Ann. appl. Biol. (1980), 96, 11 1-1 18 Printed in Great Britain

111

Effect of simulated rain on retention, distribution, uptake, movement and activity of difenzoquat applied to Avena fatua

BY J. C. CASELEY AND D. COUPLAND Agricultural Research Council Weed Research Organization, Begbroke Hill, Yarnton,

Oxford, OX5 1PF

(Accepted 28 March 1980)

S U M M A R Y

In glasshouse studies the degree of control of Avena fatua increased as the period between application of difenzoquat and the onset of simulated rain was prolonged. 0.5 mm of ‘rain’ removed 29% of the herbicide deposit without adversely affecting performance at the recommended dose of 1 kg/ha. A further 30% was removed by 2.0 mm of ‘rain’, resulting in a marked reduction in acrivity. With lower amounts of ‘rain’ (0.16 mm), some of the spray deposit was redistributed from the leaf lamina to the leaf base/ligule area. The rate of penetration of 14C-difenzoquat was much greater when applied to the inner surface of the leaf sheath than when the leaf blade and outer sheath areas were treated. Translocation from the ‘inner sheath’ to other parts of the plant was up to 100 times greater than from other areas. It is suggested that the performance of difenzoquat is not reduced by low amounts of rain because: (1) the spray deposit is removed principally from the leaf blade, whilst in the more responsive liguleheaf sheath area the herbicide remains in solution, (2) the recommended dose of 1 kg/ha allows for some loss of active ingredient without reduction in performance. The practical implications of the work are discussed and further topics for research are outlined.

I N T R O D U C T I O N

Difenzoquat is a water-soluble herbicide widely used for the control of Avena fatua in barley and wheat (Wingfield & Caldicott, 1975). In common with other foliage-applied compounds, its performance can be diminished by rainfall (Baldwin & Livingston, 1976) but quantitative information has hitherto been lacking. The work reported here consisted of two groups of experiments. The first dealt with the effects on spray retention and distribution and herbicide performance of different amounts of simulated rain applied at several time intervals after spraying. In the second group, the consequences of redistribution on penetration and movement of the herbicide were followed using 14C labelled difenzoquat.

M A T E R I A L S A N D M E T H O D S

Plant material Seed of Avena fatua (L.) was obtained in autumn 1972 from the field. For consistency of

plant material between experiments only large (20-25 mm) brown-husked seeds were selected. These were sown on moist filter paper and germinated at 15 OC. When the radicles had just emerged, the young seedlings were planted in a soil mix (one seedling/9 cm diameter pot, at a depth of 2.5 cm) and put in an unheated glasshouse where the temperature was 16 8 “C and the humidity 60 f 20%. The soil mix consisted of 1 part peat, 1 part sand and 4 parts of Begbroke sandy loam. The seedlings were not given any additional nutrients until growth stage 12 (Zadoks, Chang & Konzak, 1974), when weekly feeding was commenced using a proprietary @ 1980 Association of Applied Biologists

112 J . C . C A S E L E Y A N D D . C O U P L A N D

liquid feed. Plants were treated at growth stage 13 for the retention, distribution and activity studies, and at stage 14 for the penetration and movement experiments.

Retention, distribution and activity studies For all treatments, herbicide solutions contained technical difenzoquat (69% active ingredient)

and 5 g/l Agral surfactant. Herbicide solutions were applied with a laboratory pot sprayer fitted with a single Spraying System 8001 ‘Tee-jet’ nozzle operating at 2.11 bar. The speed of the nozzle carriage and its height above the foliage were adjusted to deliver 200 litredha.

Following herbicide treatment, the plants were kept in the glasshouse for 3 wk, after which the fresh weight of foliage was recorded. The plants were arranged in a fully randomised design and the numbers of replicate plants appear in the tables.

Simulated rain was applied by a second laboratory pot sprayer fitted with a constant speed boom carrying six 8001 ‘Tee-jet’ nozzles at approximately 2 m above the foliage. The pressure was set at 3.52 bar to deliver a fine spray. The-amount of ‘rain’ applied was dependent upon the number of times that the spraying boom passed over the plants. Thus ‘rain’ duration differed according to the amount of ‘rain’ applied.

