TRANSLOCATION, DISTRIBUTION AND METABOLISM OF TRIFORINE IN PLANTS A. FUCHS Laboratory of Phytopathology, Agricultural University, Wageningen, The Netherlands, and

W. OST Celamerck GmbH & Co, KG, lngelheim, Germany

After short-term root-treatment of plants with SH-triforine (labelled in the piperazine ring) or x4C-triforine (labelled in the side chain), the label was translocated primarily to the leaves present at the time of treatment, without any redistribution of the label taking place after termination of the treatment. Autoradiography showed the radioactivity to be very evenly distributed over the leaf blades, without any appreciable accumulation in leaf margins. In infected leaves, however, there was a tendency for increased accumulation at the infection sites. In dilute aqueous solution triforine was decomposed rather rapidly, the chemical half-life being three days only. From these solutions several non-fungitoxic degradation products have been isolated and their chemical structures determined. In plants, decomposition of triforine was considerably slower than in aqueous solutions, the half-life of the compound varying depending on plant species. In plants, at least four conversion products could be demonstrated, one of which is piperazine. Whether the other three compounds are identical with or structurally related to the breakdown products found in aqueous solution is not yet known. Its low persistence and low animal toxicity suggest that triforine does not offer serious hazards to the environment.

Introduction Several reports on uptake, translocation and m e t a b o l i s m o f triforine in plants have been published recently (Fuchs 1971; Fuchs e t al. 1971, 1972; Ost e t al. 1971; Ebenebe 1972; Eichler 1972; Von Bruchhausen and Stiasni 1973; E b e n e b e et al. 1974). F r o m these reports, it has become evident that triforine is r e a d i l y taken up via plant roots, and translocated to stems and leaves. A n apparent lack o f a c c u m u l a tion o f triforine in the roots (Fuchs 1971; E b e n e b e 1972; E b e n e b e e t al. 1974) and a rather high turnover rate o f the fungicide in aerial plant parts (Fuchs e t a l . 1972) seem to account for an " e a r l y " m a x i m u m o f triforine concentrations in the shoots, which, d e p e n d i n g on the e x p e r i m e n t a l conditions used, was found 2-8 d a y s after administration o f the fungicide.

Presented at the Third International Congress of Pesticide Chemistry (IUPAC), Helsinki, Finland, 1974. Archives of Environmental Contamination and Toxicology Vol. 4, 30-43 (1976) ~1976 by Springer-Verlag New York Inc.

30

Triforine in Plants

31

In aerial plant parts triforine was found to be metabolized to at least four metabolites (Fuchs et al. 1972; Von Bruchhausen and Stiasni 1973), one of which was identified as piperazine. In the experiments to be briefly described here, translocation and distribution as well as metabolism of triforine in plants were examined more closely by using both 3H- and l~C-labelled triforine, whereas analysis of each single plant part provided a detailed picture of these phenomena. These processes were studied in relation to such parameters as mode and duration of application, age of plants, etc. In addition, metabolism of triforine was compared with non-enzymatic degradation of the fungicide in aqueous solution, in order to further elucidate the chemical nature of the breakdown products, thus permitting more exact conclusions regarding the fate of triforine in plants. A more detailed description of the results will be given elsewhere (Fuchs and De Vries 1974, Fuchs et al. 1974).

