Study On Antifungal Activity, Inducing Resistance, And Their Mechanisms Against Fulvia Fulva (Cooke) Cifferri By Chitosan

In this research, the antifungal activity and mechanism of chitosan were investigated against Fulvia fulva (Cooke) Cifferri. The results showed the main antifungal phages of chitosan against Fulvia fulva, the effects of different factors on the antifungal activity, and the mechanism of different molecular weight (MW) chitosan were speculated. The effects of different MW chitosan on inducing resistance of tomato to tomato leaf mould and their Physiology-biochemistry mechanism were investigated. The influence of chitosan on the growth and development, yield and quality of tomato were studied and offer information for application of chitosan as stimulator and bio-pesticide for protecting tomato. 1 The preparation of chitosan samplesA serial different MW chitosan samples (3 KDa-1540 KDa) with the same degree of deacetylation (DD) (80%), were obtained by the method of acetic acid hydrolysis. Several different DD (73%-95%) with the same MW(1600 KDa) were obtained by the method of acetic anhydride acetylation.2 The antifungal activity and mechanism of chitosan against Fulvia fulva (Cooke) Cifferri in vitroIn fact, the chitosans inhibited the germination of spore, suppressed fungal colony growth, however it hadn't distinct suppression to sporulation. The effects of concentration, deacetylation degree, MW, solvent, and pH of medium on the antifungal activity and mechanism of chitosan against Fulvia fulva in vitro have been investigated by the method of poison media technique. The results indicated that the chitosans have strong anti-fungal activities, and the activity increased with the increase of concentration, decrease of pH of medium, lactic acid and acetic acid were the better solvent, the 86% of deacetylation degree and the middle MW(213KDa-499 kDa) of CTS have better effect than others. The comparative of antifungal activity between chitosan and other fungicides was studied, the results showed that the antifungal activity of relative low concentration chitosan is lower than other fungicides, but 2 mg/ml chitosan is equal with triadimefon and carbendazim. Antifungal mechanism of chitosans with different MW against Fulvia fulva causing leaf mould in tomato plants were studied in vitro by the optic microscope, scanning electronic microscope (SEM) and confocal laser scanning microscopy (CLSM) coupled FITC (fluorescein isothiocyanate)-fluorescence detection system, We found the chitosans can induced the hyphal swelling through the optic microscope, and the SEM observation showed that the surface of hyphal which was treated with chitosan of low MW (8.2 kDa) was smooth, but it was rough with the high MW (1320 KDa) chitosan; CLSM showed fluorescence of the FITC-labeled chitosans whose MW were below 500 KDa could enter into the inner of mycelia, however, 1320 KDa chitosan was blocked off the outer of mycelia. Chitosan can control the pathogen by affecting the rate of penetration and the activity of chitinase.3 The inducing resistance and mechanism of chitosan to tomato leaf against tomato leaf mouldIt could induce the tomato plants resistance to tomato leaf mould when the tomato seed was soaked or the tomato seedings were leaf sprayed by chitosan with different MW and concentration. Chitosan treatment of tomato before inoculation reduced the rate of diseased leaf and diseased index of tomato cultivars. The relative immunization efficiencies of tomato cultivar increased firstly and then decreased with the increased of chitosan concentration and MW. The chitosan with 213 KDa MW and concentration of 5 mg/ml made better immunization efficiencies than others, but the efficiency of leaf spraying was better than that of seed soaking, it's 53.77% and 29.96% respectively.The activities of POD, PPO, PAL, Chitinase andβ-1,3-glucanase in tomato seeding leaves could been induced more strongly when the tomato seed was soaked or the tomato seedings were leaf sprayed by chitosan with different MW and concentration. But the activities of these enzymes induced by chitosan with different MW, concentration, and with different treatment style showed different. Changes of these enzymes in leaves of tomato cultivar with the times after spraying chitosans were different with different MW and concentration.