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.     

INVESTIGATIONS ON FUNGICIDES. I. FUNGICIDAL AND SYSTEMIC FUNGICIDAL ACTIVITY IN CERTAIN ARYLOXYALKANECARBOXYLIC ACIDS

Fifty-four aryloxyalkanecarboxylic acids have been examined as systemic fungicides. Of these, four which produced negligible damage in broad-bean seedlings yet conferred systemic fungicidal protection to broad beans against Botrytis fabae and to tomatoes against Alternaria solanixvere studied in detail. The direct fungicidal activity of twenty compounds was assessed against B. fabae and Pythium ultimum. No correlation between fungicidal and systemic fungicidal action toward B. fabae was established. Other possibilities regarding their mode of action as systemic fungicides are discussed. The systemic fungicidal activity of a-phenoxyziobutyric and 2:4:6-trichlorophenoxyacetic acids in beans and of the latter compound in tomato plants was found to depend upon seasonal factors, but for 3-phenoxybutyric and 5-phenoxycaproic acids such variation was not observed. Results obtained in the bean test by two methods of assessment were found to correlate. The potato petiole test (van Raalte) for assessing systemic fungicidal activity was studied and modified. Evidence was obtained that the results might be unreliable owing to toxic effects produced by the compounds on the petiole tissue. Attempts were made to avoid this complication, but when phytotoxicity was eliminated, little fungicidal activity was shown.     

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 .     

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.     

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.     

FUNGICIDAL ACTIVITY AND CHEMICAL CONSTITUTION: VII. A STUDY OF THE STRUCTURAL SPECIFICITY OF SEVEN FUNGICIDES

The relative saturations of the following fungicides, in dilute McIlvaine buffer containing sucrose, required for 50% inhibition of spore germination of Sclerotinia laxa were determined as under: phenylmercuric chloride, 0.0002; nystatin, 0.028; dichlone, 0.034; copper-8-quinolinolate, 0.039; 2:4-dichloro-6-( o -chloroanilino)-s-triazine, 0.131; captan, 0.132; bis-(5-chloro-2-hydroxyphenyl)methane, 0.234. These values are discussed in relation to current views on the fungitoxicity of the compounds.     

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.     

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.     

Splash dispersal of fungus spores and fungicides in the laboratory and greenhouse

A laboratory technique is described for the production of drops of simulated rain in which fungal spores were suspended. When such drops containing conidia of Botrytis fabae impacted on a target leaf the secondary droplets produced infections on receptor broad bean leaves. The capacity of fungicides applied to the target leaf to redistribute in secondary splash droplets was examined in terms of the infectivity of the spores in the droplets. The extent to which a copper fungicide reduced infection on the receptor leaves was related to the level and tenacity of the fungicide deposit on the target leaf. The effect of wetting agents on the redistribution of this fungicide could probably be explained by their influence on the tenacity of the initial deposit. In general the capacity of different fungicides to inhibit infection by the secondary droplets was related to the inherent toxicity of the fungicides to B. fabae. Implications of the dispersal of spores and fungicides by rain splash are briefly considered with reference to field conditions.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of l-phenylpyrrolidine-2,5-dione derivatives were investigated on Sclerotinia sclerotiorum by the agar dilution method. In addition, several representative compounds were tested for antimicrobial spectra in vitro with 15 pathogenic microbes and for foliage protection activity in green house tests with rice blast, rice brown spot, rice sheath blight and kidney bean stem rot. It was found that 3,5-dihalo-substituents on the benzene moiety are essential to high antifungal activity against Sclerotinia sclerotiorum. Generally, l-(3′,5′-dihalophenyl)pyrrolidine-2,5-diones are active against Corticiaceae, Dematiaceae, Pleosporaceae and Sclerotiniaceae, especially active against Sclerotinia sclerotiorum and Botrytis cinerea (the conidia form of Sclerotinia fuckeliana). N-(3,5-Dichlorophenyl)itaconimide showed a peculiarly broad antimicrobial spectrum. In green house tests, these compounds showed high activity against rice brown spot, rice sheath blight and kidney bean stem rot. Results of green house tests on the above-mentioned diseases correlate fairly well with those of in vitro tests.     

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.     

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.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES: CHLORO-SUBSTITUTED PHENYLACETIC ACIDS

The plant growth-regulating activity of all the mono- and di- chloro-substituted phenylacetic acids, together with the 2:3:6-trichloro- derivative, has been determined in the wheat cylinder, pea segment and pea curvature tests. Each of these compounds showed activity, the 2:3-dichloro- and the 2:3:6-trichloro- derivatives being extremely active. The 3:5-dichloro- acid was inactive in the wheat test but showed moderate activity in the pea tests.
All these findings are discussed in relation to the plant growth-regulating activity of corresponding benzoic and phenoxyacetic acids.     

