Study On The Biology And Ecology Of Annual Bluegrass(Poa Annua L.)and Its Mechanisms Of Tolerance To Fenoxaprop-P-Ethyl

Annual bluegrass (Poa annua L.), a kind of worst grass weed in wheat (Triticum aestivum L.) and oilseed rape (Brassica campestris L.) fields in the middle and lower reach of Yangtze valley in China, was chosen as a main research object in this paper. The biological and ecological characteristics of P. annua and its mechanisms of tolerance to herbicide fenoxaprop-P-ethyl were studied to elucidate why the occurrence of P. annua were increased gradually and to make theoretic complementarity for herbicide tolerance mechanism of P. annua. In general, this paper did some exploration in following aspects: the characterization of seed dormancy and germination of P. annua; the characteration of fructification of mature P. annua plants, the occurrence dynamics, the population biomass and the influence of different kind of preceding crops and cultivation on plant biomass; the susceptibility of ACCase (acetyl coenzyme A carboxylase) to herbicide fenoxaprop-P-ethyl from P. annua; the relations of herbicide tolerance of P. annua with ACCase activity, amino acid sequence of ACCase CT domain, relative expression level of ACCase genes, GST activity, contents and activity of cytochrome P450monooxygenase, and anti-oxidant indexes.1.Biological and Ecological Characteristics of P. annuaThe mature seeds have about20days dormancy after being collected from fields. The dormancy of seeds could be broken by being buried underground5cm, buried ungerground5cm with water, soaked in the water at4℃after being disinfected using0.5%sodium hypochlorite (NaCIO). The seed dormancy of P annua could be broken by treated with chemicals such as250mg·L-1GA3,500mg·L-1KNO3and31.25mg·L-1Ethopon for24h. The optimum temperature range for seed germination was10～25℃and light was not necessary. Seed germination was sensitive to osmotic potential. But it was quite tolerant to salinity. More than80%of seeds could be germinated at pH values ranged from4to10. Seedling emergence was higher (55%) when seeds were sown at the depth of0～2.0cm.Few seedlings emerged when seeds were planted at a depth of3.0cm. Information gained in this study will load to a better understanding of the requirements of P. annua gerimination and seedling emergence.Fruiting traits of P. annua were investigated before the seed exfoliated in wheat fields rotated with rice. Its average plant height was33.96±1.89cm, average tillering nodes were6.7±1.2, average productive tillering nodes were5.6±1.2, average grain number per spike was1004±155and average1000-grain weight was0.2944±0.02g. Ecology exploration of P. annua showed that a main emergence fastigium was observed in winter with majority seedlings. The zooming period of plant height and fresh weight of P. annua occurred at15WAP (weeks after planting), which was three weeks later than wheat, and suggested that chemical control and fertilization should be performed within15WAP.The seedling emergences of P. annua in the wheat fields were varied when it occurred in the different previous crop and different tillage methods. The seedling emergence was lowest when it occurred in the wheat fields, which corn (Zea mays) was the previous crop and the wheat was planted after shallow tillage.2. The Sensitivity of P. annua to Herbicide Fenoxaprop-P-ethyl and Other HerbicidesThe susceptibility of P. annua to fenoxaprop-P-ethyl were identified by seed-bioassay and whole-plant test, which were collected from different fields, and similar results were obtained by both of methods. The biotypes, which had never been applied with fenoxaprop-P-ethyl, had very high ED50values which were not impossibily applied in the fields. This indicated that P. annua was tolerant to fenoxaprop-P-ethyl.The sensitivity level of Alopecurus aequalis, A. japonicus, Beckmannia syzigachne, Sclerochloa kengiana, and Polypogon fugax to fenoxaprop-P-ethyl were evaluated by whole-plant bioassay, which were collected from Zijin Mountain (Nanjing, Jiangsu), where fenoxaprop-P-ethyl had never been applied. The A. aequalis was the most sensitive to fenoxaprop-P-ethyl among the five grasses.The sensitivities of P. annua and susceptible reference weed A. aequalis to other ACCase inhibiting herbicides were evaluated by whole-plant bioassay. P. annua and susceptible weed A. aequalis were collected from Zijin Mountain (Nanjing, Jiangsu), where fenoxaprop-P-ethyl had never been applied. P. annua was found to be tolerant to