Apple Valsa canker (AVC), primarily caused by the pathogenic fungus Valsa mali , is a devastating disease of apple. The development and application of high efficiency and low toxicity fungicides are of great significance for disease control. Natural active substances serve as a vital foundation for the development of novel green fungicides. In previous studies, the endophytic fungus Aa-Lcht was confirmed to contain specific inhibitory effect against V. mali . This study confirmed that Aa-Lcht fermentation broth (FB)’s suppression of V. mali growth and conidial germination, exhibiting AVC prevention. Through extraction, isolation, and purification, one active substance with high inhibitory effect on V. mali was obtained and identified as altenusin. Its concentration value for 50 % of maximal effect (EC 50 values) against V. mali mycelium growth was 3.118 μg/mL. Before V. mali infection, the lesion length of apple twigs and lesion area of fruits pretreated with altenusin decreased by 47.27 % and 80.52 %, respectively. Further research revealed that the hyphae of V. mali treated with altenusin exhibited irregular thickening of cell walls, severe vacuolation within the cells, and protoplasmic exudation. Meanwhile, transcriptome analysis indicated that altenusin primarily inhibited V. mali by interfering with the normal hyphal cell metabolism, substance degradation, peptidase activity, and proteolysis functions. Additionally, altenusin could also significantly up-regulate the expression of immunity resistance-related genes MdCYP81F2 , MdPR2 , MdPR4 , and MdPR5 in apple. Summarizing the above, the altenusin provides valuable insights for the development of novel green fungicides for controlling AVC disease, contributing to the safe and healthy development of apple production.
Two new metabolites, an α-pyridone derivative, 3-hydroxy-2-methoxy-5-methylpyridin-2(1H)-one (1), and a ceramide derivative, 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide (2), and a new natural product, 3-hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide (3), along with 15 known compounds including chaetoglobosin C (7) and chaetoglobosin F (8) were isolated from the solid culture of the endophytic fungus Botryosphaeria dothidea KJ-1, collected from the stems of white cedar (Melia azedarach L). The structures were elucidated on the basis of spectroscopic analysis (1D and 2D NMR experiments and by mass spectrometric measurements), and the structure of 1 was confirmed by X-ray single-crystal diffraction. These metabolites were evaluated in vitro for antimicrobial, antioxidant, and cytotoxicity activities. Pycnophorin (4) significantly inhibited the growth of Bacillus subtilis and Staphyloccocus aureus with equal minimum inhibitory concentration (MIC) values of 25 μM. Stemphyperylenol (5) displayed a potent antifungal activity against the plant pathogen Alternaria solani with MIC of 1.57 μM comparable to the commonly used fungicide carbendazim. Both altenusin (9) and djalonensone (10) showed markedly DPPH radical scavenging activities. In addition, stemphyperylenol (5) and altenuene (6) exhibited strong cytotoxicity against HCT116 cancer cell line with a median inhibitory concentration (IC50) value of 3.13 μM in comparison with the positive control etoposide (IC50 = 2.13 μM). This is the first report of the isolation of these compounds from the endophytic B. dothidea.
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