Plant Protection Quarterly Vol.9(1) 1994 15 this study. The objective of this study was, therefore, to evaluate the effects of these Effects of glufosinate-ammonium and terbuthylazine two herbicides on germination and seed- ling growth of two weed species preva- on germination and growth of two weed species lent in plantation crops of Malaysia.

Ismail Sahid and K. Kalithasan, Department of Botany, Universiti Materials and methods Kebangsaan Malaysia, 43600 UKM, Bangi, Malaysia. Weed seeds Seeds of a broadleaf weed, A. gangetica, Summary and a grass species, P. conjugatum, were Effects of glufosinate-ammonium and control 100% of the weed seed pop- collected from a rubber estate near Sungai terbuthylazine on germination and ulations. Clearly, a weed control measure Buluh, Selangor. After air-drying, har- growth of two weed species were evalu- has to severely reduce the weed seed vested seeds were kept under laboratory ated in the laboratory and in the green- population in the soil to be effective. Re- conditions in air-tight glass bottles until house. Germination of Asystasia gang- search on the effects of herbicide on seeds used. etica (L.) T. Anders. and Paspalum in soil may contribute to knowledge on conjugatum Berg. declined considerably the effective control of weed seed Herbicides when seeds were germinated in petri populations. Glufosinate-ammonium (ammonium-DL dishes containing either glufosinate-am- The effects of herbicidal treatment on -homoalanin-4-yl(methyl) phosphinate) monium or terbuthylazine at 250 mg L-1. seed reserves in the soil or on subsequent was obtained from Hoechst (Malaysia) Glufosinate-ammonium also caused a growth of weed seedlings form an impor- under the trade name Basta®; it contains marked reduction in radicle elongation tant aspect of weed control. However, not 150 g a.i. L-1. Terbuthylazine (2 tert. of Asystasia, whose fresh weight was many studies have reported on the effects butylamino-4-chloro-6-ethylamino-5- reduced proportionately with increasing of herbicide on seed germination; it seems triazine) was supplied by Ciba-Geigy concentrations of either glufosinate- that only paraquat, glyphosate, prona- (Malaysia) under the trade name ammonium or terbuthylazine. Terb- mide, propham and metribuzin (Young Gardoprim®; it contains 500 g a.i. L-1. uthylazine was more effective than et al. 1984, Salazar and Appleby 1982) glufosinate-ammonium in reducing ger- have been studied for such effects. Laboratory experiment mination and growth of both weeds Glyphosate or paraquat applied directly Fifty seeds each of the two weed species when applied either to seeds on the soil to seeds on the soil surface may reduce were placed on 9 cm filter paper in a se- surface or to the soil prior to sowing. germination and/or seedling growth of ries of petri dishes that contained 8.0 mL Storage of seeds after treatment with several grass species (Salazar and of the herbicide solution or distilled wa- either herbicide reduced the inhibitory Appleby 1982, Egley and Williams 1978, ter. The experimental design was a effect of the herbicide on germination Klingman and Murray 1976). randomized complete block with five rep- and growth of the weeds. Residual Glufosinate-ammonium is used in lications per treatment. The concentra- terbuthylazine in soil 14 days after treat- plantation crops such as cocoa, oil palm tions of glufosinate-ammonium or ment could still reduce germination and and rubber as well as in vegetable crops terbuthylazine used were 0 (control), 25, growth of the weeds. and on non-crop land to control a broad 50, 100 and 250 mg L-1. spectrum of grasses, broadleaf weeds, The petri dishes were placed in dark Introduction sedges and ferns (Kuah et al. 1989, germination chambers at a constant tem- Asystasia gangetica and Paspalum conjug- Rodrigo 1988, Kassebeer et al. 1983). It is perature of 27°C. On days 3 through 10 atum are two weeds common in cocoa, rapidly adsorbed to soil particles, effec- daily germination counts were made. rubber and oil palm plantations. A ma- tively preventing degradation of the her- Seeds were considered germinated when ture A. gangetica plant produces several bicide, or degraded by soil microorgan- the radicle had protruded through the fruits which contain four seeds each. The isms, releasing carbon dioxide (WSSA seedcoat. Radicle length and fresh weight ripe fruits usually burst with considerable 1989). of 10 seedlings were recorded after 10 force, scattering the seeds one to three Terbuthylazine, a broad-spectrum s- days. The experiment was repeated twice metres away from the mother plants (Lee triazine