Ann. uppl. Bwl. (1987), 111, 173-183 Printed in Great Britain

173

The relationship between the entry of isoproturon into Alopecurus myosuroides and its effect on growth and

COz fixation

BY A. M. BLAIR LARS Weed Research Unit, BroomS Barn Experimental Station,

Higham, Bury St Edmunds, Sufsolk IP28 6NP, UK

(Accepted 6 February 1987)

S U M M A R Y

Alopecurus myosuroides (blackgrass) was grown in nutrient and with isoproturon added to the solution when plants had three leaves. The transpiration stream coefficient factor was calculated over a period of 96 h after treatment to be approximately 1. On this basis isoproturon entry into A. myosuroides was estimated using data for water loss through plants and there was a linear relationship between herbicide entry and plant weight 14 days after treatment. Treatment for at least 7 days with isoproturon at a dose of 1 pg a.i./15 ml nutrient solution (= 0.32 x 10-6~) before returning plants to fresh nutrient was necessary for damage to be still evident after 14 days. This time period corresponded with the reduction of C 0 2 exchange to zero as measured by infra-red gas analysis. Some recovery occurred from 6 h treatment with 120 pg a.i. isoproturon/l5 ml nutrient solution when assessed after returning plants to untreated nutrient for 14 days. A quicker depletion in C 0 2 uptake by blackgrass occurred when both the primary and secondary roots were treated compared with secondary alone but eventually the levels reached were similar. The ‘CALF’ model predicted much higher concentrations of isoproturon than appeared necessary to damage A. myosuroides. This suggests a major influence of climate on the plant and this and other interactions are discussed.

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

Soil moisture influences the performance of soil acting herbicides (Walker, 1971) including isoproturon (Blair, 1985) by affecting the availability and location of the herbicide in the soil in relation to sites of entry into the plant. Blair (1978) concluded that much of the activity of isoproturon resulted from uptake from the soil at least under the glasshouse and controlled environment conditions examined.

The object of the present experiments was to measure the amount and rate of herbicide activity by exposing parts of the A . myosuroides (Huds.) root system for varying times to different concentrations of isoproturon. Plant dry weight was recorded and speed of herbicide entry and arrival at the site of action in the plant was indicated by measuring C 0 2 exchange. In order to eliminate the influence of herbicide adsorption onto soil, A . myosuroides plants were grown in a nutrient culture system. Using this information the amount of herbicide required to damage the plant can be estimated. These results are considered in relation to the amount of isoproturon predicted by the ‘CALF’ model (Nicholls, Walker & Baker, 1982) to be present in the field soil profile uiing measurements for the autumns of 1983 - 84 from the meteorological site at the Weed Research Organization, Oxford. This model has been used to predict the amount and location of herbicide in the soil, e.g. monuron (Nicholls, Briggs &

0 1987 Association of Applied Biologists

174 A . M . BLAIR

Evans, 1983), although too much movement was predicted following heavy rainfall. The eventual aim is to link the knowledge of the amount of isoproturon required to kill the plant with the predicted availability of herbicide.

The work forms part of a programme to understand both the factors which contribute to the variable performance of substituted urea herbicides in the field and the wider principles of how climate affects herbicide performance.

MATERIALS A N D M E T H O D S

A. myosuroides seeds were pre-germinated in an incubator at 20°C for 24 h on moist filter paper in the light followed by 48 h in the dark. The germinated seeds were then transferred into a system modified from that described by Pestemer, Stalder & Potter (1983). Seedlings were planted at 12 mm depth in vermiculite in a polystyrene container (10 cm diame- ter x 5.5 cm depth). This container rested on the rim of a beaker (800 ml) darkened with metallised polyester film. The vermiculite (covered on surface with black alkathene beads to exclude light) was kept moist by a wick dipping into 300 ml of a quarter strength Hewitt’s solution (Hewitt, 1966). The plants were kept in a Votsch controlled environment room (16/10”C, 75/86% r.h. day/night) for 2 wk. Day length was 14 h with light (max. 95 Wmd2) provided by cool white fluorescent and additional tungsten lamps. After the 14-day period seedlings were floated in water and teased from the vermiculite and transferred to a double- box system. This comprised two polystyrene boxes (3.5 x 5.5 x 2 cm) held together with black adhesive tape such that primary and secondary roots could be introduced into separate boxes and treated independently. The lids of the boxes were darkened with black tape and the plant supported by cotton wool in a notch cut in the lid of one box. Nutrient solution (15ml/box) was added and the plants kept for 5 - 7 days in the Votsch room prior to isoproturon treatment when they had three leaves.

