Ann. appl. Biol. (1975), 80, 191-198

Printed in Great Britain

Investigations on fungicides

XVIII. The fungitoxicity and systemic antifungal activity of certain bis-[(l-formamido-2,2,2-trichloro)ethyl-amino]-alkanes and related

compounds

BY G. A. CARTER, K. CHAMBERLAIN AND R. L. WAIN Agricultural Research Council Plant Growth Substance and

Systemic Fungicide Unit, Wye College ( University of London), Ashford, Kent, TN25 5 A H

(Accepted 20 December 1974)

SUMMARY

A number of bis-[(~-formamido-z,z,z-trichloro)ethylamino]-alkanes and their dioxy and dithio analogues were examined for direct and systemic antifungal activity. All compounds showed very low in vitro fungitoxicity and only slight protectant anti-mildew activity when applied as a foliar spray. Certain diamino and dioxy derivatives were, however, systemically active against powdery mildew fungi when applied as a root drench to seedlings of wheat or cucumber; the dithio analogues were inactive. The level of systemic activity shown by these compounds was markedly influenced by the nature of the hydrocarbon ‘bridging’ group.

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

Chloraniformethan (I) is a powdery mildew fungicide showing some movement within plant tissues (Davis, Weeden & Martin, 1971). The chemically related fungi- cide Triforine (11) is also systemically active against powdery mildews (Schicke & Veen, 1969). Previous studies in these laboratories (Carter, Summers & Wain, 1972; Carter, Chamberlain & Wain, 1973, 1975) indicated that in compounds of this type the terminal formamide group in the molecule is essential for anti-mildew activity. Our investigations also showed that alkoxy- and alkylamino-trichloroethylformamides (111; X = NH or 0, Y = an alkyl group) are systemically active against powdery mildews when applied to the roots of wheat or cucumber seedlings. In the present study we examined the relationship between structure and antifungal activity in compounds of type IV where X and X’ = NH, 0 or S and Y is a polymethylene or methyl substituted polymethylene group.

G. A. CARTER, K. CHAMBERLAIN AND R. L. WAIN CCI9CH.NH.CHO

CCl,.CH.NH.CHO

CCI&H.NH.CHO CCI,.CH.NH.CHO I

X I

X I I Y

I Y

7 CC13.CH.NH.CH0

IV Ill

CCI3.CH.NH.CHO I

CI

V MATERIALS

The chemicals listed in Table I were prepared from N-( 1,~,~,2-tetrachloroethyl)- formamide (V) and the appropriate diamine, diol or dithiol by the method described by Ost, Thomas, Jerchel & Appel(1969) and Ost, Thomas & Jerchel (1971). Triforine was also included in the tests both as a commercial 20% emulsifiable concentrate formulation (Cela W 5 q ) and as the pure (unformulated) compound.

M E T H O D S

Direct antifungal activity was assessed by in vitro measurements of inhibition of spore germination and by the application of protective sprays to wheat seedlings. Therapeutic activity was measured using inoculated leaf disks and systemic activity by applying the test compounds to the roots of wheat or cucumber seedlings. Details of the test methods are given by Lehtonen, Summers & Carter (1972) and Carter et al.

Spore germination tests All compounds were tested for their ability to inhibit the germination of conidia of

Alternaria brassicicola (Schw.) Wiltshire, Botrytis cinerea Pers. ex. Fr., Septoria (Leptosphaeria) nodorum Berk., Cladosporium cucumnum Ell. & Arth., and Asper- gillus niget van Tieghem plus uredospores of Uromyces viaae-fabae (Pers.) Schroet.

(1972).

Investigations on fungicides I93

Table I. Compounds investigated Basic formula IV

Compound I

no.

