The Effect And Mechanism Of β-Carboline Amide Derivative Against To Peronophythora Litchii Chen

Peganum harmala L. is a perennial herb, which has a class of secondary metabolites: β-carbolines that have a variety of biochemical activities and pharmacological effect, such as anxiolytic, sedative, anti-malarial, anti-tumor, anti-oxidation, anti-inflammatory, anti-viral, anti-fungal and so on. Peronophythora litchii Chen is one of the main pathogens of Litchii, which can affect seriously yield and quality of Litchii. So it need to be spent lots of resources for prevention and control P. litchii every year. And it has been generated drug resistance for some fungicides. Amide fungicides plays an important role to against Oomycetes, and some new amide fungicides has a very good control effect to against it at recently reported. Utilizing plant secondary metabolites as lead compound to develop new drug is a new focus of the world pesticide research. In this paper, it firstly measure the inhibition effect of 29 β-carboline amide derivatives to against six plant pathogenic fungi, and select a compound that have the strongest inhibition effect to against P. litchii, then have a study of its effect on the inhibition activity, morphology, biochemistry and mitochondrion function of P. litchii, and explore its mechanism. The results are as follows:The β-carboline amide derivatives are measured their activity to against six different plant pathogenic fungi by using growth rate inhibition method. The results show that the derivatives all have demonstrated inhibition activity to against the plant pathogenic fungi and some compounds have significant inhibition effect against some pathogenic fungi at the concentration of 100 μg/m L, in which the activity of compound F4 show a significant effect to against P. litchii and its inhibitory rate are more than 95%.Further study find that compound F4 have demonstrated a significant preventive effect(92.59%) and treatment effect(59.26%) to against P. litchii by using detached leaf test at the concentration of 100 μg/m L. At the high concentration(86.22 μg/m L), sporangia formation inhibition rate, sporangia germination inhibition rate and germ tube elongation inhibition rate of P. litchii are 99.4%, 72.46% and 80.29%, individually, all have significant effect. It shows good application potential, so compound F4 is chosen as representative to in-depth study the mechanism to against P. litchii.Using microscopy techniques to measure morphological effect of P. litchii by exposing on compound F4. We can observe irregular morphologies of P. litchii by using optical microscopy. For example, mycelia grow irregularly, branch reduce, surface appear swelling, trench and shrinking, cell contents reduce; germ tube of sporangia are thin, short, few branches, uneven surface and cells lack contents. Using scanning electron microscopy(SEM), we can find that mycelia have severe shrinkage, most surviving mycelia have severe swelling and shrinking, and the sporangia appear collapse and shrunken. Using transmission electron microscope(TEM), the result shows that cell wall and membrane become thicker and rough, and they generate plasmolysis accompany numbers of small lomasomes, cellular matrix is lacking and produce vacuoles and concentrate obviously to become a "black hole", mitochondrion are swollen seriously, and its membrane gap disappear and the number of ridge reduced significantly.P. litchii can demonstrate a series of changes in biochemistry after treating by compound F4. For example, the extracellular p H have serious deviation compare with control, the content of extracellular reducing sugar and malondialdehyde(MDA) are all increase, the content of soluble protein and the activity of NOX(NADH oxidase) are all decrease. And all above them will become more serious when the drug concentration and time have increased. These indicate that compound F4 can make antioxidant capacity of P. litchii decreasing, then induce reactive oxygen species(ROS) accumulate obviously, so that the cell membrane produce peroxidation damage, resulting in the destruction of the integrity and function and a series of cell lesion.Research of mitochondrion function change by exposing to compound F4 find that mitochondrial membrane appear irregular changes, the openness of membrane permeability transition pore(MPTP) increase, the mitochondrial transmembrane potential(Δ?m) and the activity of enzyme complex I-V decrease significantly. All of them have clear displayed membrane damage of the mitochondrion and its energy metabolism has destroyed. This suggests that the compound F4 may act upon the mitochondrion of P. litchii, then respiratory chain complex activity is blocked and energy metabolism is disrupted or inhibited, resulting in the growth inhibition of P. litchii.In summary, the mechanism of compound F4 against to P. Litchii, at an exterior level, it can inhibit the growth of mycelia, production and germination of sporangia, causing the morphology change of cell surface and internal, especially, the mitochondrion morphology; at biochemical level, it can cause a series of changes, such as extracellular p H, content of extracellular reducing sugar, MDA, ergosterol, soluble protein and activity of NOX; however, the most important is that it can cause morphology and function damage of mitochondrion, resulting in respiratory chain complex activity is blocked, inhibit energy metabolism, and finally, the growth of P. Litchii is inhibited.