Proe. Indian Acad. Sci., Vol. 86 B, No. 3, September 1977, pp. 143-150. ~) Printed in India.

Alachlor: A new potent antitranspirant on maize

M SANTAKUMARI, C S REDDY and V S RAMA DAS Department of , Sri Venkateswara University, Tirupati 517 502

MS received 21 April 1977

Abstract. Foliar application of alachtor, 2-chloro-2-6-, diethyl-N-(methoxy methyl) acetanilide, at 20 mg/1 on two-week-old plants of Zea mays L. produced remarkable partial inhibition of stomatal opening and significant reduction in the rate of transpira- tion. Antitranspirant activity of a single foliar application of alachlor persisted for twenty days after treatment. The rates of and apparent photosynthesis with alachlor treatment were compared with those of phenylmercuric acetate (PMA) treatments. Treatment with alachlor produced higher rates of photosynthesis, increased growth rate and yield, while PMA-treated plants exhibited a reverse trend. Alachlor thus proved to be an effective yield increasing antitranspirant under the experimental conditions used.

Keywords. Alach~or; antitranspirant activity; foliar application

1. Introduction

During the past few years several attempts have been made to discover an ideal chemical antitranspirant for use on crops in dry regions of the world (Zefitch 1969, Gale and Hagan 1966; Poljakoff-Mayber and Gale 1973). Alkenylsuccinic acids are known to induce partial stomatal closure (Zelitch 1964). Most of the known chemical antitranspirants have undesirable toxicological side effects. Phenylmercuric acetate (PMA) has been extensively tested and although it conserves water, it also causes a substantial reduction in radial trunk growth, (Waggoner and Turner 1971) inhibited photosynthesis (Waggoner and Pallas 1968) and photophosphorylation (Nozaki et al 1961) and produced damages to foliage of barley and maize (Majernik 1970). Recently, wtfile studying the stomatal behaviour towards herbicidal treatment, it was noticed in this laboratory that an a-chloroacetamide herbicide, alachlor (2-chloro- 2, 6-diethyl-N-(methoxy methyl) acetanilide) could induce partial stomatal closure in isolated epidermal strips (Das and Santakumari 1975). This observation has led us to test the effectiveness of foliar application of this chemical as compared to PMA, as an antitarnspirant on maize under field conditions.

2. Materials and methods

Experimental plots (10 × 4 M) with loam soil and mixed manure in 3 : 1 propor- tion were prepared in the University Botanical Gardens. Soil moisture levels were

143 144 M Santakumari, C S Reddy and V S Rama Das maintained at 20 per cent by regulated watering. Seeds of maize (Zea mays L. C V, Hi-Starch) were sown at 1 cm depth. When the plants were two-week-old, the anti- transpirants PMA (15 rag/l) and alachlor (20 mg/1) were sprayed without any wetting agent on to the foliage; a second spray was given thirty days later. The width of the stomatal aperture was measured on stomatal imprints obtained with nail polish (Sampson 1961). The size of the aperture was confirmed simul- taneously by measuring the pore width in the detached epidermai strips fixed imme- diately in alcohol. water potentials were determined by using methylene blue as an indicator solution (Knipling 1967). Transpiration rates were measured by the conventional pot weighing method. To minimise the loss of water by evaporation from soil, polythene paper was spread on the surface of the soil.

2.1. Apparent photosynthesis (Dry weight increase method)

A definite number of leaf discs of uniform size were taken in the morning from one side of the midrib of an intact leaf by using Ganong's leaf puncher. The leaf discs were collected in specimen bottles from different plants situated in all parts of the plot and were oven dried to a constant weight. In the evening an equal number of leaf discs were cut from the second side of the midrib and were dried similarly. The differences between the dry weight of these two samples were taken as the rate of apparent photosynthesis. Growth data including the height of the , leaf area, fresh weight and dry weights were determined at 5-day intervals after the application of chemical to the foliage. Morphological symptoms were recorded throughout the experimental period. Yield was expressed in terms of 1000- seed weight, length, diameter of the cobs and dry weight of the stem.

3. Results

Diurnal rhythm in stomatal aperture size variation of untreated plants was characteris- ed by the maximum stomatal opening at 12 noon. Partial stomatal closure with alachlor and nearly complete closure with PMA application and a recovery to the control level only 20 and 25 days after the treatment respectively were noticed with foliar application (figure 1). The extent of recovery in stomatal opening after the chemical treatment is shown in figure 2.

Table I. Effect of alachlor or PMA on the leaf water potential of two-week-old maize plants.

