| THS-1, an isolate of Trichoderma sp., has been demonstrated to be strongly suppressive to tobacco brown spot pathogen Alternaria alternata (an isolate 97Aa003) by the dural culture method and to be chitinase-producing by the DNS (3,5-dinitrosalicylic acid) method and the clear zone method on colloidal chitin-containing agar plate. The types and properties of chitinases and their roles in suppressing tobacco brown spot pathogen were conducted.THS-1 produced high yield chitinases with high activity in the liquid culture on a rotary shaker induced by colloidal chitin. The chitinases were purified from fermentation filtrate by fractionation with ammonium sulphate in 90% saturation and chromatography on Source 15Q PE 4.6/100 Column and were quantitatively measured by the DNS method. The purfied chitinases showed two bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weights were estimated to be 40kD and 70 kD, compared with 14.2-66 kD standard protein markers. The two chitinases represent two types of chitinase, and were almost consistent with endochitinase (41kD) and chitobiase (JV-acetyl-glucosaminidase, 72kD) that were identified from Trichoderma harziaum respectively.The properties of the purified chitinases (mixture of two chitinases) were conducted. The optimum temperature for chitinase activity ranged 40-55 C and optimist was 50 C, and the optimum pH values around 4.5 and 7.0 with colloidal chitin as substrate. However, they had a wide range of pH from 3.5 to 8.0.The inhibitions of spore germination of A. alternata strain 97Aa003 by fermentation filtrate (crude chitinase) and purified chitinase were determined by the Finger Tube Culture method. More than 90% of conidial germination of A. alternaria was strongly inhibited by the crude chitinase at 25.2 U within 48 hours and at 12.6 U within 12 hours. Then the conidia germination was over 50% after 24 hours for 12.6 U treatment and 12 hours for 6.3U treatment, compared with the water treatment of 100% conidia germination within 4 hours. The germinal tube treated with water grew normally, while the conidia treated with crude chitinase didn't germinate orabnormally germinated and grew, even disrupted conidial cell-wall.The conidia germination of A. alternaria was about 60%-20% within 48 hours after treatment with purified chitinases at 9.4U and approximately 85% within 24 hours and near 50% 48 hours later after treatment with crude chitinase at the similar enzyme activity unit. The inhibition of conidia germination of A. alternaria was stronger by crude chitinase than that of purified one.The pathogenicity of A. alternaria was tested by the Leaf Droplet Inoculation method which the seedling leaves of tobacco (K-326) was inoculated by conidia treated with crude chitinase at 19.5U/ml and 9.8U/ml. Lesion area was decreased from 60% to 40% after 4 days and 7 days respectively compared with the water treatment. Lesion area at 4.9U/ml treatment was decreased from 40% to 20% 4 days and 7 days later.The conclutions of the present study include that the crude chitinase produced by THS-1 strongly inhibits the conidia germination of A. alternaria and their infection to tobacco seedling leaves. The purified chitinases were composed of two enzymes with the molecular weight of 40 kD and 70 kD. The inhibition of conidia germination of A. alternaria was less effective by purified chitinases than that crude ones, indicating that some other proteins, in addition to chitinases in the crude chitinase, to synergize the chitinases in the inhibition of conidia germination of A. alternaria. |
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