Retention measurements were taken on plants sprayed with difenzoquat at 1 kglha. After the spray deposit had dried (5 min) the shoots were cut off at soil level and the herbicide was washed from the foliage by shaking in a plastic bag with 20 ml 50% ethanol. The resulting solutions were filtered through Whatman GF/A filter paper and then retained for polarographic analysis.

5

Fig. 1 . Sampling areas for retention measurements. (1) Apical f of leaf. (2) Middle 4 of leaf. (3) Basal f of leaf. (4) Ligule region at leaf base (z3 cm long). (5) Rest of leaf sheath and ‘stem’.

In the retention/redistribution experiment, plants were chosen with the last fully expanded leaf at an angle of approximately 45O to the main stem. They were sprayed with herbicide and allowed to dry as described above. They were then divided into two groups; to one, 0.16 mm of ‘rain’ was applied, the other was kept dry. When the leaves of the first group had dried, the foliage of both sets of plants was cut off at soil level and the last fully-expanded leaf divided into the sections shown in Fig. 1. For each replicate value, similar sections from three plants were combined and herbicide washed off (10 ml of 50% ethanol), filtered and analysed polarographically using a Southern Analytical cathode ray polarograph Type A 1670. The solutions to be assayed (9.5 ml) were made alkaline by the addition of 0.5 ml of 10 M sodium hydroxide. About 5 ml of these solutions was transferred to the polarograph cells, de-oxygenated with a stream of nitrogen and polarograms obtained using a reduction potential of -1.33 v with respect to a dropping mercury cathode (method according to T. Byast, WRO, personal communication).

I4C uptake and movement studies The treatment solution for these studies contained unlabelled difenzoquat (5 g a.i./l), Agral

surfactant (5 g/l) and I4C-difenzoquat (sp. act. 13.04 mCi/g, 1,2-dimethyl-3,5-diphenyl- 1H-(3-’4C)-pyrazolium methyl sulphate) to give 13 175 cpm/,ul.

Rain effects on difenroquat activity against Avena fatua 113

A Burkard automatic applicator fitted with a 1 ml Agla syringe was used to apply a single 2 pl drop to each of the treatment positions, except the ‘inner-sheath’ where a discrete drop could not be dispensed. The four treatment areas are shown in Fig. 2 and described as follows: (i) ‘Mid-leaf’, a 1 cm portion of the second leaf, mid-lamina region, adaxial surface. (ii) ‘Leaf base’, a 1 cm portion at the base of the second leaf blade just above the ligule, adaxial surface. In this case a lanolin barrier prevented any movement of solution to the ligule and leaf sheath. (iii) ‘Outer-sheath’, a 1 cm portion of the second leaf, abaxial surface. (iv) ‘Inner-sheath’ - this region cannot be defined precisely. The 2 pl of herbicide was applied in between the leaf sheath (2nd leaf) and the shoot tissues it enclosed, thus the solution came into contact with both ad- and abaxial surfaces. Application of fluorescent dye to this treatment position indicated that solutions containing surfactant spread in between the closely folded immature leaves.

Treatment area (i) Mid-leaf (ii) Leaf base (iii) Outer sheath (iv) Inner sheath

Fig. 2. Diagrams showing relationship between application area (Ifil@ and parts subsequently sampled. Key: ( 1 ) Treated part. (2) Leaf tissues distal to treatment area. (3) Remainder of foliage. In all cases the root samples and apical meristem samples were identical (see text for definition).

All positions shown in Fig. 2 and described in detail below, were sampled at 4,8,24 and 48 h after application and in addition, washings from the treated areas were taken at zero time.

