These isolates have the capability to produce melanin and melanin like pigments. Sensitivity to Zineb a dicarboximide fungicide was determined on a series of PDA plates supplemented with fungicide by using an agar disc 4 mm from an axenic culture as an inoculum. Fungicides: The fungicides used were Zineb [zinc ethylene bis dithiocarbamate ] and benelate [methyl 1- butyl-carbamoyl benzimidazolcarbamate] Ciba Geigy.
The concentrations of the fungicides used were 10 ppm up to ppm. Fungicide was diluted to give a range of concentration from to ppm, when added to the medium. This inoculum corresponded to 0. Measurement of lipid peroxide a Extraction of total lipids: The samples were extracted on the day of harvest to minimize lipid peroxidation due to sample storage.
Extraction of total lipids was done according to Folch et al. All values means of 3 determinations of extracted total lipids from samples were referred to total phospholipids. The total phospholipids were indirectly estimated from the phosphorus in dry lipid residue. Phosphorus was measured as recommended by Bartlett Effect of melanin precursors and inhibitors on melanization and growth: The isolation of melanin from the mycelial mats grown on Czapek-Dox liquid medium with or without various amounts of melanin precursors and melanin inhibitors was carried out according to the method of Ellis and Griffith Woloshuk et al.
Melanization was monitored at nm as the incremental optical density O. D with reference to comparable culture without melanin precursors or inhibitors. Cultures with melanization beyond an O. D of 1 were diluted 10 folds and measured against a similarly dilute reference culture Nicolaus et al. Isolation and characterization of melanin: Twenty air dried mycelial mats were used for melanin extraction.
Because of rapid oxidation of pigment in air, extraction and analysis were carried out under nitrogen. Sclerotuim cepivorum appeared to be more sensitive to Zineb than Fusarium oxysporum , Alternaria solani was comparatively the least sensitive. On the basis of malondialdehyde content, culturing of 3 fungi in presence of Zineb led to an increase in their lipid peroxide contents as compared to that in the absence of the fungicide. Their colour ranged from brown to black.
Furthermore the patterns of IR-absorption spectra of extracted melanins were almost the same as the patterns of fungal melanins previously reported Potgieter and Alexander, ; Chet and Henis, ; Ellis and Griffiths, Melanization and resistance to Zineb: In comparison to cultures grown without dopamine a melanin precursor Table 7 , resistance to Zineb was demonstrable and peaked at 1.
Such treated fungi produce extra melanin and acquire resistance from prolonged growth in dopamine medium. Melanin inhibitors: A clear correlation exists between the presence of some melanin inhibitors Na2EDTA, tricyclazole and chlobenthiazone in Zineb growth cultures and the retardation in fungicide sensitivity values.
Being classified as dicarboximide fungicide, Zineb may interface with cytochrome C reductase and a number of other flavin enzymes leading to accumulation of peroxy radicals and the superoxide anion.
These reactive radicals then cause the formation of lipid peroxides within the cell membrane which shall be the ultimate cause of cell death in dicarboximide-treated fungi Christopher and Nair, These free reactive radicals are removed by the activity of non-enzymatic antioxidants. These results revealed that tocopherol, acting as a free radical scavenger, alleviated the inhibitory effect of Zineb.
This action supports the hypothesis that the net fungitoxic effect of Zineb is free radical formation. A similar effect was also seen with some related dicarboximide fungicides Christopher and Nair, In contrast, the toxicity of benelate was not affected by the addition of this antioxidant in the medium of the tested pathogens.
Benelate is a benzimidazole fungicide which binds to the B-subunit of the tubulin protein, involved in cell division Davidse, , lipid peroxidation is a complex process involving the formation and propagation of lipid radicals leading to the eventual destruction of membrane lipids. It has been recorded that dicarboximide fungicides induce lipid peroxidation by means of oxygen activation in fungi Radice et al. Such higher content of lipid peroxides in Zineb-grown cultures, when assessed on the basis of malondialdehyde level, ensures and puts a further evidence that Zineb toxicity to fungi is through the formation of free oxidant radicals.
In this study, increased melanization caused by inclusion of dopamine, melanin precursor, in Zineb-growth cultures led to a decrease in the sensitivity of the melonatic pathogens towards the fungicide. An important use of melanin may be its ability to concentrate and immobilize a quantity of reducing equivalents just outside, the surface of the cell, while the oxygen free radical species appear to be harmlessly absorbed by melanin Kwon-Chunge et al. Moreover electrically charged oxidants are excluded from the cytoplasm and react primarily with melanin Korytowski and Sarna, Indeed melanin has been shown to neutralize potentially microbicidal strong oxidants similar to these produced by leukocystes in response to infection Jocobson and Tinnell, ; Wang and Casadevall, On the other hand, the present results showed that the melanin inhibitor, Na2 EDTA, really inhibited melanin formation by the three tested pathogens.
Thus the fungal cell lost partly the capability of melanin to scavenge the free radical s generated by carboximide and it is likely that the free radical s freely cross the cell membrane to attack intercellular targets. The evidence for a relationship between melanization and resistance to oxidant thus seems very strong. From the results, the antioxidant when associated with the carboximide in growth media of the tested fungi, may play its role on two axes. Melanization of the appressorium is crucial for subsequent penetration into host epidermal tissue.