Herbicide solutions as appropriate were weighed into each box and also at any subsequent change of solution. At the end of each experiment plant and boxes were weighed and the solutions recovered from each box weighed. Fresh and dry weights of shoots and both primary and secondary roots were recorded 14 days after initial treatment unless otherwise recorded. C 0 2 exchange was recorded in some experiments using the infra red gas analysis (IRGA) system described by Merritt & Simmons (1985). Radio-labelled isoproturon (specific activity 35.15 pCi/mg), was added to the nutrient solution in one experiment to measure the transpiration stream coefficient factor (TSCF) which is defined as :

concentration in transpiration stream/concentration in external solution (Shone & Wood, 1974). The plants were divided 24 or 96 h after treatment into individual leaves, tillers and primary

or secondary roots. Leaf samples were cut into pieces which were solubilised in 1 ml of soluene solution (diluted 1 :1 with toluene) for 24 h and decolourised with 300 pl of a freshly prepared solution of benzoylperoxide in toluene for 24 h. Triton scintillant (5 ml) was then added and samples kept in the dark for 24 h before counting using a Packard Tricarb scintillation spectrometer. Root samples were oxidised at 800°C in an oxygen train type oxidiser and I4CO2 trapped in 10 ml Reich’s scintillant for subsequent counting as before.

The ‘CALF’ model described by Nicholls et al. (1982) was used to predict the distribution of isoproturon in the soil profile. This model uses laboratory generated data on herbicide adsorption and rates of degradation at different temperatures and soil moistures, rainfall, evaporation and air-temperatures. Three initial soil moisture profiles were selected, one in which it was dry throughout the top 20 cm and two which were moist in the top 10 cm but one of which dried below this depth (Fig. 1). The isoproturon movement predicted by the ‘CALF’ model was computed for the drier autumn 1983 (Fig. 2) following a very dry summer and for that of 1984 (Fig. 3).

Efect of isoproturon on blackgrass

0 3.5-

0 30.- - - E E

a

. - - 0.25.-

c .-

5 0.20.-

0,15.*

0

175

t.-.-r,- -=.‘., 0-0-

.-=-n-.-

-q=-.+-m- -1

‘a

\. \a

\ +-&*A-A-A-A-A-.-

I

.A-A-A-~

Days Fig. 2. Maximum (-) and minimum (----) temperature and rainfall (m) at Weed Research Organisation, Oxford in September 1983.

Data were subjected to analysis of variance where appropriate and standard errors are given in the Tables. A logistic growth curve was fitted to data (Fig. 4) and standard errors given for each assessment time. A linear regression was fitted to data (Fig. 5) using ‘Genstat’ and correlation coefficient (r) for the line given. The output from the IRGA experiments took the form illustrated by Merritt & Simmons (1985). A line was drawn joining the daytime values (Fig. 6) and a parallel curve analysis carried out using a maximum likelihood programme (Ross, 1980).

176 A . M. B L A I R

Days

Fig. 3. Maximum (-) and minimum (----) temperature and rainfall (m) at Weed Research Organisation, Oxford in September 1984.

- 0.2 -

c 9 0.1.5- a c - . a n 3 i2 0.1 - a

0.05 - I 0 I I I '

8 16 24 Days

S.F. I

Fig. 4. Plant dry weight of A. myosuroides after treatment with isoproturon (1 pg/15 ml) for various periods and assessed after 24 days.

Effect of isoproturon on blackgrass 177

I I 1 I I 1 I

Isoproturon (pg) calculated to enter plant

1 1 I 0.45 0.75 1 .05 1.35 1.65 0.15

Fig. 5 . The effect on plant dry weight (%untreated) of treating A. myosuroides with isoproturon for < 72 h.

L

I I I I I I 1 1 I 1 1 I 1 24 72 I20 168 216 264

Time (h) post-treatment

Fig. 6. CO, exchange following treatment of A. myosuroides with isoproturon. The day-time values are joined up to give a line (-) for each treatment. (Untreated ; 120 pg isoproturon/lS ml nutrient/6 h A; 1 pg/IS m1/14 days 0.)

