Pestic. Sci. 1991, 32, 427438

Field Effectiveness and Biodegradation of Cyclic Imides in Lettuce Field Soils

Christian Martin

Societe Cooperative de Recherche et d’Experimentation Agricole des Pyrenees Orientales, 19 Avenue de Grande Bretagne, 66025 Perpignan, France

Pierre Davet*

Laboratoire de Biologie et Pathologie Vegetales, Institut National de la Recherche Agronomique, 34060 Montpellier, France

Danielle VCga & Camille Coste

Groupe d’Etudes et de Recherches Appliquees Pluridisciplinaires, UniversitC de Perpignan, 66025 Perpignan, France

(Revised manuscript received 4 February 1991 ; accepted 12 February 1991)

ABSTRACT

When 42field trials, carried out from 1975 to 1989 in the Perpignan region (France) for control of lettuce drop caused by Sclerotinia minor, were compared, a decrease in the field effectiveness of cyclic imides was perceptible, beginning approximately in 1985. Moreover, in 15 out of 46 commercial lettuce fields surveyed in 1988 and 1989, the effectiveness of iprodione was less than 80%, the level of acceptability for the growers. In these fields, fungicide degradation, estimated by 3,5-dichloroaniline

formation, was faster than in soils in which S. minor was adequately controlled. Statistical analyses showed that the iprodione degradation index was strongly linked to the history of fungicide treatment and was weakly correlated to soil p H or clay content. All the fields characterized by low iprodione effectiveness were associated with high levels of fungicide treatment and high degradation index. Moreover, we observed that soil from a field which had received iprodione for more than ten years did not degrade vinclozolin quickly, while soil from another part of the same field which had been treated with vinclozolin fo r eight years degraded vinclozolin faster than iprodione.

*To whom correspondence should be addressed.

427

Pestic. Sci. 0031-613X/91/$03.50 0 1991 SCI. Printed in Great Britain

428 C. Martin, P . Davet, D. Mya, C. Coste

1 INTRODUCTION

Collar rot caused by Sclerotinia minor Jagger is the main limiting factor in lettuce production in Roussillon (a region of France). Without fungicidal protection, up to 90% of the crop can be lost. Fifteen years ago, growers used to protect their fields with benzimidazoles and/or dichlofluanid, but the results were not satisfactory. Cyclic imides were tested as early as 1974 and generally still control the disease satisfactorily when applied over the soil surface and to the base of plants. Extensive use of iprodione began after 1977 and this remains the most commonly used fungicide. In the past few years there has been an increase in the use of vinclozolin and procymidone. However, in 1980, some growers reported abnormal failure of iprodione to control lettuce drop. Other growers’ complaints have been registered since that time, especially in the latter half of the 1980s, also involving vinclozolin. In most of the cases, these growers had been using one of these fungicides for at least three consecutive years in their fields.

In the present study, we used field data collected over 15 years in order to confirm the validity of these observations. Laboratory studies have demonstrated that this efficiency loss could not be attributed to the appearance of cyclic-imide-resistant S. minor strains,’ and that it could be due to the rate of microbial degradation of iprodione or vinclozolin, which is faster in soils previously treated with these fungicides than in untreated soils.’ Walker3 observed such a phenomenon in Great Britain. He showed that this microbial degradation led to the appearance of 3,5-dichloroaniline, which could be detected by a colorimetric m e t h ~ d . ~ This paper examines whether the enhanced degradation phenomenon observed in the laboratory may actually explain failures in the practical control of lettuce drop in the field.

2 MATERIALS AND METHODS

2.1 Field trials with cyclic imides

2.1.1 Collecting data To study the change of efficacy of cyclic imides with time, we used the results of 42 comparative trials carried out from 1975 to 1989 around Perpignan by an extension service (the Societe Cooperative de Recherches et d’Experimentations Agricoles des Pyrenees-Orientales).’ These trials always included at least two of the three commercialized cyclic imides.