Washings from the treated area. For the ‘mid-leaf’, ‘leaf-base’, and ‘outer-sheath’ treatment positions, the leaf was cut approximately 5 mm below the treated area and the lower portion inserted into the neck of a 10 ml volumetric flask. The herbicide was washed from the leaf surface with 10 ml of a dilute herbicide solution (1 g/l, to facilitate exchange) containing Agral surfactant ( 5 gh). For the ‘inner sheath’ position the shoot was cut at soil level and at the ligules of the first and second leaves. The stem tissues, containing the treated part isolated in this way, were then carefully separated and all their surfaces washed thoroughly as described above. For all washings, a 1 ml subsample was added directly to 10 ml of Triton X-100 scintillation cocktail (Turner, 1968).

Treated part. Since the presence of the surfactant caused the drops of herbicide solution to spread over the leaf surface, approximately 5 mm above and below the treatment area was included in all samples, except for the ‘inner-sheath’ position. In this case all the leaf tissues subjected to the washing treatment were included.

Leaf tissue distal to the treatment area and remainder of foliage. The sample material varied according to treatment position and is shown in Fig. 2.

Apical meristem region. This sample consisted of all shoot tissues below soil level and was taken from the same region for all treatment positions.

Roots. All soil particles were washed from the roots which were then blotted dry. All plant samples were stored at -18 OC until burnt in a Harvey Biological Oxidiser. The

14C0, from the oxidiser was trapped in a proprietary CO, absorbing scintillation cocktail

114 J . C . C A S E L E Y A N D D . C O U P L A N D

( O x y ~ o l - ~ ~ C , ICN Pharmaceuticals Ltd) and all samples were counted at approximately 75% efficiency.

R E S U L T S

Effect of time interval between spraying difenzoquat and the incidence of ‘rain’ (Table I > Both levels of ‘rain’ applied 5 rnin after spraying difenzoquat at 0.5 kg a.i./ha significantly

reduced herbicide activity. When the interval between spraying and ‘rain’ was increased to 30 and 240 min, only the higher level of ‘rain’ reduced activity. At 1 kg a.i./ha (the recommended

Table 1. Effect on the performance of difenzoquat of simulated rain applied 5 rnin, 30 min and 4 h after spraying

Difenzoquat kg/ha . . . Amount of Time between ‘rain’ (mm) application and ‘rain’

0.5 5 min 30 rnin

4 h 2.0 5 min

30 min 4 h

‘No rain’ - S.E. &

0 0.5 1 .o 2.0 Foliage fresh weight (9)

I 0.92, 0.68 0.80 0.84 0.80 0.64 2.16, 1.44, 2-08, 1.20, 1.00* 0.68

4.00 0.64 0.64 0.09 0.12

7

0.76 0.60 0.60 0.84 0.88 0.72 0.44 0.16

Values are means of eight replicates. Values followed by * are significantly different from the ‘No rain’ treatment at P = 0.05.

dose for this herbicide) only 2.0 mm of ‘rain’ applied at the 5 and 30 rnin intervals had an adverse effect, while at the 2.0 kg a.i./ha dose none of the rain treatments significantly reduced herbicide performance. Delaying the onset of ‘rain’ treatment resulted in a higher level of herbicide activity. This effect could be seen within 7 days of spraying, since the contact effects of difenzoquat, notable severe leaf chlorosis, were greatest on the ‘no rain’ plants and least on those sprayed at the lowest dose and subjected to 2.0 mm of ‘rain’ at the 5 and 30 min intervals.

Effect of simulated rain on retention of difenzoquat (Table 2) ‘Rain’ was applied at 0.5 mm and 2.0 mm when the herbicide spray deposit had dried,

approximately 5 rnin after spraying. As can be seen from Table 2, these amounts of ‘rain’ removed an average of 29 and 60% of the herbicide respectively.

Table 2. Retention of difenzoquat on foliage after 0,0.5 and 2.0 mm of simulated rain

Difenzoquat retention Oldg dry wt)

% ‘Rain’ (mm) f removed

0 228.9 (6.7) 0.5 162.2 (8.3) 29.1 2.0 91.5 (7.2) 60.0

Values are means of 10 replicates, S.E. in parentheses.