Materials and methods 3H-triforine (uniformly 3H-labelled in the piperazine ring) or ~4C-tdforine (uniformly 14C-labelled in the trichloroethyl moiety of side chains) was administered via the roots of plants belonging to different species, either by immersing the plants with their roots in aqueous solutions or by applying the fungicide as soil drenches. After appropriate periods of " i n c u b a t i o n " , plants were harvested and divided into roots, hypocotyls, cotyledons, epicotyls, internodes, leaves, leaflets, etc. After fresh weight determinations had been made, all parts were extracted in 10 to 50 ml of methanol, depending on fresh weight. Some of the ~4C-triforine-treated plants were left intact and dried between filter papers as evenly and quickly as possible; then, autoradiographs were prepared by exposing Kodak No-Screen Medical X-ray film to the dried plants. Radioactivity in the extracts of all separate plant parts was measured by liquid scintillation counting (LSC) and expressed as dpm/mg fresh weight. The degree of accumulation of radioactive compounds (triforine + possible breakdown products) in each single part was calculated relative to average radioactivity (expressed as dpm/mg fresh weight) of total aerial plant parts, this ratio providing the best approximation of the distribution of the label over the various plant parts. In addition, in some cases the amount of radioactivity present in specific plant parts (e.g., primary leaves, first two leaves present at the time of application of radioactive triforine) was also given relative to total amount of radioactivity in aerial plant parts. In order to transform the data on distribution of radioactive compounds in general into data on distribution of triforine, extracts were further subjected to thin-layer chromatography (solvent butanol/acetic acid/water 4:1:1). TLC's were cut into ten equal pieces, with Rr-values 0.00 to 0.10 to 0.90 to 1.00, and radio-activity in them was measured by LSC; in the case of 14C-triforine, analysis of TLC's also proceeded by radioscanning. The analytical procedures used separated triforine from its degradation products and thus enabled calculation of total amounts of radioactivity present as 3H- or 14C-triforine in all single plant parts, also in relation to the

32

A. Fuchs and W. Ost

incubation period, and thus permitted conclusions on the rate of conversion of triforine in plants. Chromatographic behaviour of degradation products was compared with that of non-metabolic, hydrolytic breakdown products of triforine. These products were obtained by incubating non-buffered aqueous solutions of triforine (30 /~g/ml) at room temperature in closed vials in darkness and in diffuse daylight. The decrease in concentration of the parent compound and the formation of ionogenic chlorine were followed quantitatively for four weeks and seven months, respectively. The appearance of other breakdown products was examined periodically for up to one year, using TLC as the method of analysis. The main hydrolysis products were isolated after freeze-drying of the aqueous solution, and after chromatographic purification identified by mass spectrometry and by comparison with authentic reference substances.

Results From Tables I and II it is evident that, after short-term treatment o f bean and tomato seedlings with 3H-triforine, radioactivity was rapidly accumulated in the leaves present at the time of treatment, without subsequent redistribution into the later expanding leaves. These conclusions are based on the following observations: a) The degree of accumulation of radioactivity in the roots (relative to average radioactivity in aerial plant parts) dropped rapidly after termination of the treatment. b) In the primary leaves (bean) or the cotyledons and first leaves (tomato) the degree of accumulation of radioactivity increased continuously, not, however, because of a continued supply with radioactive substances, but because of a decrease of the average radioactivity in the aerial parts due to the growth of plants; since all new growth was virtually devoid of radioactivity, the first leaves became relatively " r i c h e r " in radioactive label, c) The percentage of total radioactivity in the first expanded leaves increased rapidly to almost 90% of the total radioactivity in aerial plant parts. An entirely different distribution pattern of radioactivity was found after shortterm treatment of " a d u l t " plants; here, radioactivity was much more evenly distributed over all plant parts, in a concentration gradient from roots to youngest leaves in barley and bean plants (Tables III and IV; see: application time: 6 hr). Uptake of 3H-triforine by barley plants showed a rapid saturation at about 60% of the administered label (Fig. 1). The total activity in the roots hardly increased after six hr (Table III), the rate of translocation of label to stem and leaves exceeding the rate of uptake from 12 hr onwards. Nevertheless, after 42 hr about 70% of the label was still retained in the roots. Radioactivity was translocated to the leaves at the expense of label in roots and stems, with preference for the first leaf up to 18 hr; after 42 hr the highest degree of accumulation (also in absolute amounts o f radioactivity) was found in the third leaf. Degrees of accumulation of radioactivity in the tillers were almost time-independent over the entire application period, the highest accumulation being found in the " s e c o n d " tiller.