herbicide, is presently being and the means were averaged. All data 1981). P. conjugatum spreads quickly by evaluated in plantation crops in Malaysia. were subjected to an analysis of variance means of its stolons as well as by seeds. With adequate rainfall or irrigation, with mean separation by Duncan’s multi- Approximately 1500 seeds are produced terbuthylazine is leached to deeper soil ple range test at 5% probability level. per plant (Dirven 1971). layers and is taken up by deep-rooted Such production of seeds contributes weeds. Terbuthylazine may move deeper Greenhouse experiments heavily to effective weed dispersal and es- into the soil, not necessarily because of its Four experiments were conducted in tablishment in plantations. Most agricul- greater mobility, but because it persists the greenhouse to determine the effect of tural soils contain a large reservoir of longer (Bowman 1989). In the soil it pre- glufosinate-ammonium and terbuthyl- weed seeds which germinate over a pe- vents the germination of seedlings for azine on seed germination and seedling riod of time. Weed seeds shed onto the several months after application (WSSA growth of the two weed species. The her- soil surface may remain there or be incor- 1989). bicide was sprayed using a laboratory porated into the profile by natural or arti- Past research evaluating the effect of sprayer that delivered a volume of 500 L ficial means (Pareja et al. 1985). Potential glufosinate-ammonium and terbuthyl- ha-1 at a pressure of 200 kPa. The dose ap- weed problems in most cultivated crops azine on the germination seeds of plied was 0 (control), 0.5 and 1.0 kg ha-1. including rubber and oil palm depend at A. gangetica and P. conjugatum is very lim- The soil used in the experiments was a least in part upon the weed seed bank. ited; most reports have considered only sandy loam (40% sand, 40% silt and 20% Norris (1985) suggests that weed control the control aspects at post-emergence clay) with an organic matter content of should exceed 99.99% efficiency to keep stages. Glufosinate-ammonium and 1.1% and a pH of 6.2. No artificial light the seed bank static and proposed that terbuthylazine, which have different ac- was supplied and the temperature in the an ideal weed control measure should tions on weed growth, were chosen for greenhouse was 27±3°C. 16 Plant Protection Quarterly Vol.9(1) 1994 Table 1. Germination, radicle length and fresh weight (% of control) of petri dish grown Asystasia and Paspalum 10 days after exposure to herbicides. Treatment Germination Fresh weight Radicle length (mg L-1) Asystasia Paspalum Asystasia Paspalum Asystasia Paspalum Control 100 a 100 a 100 a 100 a 100 a 100 a Glufosinate-ammonium 25 72 b 49 d 48 b 65 bcd 5 c 60 b 50 68 b 15 e 41 bc 55 cd 4 c 32 d 100 36 c 7 ef 33 c 30 e 3 c 12 e 250 11 d 1 f 20 d 5 f 1 d 3 f Terbuthylazine 25 95 a 100 a 100 a 75 b 84 a 52 bc 50 17 d 97 ab 95 a 70 bc 84 a 44 bc 100 15 d 93 b 41 bc 55 cd 84 a 40 cd 250 6 d 83 c 20 d 55 cd 52 b 52 bc Means within a column followed by the same letters are not significantly different at the 5% level by Duncan's multiple range test.

The four experiments (methods of ap- Results and discussion The results of this experiment indicate plication) were as follows: that at higher concentrations, glufosinate- i. Herbicide applied directly to seeds. Laboratory experiment ammonium caused drastic reduction of Fifty seeds of each species were placed After 10 days, each herbicide reduced radicle elongation of both species, but es- in separate petri dishes, sprayed with germination of Asystasia proportionately pecially of Asystasia. Glufosinate-ammo- the appropriate herbicide solution and with increasing herbicide concentration nium not only reduced germination of dried at 20°C for three hours. The seeds (Table 1). Seed germination of Paspalum Paspalum and Asystasia at higher concen- were then placed firmly on an un- declined significantly in the presence of trations, it also consequently reduced treated, moist soil surface in individual 25 mg L-1 glufosinate-ammonium and fresh weight and radicle elongation. At plastic trays (30 cm × 30 cm × 10 cm). prevented seed germination at 250 mg L-1. concentrations as low as 50 mg L-1, ii.Herbicide applied to seed and soil. Terbuthylazine at 250 mg L-1 caused a terbuthylazine caused a marked reduc- Seeds of each species were placed on slight reduction in the germination of tion of germination of Asystasia. the surface of moist soil in similar trays Paspalum. In the case of Asystasia, terb- and then sprayed with the appropriate uthylazine at 50 mg L-1 caused a greater Greenhouse