2.1.2 Fungicides Commercial 500 g kg- WP formulations of iprodione (3-(3,5-dichlorophenyl)-N- isopropyl-2,Cdioxoimidazolidine-1-carboxamide; vinclozolin (RS)-3-(3,5-dichloro- phenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione) and procymidone (N-(3,-5,- dichloropheny1)- 1,2-dimethylcyclopropane- 1,2-dicarboxirnide) were used: ‘Rovral’, ‘Ronilan’ and ‘Sumisclex’, respectively. These fungicides were sprayed according to standard recommendations (3 x 1000 litre ha-’ treatments with a dosage of 0.75 g a.i. litre-’). Each treatment included four replications; each replicate concerned 70-100 lettuce plants.

Biodegradation of cyclic imides in lettuce field soils 429

2. I .3 Estimating Jield effectiveness of treatments Field effectiveness (FE) represents the decrease in death rate resulting from the treatment, compared to a non-treated control. It was calculated by the following formula:

1 death rate in the treated zones FE=100 I--- ( death rate in the non-treated controls

Death rate is the percentage of plants killed by S . minor, assessed at harvest

2.2 Survey of growers’ fields

2.2.1 Fields and treatments Forty-six commercial fields, representing the main lettuce-producing areas around Perpignan, were observed in 1988 or 1989. The crops were treated by the farmers with a commercial formulation of iprodione, according to the standard recommendations described above.

2.2.2 Soil sampling At planting time, soil was collected from the top 8 cm in each field. Each sample included ten subsamples from the same field. The soil was sieved through a 2-mm screen. Soil pH and percentages of clay and organic matter were determined after each sampling. Differences between organic matter contents were slight (mean = 1.75 (+0.27)%). Samples were kept in plastic bags at 5°C until use.

2.2.3 Estimating fungicide degradation

2.2.3.1 Sample incubation. Air-dried soil samples were mixed with aqueous suspensions of a commercial formulation of iprodione in order to obtain a final soil moisture content of 20% and a concentration of 50 mg a.i. (kg dry weight)- ’. The mixture was distributed in eight 100-ml flasks (25 g per flask) which were tightly closed and incubated at 28°C.

2.2.3.2 Dosage. We followed Walker’s method4 and used the titration of 3,5-dichloroaniline as the criterion for iprodione degradation. Acetone extraction and the colorimetric test were carried out according to Walker’s procedure. After reading of optical density at 530 nm, 3,5-dichloroaniline formation was estimated by comparison with a standard calibration curve. Titration of duplicate samples of 3,5-dichloroaniline was done after intervals of 2, 3, 7 and 14 days.

2.2.3.3 Degradation index. For each sample, the quantity of 3,5-dichloroaniline present in soil was plotted against incubation period. The area between the curve and the horizontal axis was calculated using a planimeter. The value obtained takes both the speed (slope of the ascending part of the curve) and the intensity (maximum height reached) of 3,5-dichloroaniline formation into account, which was considered as representing iprodione degradation and named degradation index. Hence, soils could be classified according to their degradation index.

430 C. Martin, P. Davet, D . Wga, C. Coste

2.2.4 Soils ranked according to their history of treatment Soils were ranked into four classes according to frequency of fungicide sprays during the five years preceding sampling. Classes were as follows:

0: never sprayed with a cyclic imide before sampling; 1 : no more than two sprays during the previous year; or several earlier sprays,

2: three to four sprays during the previous year; 3: more than four sprays a year for at least three years.

but no cultivation for one or two years before sampling;

2.2.5 Soils ranked according to ,field efectiueness of iprodione In some of the 46 fields, field effectiveness could be calculated as described in Section 2.1.3. In the others, controls could not be introduced and it was then estimated by comparison with these reference fields. A value above 80% was considered good : this reference value corresponds approximately to the mean field effectiveness calculated from all of the cyclic imide trials carried out from 1975 to 1984. A value lower than 80% was considered bad.

