Primary Study On Inhibiting Mechanism Of Propamidine Against Botrytis Cinerea

Aromatic diamidines, and in particular bis(amidinophenoxy)-alkanes, have 70 years history in the treatment of human diseases and show diverse pharmacological activity. Propamidine is an antimicrobial agent used clinically for the treatment of Acanthamoeba keratitis and other corneal infections. But limited oral bioavailability and both acute and chronic toxicity have slowed down the studies on development of this class of compounds. The antifungal activity of pentamidine against Botrytis cinerea was found incidentally by NZYM Inc. in 1990s, a large number of analogues have been synthesized and propamidine have been used in the field for treating crop's disease. Propamidine is a new type, low toxicity, high efficiency and without side effects of pollution antiseptic which has the particular protective and therapeutic action to many plants pathogeny. It is used to prevention and cure many field, vegetable of shed, fruit tree and economical crop's disease which caused by Botrytis cinerea Pers. Propamidine has the diplex protective and therapeutic action which can absorb, distribute and metabolize in the plant.Although propamidine has strong antifungal activity and has been used to treat crop's disease, few or no literatures reported the inhibiting mechanism of propamidine against botrytis cinerea now. At present, the inhibiting mechanism of propamidine against Botrytis cinerea Pers. will be studied using electron microscopy and biochemistry techniques. The main results and conclusions were given as follows:1. Effects of propamidine treatment on mycelial shape and ultrastructure of Botrytis cinerea PersoonThe studies revealed that propamidine not only inhibited the hyphal growth, but also caused a series of marked hyphae morphological and cell structural alterations. These changes are generally irregular swelling, excessive branching and many tumors on the surface of hyphale, considerable thickening of the hyphal cell walls, particularly at the hyphal tip region, increasing vacuoles and electrondense bodies, excessive mitochondrion irregular swelling, and so on. Above cytological change resulted in the necrosis of hyphal cell.Scanning electron micrographs displayed that the quantity of conidium decline and conidia collapsed practice of propamidine at the beginning of disease; don't influence the quantity of conidium but conidia collapsed practice of propamidine after of disease. Transmission electron micrographs revealed that Botrytis cinerea Pers. Can't invade tomato of epidermal organise practice of propamidine at the beginning of disease; but Botrytis cinerea Pers. Can invade tomato of epidermal organise collapsed practice of propamidine after of disease. propamidine can inhibited the hyphal growth, the tip of the hyphae were shrunken, considerable thickening of the hyphal cell walls, particularly at the hyphal tip region, cytoplasm inhomogeneity and inclusion in cell exosmosis, mitochondrion manifold, vesiculated hypha cell, cell empty and so on spray propamidine at various periods. Above cytological change resulted in the necrosis of hyphal cell. The studied showed that propamidine had distinct protective and therapeutic action influence.2. Effects of propamidine treatment on energy metabolize of Botrytis cinerea PersoonThe electric conductivity in Botrytis cinerea solution which was treated with different concentrations propamidine was measured using digital conductivity meter. We discovered that the electric conductivity gradually increased as the concentration of propamidine increased. The result compares with the observed outcomings usingelectron micrographs are very similar. These indicated that the structure of cell membrane was destroyed when Botrytis cinerea was treated with propamidine.Mitochondria, whose primary function is to produce energy, play a central role in cellular metabolism and in the control of bital functions. Mycelium respiration was first examined. It may be noted that the respiratory activity of treatment mycelia with different concentrations propamidine were less that of the control, but they have sameness of order of activity change. For the propamidine treatment, the State 3 respiration, state 4 respiration, respiratory control ratio and ADP/O ratio were significantly lower when compared to the corresponding controls at various contentration points.3. Effects of propamidine treatment on substance metabolize of Botrytis cinerea PersoonDecrease of respiratory activity or energy synthesize will changes the substance catabolism. To improve this, we measured the content of total sugar, reducing sugar, protein and fat of Botrytis cinerea treat with different concentrations propamidine. The results were shown as follows: 1) the content of total sugar, reducing sugar and protein initially increased and then decreased as the concentration of propamidine increased. 2)the content of fat always was increased when the propamidine concentrations was increased. 3) the content of total sugar, reducing sugar, protein and fat in Botrytis cinerea treat with different concentrations propamidine were more than the content of control's. These results show that substance catabolism was inhibited when the Botrytis cinerea was treated with different concentrations propamidine.4. Effects of propamidine treatment on mitochondrial complexes activity of Botrytis cinerea PersoonTo examine how propamidine influences mitochondrial individual respiratory chain enzymes activities, mitochondria were isolated. The mitochondria of various concentration propamidine treatment were assayed indicidually to determine activity related to respiratory chain enzymes and oxidative phosphorylation. Activities from the excised control were used as the basis for comparison and set at 100%. Comparisons were first carried out to determine if there were significant changes in controls following treatment and, second, to determine if there were significant differentces between th control and the treatment at various contentration points. The results shows that the activity of mitochondrial respiratory chain enzymes have not been changed whether the Botrytis cinerea was treat with propamidine or not. This result indicated the propamidine did not combined with mitochondrial respiratory chain enzymes for changing their activity.When we first treated Botrytis cinerea with different concentrations of propamidine, and then extracted all of mitochondrial respiratory chain enzymes for detecting their activity. The results were shown as follows: 1)when Botrytis cinerea was treated with increasing concentrations of propamidine for one day, the activitys of complexâ… ,â…¡,â…£and ATP enzyme were increased. But the activity of complexâ…¢was decreased . 2)when Botrytis cinerea was treated with increasing concentrations of propamidine for three days, the activitys of complexâ… ,â…¡,â…¢,â…£and ATP enzyme were decreased. These results indicated that the propamidine may be inhibited the activity of complexâ…¢.In summarize, we can draw the conclusion about the inhibiting mechanism of propamidine against Botrytis cinerea. The propamidine will enter into the organism cell when the Botrytis cinerea was treated with propamidine. Then the propamidine will be combining with a gene region which encoded a subunit of complexâ…¢, and inhibiting the expression of this gene. At last, the decreased activity of complexâ…¢will be acrousing respiration weaken, energy synthesis lower, mitochondria increased and cell membrane broken. These changes will deform the mycelium shapes, control the growth.