INVESTIGATIONS ON FUNGICIDES II. ARYLOXY- AND ARYLTHIO-ALKANECARBOXYLIC ACIDS AND THEIR ACTIVITY AS FUNGICIDES AND SYSTEMIC FUNGICIDES

A wide range of aryloxyacetic acids and corresponding acids with alkyl groups in the side chain, their arylthio- analogues and the antibiotic griseofulvin have been assessed in the plate test for fungistatic effect on six fungi, and as systemic fungicides against Botrytis fabae in broad beans and Alternaria solani in tomatoes. The results indicate that in general, arylthio- derivatives are more fungicidal than their aryloxy- analogues. The systemic fungicidal performance of x-(2-chlorophenylthio)propionic acid in the tomato test at 1–100 p. p. m. was found to be of the same order as that shown by griseofulvin at 50–500 p. p. m. Variable results were obtained with griseofulvin in the tornato test and its performance in the bean test was consistently poor. Further evidence is presented which indicates that the protection conferred by certain compounds may not be due to activity per se .     

STUDIES ON SYSTEMIC FUNGICIDES. I. FUNGICIDAL PROPERTIES OF THE ARYLOXYALKYLCARBOXYLIC ACIDS

A method is described for assessing the systemic activity of compounds in checking the infection of broad bean ( Vicia faba ) by the fungi Botrytis cinerea or B. fabae. Treatment consisted in allowing the roots of young seedlings to stand in a solution containing 10 p.p.m. of the chemical for 2–3 weeks. The plants, together with controls, were then inoculated and when symptoms had had time to develop, the degree of chocolate spot infection was assessed. Several methods of disease assessment were examined and are critically discussed.
Certain phenoxyalkylcarboxylic acids tested by this method consistently gave a reduction in the mean size of fungus lesions on the bean leaves, clearly indicating systemic fungicidal action. The most promising substances were 2:4:6-trichlorophenoxyacetic, pentachlorophenoxyacetic and pentachlorophenoxy iso butyric acids. Further experiments with these compounds involving soil treatment, stem injection and spray application are described, and in most cases systemic fungicidal activity was clearly demonstrated. Certain compounds caused visible damage to the plants or resulted in a reduction in growth.
The results presented indicate that phenoxy acids can be fairly readily translocated in bean plants and that they tend to accumulate in actively growing tissues. It is considered unlikely, however, that they persist for long periods in plant tissue.
In the soil, the compounds appeared to remain effective for a considerable time, particularly the less soluble pentachloro acids, suggesting that soil application might provide a safe and useful method of treatment.     

INVESTIGATIONS ON FUNGICIDES

Twenty (aryloxythio)trichloromethanes were examined for in vitro fungicidal activity against six fungi. All compounds showed a direct fungistatic effect and some exhibited a marked fumigant action. Good protectant action against Alternaria solani on tomato and Botrytis fabae on broad bean was obtained with certain compounds, but none was better than the three standard protectants used for comparison. When supplied to plants through their roots, eight conferred significant systemic fungicidal protection against Alternaria solani in tomato, but there was no significant protection against Botrytis fabae in broad bean. In preliminary tests the 2:4:5-trichlorophenoxythio analogue gave promising results when examined as a fumigant fungicide for eradication of downy mildew in lettuce plants, for reducing lenticel rot in stored apples and for preventing blue-mould in oranges.     

STUDIES ON SYSTEMIC FUNGICIDES

A number of mono-, di-and trichloro-β-naphthols and the corresponding naphthyloxy- n -carboxylic acids were prepared. They were tested as systemic fungicides against chocolate spot of broad bean ( Vicia faba ) caused by Botrytis fabae and as fungicides in vitro by their effect on the germination of spores of Sclerotinia laxa and the growth of the mycelium of Botrytis fabae.
The systemic fungicide tests showed no difference in the activity of the compounds associated with different side chains, though there were slight but significant effects associated with different chlorine substitutions in the naphthalene nucleus. The 3:4-dichloro-2-naphthol derivatives were more effective than the other compounds tested.
Fungicide tests with the 1:3-dichloro-2-naphthol derivatives suggested that there was some increase in toxicity to the spores of Sclerotinia laxa with increasing length of side chain. The effectiveness of the various chlorine-substituted compounds in suppressing growth of Botrytis fabae was in the following descending order, 1:3:4-trichloro-, 1:3-dichloro-, 1:4-dichloro-and 3:4-dichloro-compounds. The concentrations required in agar culture to induce reductions of B. fabae growth comparable to those found in the leaf could reasonably be expected to occur in the bean plants treated with the 2-naphthyloxyacetic acids at 10 p.p.m.     