13.17 1.05 1.12

Degree of accumulation of radioactivity in relation to average radioactivity in aerial plant parts

85.5 c

Percentage of radioactivity in primary leaves present as 3H-triforine

a Immediately after administration of 3 H-~riforine; hint. = internode; tin roots.

41.6

Percentage of radioactivity in primary leaves (with respect to total radioactivity in aerial plant parts)

roots int. b 1 + 2 primary 1.

2+0

9a

64.6

86.4

0.42 0.41 2.40

2+1

22

52.3

84.6

0.59 0.63 3.69

2+2

33

Age (days)

41.5

86.0

0.70 1.28 5.95

2+3

43

Distribution of radioactivity and metabolism of triforine after administration of 3H-triforine to bean plants, cv. Dubbele Witte z.dr. (Age of plants at time of administration o f triforine: 9 days)

Number of leaves (primary + trifoliate leaves)

Table I.

40.2

89.0

0.76 1.32 14.58

2+5

54

-~"

--.

,--t

87.3

Percentage of radioactivity in second leaf present as 3H-triforine

53.6

86.8

1.01 1.10 1.13 1.88 2.14

2+3

33

16.0

87.2

1.52 4.82 3.79 8.08 4.70

2+5

49

almmediately after administration of 3H-triforine; b c o t . = cotyledon; edropped; donly in leaf 1 and 2

74.5

Percentage of radioactivity in cotyledons and first and second leaf (with respect to total radioactivity in aerial plant parts)

8.20 0.72 0.56 1.27 1.41

Degree of accumulation of radioactivity in relation to average radioactivity in aerial plant parts roots cot. b 1 cot.b2 leaf 1 leaf 2

2+2

Number of leaves (cotyledons + true leaves)

26 a

Age (Days)

10.6

88.9

2.11 10.39 10.06 11.20 4.00

2+6

58

Table II. Distribution of radioactivity and metabolism of triforine after administration of 3H-triforine to tomato plants, cv. Bonnet Beste (Age of plants at time of administration of triforine: 26 days)

3.4

85.1 d

3.17 _e _c 14.09 8.02

0+9

77

rn e~

~r

>

Triforine in Plants

35

In bean plants, uptake of ~H-triforine took place much more slowly than in barley plants, after 18 hr only a third of the total amount administered being taken up. As can be derived from the low degree of accumulation in the roots after 42 hr, aH-triforine, however, was readily translocated to the stems and leaves, only 50% being still retained in the roots at that time (Table IV). In the stems, radioactivity was initially present in a concentration gradient from roots to tips; after prolonged " i n c u b a t i o n " , a maximum in the degree of accumulation was found in the second internodes. With regard to the leaves, the label was preferentially translocated to the youngest leaf present. Since in the latter experiments, even after 42 hr of "incubation", from 75 to 90% of the radioactivity was present as 3H-triforine, the data given can be assumed to reflect the actual distribution of the parent compound. However, in experiments as described in Table I and II, the amounts of radioactivity present do not represent the

Distribution of radioactivity after administration of 3H-triforine to "adult" barley plants, cv. Cambrinus, in relation to duration of uptake;administered 13 830 600 dpm per plant (Age of plants at time of administration: 33 days)

Table IlI.

Degree of accumulation of radioactivity in relation to average radioactivity in aerial plant parts

Amounts of radioactivity (in dpm/mg fresh weight) per plant part

Applicationtime(hr) 6 roots "stem" leaf 1 leaf 2 leaf 3 leaf4 leaf 5 leaf 6 leaf 7 tiller 1 tiller 2 tiller 3

28.03 2.53 3.24 1.74 1.47 0.96 0.50 0.22 0.36 0.85 I. 19 0.88

AppHcationtime(hr)