I 2 3 4

5 6 7 8 9

I 0 I1

I 2

I3 I 4 I5

16 77 I8

X

NH NH NH NH

0 0 0 0 0 0 0

S S S S

0 S 0

x Y M.P. (“c)* Bis-[( I -formamido-z,~,2-trichloro)ethylamino]alkanes

NH (CH32 142-144 NH (CHa13 Oil NH (CHah 141-144 NH CH(CH3)CHz oil

Bis-[( I -formamido-~,z,z-trichloro)ethoxy]alkanes 0 (CH& Oil 0 (CH2)3 Oil 0 (CHZ)4 Oil 0 (CHZ), Oil 0 CH(CH3)CHp Oil 0 CH(CH.d(CHz)a Oil 0 CH(CH,)CH( CH,) Oil

Bis-[( I -formamido-z,z,z-trichloro)ethylthio]alkanes S (CHZh. 163-165 S (CHa)3 147-150 S (CHZ)4 151-1 54 S CH(CHJCH2 149-152

Miscellaneous

NH (CHz)a 146-148 NH (CHa), 113-1 I7 S (CHA Oil

* Where an oil is indicated it implies a very viscous undistillable compound. t Ost et al. (1969). $ Ost et al. (1971).

Protectant activity The compounds were dissolved in 50% aqueous acetone and applied as a foliar

spray to 7- to 10-day-old seedlings of wheat immediately prior to inoculation with either powdery mildew, Erysiphe graminis DC f sp . tritici Marchal or brown rust, Puccinia recondita Rob. ex Desm f.sp. tritici Eriks. & Henn.

Leaf disk test Disks were cut from leaves of cucumber inoculated on one surface with spores of

mildew, E . cichoracearum DC, or anthracnose, Colletotrichum lagenarium (Passer.) Ell. & Hals., and from leaves of broad bean inoculated with U. viciae-fabae. These disks were floated, inoculated side uppermost, upon aqueous solutions of the test compounds containing 500, 100, 20 or 4 mg/l and the extent to which symptoms of disease and phytotoxicity developed was recorded.

Root application Five seedlings of wheat (cv. Eclipse) were grown in 80 cc plastic cups containing

either sand or EFF compost. Single seedlings of cucumber (cv. Sutton’s Improved Telegraph) were grown in pots of sand. Treatment began when the wheat seedlings

I94 G. A. CARTER, K. CHAMBERLAIN AND R. L. WAIN were 3 days old and the cucumber seedlings 10 days old. During the treatment period, 7 days for wheat, 10 days for cucumber, each pot received 2 x 20 ml of solution, controls receiving water. Sand grown plants were also given a complete mineral nutrient solution.

From 24 h after the second root treatment, plants were continuously exposed to infection by E. graminis (wheat) or E. cichoracearum (cucumber) and mildew develop- ment assessed at intervals. Some wheat plants were inoculated with a suspension of uredospores of P. recondita and incubated for 48 h under high humidity, rust develop- ment being assessed 10 days later.

RESULTS

Activity in spore germination tests All experimental compounds showed very low fungitoxicity being inactive towards

spores of Alternaria, Botrytis, Septoria, Cladosporium and Aspergillus when applied at 1000 mg/l. Certain compounds inhibited germination of Uromyces uredospores at 250 mg/l(13, 18),* 500 mg/ l (~o) or 1000 mg/l(7, 11, 17). Triforine, although inactive against the other test fungi, showed high activity against spores of A . niger (inhibitory at 25 mg/l) and slight activity against those of C. cucumerinum (inhibitory at 250 mg/l).

Protectant activity When applied as a foliar spray at IOO mg/l Cela W524 was fully effective against

powdery mildew of wheat and reduced brown rust infection by 75 %. Unformulated triforine was not effective against the rust but reduced powdery mildew infection by c. 50%. None of the experimental compounds was effective against rust but five ( I , 5 , 9, 11, 18) reduced mildew infection by ~ O - S O ~ ~ , the remainder being inactive.

Activity in leaf disk tests Unformulated triforine inhibited the development of cucumber mildew at IOO mg/l,

inhibited broad bean rust at 500 mg/l but was inactive against cucumber anthracnose. At 500 mg/l no experimental compound was effective against either anthracnose or rust whilst the level of activity against cucumber mildew varied from inactive at 500 mg/l to fully inhibitory at 20 mg/l (see Table 2). No symptoms of phytotoxicity were observed in any of the treated disks.