FUNGICIDAL ACTIVITY AND CHEMICAL CONSTITUTION

2:3–Dichloro-1:4-naphthahydroquinone and several derivatives were prepared and tested in aqueous acetone solution against four fungi using a spore germination technique. The hydroquinone showed the same order of fungicidal activity as 2:3-dichloro-1: 4-naphthaquinone, but was no less phytotoxic. Whilst the dibenzoyl ester and the dimethyl ether proved virtually inactive, the diacetyl ester was found to be as effective as 2:3-dichloro-1:4-naphthaquinone against Sclerotinia laxa, Botrytis fabae and Cladosporium fulvum , and also less phytotoxic to plum fruitlets and tomato and broad bean foliage. Reduced fungicidal activity was shown when the substance was formulated as an aqueous suspension suitable for field application.
The rates of alkaline hydrolysis of these esters have been determined, and a relationship between ease of hydrolysis and fungicidal activity is apparent. It is suggested that these compounds, whilst not inherently active, are similarly hydrolysed to 2:3-dichloro-1:4-naphthahydroquinone in the presence of a fungal esterase.
* Byrde, R. J. W., Crowdy, S. H. & Woodcock, D., 'Studies on Systemic Fungicides. III', Ann. appl. Biol . 40 , 152, is regarded as Part I of this series.     

FUNGICIDAL ACTIVITY AND CHEMICAL CONSTITUTION:

Nine derivatives of pentachlorophenol have been prepared and tested for fungistatic activity against the mycelia of Botrytis fabae, Sclerotinia laxa and Stereum purpureum and for phytotoxicity towards tomato, apple and plum. The most fungitoxic of these compounds was the acetyl ester. Its phytotoxicity, although much less than that of pentachlorophenol itself, nevertheless precludes its effective use as an eradicant fungicide on foliage.     

Observations on the vapour phase activity of some foliage fungicides

By means of a Botrytis fabae/Vicia faba bio-assay technique it has been demonstrated that phenyl mercury chloride, maneb, mancozeb, dichlo-fluanid and oxythioquinox protect areas of leaf beyond the visible limits of the fungicide deposits. The evidence suggests that the extended areas of protection are due to the release of fungicidal vapours. For a given dose of mancozeb the area of protection was related to the number of conidia of B. fabae dusted on to the leaves and for a given inoculum density it extended with increasing fungicide dose applied in standard drop sizes. When the same dose of fungicide was applied in increasing volumes of water, producing widening areas of deposit, the area of protection also increased. Fungicide deposits aged on leaves for up to 4 weeks continued to release toxic vapours. Contact between the fungicides and leaves or between fungicides and spores was not necessary for the demonstration of the phenomenon since vapours diffused from deposits on glass and inhibited the germination of spores in water droplets placed at a distance from the fungicide source. For a given distance separating the fungicide and the spores inhibition increased with increasing fungicide dose. For a standard fungicide dose, inhibition decreased with increasing distances between the fungicide and the spores. The fungicidal vapours inhibited the germination of spores of test fungi other than B. fabae. The practical implications of these observations are examined in the light of evidence that vapour phase protection can occur on leaves incubated in large cabinets; on leaves pre-incubated at unsaturated humidities; and on leaves incubated in a moving stream of air.     