Rain effects on difenzoquat activity against Avena fatua 115

Redistribution of difenzoquat over the leaf suface following 0.16 mm ‘rain’ (Table 3 ) There were no visible signs of run-off following the application of this amount of ‘rain’. On

many of the plants. liquid was observed to have collected in the angle made by the leaf with the main stem. Table 3 shows that almost all of the herbicide lost from the lamina section was redistributed to the leaf basehgule area. There was no change in the amount recovered from the ‘stem’ section.

Table 3. Redistribution of difenzoquat on last fully-expanded [eaf by 0-16 mm ‘rain’

Amount of difenzoquat recovered (pup)

rp~-.-.---, 96 reduction (-) Section No ‘rain’ ‘rain’ or increase (+)

Apical 1/3rd of lamina 5.3 4.3 -20.8 Middle 1/3rd of lamina 7.7 7.4 -3.9 Basal 1/3rd of lamina 6.3 5.4 -14.3 Leaf basehgule area 2.0 4.2* + 110.0 Rest of leaf sheath and stem 0.6 0.6 0 Total 21.9 21.9

Values are means of four replicates. * indicates significant difference from no rain treatment (s.E. = f 0.446).

Uptake and movement experiment (Table 4 )

The aqueous washes imposed immediately after application of the 14C-difenzoquat removed all detectable traces of the herbicide from the ‘mid-leaf’, ‘leaf-base’ and ‘outer-sheath’ areas but a significant number of counts were not recovered from the ‘inner-sheath’. This ‘treated part’ had 3643 cpm remaining after washing at zero time (14.3% of the total activity applied). After 4 h, 26% of the applied herbicide was recoverable from the ‘inner-sheath’ compared with over 96% for all the other application positions: by 48 h recovery at the former site was 4%, but the lowest value for the latter was 56%.

Following application to the ‘inner-sheath’, accumulation in the ‘treated part’ occurred very rapidly and was highest (64%) at 4 h, but declined to 36% at 48 h after application. However, in the other treatment positions uptake and accumulation continued throughout the 48 h to a maximum of 40%. After 4 h, significant translocation of herbicide from the treated part to all other untreated sample regions had occurred only following application to the ‘inner-sheath’. Of the other treatment positions, only the ‘leaf-base’ and ‘outer-sheath’ areas gave rise to subsequent traces of activity in the roots. After 48 h, 59.7% of the herbicide applied to the ‘inner-sheath’ position had moved into other sample areas compared with 4.3, 3.0 and 4.3% respectively from the ‘mid-leaf’, ‘leaf-base’ and ‘outer-sheath’ positions.

D I S C U S S I O N

The effects of rain on the performance of a foliage-applied herbicide, such as difenzoquat, will depend upon several factors including: the amount of rain (intensity x time), the interval between spraying and the onset of rain, and the rate of herbicide uptake; these in turn may be influenced by the quantity, formulation and position of herbicide deposited on the foliage. Prolonging the period between spraying and the incidence of rain increases the opportunity for penetration into the plant where it will be no longer readily susceptible to removal by rain. This is illustrated by the decreasing amount of ‘‘C-labelled herbicide recoverab!e in the aqueous wash with increasing time after application (Table 4) and by the greater reduction in foliage fresh weight of plants subjected to rain at increasing intervals after difenzoquat treatment (Table 1).

116 J. C. CASELEY A N D D. C O U P L A N D

Table 4. The eflect of site of application on uptake and translocation of I4C difenzoquat

Activity in samples (as % total activity recovered) Position of treatment

A \

Sample

Aqueous washes

Treated part

Leaf tissues distal to treatment area

Remainder of foliage

Roots

‘Apical meristem’

% recovery

Time (h)

