The Extraction, Purification And Identification Of Toxin Of Pseudocercospora Oxalidis

Oxalis rubra (Oxalis corymbosa DC.Prodr.) originally produced in the American tropical region. It has become an important invasive species in China. It has strong resistance, reproduction ability and growth capacity. Bulb propagation is the main reproductive way. In China, both north and south, Oxalis rubra has badly spreaded, and the grain production has been severely affected, like pepper, corn, peanut, soybean and watermelon, the loss rate in some areas reached 100%.It also can cause serious damage on the lawn in Chongqing Beibei, Oxalis rubra has become the dominant species in the Zoysia lawn. The occurrence frequency of most of the year period is over 0.5.The mainly way to control Oxalis rubra is the chemical control. Chemical control, to some extent, can control the occurrence of weed, but the damage to the environment and the pollution is not to be ignored, even being a great threat to our health of human beings.Oxalis grass leaf spot is an important disease of Oxalis rubra. The central part of the lesion showed light brown, the border is dark brown and light yellowish brown halo ring surround the spot. This serious disease, in some areas of Chongqing, caused the death of Oxalis grass in large area, showing a good potential as a biological herbicide. The study was carried out to isolate the Oxalis leaf spot pathogens firstly, secondly to culture toxin production, to conduct the toxin extraction and purification, and then do the identification of high performance liquid chromatography. The purpose of study is to make a reliable experimental basis for Oxalis leaf spot fungus to be as productive bacteria for future biological control herbicide.The primary results were as follows:In the duration of Oxalis grass leaf spot disease in the grass, the typical disease leaves were selected, then the pathogens were isolated and observed. By the method of determining the pathogenicity of these strains, the result showed that strain SX-01-01 was the most pathogenic strain in all Pseudocercospora oxalidis strains.Through induction of toxin production in Fries'3 Liquid medium, the strain SX-01-01 was fermented. At the third days of fermentation, brown mycelial ball began to appear. The surface of mycelial ball was covered with small thorn, which was soft, and also could swing with the media in the medium, but it had poor toughness, easy taken off. The small thorn with conical shape in appearance, also with wide base and more fuscous as dark brown, the top was small and the color was light brown. Mycelium at day 9 reached the maximum.When observing the surface spinule of mycelium under the microscope, it was found that the composition of small thorns was mycelium. There was dense mycelium in small thorns, which was the skeleton of the thorns.Skeletons had wide base and conical tip, radiating loose mycelium to the surrounding. From the results from the micrograph of internal mycelium, it was found the pellet surface mycelium was dense, inward gradually became sparse, the center of mycelium had no hyphae but liquid medium.When the fermentation was over, it was found that some abnormal mycelium existed in the medium.These hyphae was much shorter and thicker, showing moniliform, and some with little branches. Then, the bioassay of fermentation roughly purified was conducted on detached leaves.The results showed that the strain SX-01-01 could produce some toxins to Oxalis grass in liquid medium of modified Fries'3 after the fermentation. Using three kinds of extractants such as petroleum ether, dichloromethane, chloroform to extract crude toxin, and the polarity of the three extractive solvent were 0.01,3.40,4.40, respectively. After extracting, the bioassay of water phase and oil phase was done respectively. It was found the toxins were easily soluble in polar organic solvents, especially the toxin in dichloromethane's extraction showed the best in these kinds of solvent. And then, the oil phase of dichloromethane's extraction was analyzed by column chromatography, nine bottles of the merger were separated. After the bioassay of the 9 combined bottles, it was found No.3,4,8 and 9 bottle were all toxic to the leaves. But the toxicity was different, No.8 bottle showed the strongest, followed by 3,4, No.9 was the worst. No.1, 2,5,6 and 7 bottle and control (CK) had no chlorosis phenomenon. The results indicated that column chromatography separation of toxins played a better role, and the separation effect was obvious.Combined bottle No.3,4,8 and 9 had toxic effect on the Oxalis grass for it could produce toxic substances.Detecting of combined bottle No.3,4,8 and 9 with high performance liquid chromatography, six wavelengths were used. It was found that on the wavelength at 200nm,220nm,300nm and 320nm, the number of measured peak of spectrum were less on 250nm and 270nm,and peak of 250nm wavelength was lower on the 270nm. Wavelength of 270nm was used for the test of these separated bottles with toxic liquid spectrum detection. Chromatographic peaks of these separated bottles removed these peaks which had the same peak time with control (CK), then draw toxins out of possible peak. The results showed that peak time might be 8.90,9.60 to the separation of toxic substances in bottles 3,the separation of bottle 4 in peak time of toxins might be 7.10, 8.90 and 9.60, the separation of toxic substances in bottle 8 in peak time might be 3.70, 3.90,3.95 and 9.20, while the separation of toxic substances in bottle 9 might be 3.40, 7.90 and 9.20 in the peak time.Because the liquid peaks of the separation in bottles No.3,4 and No.8,9 didn't show repeatedly, it suggested that their separation substance contain two or more kinds of toxins.After these tests, further evidence of the Pseudocercospora oxalidis Goh & Hsieh was provided as to be a potential biological herbicide, with prospects for possible biological control. The feasibility for separation of the toxin produced by Pseudocercospora oxalidis Goh & Hsieh was proved through the above methods in the laboratory. Our experiments provided a base for future research and development of the toxin to be the final herbicide.