Activity in root application tests Activity against rust on wheat. Cela Ws24 and unformulated triforine were fully

effective against brown rust when applied at the rate of 4 mg/pot to the roots of sand- grown wheat seedlings. All experimental compounds were completely inactive.

Activity against powdery mildews. The results obtained in tests for systemic activity against powdery mildew on wheat and cucumber are listed in Table 3.

Initial applications at rates of 4, 2 or I mg/pot to wheat seedlings growing in sand indicated that whilst many diamine (1-4) and diol (5-1 I) derivatives showed marked systemic activity 7 days after treatment, the dithiol analogues (12-15) were largely

* Numbers in parentheses refer to compounds listed in Table I .

Investigations on fungicides I95 inactive. In a second series of experiments lower application rates were included and mildew assessments made over a longer period of time. As before the dithio com- pounds proved inactive but certain diamino (1,2), dioxy, (6,7) and related compounds (16,17) showed systemic activity comparable to that of unformulated triforine or Cela W524.

Table 2 . Therapeutic activity against powdery mildew of cucumber in leaf-disk tests

Minimum Minimum concentration (mg/l) concentration (mg/l)

required to required to Compound no. inhibit mildew Compound inhibit mildew

I

2

3 4

5 6 7 8 9

I 0 I 1

20 1 0 0

5 0 0 100

20 20

I 0 0 > 500

20 I 0 0 I 0 0

12

13 14 '5 16 ' 7 18 Triforine

5 0 0 > 500 > 5 0 0

500

20 20

100 20

A preliminary test in which compounds, I, 2, 4 and 5 , together with Cela w524, were applied at rates of 8 ,4 or 2 mg/pot to wheat seedlings growing in EFF compost showed that these compounds were less effective than when applied to sand. A second experiment in which all compounds were applied to compost-grown plants at a rate of 8 mg/pot confirmed that systemic activity was less than in sand although the relative effectiveness of the compounds remained the same.

I n a single experiment in which all compounds were applied at a rate of 2 mg/pot to sand-grown cucumber seedlings the dithiol derivatives again proved inactive. Many compounds that showed notable systemic activity in wheat seedlings were also highly active in cucumbers (I, 2, 4, 5 , 6, 9, 16) but others (3, 7, 10, 17, IS) were much less effective, as was Cela w524.

D I S C U S S I O N

Antifungal activity within this group of compounds was largely restricted to in vivo activity against powdery mildews, in vitro toxicity to other fungi being very low. When applied to the roots of wheat seedlings the most effective compounds (I, 2, 16, 17) showed a level of systemic anti-mildew activity comparable to that of Cela w524 whilst compounds I , 2,4, 5,6 ,9 and 16 were all more effective than Cela when applied to cucumber seedlings. The antifungal activity of these compounds was, however, more restricted than that of triforine in that they were not very effective against mildew when applied as a foliar spray, they were not systemically active against brown rust of wheat and they showed no in vitro fungitoxicity towards spores of A. niger. This specific in vivo anti-mildew activity was similar to that shown by the alkoxy- and alkylamino-trichloroethyl formamides examined in earlier studies (Carter et al. 1973, '975).

G. A. CARTER, K. CHAMBERLAIN AND R. L. WAIN

Table 3. Systemic activity* against powdery mildew following application to the roots of seedlings of wheat or cucwnber

Wheat Cucumber

Sand grown Soil grown r 7 -

I 8 4 2 I

0.4 0'2

2 8 4 2 I

0.4 0'2

3 8 4 2 I

4 8 4 2 I

5 8 4 2 I

6 8 4 2 I

0.4 0'2

7 8 4 2 1

0.4 0'2

8 8 4 2 I

9 8 4 2 I

0.4 0'2

Test I1 Test I Test I1

- - - -

of < I f

100 - - -

Sand grown -

Investigations on fungicides I97

Table 3 (cont.) Wheat Cucumber

7 Sand grown Soil grown Sand grown

c - 7- , , Com- Dose Test I Test I1 Test I

no.

