Morphological and Molecular Characterization of Botrytis fabae Isolates Causing Chocolate Spot Disease on Faba Bean

Abstract

Chocolate spot disease caused by Botrytis fabae is a major constraint to faba bean production, leading to significant yield losses under conducive environmental conditions. The present investigation aimed to characterize B. fabae isolates through integrated morphological and molecular approaches to understand pathogen variability associated with disease outbreaks. Diseased leaf samples exhibiting characteristic reddish-brown necrotic lesions were collected from faba bean-growing regions and subjected to pathogen isolation on potato dextrose agar. Pure cultures showed typical Botrytis colony morphology with grey mycelial growth and profuse sporulation. Microscopic examination revealed branched conidiophores bearing globose to ovoid conidia consistent with B. fabae descriptions. Molecular identification was performed using ITS rDNA sequencing, which confirmed the identity of isolates as Botrytis fabae with high sequence similarity to reference strains. Phylogenetic analysis grouped the isolates within a distinct clade closely related to other Botrytis species infecting legumes, indicating genetic relatedness yet clear species-level separation. Pathogenicity assays conducted on healthy faba bean plants reproduced typical chocolate spot symptoms, thereby fulfilling Koch’s postulates. Variability in virulence among isolates was observed, suggesting the presence of pathogenic diversity within field populations. Disease development was favored under high humidity and moderate temperature conditions, which accelerated lesion expansion and sporulation. The study highlights the importance of combining morphological traits with molecular tools for accurate identification of B. fabae, particularly in regions where multiple Botrytis species coexist. These findings provide essential baseline information for disease monitoring and support the development of targeted management strategies, including resistant cultivar deployment and timely fungicidal interventions. Overall, the study enhances understanding of the epidemiology and genetic identity of B. fabae in faba bean production systems.