Ann. appl. Biol. (1979), 91,203-210 Printed in Great Britain

203

Investigations on fungicides. XXI. The fungitoxicity and systemic antifungal activity of some

aromatic quaternary ammonium salts

BY G. A. CARTER*, A. J. HEYNSt, B. E. KNIGHTS AND R. A. WAINfi Agricultural Research Council Plant Growth Substance and Systemic Fungicide Unit, Wye

College (University of London), Ashford, Kent TN 25 5AH

(Accepted 16 November 1978)

SUMMARY

Root application of certain quaternary ammonium salts, most notably tri-n-butyl- and tri-n-pentyl-4-chlorobenzylammonium bromides, to wheat seedings grown in water culture enhanced their resistance to powdery mildew (Erysiphe graminis) but not to brown rust (Puccinia recondita). Anti-mildew activity was much less apparent if the compounds were applied to sand- or soil-grown seedlings. Systemic activity is considered in relation to the fungitoxicity, phytotoxicity and growth-retardant activity of these compounds.

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

The test compounds were of interest as potential systemics since many are active as growth retardants (Knight, Taylor & Wain, 1969), and must, therefore be taken up by the plant and be physiologically active. Furthermore, other growth retardants have been shown to possess activity in vivo against fungal pathogens (e.g. Sinha & Wood, 1964, 1967; Tahori, Zeidler & Halevy, 1965; Smith & Corke, 1966; van Andel, 1968; Corke, 1969; Smith, 1969; Buchenauer, 1971; Buchenauer & Erwin, 1973, 1976; Erwin, Tsai & Khan, 1976).

MATERIALS

The test compounds were a series of aromatic trialkylammonium bromides

Ar-CH,N+R, Br-

Full details of their synthesis and purification are given by Knight (1966). In some tests, other established growth retardants were included (Table 4).

METHODS

Fungitoxicity Spore germination tests. Duplicate 0.1, 0.075, 0.05, 0.025, 0.01 and 0.005 ml aliquots of

freshly prepared solutions (2 g 1-I) of each compound in ethanol were pipetted into the depressions of microscope cavity slides. When the ethanol had evaporated, 0.2 ml of a suspension of spores was added to each depression, giving a concentration range of from 1000 to 50 mg 1-'.

Present address: Long Ashton Research Station, University of Bristol, Bristol, BS18 9AF. t Present address: Fruit and Fruit Technology Research Institute, Stellenbosch, S. Africa. $ Present address: Development Department, Fisons Pest Control Ltd, Cambridge. 4 Present address: University of Kent, Canterbury.

204 G . A . C A R T E R e t d .

The spore suspension was prepared by washing conidia of Alternaria brassicicola (Schw.) Wiltsh., and Botrytis cinerea Fr. from 7-10-day-old colonies on potato dextrose agar plates, and uredospores of Uromyces fabae (Grev.) Cooke from infected broad bean leaves. These spores were centrifuged and washed three times in distilled water; they were then re-suspended in sufficient sucrose solution (2 g 1-l) to give 50 spores within the field of a x 10 microscope objective. After incubation in a moist chamber at 25 " C for 16 h the percentage germination was assessed. Germination was considered to have occurred when a germ tube at least equal to the spore diameter was observed. Germination on control slides was from 90-98% for Alternaria and Botrytis and from 75-90% for Uromyces. All residues had apparently totally dissolved in the germination medium.

The fungitoxicity of the test chemicals is expressed as the minimum inhibitory dose, i.e. the lowest concentration completely inhibiting germination. Compounds completely inhibitory at 50 mg I-' were re-tested at a lower concentration range. It is possible in these, as in other tests, that owing to the readiness with which quaternary ammonium compounds undergo H-bonding to glass, plastic and paper that the actual concentration available to the test organism was some- what less than that of the applied solution.

Tests against Erysiphe graminis DC were made by the method of Wain, Sobotka & Spencer (1963) except that the test compounds were in aqueous solution and the conidia were incubated overnight at 15-18 OC. Compounds were tested only at 100,250 and 500 mg I-'. Germination on control agar varied from 65-80%.

