Effects Of Glyphosate On The Soil Microecosystem And Research Of Glyphosate-degradation And Glyphosate-resistance Fungi

Glyphosate is a kind of herbicide with high-effect and low toxicity and it is widely used in the world. But along with the excellent weeding effect, it also possesses some shortage. A long-time usage of glyphosate may destroy the soil micro ecosystem of farmland, and its broad-spectrum feature could kill the crops. So, the researches on repair of glyphosate-contaminated microorganism and the genetically modified anti-glyphosate plants are of great significance. Our works are mainly about hereinafter aspects:First, we studied the effects of glyphosate have on micro ecosystem of soil;Second, we screened and identified a fungi strain JLC 31721 with high ability of glyphosate degradation and a strain JLC 31764 with the extreme tolerance of glyphosate;Third, we optimized the culture condition of JLC 31721 in order to find the highest degradation effect;Fourth, we cloned a part of glyphosate tolerance related gene"aroM"of JLC 31764.By the determination the quantity of bacteria, fungi and actinomycetes, the measure of activity of urease, catalase and acidic phosphatase in the soil treated with glyphosate on a range of concentrations, we have estimated the secure concentration of glyphosate usage. We found that if the concentration of glyphosate is above 10mg/kg soil, the quantity of microbes and the activity of enzymes would be affected evidently. After treated by the herbicide, the quantity of bacteria and fungi in soil decreased, then it rebounded and finally surpass the control group's level after a short time. It reflects that the glyphosate could restrain the quantity of bacteria and fungi first and activate it later. The quantity of actinomycetes decrease sharply after treated with the herbicide, and this restriction would last, which means the glyphosate have high toxic effect on actinomycetes. The activity of the three enzymes in soil shown a similar change as the quantity of bacteria and fungi do, which is restrained first and then activated. But the results have shown that the urease is more sensitive to glyphosate than catalase and acidic phosphatase.By enrichment and screen of the microbes from abundant soil samples contaminated by glyphosate for a long time, we obtain a fungus named JLC 31721 which has the ability to degrade 65.8% of 500mg/L glyphosate in 7days; and a fungus named JLC 31764 which can grow naturally in the medium with 600mmol glyphosate. After morphological identification and rDNA ITS sequence comparison, we confirm the two strains as Aspergillus flavus and Paecilomyces lilacinus.In order to increase the degradation ratio of JLC 31721, we optimized the culture condition. The optimized parameters include adding carbon source and phosphor source, PH value, temperature, ventilator capacity and original glyphosate concentration. By determining the degradation ratio in different conditions, we got the optimum culture condition as follow: glyphosate as the only phosphor source in the culture broth; adding some glucose as supplementary carbon source; the broth hold 1/5～1/3 volume of the triangular flask; cultured in PH 6.0, temperature 28℃and original glyphosate concentration 50mg/L. After 7 days culture, JLC 31721's degradation ratio could rise to 90%. We also examined the glyphosate degradation kinetic curve of JLC 31721 cultured in the optimum condition. The results show that during 0-48h period, the degradation ratio was extremely low, only about 20%; during 48-72h, the degradation ratio increased sharply to 80%; then the increasing trend slowed down, 120h later it maintained on the level of 90%.The strain JLC 31764 shows supreme resistance ability to glyphosate, which reflect its glyphosate-act-related gene AroM may have mutated, this gene could be used to construct anti-glyphosate transgenic plants, which have high applied value. As the aroM sequence of Paecilomyces lilacinus hasn't be reported yet, we referred to other fungi's sequences to design a pair of degeneracy primers. We cloned a 400bp DNA fragment from the cDNA of JLC 31764 strain by using this pair of primers. Then we ligated the fragment to pMD 18-T vector for sequencing. The sequencing results show that the fragment have some homology to the AroM gene of Sclerotinia sclerotiorum and Pichia pastoris, but the homology is low (below 30%), so we can not confirm if the fragment is a part of aroM gene of JLC 31764. We will continue to clone and analyze the cDNA which the fragment in to confirm if it is the aroM gene of JLC 31764, meanwhile, we will design other degeneracy primers to clone the aroM gene of JLC 31764.