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

本研究的目的是检测和鉴定白桦抗真菌活性物质。用桦属的溃疡真菌感染的日本小植株，孔菌首先以白桦无菌苗8号为材料进行大量繁殖，I.将拟南芥IO-U1和IO-B2菌株接种到它们（处理），然后在无菌和未损伤的小植株（对照（1））和损伤的小植株（对照（2））之间比较处理的生长量。结果表明，该菌的侵染抑制了白桦试管苗的生长，尤其是主根的生长。其次，用有机溶剂提取对照（1）、（2）和处理植株，然后用气相色谱-质谱仪分析所获得的提取物，以比较它们之间的提取物。结果，虽然没有检测到植物抗毒素，但检测到了一种未鉴定的化合物，该化合物被认为是植物预期素。ad中 关于我们 此外，β-谷甾醇，这被认为是一种植物预期，也定量提取物。结果，自由形式的量增加的伤害，而真菌感染的量减少。另一方面，损伤和真菌感染都增加了一种形式的量。从这些结果可以设想，β-谷甾醇可能是转化为另一种植物预期素的前体。作为第三实验<rd>，采用液体和固体琼脂培养基进行β-谷甾醇的抗真菌试验。结果表明，β-谷甾醇浓度为100 μg/mL时抑菌效果最好。因此认为β-谷甾醇在一定程度上起着植物前体蛋白的作用。作为第4实验<th>，从对照（1）、（2）和处理植株制备粗酶溶液，然后使用二维电泳进行蛋白质组分析。结果。在pH 3 ~ 7和分子量小于100 kDa的范围内，所有粗酶液中均出现大量蛋白质斑点。此外，9个斑点消失或变淡，4个斑点变深。真菌感染后1天，处理组的2D电泳凝胶上新出现一个斑点。另一方面，在真菌感染后6天，在处理的2D电泳凝胶上，6个斑点消失或变淡，2个斑点变深。这些蛋白质被认为参与了感染真菌的桦树幼苗的代谢变化。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