Systemic activity

(a) Wheat grown in water culture In a wheat internode bioassay test for growth-retardant activity (see Knight et al., 1969),

seedlings of wheat (Triticum aestivum L. cv. Eclipse) were grown in water culture and treated at the roots for 13 days with the test chemical. At the end of this period two seedlings were taken at random from each treatment and five segments, each approximately 1 cm long, were cut from the centre of both the first and second leaves. The 10 leaf segments from one plantheatment were inoculated with brown rust by brushing dry uredospores of Puccinia recondita Desm. over their adaxial surface prior to floating them on an aqueous solution containing kinetin (5 mg I-') to delay senescence.

Leaf segments from the second plant were inoculated with powdery mildew (E. graminis) by removing the lids from the dishes within which they were floating and shaking heavily mildewed wheat seedlings above them.

Inoculated leaf segments were kept in a growth room at 18 OC and under a day-length of 14 h at c. 400 lux. Seven days after inoculation, disease development on each segment was assessed on an arbitrary scale of 0 (no visible symptoms) to 5 (infection as control).

(b) Wheat grown in sand or soil Wheat seedlings, pre-germinated at constant temperature until their coleoptiles were 2-3 cm

long, were potted into air-dried soil or sand, five seedlings per 80 ml capacity plastic cup. Each cup then received 20 ml of a test solution. Seedlings grown in soil received a further 20 m15 days later and were inoculated with mildew on the seventh day after treatment commenced. Sand- grown seedlings received a further 20 ml of solution after 7 days; on the 9th day they were given a standard mineral nutrient solution and were inoculated with mildew. All plants were inoculated in a settling tower using conidia shaken from heavily infected plants.

Infection was assessed 7-10 days after inoculation by counting the number of mildew pustules developing on both surfaces of the first leaf.

Fungitoxicity of quaternary ammonium salts 205

(c) Systemic activity of tri-n-butyl-4-chlorobenzylammonium bromide in soil, sand and water

Wheat seedlings, grown in soil or sand, were root-treated with tri-n-butyl-4-chlorobenzyl- ammonium bromide as described above. Other groups of five seedlings were supported on gauze stretched over small Petri dish bases containing 40 ml of a test solution plus mineral nutrients.

Batches of seedlings grown in soil, sand or water-culture were inoculated with mildew 7, 14 and 21 days after treatment commenced, the method of inoculation and assessment of infection being as described above. At the time of inoculation, phytotoxic damage to the shoot (and to the roots of plants grown in water culture) was assessed on an increasing scale of - to + + +. (d) Measurement of phytotoxicity

The effect of the test compounds on the root growth of wheat seedlings growing under standardised conditions was used as a measure of their phytotoxicity (see Carter & Wain, 1964).

Dilutions of aqueous solutions of most compounds were prepared and 4 ml aliquots pipetted on to 9 cm diameter seed testing papers placed in Petri dish lids. Compounds of low water solubility were dissolved in acetone, 4 ml of this solution being added to each paper, the solvent allowed to evaporate and 4 ml of water added. These less water-soluble compounds may not have fully dissolved in the water but as the germinating seeds were in direct contact with the impregnated paper it seems reasonable to assume that the compounds were able to exert their full effect upon root growth. A single row of 10 uniformly-germinating seeds of wheat (cv. Eclipse; was then arranged around the periphery of each paper. The phytotoxicity of each salt was expressed by the concentration required to inhibit the growth of seedling roots by 50% over a 48- h period.

(e) Tests using dwarfFrench bean In tests for growth-retardant activity, pre-germinated seedlings of dwarf French bean

(Phaseolus vulgaris L, cv. Canadian Wonder), were root-treated with the test chemicals. Internode measurements were made approximately 2 wk after treatment when the first trifoliate leaf had developed and the first internode had apparently ceased to elongate. At this stage the under surface of both primary leaves and the first trifoliate leaf was inoculated by spraying with a dense suspension of the uredospores of Uromyces appendiculatus (Pers.) Unger.

After 48 h incubation in high humidity the inoculated plants were returned to the greenhouse. Fourteen days later a uniform and heavy infection of rust had developed on control plants; the intensity of infection was then assessed for each leaf on an arbitrary scale of 0-5.

culture

RESULTS

Spore germination tests Most compounds showed low fungitoxicity, having a minimum inhibitory dose of > 100 mg 1-l

against all test organisms. However, antifungal activity increased within a series of trialkyl-4- chlorobenzylammonium bromides reaching a much higher level for the n-hexyl, n-heptyl and n- octyl analogues (Table 1).