178 A. M. BLAIR

RESULTS

Preliminary experiments showed that plants grown for 14 days without a change of nutrient solution responded similarly to those grown in solutions changed every 3 days. Hence, in all subsequent experiments the solutions were not changed after treatment.

Considerable variability of A. myosuroides growth habit was evident in all experiments with some plants being very prostrate and others erect. Isoproturon activity did not appear to be affected by plant habit in any of the work described here.

Transpiration stream coeficient factor The TSCF was calculated 24 and 96 h after treating A. myosuroides and was 1.05 f 0.099

and 1.03 f 0.007, respectively, indicating passive entry. Hence, in subsequent experiments an approximate calculation of isoproturon entry into plants was made using water loss measurements from plants over the duration of treatment. The distribution of I4C varied between plants but accumulation was greatest in the second and third leaves, leaf 1 being the oldest leaf (Table 1).

The efect of time of exposure to isoproturon solution Only plants which were exposed to isoproturon (1 pg a.i./15 ml = 0.32 x ~ O + M ) for longer

than 7 days showed a reduction in final plant weight (Fig. 4) 24 days after treatment. Some recovery in plant weight could occur when plants were returned to untreated nutrient after 14 days exposure to the herbicide (data not shown).

Interaction between time of exposure and different concentrations of isoproturon In eight experiments a standard treatment of 1 pg a.i./15 m1/14 days was included and the

range of control varied from 17 - 55% of untreated within these experiments. There was, however, a statistically significant negative correlation (P < 1%) between dry weightlplant and calculated herbicide entry from the amount of water transpired for plants treated for < 72 h with higher doses of isoproturon (Fig. 5). The range of doses covered 60 pg/15 ml between 1 - 24 h, 120 pg/15 ml between 1 - 72 h and 240 ,ug/15 m1/6 h.

Treatment for 6 h with either 120 (Fig. 6)or 240 pg/15 ml had a marked immediate effect on C o t exchange by A. myosuroides whereas treatment with 1 pg/15 m1/14 days caused only a gradual decline in COz exchange. There was some recovery of COz uptake by plants treated with 120 pg/15 m1/6 h on returning plants to untreated nutrient. The slope of this line was significantly different from that of the untreated plants.

Table 1. The accumulation of 14C (dpm) in diflerent parts of A. myosuroides 24 and 96 h ajier treatment via the roots with 14C isoproturon (4053 dpmlml solution)

Treated for 24 h Treated for 96 h

Plant 1 2 3 1 2 3 *-

Leaf I 440 2 898 3 864 4

Tiller I 44 2

Root primary 264 Secondary 340

-

-

470 1232 1939 1019 606 3456 1075 1430 6419

- 1494 646 196 226

857 349 336 669 427 408 1180

-

- -

2400 4234 4458

240 I422

87 625 946

1663 2904 5018 1124 1540

71 209

1594

- not present at time of assessment.

Effect of isoproturon on blackgrass 179

Table 2. The effect of isoproturon applied to different regions of the root system of A. myosuroides

Region treated Dry wt in mg (% untreated) (1 ~ d 1 5 ml/ r A

-l

1 1 days) Foliage Primary root Secondary root

None 71.0 12.0 30.0 All roots 32.7 (46.5) 7.0 (58.3) 8.0 (26.7) Primary roots 65.7 (93.0) 9.0 (75.0) 23.0 (76.7) Secondary roots 36.0 (50.7) 6.0 (50.0) 17.0 (56.7) S.E. f 7.34 1.05 2.97

(6 D.F.) (6 D.F.) (6 D.F.)

Uptake of isoproturon by diferent parts of root system Where all roots were treated with 1 pg/15 ml/ 11 days, damage to A. myosuroides was similar

to that where secondary roots only were treated but greater than that where primary roots only were treated (Table 2). Secondary root weights were markedly reduced where all roots were treated. A faster depletion of COz uptake by A. myosuroides occurred when all roots were treated than when treatment was confined to secondary roots only; the final depletions were similar. The calculated amount of isoproturon entering the plant increased in the order primary < secondary < all roots treated. The contribution to damage made by isoproturon entry through the primary roots was small and was not directly proportional to either the amount of root present or the calculated herbicide uptake.