2.3 Field comparison of iprodione and vinclozolin

2.3.1 Collecting data Fortuitously, a large, very homogeneous field was located in Los Horts, near Perpignan, which had been divided into four equal parts belonging to different owners. Each of these parts measured approximately 40 x 50 m. In three of them, lettuce had been cultivated every year for about 20 years. In two of these plots, iprodione had been sprayed very frequently for at least ten years. In the other plot, which belonged to another grower, only vinclozolin had been regularly used for eight years. Finally, the fourth plot had not been cultivated for many years and had never been sprayed with cyclic imides.

2.3.2 Soil sampling and measure of fiingicide degradation Ten subsamples from the top 8 cm of soil were taken from each of the four plots, mixed and prepared as described above. Aliquots of every sample were incubated with commercial formulations of either iprodione or vinclozolin at a concentration of 50mg a.i. kg-l. Soil moisture was adjusted to 20% and treated soils were maintained at 28°C. After 2, 3, 7 and 14 days, 3,5-dichloroaniline was extracted and titrated, and the degradation index was calculated. Every measurement was duplicated.

2.4 Data analysis

The numbers of trials with good or bad efficacy were compared by contingency tables. A correlation matrix of field data was calculated, and a principal components analysis was realized, using the STAT-ITCF program.6 The principal components analysis was performed with reduced centred data (data divided by their respective standard deviations).

Biodegradation of cyclic imides in lettuce field soils 43 1

TABLE 1 Field Effectiveness of Cyclic Imides, Calculated from Results of SCREAPO Trials Carried Out since 1975 in the Region of

Perpignan (Roussillon), France

Year Field eflectiveness (YO) Dead Iprodione Vinclozolin Procymidone plants in

controls W)

1975 1976 1977 1977 1978 1978 1979 1979 1979 1979 1979 1979 1979 1979 1980 1980 1981 1984 1984 1984 1985 1985 1985 1985 1986 1986 1986 1987 1987 1987 1988 1988 1988 1988 1988 1988 1989 1989 1989 1989 1989 1989

95.00 94.93 63.18 65.90 96.32 40.50 97.54 60.83 91.87 85.9 1 92.83 57.47 86.09 93.33 43.91 98.99 91.08 96.27 85.40 82.92 85.00 83.94 51.71 56.36 83.79 -

-

27.49 90.97

82.37 25.82 67.07 38.99 48.17 48.95 82.37 25.82 67.07 38.99 48.17 48.95

-

76.43 98.62 61.99 68.20 96.54 72.73 97.54 76.04 96.80 84.28 90.5 1 77.01 88.70 92.33 36.73 87.85 74.96 97.33 98.49 - - - -

26.9 1 90.1 7 55.05 51.11 - -

87-79 - - - - - - - -

- - -

-

- -

67.95 68.97 93.29 72.73 96.55 72.35 90.89

95.78 82.18 96.23 95.67 36.53 93.93 98.98 98.13 96.99 98-33 90.00 93.89 80.79 66.18 90-65 49.39 40.54 69.16 86.45 9 1.07 91.18 88.51 78.65 49.73 63.60 85.77 91.18 88.51 78.65 49.73 63.60 85.77

-

14.00 21.70 75.50 78.30 46.20 60.50 40.60 21.70 28.00 3 1.03 79.50 47.40 17.40 34.50 30-00 50.10 24.70 63.90 37.50 86-30 24.00 8.00

44.20 96.30 27-50 90.60 77.59 18.48 1550 46.70

9.93 45.00 41.00 62.67 2 1.65 11.95 9.93

45.00 41.80 62.67 2 1.65 11.95

432 C. Martin, P . Davet, D . V&a, C. Coste

3 RESULTS

3.1 Field trials with cyclic imides

Field effectiveness values for all three fungicides (Table 1) were generally high during the first ten years following the first 1975 trials, with very few poor performances (for example, the value for iprodione was 40.5% in a 1978 trial, and for vinclozolin and procymidone, 36.7 and 36.5%, respectively, in a 1980 trial). Mean field effectiveness values, calculated over the period from 1975 to 1984, were slightly above 80% (averages of 81, 82-8 and 856%, respectively, for iprodione, vinclozolin and procymidone) and they did not significantly differ from each other.