1 0

1 1

I 2

'3

I4

75

16

I7

I8

8 4 2 I

0.4

8 4

0'2

2 I

8 4

8 4

2

2 I

8 4 2 I

8 4

8 4

2

2 I 0.4 0'2 ~ - 8 4 2 I

0.4

8 4 2 I

0'2

0.4 0'2

Triforine 8 1

I

0.4 0'2

Cela 8 w524 2

I

0'4 0'2

441 - _ _

* Figures are infection ratings as a percentage of control. -f. Figures in parentheses show post-treatment period in days. 1, Indicates symptoms of mild or severe phytotoxicity.

G. A. CARTER, K. CHAMBERLAIN AND R. L. WAIN A comparison of the systemic activity of these compounds shows that, as compared

with their diamino and dioxy analogues the dithio compounds (12-15) were almost totally inactive. In general the diamine and diol derivatives showed a similar level of activity; although individual pairs of analogues differed in their effectiveness on con- sistent pattern of relative activity emerged. Amongst the three compounds which contain two different hetero-atoms in the molecule (16, 17, IS) systemic activity was highest when a sulphur atom was absent (16) and lowest when an amino group was lacking ( I 8).

Within both the diamine and diol derivatives, systemic activity decreased if the 'bridging group ' ( Y ) was extended beyond two or three methylene groups. In most instances the substitution of a branched chain also decreased systemic activity (com- pare 4 with I ; 11 with 5 ; 10 with 6).

Almost all compounds which at 20 or ~ o o m g / l inhibited the development of mildew on cucumber leaf disks showed good to moderate systemic activity when applied to the roots of seedlings of wheat or cucumber. This suggests that these com- pounds, or metabolites produced in vivo, are readily translocated. The dithio com- pounds, however, showed little or no activity in leaf disk tests and thus lacked intrinsic in vivo antifungal activity whether or not they were translocated.

R E F E R E N C E S

CARTER, G. A., CHAMBERLAIN, K. & WAIN, R. L. (1973). Investigations on fungicides. XV. The fungitoxicity and systemic antifungal activity of N-(z,2,2-trichlor0-1 -methoxyethyl)- formamide and related compounds. Annals of Applied Biology 75, 49-55.

CARTER, G. A., CHAMBERLAIN, K. & WAIN, R. L. (1975). Investigations on fungicides. XVII. The systemic anti-mildew activity of alkoxy, alkylamino- and alkylthiotrichloroethyl for- amides. Annals of Applied Biology 79, 313-320.

CARTER, G. A,, SUMMERS, L. A. & WAIN, R. L. (1972). Investigations on fungicides. XIV. Fungitoxicity of N-(z,~,2-trichloro-1-methoxyethyl)-formamide and related compounds. Annals of Applied Biology 70, 233-243.

DAVIS, M. E., WEEDEN, S. M. & MARTIN, T. J. (1971). Studies of powdery mildew control in spring barley using chloraniformethan. Proceedings of the 6th British Insecticides &3 Fungicides Conference, Brighton, pp. 42-5 I .

LEHTONEN, K., SUMMERS, L. A. & CARTER, G. A. (1972). Fungitoxicity of acid and alkaline hydrolysis products of drazoxolon and related arylhydrazonoisoxazolones. Pesticide Science 3, 357-364.

OST, w., THOMAS, K. & JERCHEL, D. (1971). German Patent No. 2030464. OST, W., THOMAS, K., JERCHEL, D. & APPEL, K. R. (1969). German Patent No. 1901421. SCHICKE, P. & VEEN, K. H. (1969). A new systemic CELA w524 N,N'bis-(formamide-z,z,z- trichloroethy1)-piperazine with action against powdery mildew, rust and apple scab. Pro- ceedings of the 5th British Insecticides & Fungicides Conference, Brighton, pp. 569-575.