Fluazifop-P-butyl, a selective graminicide, has been widely used to control annual grass weeds for more than three decades in Taiwan, and a resistant (R) biotype of goosegrass has consequently appeared. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS) was applied to analyze metabolites of fluazifop-P-butyl in a R biotype of goosegrass. Six signals, including mass-to-charge ratios (m/z) 512, 432, 423, 415, 314, and 160, in positive scanning mode, and four signals, including m/z 788, 623, 593, and 162, in negative scanning mode, were found in the metabolites of the R and S biotypes, respectively. All of the signals of these metabolites in the R biotype showed higher intensity than those of the S biotype, except for m/z 162. Based on the molecular weights of the fragments (MS2 signal) from the molecules (MS1 signal) and comparison with the Metabolite Link Metabolomics database, MassBank database, and related references, one reduced form of fluazifop acid, i.e., 2-[4-(5- trifluoromethyl-2-pyridyloxy) phenoxy] propanol (MW 313); two types of intermediates of MW 163, i.e., 5-trifluoromethyl-2-pyridone and 5-trifluoromethyl-2-hydroxypyridine (or 2-hydroxy-5-trifluoromethyl pyridine); and five possible conjugated compounds containing a common core fragment (MW 255) from fluazifop acid were identified. In addition, another compound, likely degraded from one of the five conjugated compounds, was also detected. Accordingly, the metabolic pathway of fluazifop-P-butyl in goosegrass is described in this study. An enzyme kinetic study on glutathione S-transferase showed that the R biotype has higher affinities to the substrates reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene, with S/R Km ratios of 3.0 and 2.4, respectively. No difference in Vmax was found, revealing that the S biotype has a strong ability to bind GSH and herbicide or target molecules and showed susceptibility to fluazifop.

The distribution, growth, and seed germination ability of the exotic lead tree in Penghu Islands, Taiwan, were studied. Survey results have shown that all lead tree plants existing throughout these islands belonged to the Hawaii type. Subsequently a comparison of germination requirements between this and the rare Salvador type, which distributes only in the island of Taiwan, was carried out. The objective of this research was to compare the germination requirements of the Salvador- and Hawaii-type lead tree plants. Lead trees in Penghu Islands have formed a pure forest with a height ranging from 2 to 4 m. All growth stages of the lead tree, i.e., vegetative, flowering, podding, and depodding stages, were found simultaneously in fall, but only vegetative and podding stages were observed in summer. Germination of both the Hawaii- and Salvador-type lead tree was evaluated under different environmental conditions, including temperature, pH, osmotic potential, burial depth, and lighting condition. The Salvador-type exhibited more than 60% seed germination after 14 d at temperatures between 20 and 35 C, whereas the seed germination of the Hawaii type increased from 20 to 60% with temperature increases from 20 to 35 C. The optimal temperature for seed germination of these two types of lead tree was 35 C. Increasing burial depth beneath the soil surface and darkness, as well as an osmotic potential below −0.4 MPa, also reduced seed germination. However, the germination of both types was not affected by pH ranging from 4.0 to 9.0. On the basis of the climatic and soil environment conditions in Penghu Islands, it is reasonable to predict that the summer season, in which lead tree seeds readily germinate on the ground or in shallow soils, is the optimal time for controlling this plant.

Out of 1,343 mutant lines of rice mutated by sodium azide from the parental Japonica-type variety ‘Tainung 67’ (TNG67), a paraquat-susceptible line 1192 and a paraquat-tolerant line 72 were selected using whole seedlings at the four-leaf stage and leaf segments at the tillering stage as test materials. Further selection from progenies of these two mutant lines yielded the susceptible 1192-11 (S) and tolerant 72-16 (T), which were studied herein. Chlorophyll fluorescence, electrolyte leakage, and lipid peroxidation were measured for leaf segments of rice following treatment with 0.1 mM paraquat. A comparison of these responses among the three rice lines (TNG67, 72-16, and 1192-11), revealed a higher tolerance to paraquat in the tolerant mutant line 72-16 and the parental variety TNG67 than in the susceptible mutant 1192-11. Analysis of the antioxidative system in paraquat-treated leaf segments showed that the reduced form of glutathione (GSH) and the ratio of GSH to total glutathione increased by 3.5-fold within 6 h after treatment (HAT) and up to 5-fold 9 HAT in the T line, as compared with the S line. In view of the high activities of both dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in paraquat-treated leaves of TNG67 and the T line, the antioxidative effect of the ascorbate–glutathione cycle is hereby proposed to play an essential role in paraquat tolerance of rice. Pretreatment of rice segments with spermine enhanced DHAR and GR activities as well as paraquat tolerance of the S line. These results suggest that the activity of ascorbate–glutathione cycle induced by spermine is involved in rice tolerance to this herbicide. Although kinetics studies showed no significant difference among the three rice lines in paraquat inhibition of GR, a lower affinity of enzyme to substrate (Km) in TNG67 and the T line and a higher maximal reaction rate (Vmax) in the T line for the oxidized glutathione substrate (GSSG) were detected. These observations further implicate the importance of glutathione reductase in paraquat tolerance of rice.