Synthesis and biological activities of 2-oxocycloalkylsulfonamides

A series of novel 2-oxocycloalkylsulfonamides (4) were synthesized and their structures confirmed by IR, (1)H NMR, and elemental analysis. The bioassay showed that they have fair to excellent fungicidal activities against Botrytis cinerea Pers and Sclerotinia sclerotiorum. Among them, compounds 4A(10), 4A(11), 4A(12), 4B(2), and 4B(3), the EC(50) values of which were 2.12, 3.66, 3.96, 2.38, and 2.43 microg/mL, respectively, displayed excellent fungicidal activity against B. cinerea Pers, and are comparable with commercial fungicide procymidone (the EC(50) value is 2.45 microg/mL). 3D QSAR against B. cinerea Pers was studied, a statistically significant and chemically meaningful CoMFA model was developed and some compounds which have a high predicted activity were forecasted. In addition, the bioassay also showed that the compounds have good inhibitory activities against human tumor cells HL-60, BGC-823, Bel-7402 and KB. It is interesting to point out that the antitumor activities of compounds 4 are in accordance with their fungicidal activity to a great extent: compounds having relatively best antitumor activities (4A(10), 4A(11), 4A(12), and 4B(3)) also displayed excellent fungicidal activity.     

Effect of extracts from in vitro-grown shoots of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Mol. on <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.     

Negatively Correlated Cross-resistance and Synergism between Phosphoramidates and Phosphorothiolates in Their Fungicidal Actions on Rice Blast Fungi

Alkyl aryl esters, dialkyl esters and diaryl esters of N-methyl-N-phenylphosphoramidic acid were synthesized and tested for cross-resistance and joint action with phosphorothiolate (PTL) fungicides using rice blast fungi, Pyricularia oryzae. Negative correlation in crossresistance was found between PTL fungicides and most of the amidates. Synergism in the fungicidal action was also found between them for wild types of the fungi, when tested by crossing filter paper strips impregnated with fungicides on agar plates inoculated uniformly with the test fungi.     

Laboratory Examination of Organic Fungicides Against Zoopathogenic Fungi in Soil

Seven commercial and five experimental organic fungicides were tested against Histoplasma capsulatum, Cryptococcus neoformans, Allescheria boydii, and Sporotrichum schenckii in a series of laboratory studies. Preliminary agar dilution plate tests indicated that Lanstan, DAC 649, DAC 469, DAC 2787, He 3944, maneb, and nabam, in order of decreasing activity, inhibited all test fungi. Terraclor, Dexon, and He 13274 were active only in high concentration. The best fungicidal properties were exhibited by Lanstan, Vapam, and DAC 649; maneb, nabam, DAC 469, and DAC 2787 were fungicidal but to a lesser degree than the former compounds. In subsequent tests against H. capsulatum and C. neoformans in artificially infested soil or by use of the buried plug technique, Lanstan, Vapam, and DAC 649 showed good activity and merit further study.     

Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus Link. isolated from stored rice

AIMS: To develop a natural fungicide against aflatoxigenic fungi, to protect stored rice, using the essential oil of lemongrass. METHODS AND RESULTS: Aspergillus flavus Link. was isolated from stored rice and identified as an aflatoxigenic strain. Lemongrass oil was tested against A. flavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg ml(-1), respectively. Aflatoxin production was completely inhibited at 0.1 mg ml(-1). The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at the concentrations of 2.80 and 3.46 mg ml(-1), respectively. CONCLUSION: Lemongrass oil could be used to manage aflatoxin formation and fungal growth of A. flavus in stored rice. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, fungicides are not used to control fungal pests or mycotoxin production on stored rice. Rice treated with the essential oil of lemongrass could be used to manage fungal pests as well as the insect pests in stored rice. The essential oil is chemically safe and acceptable to consumers, as synthetic chemical fungicides can cause adverse health effects to consumers.     

Investigations on fungicides: The systemic fungicidal activity of certain N-carboxymethyl dithiocarbamic acid derivatives

The systemic activity of thirty-three N-substituted S-esters derived from dithiocarbamic acid was investigated by assessing their ability to reduce the infection of broad-bean seedlings by Botrytis fabae and of wheat seedlings by Erysiphe graminis, following application to the roots or cut shoots of the host. Marked systemic activity against mildews was shown by the N-carboxymethyl dithiocarbamates, by S-carboxymethyl-N, N-dimethyl-dithiocarbamate (G₃₃) and by procaine and 6-azauracil. The effect was not very host-specific since most compounds showing high activity in wheat also showed activity in pea, cucumber and, to a smaller extent, apple. All the materials tested showed a much lower level of systemic activity in broad-bean seedlings against B. fabae. Measurements were also made of the uptake, translocation, phytotoxicity and the stability within plant tissues of some of these compounds. The degree of systemic activity which they show is discussed in relation to these and other properties of the compounds.     