Days after chemical treatment Treatment 5 10 15 20 25 30

Control --6"1 --6"7 --6"0 --5"9 --5"6 --6"3

Alachlor (20mg/1) --4"2 --4"0 --3"9 --4"0 -4'1 --4"7

PMA (15 rag/l) --4"0 -3"7 --3"6 -3"5 --2"8 --2'6 Antitranspirant action of alachlor 145

O LIGHT INTENSITY 50 ~, • TEMPERATURE O.

o_~ o' 21

" o~-°'-°--~ %'o--0-

8~- A CONTROL _= ~ /\ "o :~:: "'°" /\ ~.4 I,

=o o .~-4.;,-%-,, J/\\.Vo.O-..~ ~~.6=~.=--- \ 12 4. 8 12 4. 8 12 MID NOON MID NIGHT NIGHT TIME OF THE DAY

Figure 1. Effectof alachlor (20 mg/l) or PMA (15 mg/D on the diurnal rhythm of stomatal opening of maize plants grown under natural light intensity and temperature. Leaf water potentials were found to increase in alachlor-treated and PMA-treated plants when compared to control (table 1). (Control plants showed the leaf water potential of - 6.7 whereas alachlor and PMA treated plants exhibited - 4.7 and - 2-5, respectively). Alacklor and PMA markedly reduced transpiration rates by alachtor and PMA treatments (figure 3). Photosynthetic rates increased with alachlor treatment but decreased with PMA (figure 4). The physical growth characteristics including fresh weight, dry weight, leaf area and height of the plant exhibited an increase with alachlor treatment and a decrease with PMA as compared to control (figure 5). Associated with these characters cob height and diameters were also higher in alachlor treated plants when compared to the control (figure 6). Seed shape and size decreased with PMA, while they increased with alachlor (figure 7). This has finally resulted in increased yield in alachlor treated plants (table 2). 146 M Santakumari, C S Red@ and V S Rama Das

6~\ • ALACHLOR o I1\ 0 PMA U

o

p- < 2 o

0

0 ~0 .._0 .....0...,..0__0...-,0 ~ 0.---0~0 0 "---,0,~,0

0 4 8 2 16 20 24. 28 2 36 DAYS AFTER CHEMICAL TREATMENT

Figure 2. Effect of alachlor or PMA on recovery of the stomatal opening at 12 noon on maize plants.

I00

.J ~~.... ~• ~4L/-I~rlLUl~,ALACHLOR. vO r'nn~,PMA.

z ~- eO ...-o. 0 o = n 6 0 ="O~O~ O Ln Z

i- °~°~°"'°~O~.o.__ ~ ul o...-O~o Q. 20 o n I I L t t I. • J o 4. e 12 16 20 OAYS AFTER CHEMICAL TREATMENT

Figure 3. Effect of alachlor or PMA on the transpiration of maize plants. Antitranspirant action of alachlor 147

• ALACHLOR. o PMA 180

~ 140

LU "I" U ~" ~ O0

o:~ t~¢:u 60io ",.o.~°--°~o--.o-'°~o~ '" F 0"~0 201 I I I i 1 I ~ t J ~_~.~, 0 4- 8 12 16 20 DAYS AFTER CHEMICAL TREATMENT

Figure 4. Effect of alachlor or PMA on the photosynthesis of maize plimts.

5000 ~00 CONTROL 4.000 • ALACHLOR .00 0 PMA / Z o 3000 ./ ;OO 3 ~.OC zc/) 2000 t,g ¢¢ ¢1. I000 tO0

0 0 20 30 40 50 20 30 4.0 50 ?.50 500 .---,H--* ( 20C •/ u ~O0

Q. t~ t=.l ,.r ,. // _,t 300 l- b. 0 ,oo ./'~/ ' ~.OO .!-. • .41 Sof W 10O Z cE ~1' I I I ! I I 20 30 40 50 20 30 4.0 50 AGE 0 F THE PL ANTS (DAYS)

Fipre 5. Effect of atachlor or PMA~on some growth characteristics of maize plants. 148 M Santakumari, C S Reddy and V S Rama Das

Table 2. Effect of alacb_lor or PMA on yield parameters of maize plants

Average Average Straw Treatment length of diameter of weight of 1000---Seed 100 plants weight (g)** the cob {~m)* the cob (cm)* (Kg)**

Control 10-70 9"7 30"0 220"60

Alaehlor (20 rag/l) 13"05 10"3 37"5 270"27

PMA (I 5 rag/l) 8"05 8"59 25"0 160"25

*Average of 60 observations. **Average of 10 observations.

