Identification and Biological Characterizations of the Causal Agent of Leaf Spot Disease in Pseudostellaria heterophylla

<i>Pseudostellaria heterophylla</i>, an important traditional medicinal plant in China, has suffered increasing yield and quality loss due to leaf spot disease in recent years. In this study, the causal agent was conclusively identified as <i>Sclerotiophoma versabilis</i> through detailed morphological characteristics and multi-locus phylogenetic analyses based on the internal transcribed spacer regions (ITS), the 28S large subunit of the nrDNA (LSU), RNA polymerase II (<i>rpb2</i>), and ß-tubulin (<i>tub2</i>) sequences. Pathogenicity tests fulfilled Koch's postulates, thereby resolving previous taxonomic inconsistencies regarding this disease. The effects of environmental and nutritional factors on mycelial growth, conidial germination, and infection were systematically evaluated. Optimal mycelial growth occurred at 20-25 °C, pH 6-8, under continuous light. Optimal mycelial growth occurred at 20-25 °C, pH 6-8, under continuous light, while conidial germination was maximized at 20-25 °C and pH 6-7 under continuous light. Starch and glycine were identified as the most favorable carbon and nitrogen sources for the fungal mycelial growth, respectively. Infection assays indicated an incubation period of approximately 3 d and maximal disease development at moderate temperatures under low-light conditions, with 6 d-old cultures exhibiting the greatest infectivity. Microscopic observations revealed that <i>S. versabilis</i> penetrated host tissues directly or via stomata without forming specialized infection structures. These findings integrate taxonomic resolution with ecological and infection biology analyses, providing mechanistic insight into the environmental drivers of leaf spot epidemics and a scientific basis for disease-risk assessment and management in <i>P. heterophylla</i> production systems.