0 4 8

24 48

0 4 8

24 48

4 8

24 48

4 8

24 48

4 8

24 48

4 8

24 48

4 8

24 48

mid- leaf

100.0b 97.5b 91.6b 63.2b 55.5b

Oa 2.5a 8. la

33.9b 40.2b

Oa

1.9a 0.13b

2*8b

Oa O.llb 0.35b 0.64b

Oa 0.13a

0.54a

Oa Oa 0-26a 0.29a

0.42b

95.1 107.9 100-3 97.9

leaf- base

100.0b 97.8b 96.2b 70.4b 6 6 . 2 ~

Oa 2. l a 3.6a

27.lb 30.6b

Oa

1 n4ab I.Sa

Oa

0.79b

0 , l l b 0.33b 0.70b

0.13b 0. la

0.58a

Oa Oa 0.33a 0.40a

0.42b

97.9 102.5 96.5

100.3

inner- sheath

85.7a 26.0a 16.7a 7.3a 4. la

14.3b 64.2b 62.2b 54.7c 36.2b

2.2b 7 . 2 ~

1 1 . 7 ~ 1 6 . 4 ~

4.3b 8 . 9 ~

2 0 - 4 ~ 33.3c

0 . 6 1 ~ 1.2b 3-OC 3.6b

2.6b 3.7b 2.8b 6.4b

93.9 97.5 95.7 94.9

outer- sheath

100.0b 96-5b 9 7 . 8 ~ 9 1 . 6 ~ 7 8 . 3 ~

Oa 3.4a 2.1a 7.5a

17.4a

Oa Oa 0.64a 3.0ab

Oa Oa Oa 0.33a

0. lb 0.08a 0.20a 0.51a

Oa Oa 0.16a 0.45a

99.3 101. I 100-7 99.2

Values are means of four replicates. Values followed by different letters are significantly different at P = 0.05 (horizontal comparisons

only).

The retention data (Table 2) show that 0.5 mm of ‘rain’ removed 29% of the spray deposit, but four times this amount of rain washed off only a further 31%. There are probably several reasons for this. The position of the herbicide spray deposit on the plant determines to a large extent the ease with which it can be washed off by rain. Thus more herbicide was lost from the leaf lamina than from the basal parts (Table 3). Some of the steps involved in this process include: the rates of solution of dry herbicide deposits, dilution of concentrated solutions either newly formed or remaining from incomplete drying of the original deposits, and direct displacement by new rain drops impacting on the leaf or ‘overflow’ as the holding capacity of the foliage is exceeded. As these events progress, it is likely that an increasing amount of rain will be

Rain eflects on difenzoquat activity against Avena fatua 117

required to wash off an equivalent quantity of herbicide since the remaining herbicide will cover a reduced area of the plant surface and/or be in a more dilute solution.

In these experiments ‘rain’ intensity was uniform and changes in this factor might well affect the wash-off capability of a given amount of rain, since increased intensity is usually accompanied by a larger median drop size (Best, 1950) with concomitant increases in terminal velocity and perhaps an increased ability to remove herbicide from the plant.

Under the conditions of this experiment the recommended dose of 1 kg/ha had a considerable safety factor for counteracting adverse conditions, since in the ‘no rain’ treatment the 0.5 and 1 .O kg/ha doses were equally phytotoxic. However, the biological effectiveness of the herbicide remaining on the plant after rain may well be changed. If dilution of the surfactant occurs, this is likely to reduce the activity of the herbicide, since difenzoquat performance is closely related to surfactant concentration (Merritt, 1976). Following amounts of ‘rain’ of up to 0.5 mm, loss of active ingredient and surfactant may be compensated by some redistribution of the remaining solution to more vulnerable areas of the plant. The retention data in Table 3 show that 0.16 mm of rain removed herbicide from the lamina, but that some was redeposited in the liguleheaf sheath area which is known to be particularly important with regard to herbicide performance (Walter & Bischof, 1976; Coupland, Taylor & Caseley, 1978). Coupland et al. also found that re-wetting the treated plant increased the activity of difenzoquat and that herbicide applied in between the leaf sheath and the rest of the stem (analogous to the ‘inner-sheath’ position of this work) resulted in greater activity than application to other sites. The “C-difenzoquat studies presented in Table 4 support and quantify these observations.