12

18

42

6

12

18

42

20.02 2.19 3.57 1.66 1.57 1.19 0.53 0.26 0.31 0.93 1.12 0.85

11.25 1.86 2.92 1.44 1.24 1.24 0.78 0.43 0.38 1.20 1.37 0.94

5.41 1.33 1.60 2.35 2.48 1.27 0.94 0.47 0.36 0.97 1.18 0.80

1972 182 213 130 106 69 38 16 27 63 89 65

1999 219 347 161 157 119 54 26 32 93 111 86

1896 311 497 244 208 208 128 71 63 203 233 159

1162 292 347 512 532 277 200 100 77 210 254 171

6732550

7454100

7671200

7934950

total uptake (ha dpm) total uptake (in % of amount administered)

48.7

53.9

55.5

57.4

radioactivity in roots, as % of total uptake

88.1

85.0

78.6

70.1

A. Fuchs and W. Ost

36

actual amounts of 3H-triforine, and, hence, the degrees of a c c u m u l a t i o n in the various plant parts might not reflect the distribution of triforine. Therefore, in these experiments the rates of degradation of triforine were estimated from the available data using methods as indicated under " M a t e r i a l s and m e t h o d s " ; for bean and tomato plants, the biological half-lives of triforine proved to be ca. 40 and 10 days, respectively (Fig. 2). Preliminary results showed that these values should not be taken too absolutely. Although they are valid for the aerial parts indicated (Tables I and II), in the roots the biological half-lives appeared to be dissimilar.

Table IV. Distribution of radioactivity after administration of 3H-triforine to "adult" bean plants, cv. Dubbele ;r z.dr., in relation to duration of uptake; administered 13 830 600 dpm per plant (Age of plants at time of administration: 46 days) Degree of accumulation of radioactivity in relation to average radioactivity in aerial plant parts Application time 6 hr

roots internode internode internode internode

1 2 3 4

10.47 a 6.44 5.08 2.60 0.80

12 hr petiole

leaf blade

1.28 1.07 0.43

0.75 b 0.41 0.38 0.55

petiole roots internode 1 internode 2 internode 3 internode 4

6.91 a 4.07 4.67 2.85 0.95

w

leaf blade m

1.16 1.94 0.85

0.56 b 0.32 0.26 1.58

Application time 42 hr

18 hr

roots internode 1 internode 2 internode 3 internode 4

3.71 a 2.45 2.53 2.22 1.34

petiole

leaf blade

_ 1.00 0.90 0.91

_ 0.67 b 0.47 0.41 1.81

petiole roots internode internode internode internode

I 2 3 4

1.58 a 2.20 2.56 2.28 1.08

-

-

_

0.96 O.95 0.60

leaf blade w

1.01 b

0.81 O.57 1.66

aradioactivity in roots, as % of total uptake: 87.1 (6 hr), 79.0 (12 hr), 55.7 (18 hr) and 50.5 (42 hr), respectively. baverage degree of accumulation of radioactivity in primary leaves.

Triforine in Plants

37

15A m o u n t o f radioactivity administered

10 Q X

,-O ...._

E O.

O ......-.=------

O --

"lD

0

I 10

0

I 20

I 30

I 40

Duration o f application (hours)

Fig. 1. Uptake of 3H-triforine by barley plants, in relation to duration of application.

In tomato plants, for instance, after 58 days c a . 30% of the label in the roots was still in the form of 3H-triforine. However, it should be realized, that (especially for roots) such data do not merely reflect metabolic rates, but also rates of translocation of the parent compound and its degradation products into stem and leaves.

100 -

~_ 5 0

0

I 0

10

i 20

310

I 40

I~ -

50

Time in days after administration o f triforine

Fig. 2. Rate of conversion of triforine in primary leaves of bean plants and in the second leaf of tomato plants after short-term (ca. 6-hr) treatment, and in the 6th leaf of pea plants after long-term (one-week) treatment.

