Identification and biological characterization of pathogen causing sooty blotch of <i>Ardisia crispa</i> (Thunb.) A.DC.

Sooty blotch, a primary leaf disease affecting <i>Ardisia crispa</i> (Thunb.) A.DC. (<i>A. crispa</i>, AC), significantly impacts both the yield and quality of this medicinal plant. However, the specific species of pathogenic fungi responsible for this disease and their mechanisms of pathogenesis remain unclear. To elucidate the etiology of sooty blotch, it is essential to investigate effective prevention and treatment methods, and provide a theoretical basis for the effective protection of AC. Initially, the alterations in internal organelles that result in sooty blotch were examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additionally, differential genes were analyzed using differential display reverse transcriptase-PCR (DDRT-PCR) in both healthy and diseased leaves of AC. Moreover, the pathogenic fungi were separated, purified and identified from leaves infected with sooty blotch of AC, and subsequently, their pathogenicity and biological characteristics were tested. Furthermore, the inhibitory effect of pathogens was measured using the water extract of traditional Chinese medicine, based on the growth rate of the mycelium. The findings indicated that the photosynthesis rate of diseased leaves was slower than that of healthy leaves, as revealed by TEM and SEM analyses. Additionally, DDRT-PCR results demonstrated that the differentially expressed genes primarily included those related to zinc finger proteins, acyl-CoA-transferases, and chloroplasts. The phylogenetic tree and pathogenicity test results showed that the pathogens causing sooty blotch of AC were <i>Annulohypoxylon stygium</i> and <i>Diaporthe angelicae</i>. Microscopic observation revealed that <i>Annulohypoxylon stygium</i> and <i>Diaporthe angelicae</i> exhibited distinct microscopic characteristics, and a pH range of 7-10 along with a subdued light environment were more conducive to the growth of pathogens. Additionally, the water extract of <i>Lonicera fulvotomentosa</i> Hsu et S. C. Cheng and <i>A. crispa</i> (Thunb.) A.DC. had a strong antifungal action on the two pathogens of sooty blotch, and the water extract of Ardisia crenate Sims had a better antifungal action on the <i>Diaporthe</i> <i>angelicae</i>. In this study, <i>Annulohypoxylon stygium</i> and <i>Diaporthe</i> <i>angelicae</i> were reported as pathogenic fungi causing sooty blotch for the first time, and affected the photosynthesis of AC leaf, and these study provides a theoretical basis for the diagnosis and prevention of <i>A. crispa</i> (Thunb.) A.DC. sooty blotch.