experiments concentration of either glufosinate- reduction in seed germination (reduced i. Herbicide applied directly to seeds ammonium or terbuthylazine. by 83% of control) than did glufosinate- Direct application of glufosinate-ammo- iii.Storage of herbicide-treated seeds. Fifty ammonium at the same concentration. nium to Asystasia seeds reduced germina- seeds of each species were placed in Fresh weight of Asystasia decreased tion more than did similar treatment with separate petri dishes, sprayed with the proportionately with increasing concen- terbuthylazine. Treatment of Asystasia appropriate herbicide treatment and trations of either herbicide. For instance, seeds with 1.0 kg ha-1 glufosinate- dried at 27°C for 14 days. The seeds glufosinate-ammonium and terbuthyl- ammonium caused 95% reduction in ger- were then placed on the moist surface azine reduced fresh weight of Asystasia to mination, compared to 85% when of untreated soil. 48% at 25 mg L-1 and 41% of control at 100 terbuthyl-azine was used (Table 2). Both iv.Herbicides applied to soil. Soil in trays mg L-1, respectively. Fresh weight of herbicides also significantly reduced the was pre-moistened to field capacity and Paspalum declined progressively in the germination of Paspalum but not as se- then sprayed with the appropriate presence of increasing concentrations of verely as they reduced germination of treatment. Fifty untreated seeds of each glufosinate-ammonium. Similarly, terb- Asystasia. species were placed on soil surface on uthylazine did reduce the fresh weight of Both glufosinate-ammonium and terb- day 0 (immediately after treatment) and Paspalum but not by as much as did uthylazine significantly reduced the shoot on day 14. glufosinate-ammonium. dry weight and shoot length of Asystasia All plastic trays were placed in large The petri dish experiment shows that compared to those of the control (Tables 3 metal trays in the greenhouse for four glufosinate-ammonium reduced radicle and 4). Terbuthylazine caused greater re- weeks and sub-irrigated as needed. Ex- elongation of the two species more than duction in shoot dry weight of Asystasia perimental design was a randomized terbuthylazine. Radicle elongation of than did glufosinate-ammonium, but no complete block with five replications. Af- Asystasia was greatly reduced (95% of significant differences were observed be- ter this growth period, percent of germi- control) when germinated in glufosinate- tween the 0.5 and 1.0 kg ha-1 rates for ei- nation per tray and shoot length of five ammonium at its lowest experimental ther herbicide. Shoot dry weight of randomly sampled plants per tray were concentration of a 25 mg L-1. In contrast, Paspalum was also reduced when seeds recorded. Shoots were then harvested, terbuthylazine reduced radicle elongation were treated with either herbicide. Shoot oven-dried at 50°C for three days and of Asystasia significantly to 52% of control length of Paspalum was greatly reduced weighed. The experiment was conducted only at 250 mg L-1, its highest experimen- when seeds were treated with either her- twice, and the means were averaged. All tal concentration. Radicle length of bicide at 1 kg ha-1. the data were subjected to analysis of Paspalum was reduced proportionately The results of this experiment indicate variance followed by Duncan’s multiple with increasing glufosinate-ammonium that direct application of herbicides to range test at 5% probability level. concentration. On the other hand, increas- seeds may cause considerable reduction ing concentration of terbuthylazine had in germination. Consequently, numbers inconsistent effects on radicle length of of viable seeds may decline, thereby re- the species. ducing seed populations in soil. Plant Protection Quarterly Vol.9(1) 1994 17 ii. Herbicide applied to seeds and soil terbuthylazine caused a marked decrease Asystasia was observed at 0.5 kg ha-1 of When glufosinate-ammonium was ap- in germination of Asystasia and Paspalum terbuthylazine with 88% reduction, while plied to exposed seeds on the soil surface, germination by 94 and 93%, respectively. shoot elongation of Paspalum was unaf- it was not as effective in reducing seed Shoot length of Asystasia was also af- fected by the same treatment. Shoot elon- germination of either species as when ap- fected more by terbuthylazine than by gation of Paspalum was, however, signifi- plied directly to the seeds in petri dishes glufosinate-ammonium (Table 4). A cantly affected when treated with (Table 2). However, at the highest rate, marked reduction of shoot elongation of terbuthylazine at 1.0 kg ha-1.