Isoproturon in soil projle as predicted by model Taking three selected contrasting soil moisture distributions down the profile as the starting

point (Fig. 1) the predicted isoproturon location in the profile using 1983 and 1984 meteoro- logical data was determined using the ‘CALF’ model (Table 3). Since there was little difference predicted as a result of the starting profiles a and b (Fig. 1) only a is compared with c in Table 3. Twenty days after treatment there were lower concentrations of isoproturon predicted for 0 - 1 cm horizon in 1984 than in 1983. This corresponded to a very heavy fall of rain during this period in 1984.

DISCUSSION

Isoproturon location in the soil profile The ‘CALF’ model predicted that for autumn conditions most isoproturon was located in

the upper 1 - 2 cm of profile up to 20 days post-treatment. This prediction was not checked by analysis. The model assumes that the herbicide is mixed into the top 1 cm of soil in the absence of plants. Mudd, Hance & Wright (1983) showed that isoproturon persistence in the field could be predicted using the model of Walker & Barnes (1981). Siriwardana, Blair & Bartlett (198 1) demonstrated that a wheat plant did not alter the distribution of isoproturon in a soil leaching column after 36 days when compared to that in the absence of a plant.

The concentration of isoproturon predicted in the soil solution was similar under the contrasting autumn conditions examined (Figs 2,3). Hence, the amount of water available in the soil may be the major factor controlling activity.

180 A. M. B L A I R

Table 3. Prediction of isoproturon (pglml soil solution) from same post spray weather conditions ajier three different soil moisture distributions

1983 data Horizon

(cm)

0 - 1 *; a 1-2

2-3 a

C

3-4 :

0 - 1 f

a

1984 data 4-5 c

1-2 a

a 2-3

3-4 : 4-5 ;

Days

4 8 10 12 16 20 I

A >

6.53 6.94 0.296 0.044 0.01 1

0 04004

0

0 -

5.87 6.27 0.267 0.039 0.0094

0 OW03

0

0 -

6.20 6.35 0468 0.07

0 0 0 0 0

-

5.42 5.82 0.257 0.1 16 0.0089

0 04003

0

0 -

5.67 5.87 0.209 0.173 0.0018 0

0 0 0

-

5.43 5.75 0.243 0.111 0.0085

0 04003

0

0 -

4.74 5.07 0.243 0.255 0.01 16 0.00 19 0.0003 0

0 -

5.46 4.75 5.65 4.98 0.193 0.281 0.163 0.219 0.0017 04043

0 0

0 0 0 0 0 0

- -

4.34 4.65 0.338 0.229 0.0103 0@017 04003 0

0 -

2.33 2.44 1.273 1.293 0.492 0.488 0.172 0.166 0.054 0.054

starting soil moisture distribution (Fig. 1). - isoproturon predicted present at <O.OOOl pg/ml.

Entry of isoproturon from the root system in nutrient culture Under field conditions only part of the root system is likely to be exposed to isoproturon in

the short-term post-spray period since the herbicide will be concentrated in the soil surface zone. Simulation of this is difficult as we have been unsuccessful in growing A. myosuroides in the system described by Shone & Flood (1980). The nearest approach we have devised is to treat primary and secondary roots separately since secondary roots originate nearer to the soil surface.

By growing plants in nutrient solution it was possible to show using the ‘IRGA’ that net photosynthesis was stopped within 6 h of treatment with a dose of 120 pg isoproturon/l5 ml whereas symptoms took several days to become visible on the plant. In contrast a much lower dose (1 pg/15 ml) took 5 - 7 days to reduce photosynthesis. This time interval also corresponded to the period after which full recovery did not occur when plants were returned to untreated nutrient. These two cases illustrate the different time periods over which climate might influence activity, a factor also discussed by Gerber, Nyffeler & Green (1983) in their review. An IRGA system has also been used by van Leeuwen & van Oorschot (1976) to monitor the ability of winter wheat plants to recover from photosynthetic inhibitor herbicides such as chlortoluron and metoxuron, both closely related chemically to isoproturon. They returned plants to untreated nutrient when photosynthesis had dropped to about 50% of its initial value from which the tolerant varieties then recovered. Henly, Dodge & Stephens (1985) examined recovery of barley and two Bromus species from isoproturon treatment. Barley fully recovered from 24 h treatment with 0.1 mM solution of isoproturon ( 3

310 pg/15 ml) which had also reduced photosynthesis by about 50%. The Bromus species had

