JOHN, WILLIAM P. WERGIN, AND JAMES D. ANDERSON. Plant Hormone Laboratory (A. R., J. D. A), Plant Stress Laboratory (W. P. W.), and Weed Science.
Plant Physiol. (1984) 76, 297-300
0032-0889/84/76/0297/04/$01.00/0
Rhythmical Changes in the Sensitivity of Cotton Seedlings to Herbicides' Received for publication February 22, 1984 and in revised form June 4, 1984
ARNON RIKIN*, JUDITH B. ST. JOHN, WILLIAM P. WERGIN, AND JAMES D. ANDERSON
Plant Hormone Laboratory (A. R., J. D. A), Plant Stress Laboratory (W. P. W.), and Weed Science Laboratory (J. B. S.), Beltsville Agricultural Research Center (West), United States Department of Agriculture, Beltsville, Maryland 20705; and Department ofBotany, University ofMaryland, College Park, Maryland 20742 (A. R.) ABSTRACT Cotton (Gossypiam hiruutam L) seedlings that were grown under a photoperiod of 12 hours darkness and 12 hours light showed oscillations in their sensitivity to the herbicides sodium 5(2-chloro4-trifluoromethyl)-phenoxy)-2-nitrobenzoate (acifluorfen), butyl 2-(4-((5-(trifluoromethyl)-2-pyridinyl)oxy)phenoxy)propanoate (fluazifop) and 3-isopro-
pyl-lH-2,1,3-benzothiadiin-4(3H)-oe 2,2-dioxide (bentazon). Sensitivity was expressed in appearnce of necrotic areas on the cotyledons and in decreased growth of the shoot. The seedings were least sensitive in the bing and middle of the light period, then the sensitivity increased and reached its ximum during the nning and middle of the dark period and then declined. Seedlings grown from germiation under continuous light exhibited very small or no oscillations in sensitivity. The oscillations in sensitivity were entained by one cycle of darkness and light. A cycle of 12 hours darkness and 12 hours Light gered the greatest oscillations while either incrsing or decreasing the duration of the dark period resulted in smaller oscillations. Apparently, these oscillations in sensitivity to herbicides were endogenonsly controled since after entrinment they continued irrespective of the light conditions.
Humans and other animals show oscillations in their sensitivity to many chemical agents such as pyrethrum, atropin, pentobarbitol sodium, ethanol and histamine (12). There are also reports on oscillations in the sensitivity of plants to chemical agents. For example, changes in membrane potential in Samanea pulvini by sucrose (1 1) and inhibition of flowering in Lemna by ions (5) were related to the time of day that sucrose or ions were applied. Of special importance are the relations between the response of plants to herbicides and the time of day when the herbicides are applied. Many studies demonstrated such relations between herbicides with various mechanisms ofaction and many plant species (1-4, 7, 8, 14). However, it is not yet clear what contributes to the daily oscillation in the sensitivity to herbicides. The aim of the present work was to test whether such daily oscillations in the sensitivity to herbicides were rhythmical changes which must be entrained by a certain photoperiod and were regulated by an endogenous clock. 'This work was carried out under the Cooperative Agreement No. 5832U4-2-384 ofthe Agricultural Research Services, United States Department of Agriculture, and the University of Maryland. 297
MATERIAILS AND METHODS Cotton (Gossypium hirsutum L.) seeds were germinated and grown for 7 d in plastic pots (12.5 cm in diameter, 12 cm in height) filled with peat and irrigated with distfilled H20. The seedlings were grown in growth chambers at 29°C under certain photoperiods which are specified in each experiment. The light source (700 ft-c) was a combination of regular incandescent light and fluorescent light (F48T18-CW-VHO, Sylvania, Danvers, MA). The herbicides sodium 5-(2-chloro4-trifluoromethyl)-phenoxy)-2-nitrobenzoate (acifluorfen), butyl 2-(4-((5-(trifluoromethyl)-2-pyridinyl) oxy)phenoxy)propanoate (fluazifop) and 3-isopropyl-1H-2,1,3-benzothiad in4(3H)-one 2,2-dioxide (bentazon) were applied by spraying the seedlings until run-off. The injury caused by the herbicides was evaluated by degree of damage to the cotyledons as expressed by appearance of necrotic areas after several days. It was scored from 1 which represents no visible necrotic areas on the cotyledons to 5 which represents completely necrotic cotyledons. At the time of herbicide application, the seedlings had only two cotyledons and had not yet developed shoots. Resumption of growth was determined by measuring