Water culture All compounds were examined for their systemic fungicidal activity against brown rust and

powdery mildew on wheat. No compound showed appreciable activity against rust but most of them were active against mildew at non-phytotoxic concentrations.

Table 2 shows the activity of a series of tri-alkyl-4-chlorobenzylammonium bromides, the figures given being the mean infection grades of leaf segments cut from both the upper and lower leaf of the treated wheat seedlings. At lo-* M the trimethyl salt was inactive and the corre-

206 G. A . C A R T E R e t d .

Table 1 . The fungitoxicity of some trialkyl-l-chloro- benzylammonium bromides

C1 1 \ CH,-N+R,Br- (3 Minimum inhibitory dose

(mg 1-9 r

R

Methyl Ethyl n-Propy: n-Butyl n-Pentyl n-Hexyl n-Heptyl n-Octyl

Ab

> lo00 750 750 750 75 10 7.5 7.5

Bc Uf

> 1000 > 1000 > 1000 1000 > 1000 1000

750 500 25 25

1.5 1.5 7.5 1.5 10 2.5

>500 >500 > 500 >500 < I 0 0 (100 < 100 -

Ab = Alternaria brassicicola Bc = Botrytis cinerea

Uf = Uromyces fabae Eg = Erysiphe graminis

Table 2. Systemic activity in water culture against wheat powdery mildew, growth-retardant activity and phytotoxicity of certain tri-alkyl-4-chlorobenzylammonium bromides

Comuound

R = Methyl Ethyl Propyl Butyl Pentyl Hexyl Heptyl

Mildew infection (on a scale 0-5) Conc. ( x lo-* M) -

10 3.3 1 0.33

4.8 4.5 4.9 3 5 5 2.6 4.5 5 0.4 0.6 3.3 0.7 0.5 1.4

Toxic 2.1 3.6 Toxic 3 3.4

Retardant activity (96 reduction of

--, internode length 0.1 at M)

3 4 16 54 38

4.6 39 (Toxic) 5 57 (Toxic)

Toxicity (conc. in mg I-') inhibiting root

growth by 50%)

> 500 >500 200 74 70 26 56

sponding -ethyl and -n-propyl salts showed only slight activity but the -n-butyl and -n-pentyl salts were much more active. The corresponding n-hexyl and n-heptyl derivatives proved phytotoxic at concentrations above M and showed only slight systemic activity at this concentration. Phytotoxicity, as measured by root growth inhibition, increased with chain length up to the tri-n- hexyl salt.

Table 3 lists the activity of certain aryl tri-n-butylammonium bromides. The unsubstituted benzyl compound was neither markedly phytotoxic nor systemically active. Increasing halo- genation of the benzene ring or the substitution of an a- or /3-naphthyl group tended to enhance phytotoxicity but the effect on systemic activity was less consistent. Amongst the mono- substituted compounds, the 4-substituted salts were most active systemically. The 2,3- and 3,4- dichloro- salts were almost equally active but other dichloro, trichloro- and naphthyl compounds were less active, the 2,6-compound being inactive.

Certain benzyl quaternary ammonium salts were compared with their phenoxymethyl (-OCH,N+R,) and phenylethyl (-CH(CH,)N+R,) analogues. The results, not presented here, showed that a few phenoxymethyl salts were slightly more active against mildew but all were more phytotoxic than their benzyl analogues. By contrast, the phenylethyl salts were always less systematically active and less phytotoxic than their benzyl analogues.

