# Overcoming isolation barriers: in vitro cultivation and molecular identification of Cephaleuros virescens associated with African mahogany

## TL;DR

This paper describes a successful method to grow and identify Cephaleuros virescens, an algae causing leaf spots on African mahogany in Brazil.

## Contribution

The study provides the first successful in vitro cultivation protocol for Cephaleuros virescens on African mahogany.

## Key findings

- A liquid-to-solid transition method successfully isolated Cephaleuros virescens from infected leaves.
- Surface disinfestation of host tissue significantly improved algal growth.
- Phylogenetic analysis confirmed the isolate as Cephaleuros virescens with over 98% 18S rRNA gene identity.

## Abstract

Algae of the genus Cephaleuros are specialized plant pathogens prevalent in tropical ecosystems; however, in vitro studies remain constrained by the historical difficulty of establishing axenic cultures. This study aimed to isolate, identify, and establish a functional cultivation protocol for Cephaleuros associated with algal leaf spot on African mahogany (Khaya ivorensis) in the Brazilian Cerrado. Isolation was successfully achieved through a phased liquid-to-solid transition: symptomatic leaf fragments were initially cultivated in liquid Trebouxia medium under constant agitation (120 rpm) for 3 days, followed by the subculturing of algal mycelium onto Trebouxia agarized medium to obtain pure colonies of isolate H-27-03. Direct isolation on solid media failed across all tested substrates (PDA, PSA, BBM, and HLE), confirming the requirement for a liquid phase to facilitate sporangia release. Surface disinfestation of host tissue proved critical, significantly enhancing algal development (yielding a growth score of Note 2) compared to non-disinfested fragments (Note 1). Phylogenetic analysis of the 18S rRNA gene sequence (GenBank MH042530) confirmed the species as Cephaleuros virescens with >98% identity. This study represents the first report of C. virescens causing algal leaf spot on K. ivorensis in the Brazilian Cerrado. By overcoming isolation barriers through standardized nutritional and physical parameters, this work provides a fundamental framework for future research on the epidemiology, physiology, and management of this emerging forest pathogen.