the shoot weight several days after the herbicide application. RESULTS Cotton seedlings grown under a photoperiod of 12 h light and 12 h darkness showed daily scillations in their sensitivity to the herbicides bentazon, acifluorfen, and fluazifop. Maximal sensitivity to the three herbicides, as observed by development of necrotic areas in the cotyledons (scored as degree of damage) occurred about 4 to 8 h after the beginning of the dark period. Later in the dark period, the sensitivity of the cotyledons began to decrease and it was much lower during the light period (Fig. 1). The seedlings were treated with herbicides before the development of the shoots. The subsequent development of the shoots was affected by bentazon and acifluorfen, depending on the time of their application. The maximal inhibition ofshoot growth was achieved when these herbicides were applied during the first and middle parts of the dark period. During the last part of the dark period the growth inhibition began to be smaller, and during the following light period it was the smallest (Fig. 1). Fluazifop exhibited a very small effect on subsequent shoot growth. However, it almost completely stopped the growth of the cotyledons when applied in the sensitive stage during the first and middle parts of the night. Application of fluazifop during the last part of the dark period and during the light period was less effective in inhibition of cotyledon growth (Fig. 1). Application of various concentrations of bentazon to seedlings
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FIG. 1. Oscillations in the response of cotton seedlings to bentazon (1 mM), acifluorfen (I mM), and fluazifop (10 mM). Seedlings were grown under a photoperiod of 12 h darkness and 12 h light for 4 d from germination, then treated with the herbicides at various times and immediately transferred to continuous light. The degree of damage to the cotyledons was scored 2 d after treatment with acifluorfen or fluazifop and 4 d after treatment with bentazon. The fresh weight of the shoots or cotyledons was measured 5 d after the treatment with bentazon or fluazifop and 8 d after the treatment with acifluorfen. (U) Dark period, (01) light period; (0) degree of damage, (U) fresh weight. Table I. Dose Response of Cotton Seedlings to Bentazon at Their Sensitive and Resistant Stages Seedlings were grown under a photoperiod of 12 h darkness and 12 h light for 4 d from germination, then treated with various concentrations of bentazon 4 h or 16 h after the beginning of the dark/light cycle (sensitive and resistant stages, respectively). After bentazon treatment all seedlings were immediately transferred to continuous light. The fresh weight of the shoots was measured 5 d after the treatment with the herbicide. Shoot Fresh Wt at Following Time (h) Bentazon 4 16 mM % ofcontrol 84±6 72±3 0.25 80±7 48±5 0.50 41 ± 1 3± 1 1.00 0 2± 1 2.00
during the sensitive or resistant stages showed that the inhibition of shoot growth that was caused during the sensitive period was achieved in the resistant period only with a 2-fold higher concentration (Table I). Any concentration above 0.25 mm inflicted more damage to the cotyledons of seedlings in the sensitive stage. Maximal difference between the response of seedlings during the sensitive and resistant stages was achieved after treatment with I mM bentazon while the higher and lower concentrations caused smaller differences. Treatment with 0.25 mm bentazon had almost no visible effect on the cotyledons (Fig. 2). The response of the seedlings to bentazon was similar if, after the application of the herbicide, they were kept under the normal photoperiod or were transferred to continuous light. Seedlings
FIG. 2. Dose response of cotton seedlings to bentazon at their sensitive and resistant stages. Seedlings were grown under a photoperiod of 12 h darkness and 12 h light for 4 d from germination, then treated with various concentrations of bentazon 4 h or 16 h after the beginning of the dark/light cycle (sensitive or resistant stages, respectively). After the bentazon treatment all the seedlings were immediately transferred to continuous light. The degree of damage to the cotyledons was scored at various times after the treatment with the herbicide. (, 0) 0.25 mM, (A, A) 0.5 mM, (0, 0) 1 mM, (* Q0) 2 mM bentazon. Solid symbolsbentazon applied at the sensitive stage, open symbols-bentazon applied at the resistant stage.