Fungitoxicity of quaternary ammonium salts 207

Table 3. Systemic fungicidal activity in water culture against wheat powdery mildew, growth-retardant activity and phytotoxicity of certain aryl-tri-n-butylammonium bromides

Compound Ar-N+(C ,H9),Br-

Ar = Benzyl 2-chlorobenzyl 2-bromobenzyl 3-chlorobenzyl 3-bromobenzyl 4-chlorobenzyl 4-bromobenzyl 2,3-dichlorobenzyl 2,4-dichlorobenzyl 2,s -dichlorobenzyl 2,6-dichlorobenzyl 3,4 -dichlorobenzyl 3 3 -dichlorobenzyl 2,3,6-trichIorobenzyl 2,4,5-trichlorobenzyl I -naphthylmethyl 2-naphthylmethyl

Mildew infection (on a scale ( r5) Conc. (x M)

& 10 3.3 1 3.2 4.3 5 0.7 3.4 4 0.3 3 4.1 2.8 4.3 4.1 1-6 3.6 4.6 0-4 0.6 3.3 0.2 0.2 2 0.5 0.7 3 1.5 3.3 ‘4.8 1.5 1.9 4.8 4.7 4.6 5 0.4 0.7 3.3 1.7 2.2 4.8 1.3 2.2 5 1.7 4.6 5 0.6 1.9 4.4 1 1 5

Retardant activity (% reduction of internode length

at M) 1

10 10 31 27 46 36 13 31 31 2

44 6

27 50 28 31

Toxicity (conc. in mg 1-l)

inhibiting root growth by 50%)

87 54

65

74

45 41 52 48 50

35 40 24 33

A comparison of the systemic activities of tri-n-butyl-4-chlorobenzylammonium bromide with that of certain other established growth-retardants (Table 4) revealed that not all active retardants are effective against mildew.

Applications to sand and soil When the homologous series of trialkyl-4-chlorobenzylammonium bromides were applied to

the roots of sand- or soil-grown wheat seedlings, only a very limited systemic anti-mildew activity was observed. Applications of the n-butyl or n-pentyl salts as lo-‘ M solutions to sand- grown seedlings reduced the incidence of mildew by 40-50% but both higher and lower homologues were ineffective. At this concentration no compound was effective when applied to soil-grown seedlings.

In further studies, higher concentrations of tri-n-butyl-4-chlorobenzylammonium bromide were applied to seedlings grown in soil, sand or water culture (Table 5) . In soil culture, applica- tions of a M solution reduced mildew by over 70% but lower concentrations were ineffective. Applications at this concentration to sand or water cultures although effective against mildew killed the wheat seedlings within 14 days. At M this compound showed some anti-mildew activity in sand culture if inoculations were made 7 days after treatment was commenced but

Table 4. The systemic activity in water culture against wheat powdery mildew and the growth retardant activity of certain reported growth-retardants

Mildew infection (on a scale 0-5 ) Conc. (x lo-’ M) &

2-chloroethyltrimethylammonium chloride ‘Cycocel, CCC’ 4.9 4.9 4.8 Maleic hydrazide 0.3 0.9 4.8 Tri-n-butyl-4-chlorobenzylammonium bromide 0.4 0.6 3.3

Compound 10 3.3 1 Ally1 trimethylammonium bromide ‘AM AB’ 4.2

Retardant activity (% reduction of internode length

at lo-’ M) 53 64 0

54

208 G . A . CARTEReta l .

Table 5 . The systemic activity against wheat powdery mildew of tri-n-butyl-4-chlorobenzyl- ammonium bromide applied to the roots of wheat seedlings growing in soil, sand or water culture

A >-- Water culture Sand culture Soil culture

r infectiont Toxicity$ Infection Infection

Concen- Daysof c-.--A-, ,-Ap, (-A-, Toxicity r----A-, Toxicity tration treatment Leaf 1 Leaf 2 Root Shoot Leaf 1 Leaf2 shoot Leaf 1 Leaf2 shoot

lo-* M I 0 f + 0 23 + -

* *

14 * +++ +++ * * +++ 18 18 + 21 +++ +++ * + + + 20 20 +

14 16 10 + + 82 94 + 100 100 - 100 100 - 21 8 4 ++ +++ 86 94 +

14 50 42 k - 94 94 - 100 100 - 21 84 100 - - 90 100 - 100 96

- 89 - - 60 - 10-3 M I 12

- 99 - - 86 - 10-4 M I 15

-

f Infection expressed as pustule counts as per cent of control. $ Phytotoxicity assessed visually on a scale of -, f, +, ++, +++. * Plant too damaged to permit disease assessment.

when the inoculation was delayed, no effect was observed. In water culture low3 M gave good protection against mildew over the 2 l-day experimental period but severe phytotoxicity developed. At M the compound was inactive in sand culture but in water culture showed systemic activity 7 and 14 days after treatment, but inoculations made after 21 days were not inhibited. No compound showed any systemic activity against rust on Phaseolus.