38

A. Fuchs and W. Ost

Experiments on the distribution of ~4C-triforine in pea plants provided results comparable to those of the experiments with 3H-triforine. The biological half-life in pea proved to be ca. 40 days (Fig. 2). Other results will be outlined elsewhere (Fuchs and De Vries 1974). The experiments discussed so far provided an approximate idea about uptake and translocation of triforine in plants; however, they did not give any information about the distribution of triforine within single leaves. Therefore, autoradiographs of healthy as well as diseased plants, treated with ~4C-triforine, were made after one and four days of "incubation". The diseased plants were employed to examine a possible effect of fungal infection on the distribution of triforine. In the healthy plants of all species chosen (viz. bean, pea, tomato and wheat), the radioactive label proved to be very evenly distributed over the leaf blades, without any appreciable accumulation in the leaf margins. In rust-infected bean leaves, on the other hand, there was a slight tendency toward increased accumulation of label at the infection sites (Fig. 3); in rust-infected wheat leaves label seemed to be preferentially translocated to the most heavily infected leaves. Perhaps, leaf damage, leading to enhanced evaporation, conditioned this increased accumulation; this seems to hold true at any rate for the healthy leaves of tomato plants, which upon prolonged "incubation" reacted to high concentrations of triforine (50/J,g a.i./ml) with distinct lesions in the leaves with concomitant accumulation of label at these sites.

i ~ %'`

~

~

o

~

/x" .og..' ~

9 o

Fig. 3. Effect of rust (Uromyces phaseoli) infection on distribution of label in 14C-triforinetreated bean leaves. Left: bean leaf, showing typical green islands around infection sites; right: autoradiograph, showing slightly increased concentrations of radioactive label around infection sites.

Triforine in Plants

39

TLC-analysis of extracts of 3H-triforine-treated plants invariably revealed four areas on the TLC-plates carrying substantial amounts of radioactivity, with Rfvalues of 0.03, 0.50, 0.75 and 0.901, respectively (Fuchs et al. 1972). TLCradioscans of t4C-triforine-treated plants also showed four maxima of radioactivity (Fig. 4) with Rf-values of 0.12, 0.40, 0.67 and 0.90, respectively. One of the four products of each of both radioactive triforines is obviously characteristic for the type of labelling: the ZH-containing compound (Re-value 0.03) is almost certainly piperazine (Eichler 1972, Fuchs et al. 1972, Von Bruchhausen and Stiasni 1973); the 14C-containing compound (Re-value 0.12) must be a substance lacking the piperazine ring, and is thus a substance arising from (part of) the side chain(s) of triforine. One of the three other substances common to both types of labelled triforine, with Re-value 0.90, is the parent compound itself; the identity of the two remaining ones is not yet known. Probably, they are identical with or structurally related to two of the hydrolysis products, which are formed upon sterilization of triforine (cf. Fuchs et al. 1971) or arise more gradually when aqueous triforine solutions are kept at room temperature. Even under such mild experimental conditions, the parent compound proved to be hydrolyzed rather quickly, in darkness as well as in diffuse daylight, no triforine being left after four weeks of incubation.

Fig. 4. TLC-radioscan of extract of t4C-triforine-treated pea leaf. The highest peak (at left) is due to unchanged triforine. Solvent: butanol/acetic acid/water 4:1:1. 1Because of the method used, the last three are only approximate values; they vary from plate to plate between 0.40-0.60; 0.70-0.80 and 0.80-1.00, respectively.

40

A. Fuchs and W. Ost Table V. Formation of CI- in aqueous solutions o f triforine (30 lzg/ml) in relation to incubation time After (time)

% of theoretical amount

30 min 10 days 28 days 50 days 7 months

< 1% 37% 71% 86% > 95%

The chemical half-life of the fungicide in triforine solutions containing 30/.xg a.i./ml proved to be only three days. Time-course measurements of chloride formed showed intermediates with intact CC13-groups to be rapidly hydrolyzed further, with formation of inorganic chloride (Table V). Since this was present at about 50% as free hydrogen chloride, the pH of the solution decreased from 5.5 to 3.6 within three days, remaining constant afterwards.