Synthesis and Anti-Oomycete Activity of 1-Sulfonyloxy/Acyloxydihydroeugenol Derivatives

Endeavor to discover biorational natural products-based fungicides, two series (26) of novel 1-sulfonyloxy/acyloxydihydroeugenol derivatives ( 3a – p and 5a – j ) were prepared and assessed for their fungicidal activity against P. capsici Leonian, in vitro. Results of fungicidal activity revealed that, among all compounds, especially compounds 3a , 5c , and 5e displayed the most potent anti-oomycete activity against P. capsici with EC₅₀ values of 69.33, 68.81, and 67.77 mg/L, respectively. Overall, the anti-oomycete activities of 1-acyloxydihydroeugenol derivatives ( 5a – j ) were higher than that of 1-sulfonyloxydihydroeugenol derivatives ( 3a – p ). It is proved that the introduction of the acyl group at hydroxy position of dihydroeugenol is more beneficial to improve its anti-oomycete activity than that of the sulfonyl group. These preliminary results will pave the way for further modification of dihydroeugenol in the development of potential new fungicides.     

Direct fungicidal activities of C6-aldehydes are important constituents for defense responses in Arabidopsis against Botrytis cinerea

C6-aldehydes, such as (Z)-3-hexenal, (E)-2-hexenal, and n-hexanal, are volatile compounds formed by hydroperoxide lyase (HPL) and found in most terrestrial plants. They are fungicidal and bactericidal compounds, and are also signaling compounds to induce defense responses in plants. Transgenic plants having overexpressed or suppressed HPL activity (SH or ASH, respectively) showed lower or higher susceptibility against a necrotrophic fungal pathogen, Botrytis cinerea. In this study, we examined whether the modulated susceptibility was accountable to the direct fungicidal activity or to the signaling potency of C6-aldehydes. When wild-type Arabidopsis leaves were inoculated with B. cinerea, HPL expression was upregulated, and concomitantly, the amounts of C6-aldehydes increased. Higher amounts of C6-aldehydes found in inoculated SH plants inhibited growth of B. cinerea in vitro, while lower amounts found in ASH plants caused no inhibitory effect on the fungi. Thus, it was suggested that direct fungicidal activity of C6-aldehydes accounted for the modulated susceptibility. With SH plants higher amounts of camalexin could be found, but with the ASH plants no difference from wild-type plants could be found. Surplus amounts of C6-aldehydes could induce formation of camalexin as signaling compounds; however, this was not the case with wild-type and ASH plants. Accordingly, it could be assumed that direct fungicidal activity of C6-aldehydes were prominently responsible to the defense against B. cinerea but their signaling roles could be little responsible if any.     

Fungitoxic action of dimethirimol and ethirimol

The systemic fungicides dimethirimol and ethirimol were shown to be toxic to spores of Erysiphe graminis and Sphaerotheca fuliginea, in germination tests in vitro. Toxicity of dimethirimol to spores of Botrytis fabae, Phytophthora infestans, Puccinia recondita, Uncinula necator and Venturia inaequalis was relatively feeble or absent. When applied in foliage sprays or in root treatments, both compounds inhibited the emergence of germ tubes from powdery mildew spores on cucumber and barley plants. The specific disease control shown by these fungicides can be explained by their direct fungitoxic action. The effects of dimethirimol and ethirimol on powdery mildew infections and on spore germination were greatly decreased by the presence of riboflavin in the light. A rapid photo-chemical interaction between riboflavin and dimethirimol was demonstrated. Marked reversals both of disease control and of fungitoxicity were also given by folic acid, and it is suggested that folic acid overcomes a metabolic block induced in powdery mildew fungi by the fungicides.     