D I S C U S S I O N

Within the series of trialkyl-4-chlorobenzylammonium bromides there is good correlation between systemic fungicidal and growth-retardant activity. This degree of correlation, however, is not found in the substituted benzyl- and naphthyl-tri-n-butylammonium salts (Table 3). Thus, for example, the 2-halogen-substituted compounds are less active as retardants than those substi- tuted in the 3-position but the reverse is true for systemic activity. It seems possible, therefore, that the systemic antifungal activity of these compounds is not a direct consequence of their growth-retardant activity. The apparent association between the two within the homologous series could equally well arise from the effect of alkyl chain length upon the uptake by and movement within the plant.

Although some of the compounds studied are highly fungitoxic, there appears to be no obvious relationship between fungitoxicity in uitro and systemic activity against wheat mildew. Thus, in the series of trialkyl-4-chlorobenzylammonium bromides fungitoxicity reaches an optimum with the n-heptyl salt whereas systemic activity is optimal for the n-butyl and n-pentyl salts, the former having only slight activity in uitro.

Edgington (1 966) has shown that lengthening the chain of n-alkyl quaternary ammonium salts increases adsorption to plant tissues thereby reducing their mobility. If this effect operates in wheat seedlings it could explain the reduced systemic fungicidal activity which was observed for the tri-n-hexyl and tri-n-heptyl salts.

When applied to soil these cationic compounds would also be expected to be strongly adsorbed to soil colloids because of the free positive charge in their molecule. The application of aqueous solutions to the soil surface is therefore unlikely to provide an even distribution within the soil and this may well explain the lower systemic activity observed in the soil experiments compared with those in water culture.

Fungitoxicity of quaternary ammonium salts 209

As measured by retardation of root growth in wheat seedlings, the higher members of the series of 4-chlorobenzyl-quaternary ammonium salts are highly phytotoxic at the concentra- tions required for the lower homologues to show systemic activity and it seems possible that phytotoxicity is another factor limiting the activity of these compounds in viva

The present compounds show marked specificity of action in that they are effective against powdery mildew of wheat but ineffective against brown rust on the same host. Other workers (Tahori et al., 1965; Smith & Corke, 1966; Corke, 1969; Smith, 1969) have also observed that the fungicidal activity of growth retardants can be very specific; furthermore, the spectrum of activity varies from one retardant to another and there is also evidence that the development of certain diseases may be enhanced following treatment with these compounds (e.g. Sturm & Jung, 1964; Bockman, 1968; Smith, 1969).

In the present studies the well-known retardants, 2-chloro-ethyl-trimethylammonium chloride (Cycocel) and allyl-trimethylammonium bromide (AMAB) proved ineffective against wheat mildew whereas maleic hydrazide reduced infection, although showing no retardant activity.

Recent investigations (Hislop & Clifford, 1976; Hislop, Clifford, Holgate & Gendle, 1978) of the eradication of overwintering apple powdery mildew (Podosphaera leucorricha (Ell. & Ev.) S a h ) , with surface-active agents have shown that Hyamine 3500, a mixture of n-alkyl-dimethyl- benzylammonium chlorides, completely eradicated mildew but was phytotoxic.

The use of quaternary ammonium salts as systemic fungicides would appear to be limited to leaf spray applications since the compounds are rapidly immobilised in soil, but phytctovicity could be a problem. The present results, however, indicate that when compounds such as tri-n- butyl-4-chlorobenzylammonium bromide are used as growth retardants, systemic fungicidal effects against powdery mildew will operate.

R E F E R E N C E S

ANDEL, 0. M. VAN (1 968). Shifts in disease resistance induced by growth regulators. Netherlands Journal ofplant Pathology 74, Supplement 1, 113-120.

BOCKMANN, H. (1 968). Phytopathological aspects of chlorocholine chloride application. Euphytica 17, Supplement 1,271-274.

