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Pseudostellaria heterophylla, 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 Sclerotiophoma versabilis 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 (rpb2), and ß-tubulin (tub2) 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 S. versabilis 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 P. heterophylla production systems. Full article

Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in eukaryotes and modulate responses to both internal and external stimuli. Pmk1 and Mps MAPK pathways regulate stress tolerance, vegetative growth and cell wall integrity in Saccharomyces cerevisiae and Pyricularia oryzae. Here, we deployed genetic and cell biology strategies to investigate the roles of the orthologs of Pmk1 and Mps1 in Sclerotiophoma versabilis (herein referred to as SvPmk1 and SvMps1, respectively). Our results showed that SvPmk1 and SvMps1 are involved in hyphal development, asexual reproduction and pathogenesis in S. versabilis. We found that ∆Svpmk1 and ∆Svmps1 mutants have significantly reduced vegetative growths on PDA supplemented with osmotic stress-inducing agents, compared to the wild type, with ∆Svpmps1 being hypersensitive to hydrogen peroxide. The two mutants failed to produce pycnidia and have reduced pathogenicity on Pseudostellaria heterophylla. Unlike SvPmk1, SvMps1 was found to be indispensable for the fungal cell wall integrity. Confocal microscopic analyses revealed that SvPmk1 and SvMps1 are ubiquitously expressed in the cytosol and nucleus. Taken together, we demonstrate here that SvPmk1 and SvMps1 play critical roles in the stress resistance, development and pathogenesis of S. versabilis. Full article