OHC--NH--CH--CCI 3 I

N ~

,"

Unstable intermediate

I

OHC-NH-CH--CCl3 Triforine

J

/

. OH

%0-0/ I ~ N

N

I

OHC_NH_CH_CCI3 I

Substance I I

1

OH

/OH

o~C-CH

H.HCI N

H.HCI N

N

N

I

/OH

H.HCl

.C-CH

OH Substance II

O//

~

OH

Piperazine

Substance III

Fig. 5. Suggested pathway of hydrolytic breakdown of triforine in aqueous sotution (30 /~g/ml), kept at room temperature for one year.

Triforine in Plants

41

Identification of the various degradation products revealed hydrolysis to proceed as indicated in Fig. 5. The asymmetric piperazine derivative N-(1-formamido2,2,2-trichloroethyl)- N'-2,2-dihydroxyacetyl- piperazine (I) was the first product to be isolated in substantial amounts after one to three weeks of incubation. It was further degraded with formation of the water-soluble intermediate bis-glyoxylic piperazine dihydrate (II) and an N-monosubstituted piperazine derivative (III). The latter breakdown product seems to be only stable in solution, or as a salt, and is probably N-glyoxylic piperazine. Finally, piperazine itself was produced (isolated as a dihydrochloride) and, in addition, a number of other chlorine-free, water-soluble products, arising from the side chains. At elevated temperatures, hydrolysis was greatly accelerated, so that some of the above-mentioned hydrolysis products could not even be demonstrated under such experimental conditions. The terminal residues of triforine hydrolysis constitute, beside piperazine, very probably a series of low molecular weight organic acids, which partly are common in plants, such as glyoxylic, formic and glycolic acid, and CO2.

Discussion A critical quantitative evaluation of the results obtained on uptake, translocation and metabolism of triforine is hampered in various ways. For instance, in the roots the amounts (and thus concentrations) of triforine are the resultant of, a) uptake of triforine from the substrate (aqueous solution, soil), in which chemical breakdown of the parent compound will proceed continuously, b) translocation to the stems and leaves, and c) metabolic and/or non-metabolic conversion of triforine into other products. Therefore, values on biological half-life in roots can hardly be assumed to represent the intrinsic value for the biological half-life of triforine characteristic of the plant species involved. In fact, only those plant parts in which any decrease in triforine concentrations is entirely due to breakdown, can be considered to give reliable results in the estimation of biological half-life. In the experiments described in Tables I and II, the primary leaves of bean plants and the first two leaves of tomato plants seem to fulfill these requirements. In these instances, after short-term treatment, triforine attained a maximum level within a few days without further uptake or redistribution of fungicide taking place after this period. Therefore, any change in triforine concentrations can be ascribed to chemical and/or metabolic conversion. The estimated values for leaves of bean and tomato plants, which were found to be ca.40 and 10 days, respectively, might be supposed at least to approximate the intrinsic value of the biological half-life of triforine for these species. These values are so dissimilar from the chemical half-life in aqueous solution (three days), that it is difficult to ascribe the differences to factors other than environmental ones. The conclusion seems inevitable that triforine, in some way or other, is protected against breakdown within the plant. In fact, a similar conclusion was drawn by Eichler (1972), who attributed the very slow degradation of triforine in the peel of apples to " i n c l u s i o n " of the fungicide in the lipophylic material of the cuticle.

