Investigating Glyphosate Tolerance In Field Bindweed (Convolvulus Arvensis L.) And Ivy Glorybind (Calystegia Hederacea Wall.)

Glyphosate [N-(phosphonomythyl) glycine] is a non-selective, broad-spectrum herbicide, which is mainly used in the non-farming ares, the orchard, the forest and fields before crop growning, prevents and controls the annual and the perennial weeds. Reseantly, more and more glyphosate was used in the cotton and the corn fields by protective atomization. Field bindweed (Convolvulus arvensis L.) and Ivy glorybind (Calystegia hederacea Wall.) are perennial weeds, which belonging to convolvulaceae and reproducting by their rhizomes and seeds. The rhizomes are easy to break while in farming and cultivation, very difficult to clean out. Each cut-off can also reproduce and the vitality is greatly strengthened. Due to winding growth on the periphery plants, they are difficult to control and can cause severe damage to crop growth and yield loss. Glyphosate tolerant C. arvensis population is naturally existied (DeGenaro 1984). This study, both C. arvensis and C. hederacea in China are investigated in their tolerance to glyphosate by bioassay, biochemistry inspection and DNA analysis, to verify C. arvensis and C. hederacea tolerance in China. The main findings are as followings:(1) The differences of sensitivity to glyphosate in population of C. arvensis and C. hederacea were not significant. The regression curve of aerial fresh weight inhibition rate was relatively more appropriate indicator of the resistance of C. arvensis and C. hederacea to glyphosate. It was cleare that C. arvensis was tolerant to glyphosate, meanwhile C. hederacea was only tolerance to glyphosate in a certain degree.(2)Shikimate accumulation in C. arvensis and C.hederacea were slighterly higher than control 1 day after being treated with glyphosate, it began to accumulate rapidly 6 days after treatment. It indicated that both C. arvensis and C. hederacea were not sensitive to glyphosate. Shikimate accumulation in C. arvensis was significantly lower than C. hederacea when treated with the same dose of glyphosate. Therefore, C. arvensis was highly tolerant to glyphosate comparing to C. hederacea. For C. arvensis, the longer the growth was, the stronger the tolerance to glyphosate.(3) The full length cDNA of EPSPS gene from both C. arvensis and C. hederacea are 1751bp, with QRF of 1560bp, encoding 520 amino acids; the start codon located at 51~53bp, the stop codon follen at 1611~1613bp, 5' non-translation area are 98bp, achieved whereas 97.94% identical. The deduced amino acid sequence is 98.1% identic.(4) The EPSPS chloroplast transit peptide length of both C. arvensis and C. hederacea was 72 amino acid residues with 94.4% similarity, there were eight differences in transit peptides representing 57% (8/14) of differences in the total amino acid sequence. The serine at position 101 (Ser101) of C. arvensis EPSPS was a polar amino acid with polar hydroxyl group and water affinity, however, in C.hederacea leucine, a non-polar hydrophobic amino acid presented at the same location.(5) The total length of EPSPS gene of C. arvensis L. was 2953 bp, containing 6 introns and 7 exons. The length of 7 exons was 390, 310, 219, 60, 217, 63, 315 bp, respectively, of which, the largest one was exon 1 with a length of 390 bp; the smallest one was exon 4 with a length of 60 bp. The length of 6 introns was 393,220,365,172,124,105 bp, respectively, of which, the largest one was intron 1 with a length of 393 bp; the smallest one was intron 6 with a length of 105 bp.(6) The Ser101 of C. arvensis EPSPS was close to conserved active site of EPSPS. It is very likely that the polar amino acid could bind with glyphosate firstly, therefore glyphosate could not occupy the PEP binding site and the activity site was protected. Subsequently, changes in the PEP binding site might affect the response of EPSPS to glyphosate leding the tolerance of C. arvensis to glyphosate.In summary, glyphosate resistance of C. arvensis and C. hederacea in China was investigated for the first time, glyphosate resistance of C. arvensis and C .hederacea detecting critior was established, the full length of cDNA in EPSPS gene from both C. arvensis and and C. hederacea were cloned for the first time, EPSPS gene of C. arvensis and C. hederacea was registered in the GenBank (the accession number: EU698030, EU526078). Results of bioassay, biochemistry inspection and DNA analysis revealed that C. arvensis was resistant to the glyphosate. This study has not only contributed to the stipulation of the National Science and Technology R & D Programme, but paved the new way for further research in glyphosate resistant weeds thoroughly, especislly to the studies of glyphosate tolerant mechanism in C. arvensis and C. hederacea, with very important theoretical and practical significances.