①seed soaking treated: The activity changes of POD,PPO andβ-1,3-glucanase in the leaves of tomato cultivar treated by different MW chitosan were consistent, but it's different with PAL and Chitinase. The effect of CTS3 on the POD activity is stronger than that of CTS7, but CTS7 is stronger than that of CTS3 as for PPO, PAL, Chitinase, andβ-1,3-glucanase. The activity of POD, PPO, PAL,β-1,3-glucanase, and Chitinase arrived to the maximum when the concentration of CTS7 is 0.313,0.625,0.313,5,20 mg/ml respectively; and the activity of POD, PPO, PAL, andβ-1,3-glucanase arrived to the maximum when the concentration of CTS3 is 0.313 mg/ml, and Chitinase arrived to the maximum, the concentration of CTS3 is 1.25 mg/ml. ②Spraying treated: The effect on the activity of POD, PAL, andβ-1,3-glucanase of CTS5 reached to the strongest, but that of PPO and Chitinase reached to the strongest is treated by CTS1 and CTS3 respectively. The activity of POD and PAL reached to the maximum when the concentration of CTS5 is 6 mg/ml;β-1,3-glucanase and Chitinase, the concentration of CTS5 is 3 mg/ml; and PPO, the concentration of CTS5 is 10 mg/ml. The activity of POD, PPO, PAL,β-1,3-glucanase, and Chitinase arrived to the maximum leaf prayed by CTS5 at the fifth, second, third, first and second day respectively.③The effect of CTS7 on the enzyme activity is better than that of CTS3 at the concentration of 5 mg/ml by chitosan seed soaked, but that of CTS3 is better than CTS7 on the POD, PPO, Chitinase andβ-1,3-glucanase by chitosan leaf spraying, except for PAL. In conclusion, the effect on enzyme activity of chitosan leaf spraying is better than that of seed soaking.The influence of times spraying with chitosan on different enzymes varied differently. The activities of PAL, Chitinase andβ-1,3-glucanase reached the strongest treated one times by chitosan sprayed. But POD and PPO, two times and three times respectively. PPO and PAL belonged to systemic induce, and POD, Chitinase andβ-1,3-glucanase belonged to local induce. Total protein PAGE electrophoresis patterns of tomato cultivars in leaves showed that some differences mainly in the numbers and shade of the color of enzymatic bands. It showed that the expression of some gene is closed or strengthened by the inducing of chitosan. Enzyme activity staining showed that no new isoforms of Peroxidase and polyphenol oxidase were found in the leaves of tomato plants treated with chitosan. But the densities of bands are relevance with the results of enzymes activities. Our data indicates that chitosan do not induce new isoforms of POD and PPO, but improve the level of POD and PPO protein.Activated oxygen free radical was produced by tomato leaves treated with chitosan among 1-8 hours, and it burst at the fourth hour after leaf sprayed; but it was restrained after 16 hour. So we speculated that the inducing mechanism by chitosan is related with the producing of activated oxygen free radical.The effect of mixed chitosan and triadimefon was better than that of single fungicide (triadimefon and carbendazim) on tomato leaf mould. It's equal between the 10 mg/ml chitosan and 500 times triadimefon.4 The influence of chitosan on the groeth and development of tomato Effect of different MW CTS on the germination percentage, germination index and vitality index of tomato cultivar (Hezuo 908) seed was different both in the open field and in the incubator. In the field experiment, these three indexes reached the maximum when the seed was treated with CTS7 and CTS3 at the concentration of 1.25 mg/ml and 2.5 mg/ml respectively; But in the incubator experiment, at the concentration of 0.625 mg/ml and 1.25 mg/ml respectively. And the percentage of seedling establishment was higher in the open field than in the incubator.The FW and length of root and was no significant difference when the seed was treated with chitoan, but the FW and length of seedings were significant difference. The stimulate effect was the best when the seed soaked with CTS7 and CTS3 at the concentration of 2.5 mg/ml and 5 mg/ml respectively; But in the leaf spraying experiment, at the concentration of 1.25 mg/ml and 2.5 mg/ml respectively.Chitosan could make the soluble solid content, Protein content and Vitamin C content increase, and Total acidity decrease; and chitosan improve the quality of tomato; and chitosan also could control of root-knot nematode in some degree.