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FIG. 3. Effect of light conditions after the application of bentazon (1 mM) on the expression ofinjury in cotton seedlings. Seedlings were grown under a photoperiod of 12 h darkness and 12 h light for 4 d from germination, then bentazon was applied at various times during the dark period. After the treatment with the herbicide, the seedlings were transferred to continuous light (@) or kept in the regular photoperiod (A) or kept in continuous darkness (U). The degree of damage to the cotyledon was scored 4 d after the treatment with the herbicide. Seedlings that were kept in continuous darkness were transferred after 4 d to continuous light, and the degree of damage to the cotyledons was scored 4 d later (0); (U) dark period.
that were tnsferred to continuous darkness, even for 4 d after the treatment with bentazon, did not show any visible symptoms. However, after transfer to continuous light the response to bentazon was similar to that observed in seedlings that were transferred immediately to continuous light or kept under this previous photoperiod after the application of the herbicide (Fig. 3). Seedlings grown under continuous light from germination did not show oscillations in their response to the herbicides. The minimal requirements to entrain the owillations in the response to the herbicides were a dark period followed by light period, then the oscillations began in the following dark period. Figure 4 describes the response of the seedlings to bentazon after en-
299
RHYTHMICITY IN SENSITIVITY TO HERBICIDES 5
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FIG. 5. Detailed kinetics of the development of the sensitivity to bentazon (1 mM) in cotton seedlings. Seedlings were grown under continuous light for 4 d from germination, then exposed to one cycle of 12 h darkness and 12 h light. Bentazon was applied at various times during the dark period of the second cycle. After the bentazon treatment all the seedlings were immediately transferred to continuous light. The degree of damage to the cotyledons was scored 4 d and shoot fresh weight was measured 5 d after the treatment with the herbicide. (U) Dark period; (0) degree of damage, (U) fresh weight.
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(U) fresh weight.
trainment with one cycle of various dark/light combinations. Exposure to one cycle of 12 h darkness and 12 h light resulted in the largest changes in the sensitivity to bentazon as reflected by the degree of damage to the cotyledons and development of the shoots. Under such entrainment the maximal damage occurred when bentazon was applied 4 h after the beginning of the dark period. Later in the dark period the sensitivity decreased and it remained low during the light period. Prolonging, as well as shortening the dark period from 12 h resulted in smaller changes in the sensitivity to bentazon. Moreover, after exposure to dark periods longer than 12 h, the maximal sensitivity occurred at a later stage during the dark period. Detailed kinetics of the development of the sensitive phase to bentazon, after one cycle of 12 h darkness and 12 h light, showed a quick rise in the sensitivity which reached its maximum 2 h from the beginning of the dark period. After about 3 h of maximal sensitivity it began to decrease gradually and reached a low level after 4 h (Fig. 5). Seedlings that were grown under cycles of 12 h darkness and 12 h light for 4 d from germination were transferred to inverted light periods, i.e. the dark period was changed to light period and
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FIG. 6. Oscillations in the response of cotton seedlings to bentazon (1 mM) under inverted photoperiod. Seedlings were grown under a photoperiod of 12 h darkness and 12 h light for 4 d from germination, then the dark period was changed to light period and the light period was changed to dark period. Bentazon was applied at various times during the last light and dark periods. After the treatment with bentazon, all the seedlings were immediately transferred to continuous light. The degree of damage to the cotyledons was scored 4 d and shoot fresh weight was measured 5 d after the treatment with the herbicide. (U) dark period, (0) light period; (0) degree of damage, (U) fresh weight.
the light period was changed to dark period. Under these new conditions the seedlings responded to bentazon according to the chronological time and not according to the immediate light conditions (Fig. 6) As usual, the degree of damage to the cotyledons and inhibition of shoot growth were highest 4 h after the beginning of the cycle and then became lower, although the dark and light periods were exchanged (Fig. 6).