Since 1985, however, the field effectiveness seems to have decreased generally: averages for trials realized between 1985 and 1989 are 58, 62.2, and 76% for iprodione, vinclozolin and procymidone, respectively. Moreover, the number of trials where the value for iprodione was low (less than 80%) significantly increased (Table 2) while cases where that for procymidone was less than 8O% did not vary appreciably. There were not enough trials with vinclozolin to support a statistical analysis, but the tendency was the same as for iprodione.

3.2 Survey of growers’ fields

3.2. I Iprodione degradation indices Marked differences between fields could be observed (Table 3). In some of the fields, no 3,5-dichloroaniline was formed within the 14 days of soil incubation, indicating that no iprodione biodegradation occurred. In other fields, high production of 3,5-dichloroaniline was apparent as soon as the second day of the experiment, giving high degradation indices. Any intermediate situation could also be observed.

3.2.2 Correlation between field efectiveness, iprodione biodegradation, history of

The degradation index was highly correlated with the history of treatment, as is shown in the correlation matrix (Table 4). Clay content was slightly correlated with pH, but there was no correlation between pH and degradation index. Other correlations were very low.

treatment and soil characteristics

TABLE 2 Separation of Trials according to whether Iprodione Field Effectiveness was Superior or Inferior to 80% for the Years before 1985 and the Years after. The Value of x 2 is 5.15 and the Difference is