THE CHEMOTHERAPEUTIC EFFECT OF PYRIDINE-2-THIOL-N-OXIDE AND SOME OF ITS DERIVATIVES ON CERTAIN PLANT DISEASES

Limited systemic protection of broad bean against Botrytis fabae and of cucumber against Cladosporium cucumerinum has been observed as a result of the application of pyridine-₂-thiol-N-oxide and its carboxymethyl derivative. Apparently this protection is, to some extent, the result of translocation of the chemicals in the plants. It was found that the vapour phase of pyridine-₂-thiol- N -oxide plays an additional role in the chemotherapeutic activity. There is no evidence that the copper chelate of pyridine-₂-thiol- N -oxide is translocatable in plants. Root applications of these three compounds had no chemotherapeutic effect.     

Antimicrobial Activity of 3-Phenylimidazolidine-2,4-diones and Related Compounds

The structure-activity relationships of 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were investigated by the agar dilution method using Sclerotinia sclerotiorum as a test microbe. Several compounds were tested for antimicrobial spectrum in vitro with other pathogenic microbes and for foliage protective activity in green house tests with rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. It was found that the antimicrobial activity was enhanced when the 1-position of imidazolidine ring was substituted by an alkyl group but was reduced when the 5-position was substituted by alkyl groups. Generally, 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were active against Scierotiniaceae, Corticiaceae, Dematiaceae, Polystigmataceae or Pleosporaceae. In green house tests, some of these compounds showed high protective activity against rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. Results of the green house tests on the above mentioned diseases correlate well with those of in vitro tests except in the case of kidney bean stem rot.     

Investigations on fungicides: X. The antifungal activity of 2-deoxy-D-glucose

The fungitoxicity of 2-deoxy-D-glucose was examined in spore germination tests using Botrytis cinerea, B. fabae, Alternaria brassicicola, Aspergillus niger, Cladosporium cucumerinum, C. fulvum, Sclerotinia fructigena, Verticillium albo-atrum and Glomerella cingulata. It proved to be toxic, at concentrations below 10 μg./ml., to all of these fungi except G. cingulata which was resistant to concentrations up to 2000 μg./ml. The effect of a-deoxy-D-glucose on the mycelial growth of these fungi was assessed and again G. cingulata proved to be resistant to high concentrations of the compound, as also did A. niger.
The results are discussed in relation to the known inhibitory nature of 2-deoxy-D-glucose in other systems.     

葡萄孢盘菌属一新种——蚕豆葡萄孢的有性阶段

本文报道了葡萄孢盘菌属的一个新种:蚕豆葡萄孢盘菌(Botryotinia fabae Lu et T.H.Wu sp.nov.)即蚕豆葡萄孢的有性阶段;并报道了新种子囊盘形成过程;新种有汉文和拉丁文描述。     

Enhancement of adventitious root formation in mung bean cuttings by 3,5-dihalo-4-hydroxybenzoic acids and 2,4-dinitrophenol

3,5-Dihalo-4-hydroxybenzoic acids enhanced adventitious root formation in mung bean (Vigna radiata L.) cuttings. 3,5-Diiodo-4-hydroxybenzoic acid was more active than 3,5-dichloro-4-hydroxybenzoic acid, increasing the number of roots formed by about 4-fold. 2,4-Dinitrophenol also enhanced significantly adventitious root formation in mung bean cuttings. The phenolic compounds were active with or without indole-3-acetic acid. The possible mechanism by which these phenolic compounds enhance rooting is discussed.Abbreviations CCCP carbonyl cyanide 3-chlorophenylhydrazone - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DNP 2,4-dinitrophenol     

Reproductive response of generalist and specialist aphid morphs with the same genotype to plant secondary compounds and amino acids

Many studies have paid particular attention to the role of either secondary plant compounds or amino acids as determinants of host-plant range in phytophagous insects. Here we examine the relative importance of both of these classes of compound in host acceptance by generalist and specialist morphs of the black bean aphid, Aphis fabae, that are morphologically similar and genetically identical. Eleven secondary plant compounds and six amino acids with known biological activity in aphids are presented to insects within an artificial membrane system as single compounds, mixtures of same-class compounds and combined mixtures of the two classes of compound. It is found that 1) when specific single secondary compounds and amino acids are presented to generalist and specialist morphs of A. fabae, differential responses are exclusively consistent with plant-use strategy for amino acids but not for secondary compounds, 2) neither secondary compound nor amino acid mixtures give reproductive responses entirely consistent with plant range, but the response to secondary compounds is broadly consistent with plant range whereas the response to amino acids is not, 3) when secondary compounds and amino acid mixtures are combined, the response to secondary compounds generally dominates that to amino acids. Some scenarios of plant-range determination by secondary plant compounds and amino acids, suggested by results, are discussed.     

Improvement of the fungal biocontrol agent Trichoderma atroviride to enhance both antagonism and induction of plant systemic disease resistance

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.