DISCUSSION The sensitivity of cotton seedlings to three herbicides (bentazon, acifluorfen, and fluazifop) as expressed by appearance of necrotic areas on the cotyledons and decreased shoot growth was related to the time of application. Similar relations between plant sensitivity to herbicides and the time of application have previously been reported for these and several other herbicides and plant species (1-4, 7, 8, 14). In the present work we showed that such oscillations in sensitivity to the herbicides were rhythmical because first, they did not occur when the seedlings were grown under continuous light from germination; second, they were entrained by a certain photoperiod; and third, after they had
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been entrained they continued irrespective ofthe immediate light conditions. Light is required for the expression of the toxic effect of bentazon and acifluorfen (9, 10, 13). However, this light requirement was not involved in the timing of the sensitivity. The sensitivity was determined only by the time of herbicide application, irespetive of the immediate light conditions after the herbicide application. The long term response of cotton seedlings to the herbicides was determined only by the time of application, irrespective of light conditions after the herbicide application. The same pattern of response was observed with three herbicides (bentazon, acifluorfen, and fluazifop) which are known to have different primary modes of action (6, 9, 10, 13). Therefore, it seems that the mechanism which contributes to the oscillations in the sensitivity is nonspecific. Current studies are aimed to clarify this mechamsm. Acknowledgments-We would like to thank Drs. A. K. Mattoo, G. F. Dietzer, and J. R. Teasdale for many fruitful discussions. LITERATURE CITEiD 1. BOVEY RW, RH HAAS, RE MEYER 1972 Daily and seasonal response of Huisache and Macartney rose to herbicides. Weed Sci 20: 577-580 2. CAMPIRANON S, WL KOUKKARI 1976 Circadian periodic response of Phaseolus vulgaris L. to 2,4-dichlorophenoxyacetic acid. Chronobiologia 3: 137-148
Plant Physiol. Vol. 76, 1984
3. DORAN DL, RN ANDERSON 1976 Effectiveness of bentazon applied at various times of the day. Weed Sci 24: 567-570 4. GO6SELINK JG, LC STANDIFER 1967 Diurnal rhythm of sensitivity of cotton seedlings to herbicides. Science 158: 120-121 5. HALABAN R, WS HILLMAN 1971 Factors affecting the water-sensitive phase of flowering in the short day plant Lemna perpusilla. Plant Physiol 48: 760764 6. JAIN R, WH VANDEN BORN 1983 Morphological and histological effects of sethoxyudin, fluazifop-butyl and DOWCO 453 on wild oats (Avenafatua). Proc Weed Soc Am, 83: 73-74 7. KOUKKARI WL, MA JOHNSON 1979 Oscillations of leaves of Abutilon theophrasti (velvetleaf) and their sensitivity to bentazon in relation to low and high humidity. Physiol Plant 47: 158-162 8. LEE SD, LR OLIVER 1981 Efficacy of acifluorfen on broadleaf weeds. Times and methods of application. Weed Sci 30: 520-526 9. MINE A, S MATSUNAKA 1975 Mode of action of bentazon. Effect of photosynthesis. Pestic Biochem Physiol 5: 444 450 10. ORR GL, FD HEss 1982 Mechanism of action of the diphenyl ether herbicide acifluorfen-methyl in excised cucumber (Cucumis sativus L.) cotyledons. Light activation and the subsequent formation of lipophilic free radicals. Plant Physiol 69: 502-507 11. RACUSEN RH, AW GALSTON 1977 Electrical evidence for rhythmic changes in the cotransport of sucrose and hydrogen ions in Samanea pulvini. Planta 135: 57-62 12. REINBERG A 1973 Chronopharmacology. In J. N. Mills, ed, Biological Aspects of Circadian Rhythms. Plenum Press, London, pp 121-152 13. SUWANKETNIKOM R, KK HArZIos, D PENNER, D BELL 1982 The site of electron transport inhibition by bentazon (3-isopropyl-lH-2,1,3-benzothiadiazin44)3H-one 2,2-dioxide) in isolated choroplasts. Can J Bot 60: 409412 14. WEAVER ML, RE NYLUND 1963 Factors influencing the tolerance of peas to MCPA. Weeds 11: 142-148