The present work showed that low amounts of ‘rain’ washed herbicide from the leaf blade - not the most easily entered site - and redistributed some of the herbicide into the more responsive ligule and inner leaf sheath areas. Concomitantly the rain fully hydrated the plant cuticle and kept the herbicide in solution - conditions conducive to uptake (Sargent, 1965). When the amount of ‘rain’ was increased to 2.0 mm it is probable that, in addition to removal of spray solution from the leaf blade, herbicide was also washed from the ligule and leaf sheath areas. With this amount of rain, dilution of both the active ingredient and surfactant may also have contributed to decreased performance.

Results with 14C-labelled difenzoquat (Table 4) showed that the rate of herbicide penetration was greater following application to the ’inner-sheath’ than to the leaf blade and ‘outer-sheath’ positions. Furthermore, accumulation of herbicide in plant parts remote from the treated area was much higher from the ‘inner-sheath’. For example, application to this position compared with the leaf blade resulted in 22 times more activity in the apical meristem, the main site of action of this herbicide. Possible reasons for the better performance of herbicide applied to the leaf inner sheath may be summarised as follows: (a) Proximity to vascular tissues and apical meristem, (b) humid microclimate favouring herbicide entry, (c) immature, perhaps easily penetrated cuticle, and (d) qualitative and quantitative differences in wax deposition.

One practical implication of the work is that even immediately following application of the recommended dose (1 kg/ha), amounts of rain up to 0.5 mm did not have an adverse effect on weed control. It is interesting to note that at the Weed Research Organization Farm, 30% of all incidences of rain over the past 3 yr amounted to 0.5 mm rain or less (R. Simmons, personal communication). The results as presented pose at least four questions which we hope to investigate in the future:

(1) If the intensity, and concomitantly drop size and terminal velocity of the rain is altered, what is the effect on retention, redistribution and herbicide performance?

(2) Can the formulation and application of herbicides be modified to deposit more active ingredient in the inner sheath regions?

(3) Will an understanding of the differences between ‘lamina’ and ‘inner sheath’ areas increase our knowledge of the barriers to herbicide entry and movement?

118 J . C . C A S E L E Y A N D D . C O U P L A N D

(4) Does the ratio of surfactant to herbicide remain constant following dilution and redistribution by rain?

The authors thank the American Cyanamid Co. for donating the 14C-difenzoquat.

R E F E R E N C E S

BALDWIN, J. H. & LIVINGSTON, D. B. (1976). The control of Auena fatua in winter wheat by the use of herbicides applied at different timings. Proceedings British Crop Protection Conference - Weeds pp.

BEST, A. c. (1950). The size and distribution of raindrops. Quarterly Journal of the Royal Meteorological Societv 16, 76.

COUPLAND, D., TAYLOR, w. A. & CASELEY, J . c. (1978). The effect of site of application on the performance of glyphosate on Agropyron repens and barban. benzoylprop-ethyl and difenzoquat on Aima,fatua. Weed Research 18, 123-128.

MERRITT, c . R. (1976). The interaction of surfactant type and concentration with controlled drop applications of MPCA and difenzoquat. Proceedings British Crop Protection Conference -Weeds pp.

SARGENT, J. A. (1965). The penetration of growth regulators into leaves. Annual Review of Plant Ph.vsiologv 16, 1-12.

TURNER, J. c . (1968). Triton X-100 scintillant for carbon-14 labelled materials. International Journal of Applied Radiation and Isotopes 19, 557-563.

WALTER, H. & BISCHOF, F. (1 976). Uber die applikationsortabhangige Wirkung neuer Nachauflaufher- bizide gegen Flughafer (A uenafatua L.). Zeitschrift fur Pflanzenkrankheiten 83, 338-35 1 .

WINGFIELD, R. J. & CALDICOTT, J. B. (1 975). Difenzoquat, 1,2-dimethyl-3,5-diphenylpyrazolium ion, a selective herbicide for the control of wild oats (Auena spp.) in wheat and barley. Pesticide Science 6,

ZADOKS, J. C., CHANG, T. T. & KONZAK, c . F. (1974). A decimal code for the growth stages of cereals.

25-30.

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297-303.

Weed Research 14.4 15-42 1.

(Received 2 Januarv 1980)