In order to explore the physiological mechanism of paraquat resistance in tall fleabane, a widespread weed in Taiwan where resistance to this herbicide has been observed since 1980, the role of the antioxidative system was assessed. The susceptible (S) and resistant (R) biotypes of tall fleabane were distinguished clearly by the relative distribution frequency of injury index caused by 78 µM paraquat. Although malondialdehyde, an indicator for peroxidation damage to the plant, in the the R-biotype was not changed, in the S-biotype malondialdehyde increased within 2 h after treatment of 50 µM paraquat. Analysis of several antioxidants and pertinent enzymes revealed that ascorbate peroxidase activity was decreased by paraquat treatment in the S-biotype; and a lower basal level of ascorbate was present in the S-biotype as well. The maintenance of a high ratio of reduced glutathione to total glutathione, coupled with a pronounced and rapid increase of glutathione reductase (GR) activity in the the R-biotype, suggests that an active reduced glutathione/oxidized glutathione (GSH/GSSG) cycle is critical to paraquat resistance of tall fleabane. The decisive contribution of a functional GSH/GSSG cycle to paraquat resistance through an enhancement of GR activity in this weed was further confirmed by an experiment of exogenous application of ascorbate.

Biological and biochemical methods, based on glufosinate inhibitory effects on plant growth and nitrogen metabolism, were examined for their applications to detect this herbicide. Dose–response analysis of radicle growth inhibition showed that, among six vegetables tested, Chinese mustard and edible amaranth were the most sensitive to glufosinate. Field mustard and cruciferous Ching-Geeng were found to be more sensitive to this herbicide than the other four vegetables when three-leaf seedlings were tested in another bioassay. In three-leaf seedlings of Ching-Geeng, accumulation of ammonium, a biochemical marker for glufosinate toxicity because of its inhibition of glutamine synthetase, showed a linear regression to the log-transformed concentrations of glufosinate ranging from 7.5 × 10−5 to 1.5 × 10−3 M. For the detection of glufosinate lower than 7.5 × 10−6 M, a linear regression was observed between ammonium accumulation and the applied concentration, instead of the log-transformed value, of glufosinate. A similar relationship was observed between the accumulation in Ching-Geeng seedlings of glyoxylate, another biochemical marker, and glufosinate but with a narrower range than that for ammonium accumulation. The applicability of ammonium accumulation in three-leaf seedlings of Ching-Geeng to detect glufosinate residue in water and soil was confirmed by high-performance liquid chromatography (HPLC).

A study was conducted to determine the dose–response of purple nutsedge tuber sprouting and plant growth to glyphosate. Tuber sprouting on day 6 satisfactorily fitted the probit model, with a predicted effective dosage causing 50% inhibition of response (ED50) of 30 mM, which is similar to that with 25 mM of ED50 on day 3. In addition, together with the dose–response analysis of bud growth on day 6, it was shown that bud growth of purple nutsedge was more sensitive to root- or tuber-absorbed glyphosate or both than was tuber sprouting; the former had 1.25 mM of ED50. During the V1–2 stage, purple nutsedge injury, including shoot dehydration, wilting, and yellowing, appeared sequentially when glyphosate was absorbed. When compared with the dose–response of shoot greenness of purple nutsedge at the V5–7 stage under the root-absorbed glyphosate treatment, purple nutsedge at the V1–2 stage was less sensitive to root-applied glyphosate. Further studies for determining the efficacy of glyphosate applied on root and foliage of purple nutsedge at the V5–7 stage showed that injury from glyphosate occurred within 7 d after foliar treatment, and the ED50 values of glyphosate for shoot survival and leaf chlorophyll were 13.1 and 14.5 mM, respectively. However, the response of plants was less sensitive to root-absorbed glyphosate, which had an ED50 of 101 mM. This finding might be the result of direct injury of roots caused by glyphosate, resulting in the delayed and diluted effect of this herbicide from root to shoot.

