage on Malt Yeast Agar medium at 4 °C negatively affected sporulation in some species, including <i>N. luteum</i> and <i>N. stellenboschiana</i>. Overall, VMM provides a standardised and reproducible medium for inducing sporulation in the <i>Botryosphaeriaceae</i>, although notable variation persists within and between species. These findings provide a methodological foundation for future studies on the biology, pathogenicity, molecular biology, and host-pathogen interactions of these fungi.