Modifier Of Biocontrol Strain Against Amaranthus Retroflexus And Isolation Of Herbicidal Activity Compounds From Biocontrol Strain

The host specific toxin from plant pathogenic fungi has bioactivity specific to plants, therefore using the host specific toxin as herbicide candidate for controlling weed will become one of main aspects for development of herbicide in the near future.Redroot pigweed is one of the world's vicious weed. It is major weed of soybeans, corn, sorghum, cotton and peanuts in China as well. Alternaria amaranthi-3 from our laboratory has a great potential as a mycoherbicide for biocontrol of Amaranthus retroflexus. To further enhance the development potential of A.amaranthi-3, the strain were improved by physical mutagenesis, chemical mutagenesis and compound mutant technology. The herbicide active compounds produced by the strain were isolated and purified by bioassay-guided column chromatography. The following conclusions:1. 74 mutants are obtained by mutant technology. The mutant strain X-4 showed a significant difference with the original strain. The strain X-4 has faster growth rate on PDA. The aerial hypha was largely formed on culture media. Colony was cyan, and colony fringe was white. The yield of spore increased 81.2% in culture 7d, inhibition rate of the liquid fermentation broth of the strains on Amaranthus radical increased 26.39 %. The strain X-4 were cultured for seven consecutive subculture under the same condition. The colony growth rate of each generation was significantly higher than the original colonies, and growth rate of each generation was stably. The colony diameter was from 8.97cm to 9 cm after culture 7d.The, production of mycelium and toxin is also relatively stable.2. For producing toxin, the optimal culture media was PSK media. The optimal culture condition were a temperament of 25℃, the pH range from 4.15 to 6.15, and culture time from 3 to 5 days. The optimal culture process were T the temperature 25℃, shaking culture (speed 120r/min) 3d, and then static culture 2d in dark conditions.3. Ethyl acetate was optimal extraction solvent for toxin extraction. The recovery coefficient of toxin extraction reached 91.07%. The store time, store temperature and pH did not affect the activity of crude toxin.4. The toxin from strains X-4 was purified by by bioassay-guided column chromatography. The bioactivity of the elution fraction was assayed by elongation inhibition method. The results showed that 3-9 bottles of petroleum ether: ethyl acetate = 5:4 elution fraction, 1-6 bottles of petroleum ether: ethyl acetate = 4: 6 elution fraction and 4-6 bottles of petroleum ether: ethyl acetate = 5:2 elution fraction have bioactivity. Based on the distribution of active components, the culture filtrate of mutant strain X4 contained at least two active ingredients. The 3-9 bottles of petroleum ether: ethyl acetate = 5:4 elution fraction, 1-6 bottles of petroleum ether: ethyl acetate = 4: 6 elution fraction were combined as activity fractionⅠ. The 4-6 bottles of petroleum ether: ethyl acetate = 5:2 elution fraction was activity fractionⅡ. The activity fractionⅠwas anaylized by thin layer chromatography with chloroform: methanol: ammonia water = 30:20:1 of developing agent. There were 4 fluorescence spot in chromatography at 365nm. The fluorescence spot of Rf 0.35 has bioactivity. 40mg toxin crystal were obtained by thin layer chromatography and recrystallization. The melting range of toxin is 186～189℃. Its strong absorption is at 290nm.5. In this test, we had determined the activity of the purified toxin through using the seed germination, radicle growth inhibition and excised leaf acupuncture methods. The results showed that the obvious does-response relationship existed in the poision of toxin for the Amaranthus retroflexus, that was, as the concentration increased the pathogenic activity increased for Amaranthus retroflexus as well; the brown lesions on leaves caused by toxins resembled as the lesions caused by the bacteria infection, it had shown that the toxin played an important role in the pathogenic process of Amaranthus retroflexus. When the toxin concentration was 200μg.mL-1, retroflexus seeds had almost no germination with the rate of only 8.67%; retroflexus radicle was sensitive to toxins, when the concentration was 100μg.mL-1, the inhibitition rate of toxin for retroflexus radicle was 88.93%, when the concentration was 200μg.mL-1, retroflexus radicle was hardly elongation; toxins can cause retroflexus leaf necrosis. Under the toxin concentrations of 200μg.mL-1 , if treated wound retroflexus leaves for 48 h, the leaves would appear brown necrosis which resembled as the necrosis caused by the pathogenic bacteria infection.