BUCHENAUER, H. (1971). Einfluss einiger Wuchshemmstoffe (CCC, CMH, Amo-1618, Phosfon-D, B- 995) auf die Fusarium- und Verticillium Welke der Tomate. Phytopathologische Zeitschrft 72, 53-66.

BUCHENAUER, H. & ERWIN, D. c. (1973). Reduction of the severity of Verticilliurn wilt of cotton by the growth retardants N,N-dimethyl pyrrolidinium iodide and N,N,-dimethyl piperidinium iodide associated with induction of antifungal compounds. Zeitschrft fur Pfanzenkrankheiten, Pfanzenpathologie und Pfanzenschutz 80,576-586.

BUCHENAUER, H. & ERWIN, D. c. (1976). Effect of the plant growth retardant Pydanon on Verticillium wilt of cotton and tomato. Phytopathology 66, 1140-1 143.

CARTER, G . A. & WAIN, R. L. (1964). Investigations on fungicides. IX. The fungitoxicity, phytotoxicity and systemic fungicidal activity of some inorganic salts. Annals ofApplied Biology 53,291-309.

CORKE, A. T. K. (1969). Observations on diseases of blackcurrants. Journal of the Science of Food and Agriculture 20,40 1-402.

EDGINGTON, L. v. (1966). Effect of chain length of the alkyl quaternary ammonium compounds upon their use as systemic fungicides. Phytopathology 56,23-25.

ERWIN, D. c . , TSAI, s. D. 62 KHAN, R. A. (1976). Reduction of the severity of Verticillium wilt of cotton by the growth retardant tributyl [(5-chloro-2-thienyl)methyll phosphonium chloride. Phytopathology 66, 106-1 10.

HISLOP, E. c . & CLIFFORD, D. R. (1976). Eradication of apple powdery mildew (Podosphaera leucotricha) with dormant season sprays of surface-active agents. Annals ofApplied Biology 82,557-568.

HISLOP, E. c., CLIFFORD, D. R., HOLGATE, M. E. & GENDLE, P. (1978). Eradication of apple powdery mildew (Podosphaera leucotricha) with dormant-season sprays of surface-active agents. Pesticide Science 9. 12-2 1.

2 10 G . A . C A R T E R et a/ .

KNIGHT, B. E. A. (1966). Studies on the biological activity of certain quaternary ammonium and related compounds in relation to chemical structure. M.Sc. Thesis, University of London.

KNIGHT, B. E. A., TAYLOR, H. F. & WAIN, R. L. (1969). Studies on plant growth-regulating substances. XXIX. The plant growth-retarding properties of certain ammonium, phosphonium and sulphonium halides. Annals ofApplied Biology 63, 21 1-223.

SINHA, A. K. & WOOD, R. K. s. (1964). Control of Verticillium wilt of tomato plants with 'Cycocel' (2- chloroethyl)trimethylammonium chloride. Nature, London 202, 824.

SINHA, A. K. & WOOD, R. K. s. (1967). The effect of growth substances on Verticillium wilt of tomato plants. Annuls ofApplied Biology 60, 117-1 28.

SMITH, B. D. (1969). Spectra of activity of plant growth retardants against various parasites of one host species. Journal of the Science of Food and Agriculture 20,398-400.

SMITH, R . D. & CORKE, A. T. K. (1966). Effect of (2-chloroethyl)trimethylammonium chloride on the eriophyid gall mite Cecidophyopsis ribis Nal., and three fungus diseases of blackcurrant. Nature, London 121,643-644.

STURM, H. JUNG, J . (1964). Der Einfluss von Blattspritzungen mit Chlorocholinchlorid (CCC) auf Wachstum und Ertrag von Weizen. Zeitschrijl fur Acker- und PJlanzenbau 120,232-252.

TAHORI, A. s., ZEIDLER, G. & HALEVY, A. H. (1965). Effect of some plant growth-retarding compounds on three fungal diseases and one viral disease. Plant Disease Reporter 49, 775-771.

WAIN. R. L., SOBOTKA, w. & SPENCER, D. M. (1963). Investigations on fungicides. Vlll. The fungicidal properties of some thiocarbamoylthionitroparaffins. Annals of Applied Biology 5 I, 445-452.