Eflect of isoproturon on blackgrass 181

not fully recovered at the end of the experiment (7 days). Dicks (1978) working with isolated lettuce chloroplasts found that a mean concentration of 0.17 x 1 0 - 6 ~ (= 0*5pg/15ml) isoproturon reduced the Is0 value (herbicide concentration giving 50% inhibition of the Hill reaction) by 50%. The results presented in this paper for A. myosuroides show some recovery, although far from complete, from 120 pg isoproturon/l5 ml nutrient solution when returned to untreated nutrient after 6 h for a further 10 days. However, even though the slope of the recovery versus time curve was significantly different (by parallel curve analysis) from that of the untreated plants, there appears to be some ability to detoxify isoproturon. This is perhaps not surprising in view of the recent reports of resistance of some A . myosuroides stocks to this herbicide (Moss & Cussans, 1985) and the fact that selectivity between crop and weed is related, at least in part, to the ability of tolerant plants to metabolise the herbicide as in the case for chlortoluron (Ryan, Gross, Owen & Laanio, 1981). However, one would expect damaged plants to be at a severe disadvantage in competition with others. The range of response to isoproturon treatment of A . myosuroides in different experiments even when growing in a controlled environment coupled with the differences in distribution of 14C within different plants (Table 1) provides a good potential from which resistance might develop. In addition other studies in the same controlled environment (R. Allen, personal communication) have recently shown a range of responses of A. myosuroides plants grown in soil to treatment with isoproturon. Although at the end of each treatment period the concentration of isoproturon solutions were measured these were at the limit of detection of HPLC methods used (Byast, Cotterill & Hance, 1977) and thus proved very variable and hence unreliable. Over the 14-day treatment period in many of the experiments there would certainly be some degradation of isoproturon and so the calculations of herbicide entry based on TSCF = 1 would overestimate entry. However, it seems from these results that less than 0.5 - 1.0 pg isoproturon per plant would have a severe effect both on photosynthesis and on subsequent plant growth.

Estimated entry of isoproturon into plants growing in soil It can be estimated that a plant of A . myosuroides will take up approximately 0.13pg

isoproturon/day if the following assumptions are made: at field capacity soil moisture is well distributed throughout the pot (Blair, 1985); a dose of 0.4 kg a.i./ha is severely damaging under the environmental conditions used in these experiments (Blair, 1984); herbicide concentration is averaged throughout the depth of soil in the pot (0.082 g/ml); an A. myosuroides plant transpires 1.6 ml/day (A.M. Blair, unpublished). This estimated value is similar to that calculated from the nutrient culture experiments despite the assumption that all roots were exposed to a uniform average herbicide concentration in the soil. This may imply that uptake by roots from a high localised compared with a lower overall concentration results in little difference in activity. Where only the primary or secondary roots were treated with 1 pg/l5 ml/l 1 days (Table 2) there was no evidence of increased nutrient solution uptake by the untreated roots. Shone & Flood (1983) also found no compensatory increase in root weight in non-stressed compared to drought-stressed parts of root systems which contrasts with other reports of compensatory uptake of water (e.g. Lawlor, 1973).

CONCLUSION

The aim of being able to predict isoproturon performance requires a knowledge of the amount of herbicide which will damage the plant under a range of environmental conditions. If models can predict the location and availability of herbicide in the soil profile it may be possible with a knowledge of root distribution and water loss through the plant to calculate the

182 A . M . B L A I R

amount of isoproturon entering the plant as attempted for chlorotriazines (Robinson & Dunham, 1982). In the case of isoproturon, however, we also need to define any factors which influence the balance between uptake by the foliage or the soil since in this paper we have only considered uptake by the roots. The model predicts much higher concentrations of isoproturon in the upper soil horizons than appeared necessary to damage A . myosuroides in the nutrient culture studies under controlled conditions. The model is currently being tested in the field with isoproturon but the discrepancy between the model prediction presented in this paper and practical results suggests a major influence of climate on the plant and this requires further clarification.

A C K N O W L E D G E M E N T S

I thank Mrs A. M. Quantrill for assistance with these experiments, Dr J. C. Caseley for helpful discussions, R. C. Simmons for assistance in running the IRGA and processing the data, C. J. Marshall for assistance with statistical analyses, Ciba Geigy AG, Base1 for the gift of labelled isoproturon, P. H. Nicholls of Rothamsted Experimental Station for the ‘CALF’ model and advice on its use and Dr J. Brownscombe for providing meteorological data relating to soil moisture.

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E j e c t of isoproturon on blackgrass 183

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(Receiried 4 July 1986)