fenoxaprop-P-ethyl as well as haloxyfop-P-methyl, clodinafop-propargyl, fluazifop-P-butyl, cyhalofop-butyl and sethoxydim, whereas it was sensitive to clethodim and tepraloxydim.The susceptibility of P. annua to19herbicides belong to different classes was determined by pre-emergence applied methods. Ethametsulfuron-methyl, sulfosulfuron, diflufenican, flufenacet, bispyribac-sodium, and metazachlor could be used to control P. annua through pre-emergence applied.The susceptibility of P. annua to15herbicides belong to different classes was determined by post-emergence spraying methods. Ethametsulfuron-methyl, mesosulfuron-methyl, flufenacet, paraquat, glufosinate, and glyphosate could be used to control P. annua through post-emergence spraying.3.The Mechanisms of Tolerance to Fenoxaprop-P-ethyl in P annuaThe IC50(the concentration of fenoxaprop-P-ethyl (free acid) that inhibited50%of acetyl coenzyme A carboxylase (ACCase) activity) values for P. annua was1.70-fold higher than that for A. aequalis, which was susceptible to fenoxaprop-P-ethyl in fields. The presence of the Leu-1781residue, which had been reported to be involved in the resistance of gramineous weeds to ACCase inhibitors, was subsequently identified in the plastidic ACCase of P. annua. Furthermore, the relative gene expression level of P. annua ACCase genes was found to be approximately2.4-fold higher than that in A. aequalis, possibly explaining the tolerance to fenoxaprop-P-ethyl of P. annua.The1-chloro-2,4-dinitrobenzene (CDNB) were used as model substrates to detect the activities of glutathione s-transferases (GST) in P. annua and susceptible A. aeuqlis in order to clarify the relationship with GST metabolic tolerance. The activity of GST in P. annua was higher than that in A. aequalis without herbicide treatment. GST activity in P. annua increased slowly after treated with fenoxaprop-P-ethyl, then it decreased to being similar to the untreated plants after3days treatment. The GST activity in A. aequalis increased when1day after treatment, then decreased to being smaller than the untreated plants in all of the cases.The contents of cytochrome P450and cytochrome b5in P. annua and A. aequalis were determined by spectrophotometric determination. After treated with fenoxaprop-P-ethyl, the contents of cytochrome P450and cytochrome b5in P. annua were significantly increased, compared with untreated plants. However, the increasing was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of cytochrome P450monooxygenases mediating p-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-dethylase (EROD), ethoxycoumarin Oxidase (ECOD), and NADPH-dependent cytochrome P450reductase were determined by influorescence spectrophotometry. The activities of cytochrome P450monooxygenase mediating PNOD, EROD, ECOD and NADPH-cytochrome P450reductase had increased in P. annua after application of fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450reductase. The O-deetylylation and reduction catalyzed by cytochrome P450monooxygenases may be involved in the metabolism of fenoxaprop-P-ethyl. It was also suggested that the induction of cytochrome P450monooxygenase activities is the mechanism involved in the fenoxaprop-P-ethyl tolerance observed in P. annua.The effects of fenoxaprop-P-ethyl and cytochrome P450monooxygenases inhibitor piperonyl butoxide (PBO), malathion and1-aminobenzotriazole (ABT) on the weed growth were evaluated by whole-plant bioassay. It had been found that PBO and ABT synergized fenoxaprop-P-ethyl activity in P. annua, while malathion had little effects. But the ED50vulues of fenoxaprop-P-ethyl had been applicaed with PBO or ABT were still very higher than the recommended dose in fileds. The synergisms of PBO and ABT valideted that cytochrome P450monooxygense is one of the mechanisms involved in the fenoxaprop-P-ethyl tolerance observed in P annua.The activities of superoxide dismutase, catalase, peroxidase, and contents of superoxide anion radial (O2·-), reduced/oxidized glutathione (GSH/GSSG), hydrogen peroxide (H2O2), ascorbic acid (AsA), thiobarbituric acid reactive substances (TBARS), and soluable protein in P. annua and A. aequalis were detected. Significant differences of all the indexes mentioned above were observed in P. annua and A. aequalis after treated with fenoxaprop-P-ethyl. The lower oxygen damages and higher activities of SOD, POD and CAT in P. annua indicated that tolerance due to oxidative stress may be one of the mechanisms of tolerance to herbicide fenoxaprop-P-ethyl in P. annua.