42

A. Fuchs and W. Ost

Autoradiographs of 14C-triforine-treated plants evoke the impression of a homogeneous distribution of label over the leaf blades; therefore, it seems an attractive idea to suggest that triforine is primarily translocated to the lipophylic outer layer of the epidermis. There, it could not only be protected against degradation, for instance, because of adsorption to lipid cell wall material, but could also attain relatively high concentrations, high enough to protect the plant against diseases caused by obligate parasites, like mildews and rusts, and other foliar diseases caused by non-obligate pathogens. This hypothesis might also constitute an explanation for the remarkable fact that, in vitro and in vivo, activities of triforine are by no means positively correlated (Drandarevski and Fuchs 1973, Fuchs and Drandarevski 1973), and that it is found to be active against an increasing number of foliar pathogens (Drandarevski and Fuchs 1973, Fuchs and Drandarevski 1973), as has been once more shown recently (Szkolnik 1974). Comparison of the TLC data on the four conversion products of triforine left three compounds, with Rrvalues of 0.12, 0.40 to 0.50 and 0.67 to 0.75, unidentified. The fourth one, with an Rrvalue 0.03, which does not contain a 14C-labelled side chain is certainly piperazine (cf. Eichler 1972. Fuchs et al. 1972, Von Bruchhausen and Stiasni 1973). The one with the Rr-value of 0.12 must be a substance arising from the side chain, since it lacks the piperazine ring; it might be glyoxylic acid or structurally related to it. The remaining ones, which contain both the piperazine ring and at least one of the C-atoms of the side chains, could be identical with or structurally related to two of the three substances I, II and III. However, their exact chemical structures await final identification. Its low persistence in aqueous solution and in plants as well as its low toxicity to mammals, fish and bees (Schicke and Veen 1969) suggest that triforine does not offer serious hazards to the environment.

References Drandarevski, C. A., and A. Fuchs: The in vitro and in vivo antifungal activity of triforine. Meded. Fak. Landb.wetensch. Gent 38, 1525 (1973). Ebenebe, C: Wirkung des systemischen Fungizids Triforine gegen Weizenbraunrost und Welkekrankheiten der Tomate. Dissertation, Giessen (1972). Ebenebe, C., V. von Bruchhausen, and F. Grossmann: Dosage-response curve of wheat brown rust to triforine supplied via root treatment. Pesticide Sci. 5, 17 (1974). Eichler, D.: Ober das Abbauverhalten von Triforine dargestellt an einigen ausgew~ihlten Kulturpflanzen. Meded. Fac. Landb.wetensch. Gent 37, 831 ( 1972). Fuchs, A.: Opname, transport en omzetting van triforine in planten. Gewasbescherming 2, 142 (1971).

Triforine in Plants

43

Fuchs, A., M. Viets-Verweij, J. Vfrrs, and F. W. de Vries: Some observations on activity and metabolism of a new systemic fungicide, N , N ' - b i s - ( l formamido-2,2,2-trichloroethyl)- piperazine (CELA W 524). Acta Phytopath. Hung. 6, 347 (1971). Fuchs, A., M. Viets-Verweij, and F. W. de Vries: Metabolic conversion in plants of the systemic fungicide triforine(N,N'-bis-(1-formamido-2,2,2trichloroethyl)- piperazine; CELA W 524). Phytopath. Z. 75, 111 (1972). Fuchs, A., and C. A. Drandarevski: Wirkungsbreite und Wirkungsgrad von Triforine in vitro and in vivo. Z. Pfl. Krankh. Pfl. Sch. 80, 403 (1973). Fuchs, A., and F. W. de Vries: Uptake, translocation and metabolic fate of t~Ctriforine in plants. In preparation. (1974) Fuchs, A., F. W. de Vries, and A. M. J. Aalbers: Uptake, translocation and metabolic fate of aH-triforine in plants. In preparation. (1974). Ost, W., V. von Bruchhausen, and C. Drandarevski: Transport of the systemic fungicide Cela W 524 in barley plants (Part I). Pesticide Sci. 2, 219(1971). Schicke, P., and K. H. Veen: A new systemic, CELA W 524 (N,N'-bis-(1formamido- 2,2,2-trichloroethyl)- piperazine) with action against powdery mildew, rust and apple scab. Proc. 5th Br. Insecticide Fungicide Conf. 569 (1969). Szkolnik, M.: Unusual post-infection activity of a piperazine derivative fungicide for the control of cherry leaf spot. P1. Dis. Reptr 58, 326 (1974). yon Bruchhausen, V., and M. Stiasni: Transport of the systemic fungicide Cela W 524 (triforine) in barley plants II. Uptake and metabolism. Pesticide Sci. 4, 767 (1973).

Manuscript received September 28, 1974; accepted November 11, 1974