Histochemical Changes And Occurrence Of Phytoalexins In Laminaria Japonica During Host's Anti-pathogenesis Process

What are the biochemical reaction characteristics of marine algae during pathogenesis? Does the marine alga host have the similar biochemical defense compare with higher plants against pathogenic bacteria? The current data to these questions is very limited. Recent studies indicated that the marine macroalgae will produce elevated level of active oxygen species (AOS) after treated with elicitors, such as oligosaccharides, which means the existence of the first step of chemical signal similar reaction system in higher plants. What are the followed reactions process occurred after the marine alga host receiving the AOS signals, it is very interested and attracted to many researchers, therefore either this is the main contents of this thesis study.In the experimental design, commericial seaweed, Laminaria japonica, was chosen as the model host organism, and alginate degradation marine bacteria were chosen to infect the seaweed. According to their pathogenic activity, three strain of marine bacteria were selected as L2 (Alteromonas sp), G (Pseudomonas sp), Py (Pseudomonas sp). The biochemical changes of malondialdehyde (MDA), which is the lipid peroxidation end product and can be measured by thiobarbituric acid (TBA) regeant, and polyphenol content, which reflected the host non-specific chemical defense activity and can be measured by Folin-Ciocalteu method, were determined at different time intervals during host pathogenesis. The results indicated that the host's histological MDA levels treated with different marine bacteria are quite different. In Py group, the MDA level changes showed a equal based double peak, while in L2 and G group, the MDA level changes showed a stepwise double peak, with the occurrence of a weak peak of MDA at 1 h inoculation, and another higher peak at 6 h. The histological changes under light microscope indicated that the rotting spots were observed at 6.5 h, while the treated seaweed with Py did not show any significant changes. Therefore, it is reasonable to deduce that the second higher peak of lipid peroxidation is closely related with the rotting phenomena. Comparing with results to higher plants, we found that the similar AOS burst occured in virulent host pathogenesis of some higher plants. The changes of polyphenols also indicated that the highest increase was happened at 4-8 h. Nevertheless, only the L2 treated group showed an unusual double peak pattern to polyphenol accumulation.High performance liquid chromatography differential display was applied to study the biochemical changes to the testified seaweed control and treated group by using HPLC-PDA system, which composed of two Waters 515 HPLC pumps, a photodiodearray detector, and a C18 reverse column, to analyze the different solvent extract. The results again indicated that the ethyl acetate extract of L2 treated sample did exhibit a new compound with retention of 43.5 min and UV absorption maximum wavelength of 280.7 nm. The target compound was purified by semi-preparative C18 column in the same HPLC system. Direct EI-MS indicated that the compound has mass peaks as 406.3, 391.3, 313.2, 207.0, and 105.1, with possible molecular weight of 406.3. Further GC-MS analyzed after trimethylsilyl (TMS) derivation, indicated the existence of two stereo-chemical isomers, and only one hydroxyl group in the compound evidence as 478.3 and 463.3 mass peaks in the TMS derivatized compound. After molecular simulation using software and NIST spectrum retrieval, we concluded that the compound is 2,4,6-tris-(1-phenyl-ethyl)-phenol (CAS no 18254-13-2). Test showed that this compound showed moderate anti-bacterial activity. According to the definition of phytoalexin, it should be produced by pathogen induction in the host, and be synthesized for against pathogen. In our case, the results obtained in this study can conform to the definition, which is possibly the first phytoalexin in the marine plants. It is need to conduced further verification the structure, and try to find our the correlation between the compound with the biosynthe...