Table 2. Germination (% of control) of Asystasia gangetica and Paspalum conjugatum seeds. Herbicide Spray seeds Spray seeds Spray seeds store 14 Spray soil seed Spray soil seed kg ha-1 only on soil days before planting immediately after 14 days after Asystasia gangetica Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 35 c 87 b 51 d 85 b 85 b 1.0 5 e 69 c 40 e 72 c 71 c Terbuthylazine 0.5 60 b 25 d 83 b 7 d 53 d 1.0 15 d 6 e 73 c 2 d 15 e Paspalum conjugatum Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 77 c 82 b 84 b 58 b 89 b 1.0 67 d 88 b 73 c 32 c 85 b Terbuthylazine 0.5 88 b 17 c 91 ab 10 d 78 c 1.0 65 d 7 d 91 ab 2 d 54 d Means for each species within a column followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test.

Table 3. Dry shoot weight (% of control) of Asystasia gangetica and Paspalum conjugatum seedlings. Herbicide Spray seeds Spray seeds Spray seeds store 14 Spray soil seed Spray soil seed kg ha-1 only on soil days before planting immediately after 14 days after Asystasia gangetica Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 48 b 44 b 15 c 81 b 84 b 1.0 40 b 37 c 9 c 65 c 69 c Terbuthylazine 0.5 29 c 4 d 49 b 23 d 54 d 1.0 27 c 2 e 47 b 16 e 25 e Paspalum conjugatum Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 68 b 70 b 73 b 90 a 84 a 1.0 39 c 46 c 56 bc 93 a 77 a Terbuthylazine 0.5 59 b 33 c 65 b 100 a 41 b 1.0 72 b 12 d 38 c 7 b 20 c Means for each species within a column followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test.

Table 4. Shoot length (% of control) of Asystasia gangetica and Paspalum conjugatum seedlings. Herbicide Spray seeds Spray seeds Spray seeds store 14 Spray soil seed Spray soil seed kg ha-1 only on soil days before planting immediately after 14 days after Asystasia gangetica Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 70 b 80 b 55 c 81 b 94 a 1.0 59 b 70 c 41 c 72 b 97 a Terbuthylazine 0.5 54 b 12 d 78 b 59 c 97 a 1.0 67 b 6 e 78 b 22 d 66 b Paspalum conjugatum Untreated 100 a 100 a 100 a 100 a 100 a Glufosinate 0.5 50 c 75 b 75 b 75 ab 93 ab 1.0 56 bc 38 c 68 bc 50 bc 93 ab Terbuthylazine 0.5 75 b 100 a 57 c 87 ab 73 b 1.0 43 c 13 c 39 d 25 c 27 c Means for each species within a column followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test. 18 Plant Protection Quarterly Vol.9(1) 1994 The reduction in shoot dry weight of whereas Asystasia shoot length was lost its effectiveness faster in soil than the seedlings seems to follow naturally af- similarly reduced by treatment with glufosinate-ammonium. It has been re- ter decreased germination and shoot elon- glufosinate-ammonium at the higher con- ported that the activity of s-triazine herbi- gation; all these are affects attributable to centration (Table 4). cides remained almost constant for sev- herbicide injury. Growth of Asystasia was iv. Herbicides applied to soil. eral months under glasshouse conditions more sensitive to treatment with terb- When applied to soil, glufosinate- (Horowitz 1969). An initial lag period of uthylazine. Shoot dry weight of Asystasia ammonium was not as effective in reduc- slow degradation has been observed with and Paspalum decreased by 98 and 88% of ing seed germination of Asystasia as when various herbicides, attributed to the adap- control, respectively, when terbuthyl- applied directly to seeds (Table 2). Even tation of the microorganisms to the new azine was applied at 1 kg ha-1 (Table 3). at the higher level, glufosinate-ammo- compound (McCormick and Hiltbolt This result shows that terbuthylazine ap- nium significantly reduced shoot dry 1966). Soil microbes may take a longer plied to seeds on the soil surface was ef- weight only of Asystasia. Interestingly, time to recover in soil treated with fective in reducing shoot dry weight of while terbuthylazine drastically reduced terbuthylazine. Therefore its residual ac- both species. The relative ineffectiveness germination of Paspalum at both lower tivity could be detected especially when of glufosinate-ammonium was probably and higher rates, it significantly affected applied at higher than the recommended due to its deactivation by soil adsorption. shoot dry weight and shoot length at only rate (0.4 kg ha-1). Evidence for this is the Earlier reports have shown that other the higher rate. lower rate of Asystasia germination in the non-residual herbicides such as paraquat Germination, shoot dry weight and presence of terbuthylazine even 14 days and glyphosate did not affect shoot length shoot length of Paspalum were not af- after treatment. and shoot dry weight of the grass species fected as much when glufosinate-ammo- As mentioned earlier, both weed spe- (Young et al. 1984) because these herbi- nium-treated soil was stored for 14 days cies are serious weed problems in rubber, cides become inactive upon