BayCEER Workshop 2026

Diversity and Distribution of Melanized Stomatal Fungi

P 7 in Posters

Leaves are an important habitat for a multitude of microorganisms, and leaf microbiomes are important mediators of host health and disease. While conditions can be relatively harsh in more exposed areas, leaf structures like stomata may offer a favorable local environment that can create microbial hotspots. At the same time, stomata are a connection to the leaf interior, so plants have developed various protective measures, ranging from wax covers to recognition of pathogen-associated molecular patterns. Melanized fungi have been observed to frequently grow in the stomatal openings of otherwise seemingly healthy conifer leaves. These fungi can acquire sufficient biomass to block the stomatal opening and potentially degrade epistomatal wax plugs but rarely seem to enter further. This behavior may hint at interesting ecological interactions, metabolic abilities, and mechanisms of leaf colonization. However, to date these fungi have remained uncharacterized due to the difficulties of isolation and culturing of microbes from the extremely small microsite of stomata (40-50 μm diameter).  

It was possible to specifically cultivate these organisms by developing a micromanipulation-based protocol. Obtained pure cultures were identified through PCR amplification and DNA sequencing of the Internal Transcribed Spacer (ITS) region of the ribosomal RNA. These methods are now being used to study diversity and geographical distribution of melanized stomatal fungi with spatially explicit sampling at multiple scales. Whole genome sequencing with nanopore is planned to elucidate the metabolic potential of these stomata-occupying fungi. 

A second experimental approach is to use fluorescence microscopy combined with automated stoma recognition and fungus detection to gain quantitative results for the prevalence of this fungal lifestyle. Aside from geographic distribution, this approach is meant to test for differences in abundance between different zones within a single tree. 

Melanized fungus in a stoma of Picea abies.

Keywords: Plant Microbiome, Fungi, Stomata, Cultivation, Sequencing, Fluorescence Microscopy, Image Recognition