At 10.4 mM bentazon treatment, the tolerance index of susceptible (S) inbred corn, line TN89, at one-leaf stage decreased to 27% of that of the control, whereas > 90% of tolerance index of control was maintained in tolerant (T) line LU21. More than fourfold of malondialdehyde (MDA) accumulated in S line within 7 d after treatment (DAT), but only a slight accumulation of MDA was found in T line. 14C-Bentazon application experiment indicated that there was no difference in bentazon absorption between T and S lines. However, bentazon metabolism in T line was more active than that in susceptible TN89. The metabolite, 6-glucose-bentazon in T line rapidly accumulated to the maximum 3 DAT, whereas this conjugate actually decreased in S line. Assay of in vitro activity of bentazon-6-hydroxylase showed that it was decreased in both lines with development and that this activity in T line at two-leaf stage was ca. 50% higher than that in S line. It is suggested that the higher bentazon tolerance in LU21 is primarily associated with an active bentazon metabolism, partially due to a higher bentazon-6-hydroxylase activity coupled with glucosylation.

The effect of glyphosate on aromatic amino acid metabolism in purple nutsedge sprouted tubers and shoots was investigated. Glyphosate at 33.5 mM caused inhibition of bud elongation, increased total free amino acid concentration, and caused rapid accumulation of shikimic acid in sprouted tubers. However, only one aromatic amino acid, tryptophan, decreased quickly to 22% of control 3 d after treatment (DAT) and remained low afterwards. This suggests that the inhibition of bud elongation is due to the rapid accumulation of shikimic acid and the repression of tryptophan synthesis. Foliar application of glyphosate at 14.5 mM to purple nutsedge shoots resulted in the rapid accumulation of glyphosate which was rapidly converted to its metabolite, aminomethylphosphoric acid. Free amino acids in leaves were also increased by glyphosate 3 DAT. The reduction in soluble protein 5 DAT and increased acid protease activity 3 DAT suggests that the late accumulation of free amino acids partially resulted from protein hydrolysis. Shikimic acid accumulated in glyphosate-treated leaves 5 DAT, but the concentration of the three aromatic amino acids was not reduced. This suggests that glyphosate toxicity in purple nutsedge shoots was associated with the rapid accumulation of glyphosate, followed by large accumulation of shikimic acid. Aromatic amino acids deficiency was apparently not a factor in toxicity.

In order to clarify the translocation and metabolism of glyphosate in lead tree, a mixture of glyphosate (0.5 mmol) and 14C-glyphosate was injected into mini tree plants (1.5-cm trunk diameter and 120 cm tall), simulating a grown lead tree in a greenhouse experiment. Within 6 d after treatment (DAT), all leaves on the lead tree plants dropped. Analysis of nonlabeled glyphosate in both xylem and phloem every 15 d showed that glyphosate residues accumulated mainly in phloem, similar to the findings in established lead tree. In this experiment, glyphosate concentration in the phloem of three parts of the trunk, i.e., upper (15 cm above the upper injection hole), middle (between the two injection holes), and lower (15 cm below the lower injection hole) parts, increased rapidly within 45 DAT, and decreased thereafter. Obviously, significant glyphosate metabolism occurred during the latter period. Further 14C radioactivity measurement also exhibited similar time-course changes of glyphosate dissipation, except that a low level of radioactivity in the phloem of the upper part and a high level of radioactivity in the lower part were detected from 45 to 90 DAT, suggesting that more glyphosate metabolites in the phloem of the upper part might have translocated with photosynthetic assimilates and reallocated to the lower part of the mini tree. Thin-layer chromatography (TLC) analysis of metabolites derived from 14C-glyphosate also revealed that about 70% of the radioactivity recovered in the phloem of the lower part comprised the unknown metabolites M1 (retention factor [Rf] : 0.83) and M2 (Rf : 0.94), with nearly 28% of the radioactivity from sarcosine (Rf : 0.76), and less than 2% of the radioactivity from aminomethylphosphoric acid (AMPA) (Rf : 0.49) and glyphosate (Rf : 0.36). It is concluded that active metabolism of glyphosate in this 90-d simulation experiment occurred mainly from 45 to 90 DAT, and the primary metabolism was modulated by C-P lyase.