contact with before planting of seeds (Tables 2, 3 and oil palm and vegetable crops. Their seeds soil particles (Knight and Tomlinson 4). The results of this experiment on have the capability to germinate and es- 1967). It should be noted that the activity Asystasia show virtually no differences tablish themselves while on the soil sur- of certain herbicides in soil is greatly af- whether glufosinate-ammonium-treated face. Results of this study suggest that fected by such factors as soil type, soil soil was stored or not, except for a consid- these species can be controlled to some moisture and soil organic content (Knight erable increase in shoot length when extent by terbuthylazine prior to germi- and Tomlinson 1967). Increasing amounts treated soil had been stored. No signifi- nation. Application of terbuthylazine of organic matter may reduce phyto- cant difference was observed in germina- generally reduced seed germination and/ toxicity effects of herbicides; perhaps this tion, shoot dry weight and shoot length of or seedling growth, but this effect was is the case with glufosinate-ammonium. Paspalum between the two concentrations dependent upon weed species and seed- Terbuthylazine is a soil-applied herbi- of glufosinate-ammonium when treated herbicide contact. Further experiments on cide, therefore its activity in soil is higher soil had been stored before planting of persistence of terbuthylazine under field than that of glufosinate-ammonium. The seeds. In contrast, germination and shoot conditions need to be carried out, espe- results of this experiment imply that un- dry weight of Asystasia declined signifi- cially under tropical conditions and over der field conditions, application of cantly with increasing concentration of a range of soil types. terbuthylazine as pre-emergence herbi- glufosinate-ammonium applied to soil cide might reduce germination and and kept for 14 days before sowing. Acknowledgements. growth of certain weed species such as In general, incubation of terbuthy- This work was supported by research Asystasia. lazine treated soil for 14 days before grant IRPA 01-07-03-011. The authors ex- iii. Effect of seed storage on germination planting reduced the effect of the herbi- press their sincere thanks to Dr. Anis and growth cide on the germination and growth of Rahman from Ruakura Agricultural Cen- Storage of glufosinate-ammonium treated both species. However, the germination tre, New Zealand for commenting on the seeds for two weeks before planting rates and growth of these species de- manuscript. Thanks are also due to Ciba- tended to have results similar to those of creased with increasing terbuthylazine Geigy Malaysia for providing terbuthyl- the experiment where seeds were sown concentrations (Table 2). Again, seeds of azine. immediately after treatment. However, Asystasia appeared very sensitive to the percentage of germination was in gen- terbuthylazine, especially at the higher References eral higher than when seeds were not rate. This study shows that herbicidal ac- Behrendt, H., Matthies, M., Gildemeister, stored. Terbuthylazine did not have much tivity in soil, especially of glufosinate-am- H. and Gorlitz, G. (1990). Leaching and effect on germination of Paspalum (Table monium, had declined during the 14-day transformation of glufosinate-ammo- 2). Other experiments have shown that incubation period. This result lead us to nium and its main metabolite in a lay- storing seed of Asystasia for two weeks af- suspect that the rates of herbicide degra- ered soil column. Environmental Toxi- ter collection could increase their germi- dation or bioactivity differ between the cology and Chemistry 9, 541-9. nation rate (Ismail et al. 1990). The in- two compounds. The degradation of Bowman B.T. (1989). Mobility and per- crease in the germination rate after stor- glufosinate-ammonium in soil is rapid sistence of the herbicide atrazine, ing may be due either to the greater seed due to soil microbial activity (WSSA metolachlor and terbuthylazine in maturity or to the possible failure of her- 1989). Field studies demonstrate that un- plainfield sand determined using field bicides to penetrate the seeds during the der natural conditions there is no lysimeters. Environmental Toxicology storage period. translocation of glufosinate-ammonium and Chemistry 8, 485-91. Shoot dry weight of Asystasia showed into soil layers deeper than 15 cm Dirven J.P.P. (1971). De chemische significant reduction when seeds were (Behrendt et al. 1990). This clearly indi- somenstelling non enige grasso-orten treated with glufosinate-ammonium (Ta- cates the rapid biodegradation of the ac- vit de gematigde en tropische gebieden, ble 3). Shoot dry weight of Paspalum was tive ingredient (Bowman 1989). 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