The genus Lasiodiplodia includes numerous plant-pathogenic species whose delimitation is complicated by overlapping morphological traits and limited resolution of common genetic markers. Lasiodiplodia brasiliensis was described as a species closely related to L. theobromae; however, its taxonomic status remains controversial. In this study, we re-evaluated the species boundaries between L. theobromae and L. brasiliensis using an integrative approach that combined multilocus and genome-scale phylogenetic analyses with morphological comparisons. Multilocus phylogenetic analyses based on ITS, tef1-α, tub2, and rpb2 revealed an unresolved relationship between the two taxa. The L. theobromae clade had low bootstrap support, whereas the ancestral node connecting both species had high support. In contrast, genome-scale phylogenetic analysis using hundreds of single-copy orthologous genes strongly supported a single monophyletic clade encompassing isolates assigned to both L. theobromae and L. brasiliensis. Morphological analyses further revealed that conidial dimensions and other diagnostic characteristics largely overlapped between the two taxa, rendering them unreliable criteria for species separation. Considering the combined molecular and morphological evidence, our results support treating L. brasiliensis as a synonym of L. theobromae. Clarifying species boundaries within this group helps stabilize the taxonomy of Lasiodiplodia and provides a reliable foundation for accurate pathogen identification and disease management.

The genera Pestalotiopsis, Neopestalotiopsis, and Pseudopestalotiopsis share highly similar morphological characteristics. Although species within these genera are recognized as plant pathogens, their pathogenicity can differ even on the same host plant, highlighting the importance of accurate genus-level identification for effective disease management. However, reliable discrimination among these genera based solely on morphology or internal transcribed spacer (ITS) amplicon length is difficult. Therefore, molecular approaches based on ITS sequence data are required for practical and reliable genus-level identification. This study aimed to develop genus-specific PCR primers through comparative genome analysis using genus-specific gene regions identified from available genomic data. The performance of these primers was evaluated using 49 isolates obtained from banana, Japanese andromeda, loquat, rubber, and tea. The primer sets achieved an overall identification accuracy of 97%. One strain could not be assigned to Pestalotiopsis, which exhibited morphological characteristics inconsistent with the genus and was positioned outside the main Pestalotiopsis clade in phylogenetic analyses, supporting the taxonomic validity of the primer-based identification. These results demonstrate that the developed primers provide a reliable and practical tool for genus-level identification and taxonomic assignment of these morphologically similar fungal pathogens, including direct detection from infected plant tissues.