The objective of this study was to evaluate the feasibility of using 2-aminobutyric acid (2-aba) as a chemical marker for the detection of sulfonylurea (SU) herbicides in crop and soil environments. A bioassay, on the basis of the injury index to vegetable seedlings, showed that both field mustard and Chinese mustard were more sensitive to bensulfuron, imazosulfuron, and pyrazosulfuron than lettuce and cabbage in winter. In a similar study, field mustard was found to be the most sensitive species among six summer vegetables. After foliar application of bensulfuron at 0.487 μM, 2-aba accumulation reached the maximum in field mustard within 6 to 12 h, before any visible symptoms appeared, and this high level was maintained up to 72 h. Among all vegetables tested for differential SU sensitivities, maximum accumulation of 2-aba occurred, in coincidence with the onset of injury, in field mustard. The accumulation of 2-aba in field mustard showed a linear regression to the log-transformed concentrations, ranging from 10−2 to 102 μM, of each of the three SUs. However, no such relationship between the applied rate, especially at the lower rates, and the residue content of SUs in field mustard plant was found. In addition, with the extract of SU-treated paddy soil sprayed on field mustard, the accumulation of 2-aba in this plant was found to reflect indirectly the amount of SU residues in the soil.

The physiological basis of glufosinate resistance for two resistant (R) rice mutants, lines ‘R11-2’ and ‘R11-3’, was studied. Seven days after the application of 0.54 mM glufosinate, two susceptible (S) lines, i.e., variety (var.) ‘FSK’ and its inbred line ‘FSK-3’, and a reference var. Tainung 67 (TNG 67) suffered severe injury, whereas the two R lines exhibited resistance. Dose–response analysis and survival rate 14 d after treatment with 1.5 mM glufosinate also supported this observation. A 14C-glufosinate experiment showed that more labeled herbicide was absorbed by leaves of R11-2 than S lines 48 h after treatment (HAT), but the partitioning of absorbed glufosinate to each part of the shoot did not differ between R and S lines. Although a higher degradation of glufosinate in R line R11-2 was found as compared with the two S lines, i.e., 46% vs. 38 to 40%, the actual concentration of glufosinate in R line was still higher than that in S lines. Foliar application of glufosinate resulted in less inhibition of in vivo activity of glutamine synthetase (GS; EC 6.3.1.2) as well as a lower accumulation of ammonium 24 HAT in R line than in S lines. Further kinetic study of GS showed that cytosolic GS in line R11-2, with a higher enzyme-inhibition constant (Ki) value to glufosinate, was less sensitive to the toxic action of this herbicide. Therefore, a higher metabolism of, and more important, a lower susceptibility of, the target protein GS to this herbicide are suggested to contribute significantly to glufosinate resistance in these rice lines.

In order to select efficient and competitive glufosinate-degrading bacteria, two soils which had been treated with glufosinate annually for more than 5 yr were screened. Three strains tolerant to this herbicide were identified by 16S rDNA analysis as Burkholderia sacchari, Serratia marcescens, and Pseudomonas psychrotolerans. In addition, a moderately tolerant strain, P. citronellolis, was isolated from a soil which had received glufosinate treatment for only 6 mo. In culture medium containing high concentration of glufosinate, the former three strains showed significant ability to degrade this glutamine synthetase inhibitor, suggesting that glufosinate-degrading bacteria would be readily found in soils after a long-term induction or selection. A subsequent biodegradation experiment showed that 30 and 50% of glufosinate was degraded 7 and 21 d after treatment (DAT), respectively, in sterilized soils inoculated with the above-mentioned three tolerant strains. While more than 30% of the glufosinate in nonsterilized soils was degraded 7 DAT by the indigenous edaphic microbes, inoculation with the three selected strains enhanced glufosinate degradation to nearly 50%. A study on the competition from edaphic microorganisms in soils by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed that within 21 d after inoculation (DAI), the propagation of B. sacchari and P. psychrotolerans was not affected, whereas that of the less tolerant P. citronellolis was inhibited. This observation suggests that a long-term herbicide exposure is a promotive factor in generating bacterial strains having high degradation efficiency and showing vigorous propagation under the competition pressure arising from indigenous microbes.