# Standardised sporulation methods for Diplodia, Lasiodiplodia and Neofusicoccum

## TL;DR

Researchers developed a standardized method to induce sporulation in several fungal species from the Botryosphaeriaceae family, which can help in studying their biology and disease mechanisms.

## Contribution

The study introduces a standardized and reproducible method using Vogel’s Minimal Medium for sporulation in Botryosphaeriaceae fungi.

## Key findings

- Vogel’s Minimal Medium successfully induced sporulation in multiple species of Diplodia, Lasiodiplodia, and Neofusicoccum.
- Significant variation in sporulation time and yield was observed among and within species.
- Reduced sucrose in the medium affected sporulation differently across species.

## Abstract

Members of the Botryosphaeriaceae 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 in vitro 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 Diplodia
sapinea, in species of Diplodia (n = 3), Lasiodiplodia (n = 2), and Neofusicoccum (n = 26). For this purpose, we used 123 isolates recently collected in the Canary Islands (Spain), as well as 67 Neofusicoccum isolates from long-term storage. All isolates were identified through multilocus phylogenetic analysis. The results showed that D.
africana, D.
mutila, and D.
seriata were able to produce pycnidia, although only D.
seriata consistently released conidia. Lasiodiplodia
brasiliensis and L.
theobromae successfully formed pycnidia and released conidia, while 20 of the 26 tested Neofusicoccum 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 Neofusicoccum species but increased sporulation in D.
africana, D.
seriata, L.
brasiliensis, and L.
theobromae. Long-term storage on Malt Yeast Agar medium at 4 °C negatively affected sporulation in some species, including N.
luteum and N.
stellenboschiana. Overall, VMM provides a standardised and reproducible medium for inducing sporulation in the Botryosphaeriaceae, 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.