Significant at P 0.02

Field efectiveness < 80% > 80%

Before 1985 14 6 After 1985 6 13

Biodegradation of cyclic imides in lettuce j e l d soils 433

TABLE 3 Soil Characteristics of the 46 Lettuce Fields Surveyed in Roussillon

~~~~~ ~

Field no. Iprodione Clay PH History Field degradation (%> (in water) of effeectiueness"

index treatment

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

0.00 27.00 0.00

104.00 74.00 75.50 99.00

101.50 65.25 85.00 35.00 0.00 0.00

12.00 0.00 0.00

72.00 59.00 53.50 53.00 4.00 0.00

49.00 7 3 .OO 57.50 28.50 18-50 50.25 12-00 33.00 49.75 51.75 0.00

19.00 38.50 16.00 25.00 57.00 39.50 66.75 42.50 32.50 33.50 5 1.50 26.00 86.25

13.30 13.30 9.70

12.90 9.00 5.60

14.70 8.90

12.70 10.00 9.90 7.00 9.40 9.90

21.40 19.80 13.60 11.70 20.70 8.30

10.60 17.70 17.00 20.80 8.90 9.40

13.10 7.70

17.20 5.30 9.20 8.10

17.00 13.30 17.00 9.40 8.10 8.10 8.10 8.10 8.10 8.10 8.10 9.20 9.20 8.10

6.00 6.00 4.45 7.30 6.75 5.85 6.40 5.80 7-55 6.55 7.00 6.10 6.70 7.10 7.30 7.60 7.55 7.30 6.25 7.90 5.60 7.65 7.50 6.15 5.80 6.70 6.25 6.15 7-25 6.65 7.25 6.10 7.50 6.00 7.50 6.70 6.10 6.10 6.10 6.10 6.10 6.10 6.10 7.25 7.25 6.10

1 1 1 3 2 2 3 3 3 3 2 0 0 2 0 0 3 3 3 3 0 0 3 3 3 0 1 2 1 2 2 3 0 1 2 0 0 3 0 2 1 0 0 2 2 3

G G G B G G B B B B G G G G G G B B B B G G B B B G G G G G G B G G G G G B G G G G G G G B

a G: 'good', B: 'bad' (Section 2.2.5).

434 C. Martin, P . Davet, D . Vkga, C. Coste

TABLE 4 Correlation Matrix Calculated from Data in Table 3

DP HP Clay p H P o )

DI 1.000 HT 0.797 1.000 Clay (%) -0.181 - 0.024 1.000 PH -0’014 0.115 0.392 1.000

a DI, iprodione degradation index. HT, history of treatment.

Further information was obtained by a principal components analysis carried out with the values of these four parameters for the 46 fields. The correlation circle is represented in Fig. 1, after projection of variables on the plane defined by the two first axes: these two axes accounted for 80.7% of the total variation. History of treatment and degradation index appeared strongly linked to axis 1, which accounted for 86 and 90% of their variation, respectively. Clay percentage and pH were linked to axis 2, which accounted for 65 and 71% of their variation, respectively: axis 2 could therefore be considered as a scale of pH and soil adsorbing properties.

When the fields were projected onto the main plane (Fig. 2), all the fields characterized by a bad field efficiency were found on the left of axis 2, i.e. on the part of the plane where history of treatment values and degradation indices were the highest. Mean degradation index for soils with a ‘bad’ field efficiency was 71.1, compared to 26.1 in ‘good’ soils. Moreover, there were a few more ‘bad’ fields above axis 1, in the part of the plane which corresponded to higher pH and clay content. In contrast, fields which still showed satisfactory iprodione effectiveness rates were projected onto the part of the plane corresponding to low history of treatment and degradation index, and most of them were situated in the lower pH and clay content zone.

3.3 Field comparison of iprodione and vinclozolin

When iprodione or vinclozolin was added to soil samples from the plot which had not been previously cultivated for many years, no 3,5-dichloroaniline formation was observed even after two weeks. Although two of the adjacent plots had been sprayed with iprodione, they had different histories of treatment. Different levels of iprodione degradation were also observed: degradation indices were 38.5 and 73 in soil samples from the plots ranked in history of treatment classes 2 and 3 respectively. However, vinclozolin was not appreciably degraded in these soils (Fig.

Finally, in the fourth plot, which had been regularly sprayed with vinclozolin, vinclozolin was quickly degraded, as revealed by the appearance of 3,5-dichloroaniline (Fig. 3(b)) but iprodione degradation was low (degradation index = 19).

3(a)).

Biodegradation of cyclic imides in lettuce field soils 435

Axis 1 horizontal Axis 2 vertical t

1 t t I t t

I I I I t

t t t t

t ‘ I t t

t t t I

t I 1 t t I

I I I t

DI t 1 1 : t : t : t t : t : t : t t 1 t : t t t : t t t t t t t t t t : t t t t 1 1 t t 1 t t 1 t : t

I t t t t I

t t t I t t

t I I- I I t

t t 4

t I t t t I

t t I

I t t t I t

t t t t 1 I

t t t t t t

t t t I

Fig. 1. Correlation circle and projection on the main plane (defined by axes 1 and 2 of the principal components analysis) of the variables: history of treatment (HT), degradation index (DI), percentage

of clay (YO Cl) and pH. Data from Table 3.

4 DISCUSSION

