Fungal diversity in acid lime (Citrus aurantifolia) leaves developing witches’ broom disease symptoms

Phytoplasma-induced witches’ broom disease (WBD) of acid lime resulted in the death of over a million lime trees in the Middle East. In this study the pyrosequencing analysis was employed to characterize the level of fungal diversity in acid lime leaves developing WBD symptoms versus asymptomatic leaves in the same trees. The level of fungal diversity was higher in symptomatic leaves (15-16 OTUs; 0.88-1.44 Shannon diversity) as compared to the asymptomatic leaves (6-9 OTUs; 0.1-0.55 Shannon diversity). The symptomatic leaves were found to have 6-8 fungal classes and 13-14 fungal genera as compared to only 2 fungal classes and 5-7 fungal genera in the asymptomatic leaves. Microsporidetes was the most common fungal class, followed by Dothideomycetes. Five unique classes were recovered from the symptomatic branches. Systenostrema was the most common genus in all samples. Some unique genera were also recovered from the symptomatic leaves. No unique classes or genera were found in the asymptomatic leaves. Analysis based on weighted and unweighted UniFrac distances showed clustering of the three asymptomatic samples of lime separately from the two symptomatic samples. This study shows that the development of WBD symptoms in acid lime could be associated with higher fungal diversity in the leaves.