Glasshouse experiments on apple scab

Dilutions of various fungicides were compared on potted MM 109 clonal rootstocks in the glasshouse for protective (pre-infection) and curative (post-infection) activity against apple scab. The materials, as commercial preparations, included captan, copper (as ‘dry’ Bordeaux mixture), dichlofluanid, didecyldimethylammonium bromide (DDAB), dispersible sulphur, dithianon, dodine acetate, lime-sulphur (LS), phenylmercury chloride (PMC), and thiram; they were assessed on the basis of percentage active ingredient in the dilutions. When acting at normal rates of use, many of the fungicides were much more effective protectively than curatively, but this contrast was less applicable to DDAB, dodine acetate, LS, and dichlofluanid. PMC, however, was an especially notable exception in proving curatively superior at unusually low rates. In addition, it was particularly active in vapour form, and was effectively translocated not only from one leaf surface to the other, but, especially at 0.005%, from sprayed to younger unsprayed leaves on the same shoots. Other standard fungicides showed little or no translocated activity, but isobutyl-o-coumarate was effective at the growing shoot-tips in spring from a spray applied to the lower leaves. PMC reduced infection when applied once at any time during the scab-incubation period, and especially 24 hr. after infection, when the control was almost complete. Longer intervals before spraying reduced the curative action, but not in proportion to the intervals; the infection pattern showed multi-peak fungicidal activity and, after the later sprays, many lesions were practically infertile. The curative properties of PMC were matched by several other formulated phenylmercury salts compared at equivalent percentage mercury content, but the dimethyldithiocarbamate proved rather less effective. A preliminary attempt to supplement the curative properties of PMC with the protective properties of LS, by mixing these fungicides at low rates, showed evidence of fungicidal antagonism, and this needs further investigation. Several fungicides, notably the inorganic sulphurs, thiram, the phenylmercury compounds, dichlofluanid, and DDAB showed incidental activity against natural infection by powdery mildew.     

Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 2: pyridazines

Special tetrasubstituted pyridazines are potent fungicides by promoting the tubulin polymerisation, hereby disrupting the microtubule dynamics in the fungus. They are monocyclic analogs of similar substituted triazolopyrimidines and pyridopyrazines with the same mode of action. The fungicidal activity of these pyridazines was evaluated against the plant pathogens Botrytis cinerea (grey mould), Mycosphaerella graminicola (wheat leaf blotch) and Alternaria solani (potato and tomato early blight). Structure-activity relationship studies revealed the importance of a methyl and a chlorine substituent next to both ring nitrogen atoms and two aryl or heteroaryl groups in the other two pyridazine positions.     

Antibacterial activity of 15-azasteroids alone and in combination with antibiotics.

A new class of 15-azasteroid analogues has been synthesized and tested for antimicrobial activity. The compounds 1, 10, 11, 11a-tetrahydro-7-methoxy-11a-methyl-2H-naphth (1,2-g) indol (methoxyimine) and 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth (1,2-g) indol-7-ol (hydroxyimine) inhibit the growth of Bacillus subtillis and Escherichia coli at concentrations as low as 10-5 M. Addition of either compound to the growth medium casued a rapid inhibition in the transport of radioactive glucose, uracil and several amino acids. The inhibition of growth and substrate transport was reversed following removl of the steroid from the medium. The evidence is consistent with a site of steroid action at the cell periphery. Combining the methoxyimine with polyor circulin at subinhibitory concentrations produced greatly enhanced antimicrobial activity against Pseudomonas fluorescens. Similar action was observed against B. subtilis when the azasteroid was combined with vancomycin or chloramphenicol. The inhibitory action of other antibiotics such as penicillin or erythromycin was not affected by addition of the test compound. The results suggest formation of a molecular complex between the azasteroid and antibiotic which is responsible for the enhanced biological activity.     

THE EVALUATION OF SYSTEMIC FUNGICIDES BY MEANS OF ALTERNARIA SOLANI ON TOMATO

I wish to deal briefly with the method we use at the Hawthorndale Laboratories for investigating the systemic fungicidal activity of griseofulvin and other materials. Our work on systemic fungicides has necessarily depended on our working out a suitable and reliable technique.