It seems clear that the reduction of iprodione effectiveness observed in several lettuce fields can be. attributed to insufficient persistence of the compound. The fungicide, which is stable in acidic environments, undergoes physico-chemical degradation in oitro when pH is almost neutral. Mean soil pH in our study was about 6.6, which could explain some of the observed instability. In Fig. 2 there is some evidence of a pH effect, although it is also clear that the fungicide was at least as effective as the 1975-84 average (‘good’) in some high pH soils, and less effective (‘bad’) in some low pH soils. However, the main factor influencing ‘good’ and ‘bad’ performances was the history of treatment of the fields. Wherever no fungicide had been applied previously, iprodione was effective, even in soils with pH levels above 7, as was the case in soils 15, 22 and 33 (pH=7.3, 7.7 and 7.5,

436 C. Martin, P . Davet, D . V i g a , C. Coste

Axis 1 horizontal Axis 2 vert ical

G29

GO6

GI4

l t t l

038 032 t ) G40 G30

G 15

G22

G33

G27

G02G34 GO1 G26 G36 G13

G39G42 G37 G21

GI2

GO3

Visible point GI5 Hidden point GI6

Visible point G42 Hidden point G43

Visible point G31 Hidden point G44

Fig. 2. Projection on the main plane of the principal components analysis of data from the 46 lettuce fields G , ‘good’, and B, ‘bad’ iprodione field effectiveness

respectively). Persistence therefore is strongly linked to previous treatments; while traces of iprodione could be detected in virgin soils after more than four weeks, in analogous soils which had been previously treated for four or five years iprodione was found to be completely degraded after less than ten days (unpublished results). Accelerated degradation is due to the presence of a microflora which is able to metabolize iprodione and produce 3,5-dichloroaniline as an intermediary p r o d ~ c t . ~ . ~ This microflora, scanty at the beginning, increases as the treatments are repeated.

Biodegradation of cyclic imides in lettuce field soils 437

10 f

(a) Time (days)

Fig. 3. 3,5-Dichloroaniline formation in soil samples after addition of 50mg kg-' ( w ) vinclozolin or (0 ) iprodione. There was no formation in the controls (not shown). (a) Plot A of Los Horts which had previously been sprayed with iprodione for many years; (b) plot C of Los Horts, which had

previously been sprayed with vinclozolin for many years.

Our analysis also revealed a possible soil texture effect: iprodione seemed to be degraded faster in clay soils (Fig. 2). A study of this clay effect would deserve closer attention.

Until now, studies related to enhanced degradation have essentially concerned iprodione because this fungicide is the most frequently used cyclic imide in lettuce protection. But repeated use of vinclozolin also resulted, as was the case with iprodione, in an enhanced degradation (Fig. 3(b)). Comparing 3,5-dichloroaniline appearance rates in the four adjacent plots of Los Horts seems to indicate that this biodegradation phenomenon is rather specific: the microflora which biodegrades iprodione is probably not exactly the same as the microflora which biodegrades vinclozolin, and vice-versa. Walker' has already reported that vinclozolin was not quickly degraded in a soil pre-treated with iprodione. But, in

438 C . Martin, P. Davet, D. V6ga, C. Coste

his samples, iprodione quickly disappeared in a soil pre-treated with vinclozolin. Procymidone seemed more stable than the other two fungicides in laboratory trials (unpublished results). However, we do not have enough observations to evaluate its persistence in the field.

Practical experience suggests that if iprodione applications are stopped for two or three years, the situation returns to normal. But this implies stopping lettuce cultivation during this period, which is not possible for many growers who have only small areas to cultivate. If it could be confirmed that there is no, or very little, cross-adaptation to degradation between iprodione and vinclozolin, the solution could be to substitute each of these fungicides for the other, without interrupting cultivation. Quantification of Walker’s4 colorimetric test gives us an easy-to-use diagnosis tool. It can be used to warn producers against risks of enhanced degradation in their fields and to give them advice on choosing fungicides.

REFERENCES

1. Martin, C., Doct. Thesis, University of Montpellier, 1989. 2. Martin, C., Vega, D., Bastide, J . & Davet, P., Plant Soil, 127 (1990) 140-2. 3. Walker, A., Brown, P. A. & Entwistle, A. R., Pestic. Sci., 17 (1986) 183-93. 4. Walker, A., Pestic. Sci., 21 (1987) 233-40. 5. Martin, C., Rapports d’ExpPrimentation Horticulture-Phytosanitaire, SCREAPO

6. Philippeau, G., Service des Etudes Statistiques. ITCF Publications, Paris, 1986. 7. Walker, A,, Pestic. Sci., 21 (1987) 219-31.

Perpignan, 1976-90.