Formation of antifungal compounds in Betula platyphylla var. japonica plantlet by the infection of a canker fungus Inonotus obliquus

本研究旨在检测和鉴定白桦中形成的抗真菌化合物(S)。感染桦树溃烂病菌的水稻试管苗。首先培养8号桦树无菌苗进行大量繁殖，接种斜纹夜蛾IO-U1和IO-B2菌株(处理)，然后比较无菌苗和未受害苗(对照1)和受害苗(对照2)的生长量。结果表明，真菌侵染可抑制白桦试管苗的生长，尤其是主根的生长。其次，用有机溶剂提取对照(1)、(2)和处理试管苗，然后用气相色谱-质谱仪对所得提取物进行分析，比较它们之间的萃取物。结果，虽然没有检测到植物防御素，但还是检测到了一种未鉴定的化合物，被认为是一种植物预期蛋白。在ad…中此外，β-谷甾醇，被认为是一种植物预期，也在提取物中得到了定量。结果表明，自由形式细菌的数量随着伤害的增加而增加，而随着真菌感染的增加而减少。另一方面，损伤和真菌侵染均使酯化产物的数量增加。根据这些结果，推测β-谷甾醇可能是一种转化为另一种植物预期蛋白的前体。作为第三次实验，采用液体和固体琼脂培养基对β-谷甾醇进行了抑菌试验。结果表明，当β-谷甾醇的浓度为100μg/mL时，抗真菌活性最强。因此，β-谷甾醇在一定程度上被认为是一种植物期待蛋白。作为第四个实验，从对照(1)、(2)和处理植株制备粗酶溶液，然后用双向电泳法进行蛋白质组分析。结果。所有粗酶溶液在pH值为3~7、相对分子质量均小于100 kDa的范围内均出现大量蛋白质斑点。此外，9个斑点消失或变暗，4个斑点变深。在真菌感染后1天，处理组的2D凝胶上出现一个新的斑点。而在真菌感染后第6天，2D电泳胶上有6个斑点消失或变暗，2个斑点变深。这些蛋白质被认为与感染该真菌的桦树试管苗的代谢变化有关。较少

The present study aims to detect and identify the antifungal compound(s) formed in the Betula platyphylla var. japonica plantlets which are infected with a canker fungus of Betula spp., Inonotus obliquus. At first, sterile birch plantlet No.8 was cultured to multiply it in large quantities, I. obliquus IO-U1 and IO-B2 strains were inoculated to them (TREATMENT), and then growth amount of TREATMENT was compared between the sterile and uninjured plantlets (CONTROL(1)) and the injured plantlets (CONTROL(2)). As a result, infection of the fungus was proved to inhibit growth of the birch plantlets, especially growth of the taproot. In the next place, CONTROL(1), (2), and TREATMENT plantlets were extracted with organic solvents, and then the obtained extracts were analyzed by a gas chromatograph-mass spectrometer to compare the extractives between them. In result, an unidentified compound, which was suggested to be a phytoanticipin, was detected, although no phytoalexins were detected. In ad … More dition, β-sitosterol, which was suggested to be a phytoanticipin, was also quantified in the extracts. As a result, the amount of the free-form one increased by injury, while the amount decreased by the fungal infection. On the other hand, the amount of the esterified-form one increased by both injury and fungal infection. From these results, it is conceived that β-sitosterol may be a precursor which is converted to another phytoanticipin. As the 3^<rd> experiment, antifungal test of β-sitosterol was performed by using liquid and solid agar media. In result, the concentration of β-sitosterol at 100 μg/mL gave the most distinct antifungal activity. It is considered, therefore, that β-sitosterol acts as a phytoanticipin to some extent. As the 4^<th> experiment, crude enzyme solutions were prepared from CONTROL(1), (2), and TREATMENT plantlets, followed by proteome analysis with using two-dimensional electrophoresis. As a result. Many protein spots appeared within the range of pH 3〜7 and molecular weight less than 100 kDa in all crude enzyme solutions. In addition, 9 spots disappeared or became faint, 4 spots became deep. And one spot newly appeared on the 2D electrophoresis gel of TREATMENT 1 day after the fungal infection. On the other hand, 6 spots disappeared or became faint, and 2 spots became deep on the 2D electrophoresis gel of TREATMENT 6 days after the fungal infection. These proteins are considered to be involved in the metabolic changes of birch plantlets infected with the fungus. Less