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（Betula platyphylla var. japonica）病原菌侵染白桦（Betula spp., Inonotus obliquus）溃疡病菌种后白桦（Betula platyphylla var. japonica）幼苗中形成的抗真菌化合物。首先对桦木8号不育苗进行大量培养繁殖，接种I. obliquus IO-U1和IO-B2菌株（处理），然后比较处理后不育苗和未受伤苗（对照(1)）和受伤苗（对照(2)）的生长量。结果表明，真菌侵染对桦树幼苗的生长有抑制作用，特别是对主根的生长有抑制作用。然后，用有机溶剂提取CONTROL(1),(2)和TREATMENT植株，然后用气相色谱-质谱联用仪分析得到的提取物，比较它们之间的提取物。结果，检测到一种未知的化合物，该化合物被认为是植物预期素，尽管没有检测到植物抗毒素。此外，在提取物中还定量测定了被认为是植物预期素的β-谷甾醇。结果表明，损伤使游离体数量增加，真菌感染使游离体数量减少。另一方面，损伤和真菌感染均增加了酯化型1的数量。根据这些结果，我们认为β-谷甾醇可能是转化为另一种植物预期素的前体。作为3^<rd>实验，采用液体和固体琼脂培养基对β-谷甾醇进行抑菌试验。结果表明，β-谷甾醇浓度为100 μg/mL时抗真菌活性最明显。因此，人们认为β-谷甾醇在一定程度上起植物预期素的作用。在4^< >试验中，分别从对照(1)、(2)和处理植株中制备粗酶溶液，利用双向电泳进行蛋白质组学分析。因此。所有粗酶溶液在pH 3 ~ 7、分子量小于100 kDa范围内均出现大量蛋白斑点。另外，9个斑点消失或变淡，4个斑点变深。真菌感染后第1天，二维电泳凝胶上新出现1个斑点。另一方面，真菌感染后第6天，处理的二维电泳凝胶上有6个斑点消失或变淡，2个斑点变深。这些蛋白质被认为与真菌感染桦树幼苗的代谢变化有关。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