Figure 6. Effect of alachlor or PMA on the cob length and diameter of maize plants (1. Control 2. alachlor 3. PMA)

Figure 7. Effect of alachlor or PMA on the seed size and shape of maize seeds. (1. Control 2. alachlor 3. PMA). Antitranspirant action of alachlor 149

The morphological symptoms observed with alachlor treatment were also interest- ing. Alachlor treatment resulted in healthy and luxurient growth of the crop with green and expanded . In contrast to this, PMA-treated plants developed early senescence and yellow streaks after 24 hr of treatment and the leaves emerging after the treatment were pale yellow in colour. Alachlor treatment delayed the leaf senescence.

4. Discussion

The present study has established the inhibition of stomatal opening by alachlor on intact maize plants which was earlier reported in isolated epidermal stripes (Das and Santakumari 1975). A remarkable decrease in transpiration rate with alachlor treat- ment is also established. Inhibition of stomatal opening observed with alachlor resembled the action of morphactins (Das et al 1976) and inhibitors of cyclic photo- phosphorylation (Raghavendra and Das 1977). Several of the antitranspirants tried earlier have been known to cause reduction in the rate of photosynthesis, which appeared to be due in part to the increased stomatal resistance to COs diffusion, and partly to the inhibition of the photosynthetic process (Slatyer and Bierhuizen 1964). Alachlor treatment at 20 rag/1 while inhibiting the stomatal opening did not alter laCO2 fixation efficiency (unpublished observations). This has resulted in increased leaf area, plant growth, dry matter production and ultimately the yield. On the other hand, PMA treatment produced complete sto- matal closure and inhibition of photosynthetic process. Apart from the less popular succinic acids (Zelitch 1969), PMA and abscisic acid (ABA) are the two widely known metabolic antitranspirants (Gale and Hagen 1966; Jones and Mansfield 1972). The present study introduces another new metabolic antitranspirant, the amide group herbicide, alachlor. Foliar application of ABA and its esters did not alter the CO2 compensation point or dry matter production in plants (Jones and Mansfield 1972). Alachlor treatment was comparable in this respect to the action of ABA. The antitranspirant action of aJachlor persisted till twenty days after treatment thus making it a highly effective antitranspirant and would be of great help to reduce the water requirements of dry land crops. It is believed from the present study that the use of alachlor as an antitranspirant could profitably be tested with other crop plants on a field scale.

Acknowledgement

This work was supported by a grant from Indian Council of Agricultural Research, New Delhi.

References

Das V S R and Santakumari M 1975 Stomatal behaviour towards four classes of herbicides as basis of selectivity to certain weeds and crop plants; Proc. Indian. Acad. Sci. B82 108-116 Das V S R, Rao I M, Swamy P M and Raghavendra A S 1976 Suppression of stomatal opening by morphactins in isolated epidermal strips; Plant Cell Physiol. 17 77-82 150 M Santakumari, C S Reddy and V S Rama Das

Gale J and Hagen R M 1966 Plant Antitranspirants; Ann. Rev. Plant. PhysioL 17 269-282 Jones R H and Mansfield T A 1972 Effects of Abscisic acid and its esters on stomatal aperture and the transpiration ratio; PhysioL Plant 26 321-327 Knipling E B 1967 Measurement of leaf water potential by dye method; Ecology 48 1038-1041 Majernik O 1970 Responses of stomata of barley and maize to phenyl mercuric acetate; BioL Plant (Praha) 12 419-423 Nozaki M, Tagawa K and Aronon D I 1961 Noncyclic photophosphorylation in photosynthetic bacteria; Proc. Nat. Acad. Sci (Wash) 47 1334 Poljakoff-Mayber A and Gale J 1973 Physiological basis and practical problems of reducing transpiration; in Water deficits and plant growth ed. T T Kozlowski (New York: Academic Press) Vol. III Ch. 9. pp. 277-306 Raghavendra A S and Das V S R 1977 Antitranspirant activity of the inhibitors of the cyclic photophorylation; J. Exp. Bot. 28 480-483 Sampson J 1961 A method for replicating dry moist surfaces for examination by light microscopy; Nature (London) 191 932-933 Slatyer R O and Bierhuizen J F 1964 The effect of several foliar sprays on transpiration and water use efficiency of cotton plants; Agric. MaterioL 1 42-53 Waggoner P E and Pallas J E 1968 Saving the water spent in plants; U S Dept. Agric. Year Book 213-217 Waggoner P E and Turner N C 1971 Transpiration and its control by stomata in a pine forest; Bull No. 726 Conn. Agric. Exp. Stn. New Haven Zelitch I 1964 Reduction of transpiration of leaves through stomatal closure induced by alkenyl succinie acid; Science 142 692-693 Zelitch I 1969 Stomatal control; Ann. Rev. Plant PhysioL 20 329-350