If the biosynthesis of melanin is blocked by mutation or chemical inhibition, penetration fails and disease is prevented. A block in melanin biosynthesis is demonstrated by distinctive color differences from wild-type pigmentation. In an albino mutant of M. The rosy mutant is blocked at the conversion of scytalone to 1,3,8-trihydroxynaphthalene 1,3,8-THN , and the tan-colored buff mutant results from a block between 1,3,8-THN and vermelone. Each of these mutants form appressoria on host tissue but fail to penetrate and cause disease.
The chemical inhibitor tricyclazole specifically affects the conversion of 1,3,8-THN to vermelone, and at higher concentrations inhibits the pathway between 1,3,6,8-THN and scytalone.
Tricyclazole is used to prevent rice blast in other countries, notably Japan, but is not registered for use in the United States. Specific inhibitors of enzymes required for other steps in the pathway have yet to be found. Summary of the Invention It is an object of this invention to provide a screening test for the identification of agents which inhibit melanin biosynthesis for a variety of agricultural and medical uses.
It is a further and more specific object of this invention to identify agents of this type which exhibit antifungal activity. These and other objects are accomplished by the present invention, which provides a screening test for the identification of agents that inhibit melanin synthesis. In the practice of the method, test samples are incubated in a culture of a fungus that produces melanin.
Agents that are positive in the test produce altered pigmentation in the fungal growth. In the practice of this invention's method for screening for the presence or absence of melanin synthesis inhibition by a test sample, the test sample is added to a culture or culture area of a melanin-producing fungus, e. Especially preferred are DHN melanin-producing fungi that pigment either in the dark or in the light.
The culture is incubated with the test sample for such time under such conditions sufficient to observe cell growth and pigmentation in a corresponding culture or culture area containing no test sample. Preferred screening tests further employ, as a positive control, a known melanin synthesis inhibitor such as pyroquilon or tricyclazole.
Melanin synthesis inhibition is determined by observation of alterations in pigmentation in the culture or culture area containing the test sample and alterations in pigmentation in the culture or culture area containing the control. In a particularly preferred embodiment, Cladosporium cucumerinum is grown in a solidified media in a transparent plate or dish, so that test samples and positive controls are observed visually and simultaneously as regions of the same culture.
Tricyclazole is employed as a control. The cultures are grown in the dark or in the light; light is preferred in one embodiment. Actives produce a halo of white, tan or red tones around the test sample in the olive-green lawn of the culture; pigmentation changes are conveniently observed on the underside of the plate or dish.
In some embodiments, actives are tested in a secondary screen that assays for penetration of Magnaporthe grisea into plant tissue such as onion epidermal tissue.
Positive test samples incubated with epidermal pieces inhibit penetration of the hyphae arising from the melanized penetration structure, the appressorium. Detailed Description of the Invention This invention takes advantage of the distinctive color changes which occur when melanin biosynthesis is blocked in fungi that synthesize the pigment.
The screen detects compounds active at any step of the melanin biosynthesis pathway. Fungi producing melanin can be employed in the screening test of this invention.
Particularly preferred are fungi that produce 1,8-dihydroxynaphthalene DHN melanin. At least 50 fungi synthesize DHN melanin. These include, for example, species of Ascomycotina and related Deuteromycotina such as Cochliobolus carbonum, C. It is an advantage of the invention that melanin-producing fungi are readily available, isolated from natural sources or obtained as fresh cultures, as frozen cultures with or without cryoprotectants such as dimethylsulfoxide, as plugs of these, or as conidia.
Preferred fungi of this type pigment in the light or in the dark. Magnaporthe grisea is preferred in one embodiment. Because of ease of growth under laboratory conditions, producing a highly uniform conidiating lawn of growth in plated cultures, Cladosporium cucumerinum is especially preferred. The fungus pigments in the light or in the dark.
Any type of solidified or liquid medium that will support growth and reproduction of melanin-containing fungi may be employed as growth medium in the method of this invention.
Numerous fungal media are known to the skilled artisan, and include standard mycological media, for example, potato dextrose media PDM , potato dextrose agar PDA media, oatmeal agar media, Misato-Hara media containing soluble starch, yeast extract, and bactoagar, basal growth media BGM containing glucose, vitamins, minerals, and water, and the like.
Antibacterials that do not inhibit fungal cell growth such as chloramphenicol may be added to the media to reduce contamination and facilitate culturing. Preferred media do not contain chromogenic or chromophoric ingredients that mask color changes and interfere with the sensitivity of the screen; therefore, media containing oatmeal are not preferred.
Solidified media are preferred; potato dextrose agar media is preferred in one embodiment. The fungus can be incorporated into the agar prior to pouring and incubation.
In the practice of this invention, test samples are incubated in cultures of any fungal species that produces melanin. A known melanin synthesis inhibitor is employed as a positive control. Potentially active agents are identified by the observation of altered pigmentation in the presence of test sample. A preferred method comprises adding a test sample to a Cladosporium cucumerinum culture. The test sample is introduced to a disk or a well on a culture plate in a standard diffusion assay using solidified media, or introduced into one of a series of equivalent tissue culture tubes or bottles in a standard turbidity assay using liquid media.
The culture is incubated for such time under such conditions sufficient to observe cell growth and pigmentation in a corresponding culture or culture plate area containing no test sample. Pigmentation in the culture containing or surrounding the test sample is compared with pigmentation in the culture or culture area containing no test sample.
The presence of melanin synthesis inhibition is determined by observing alterations in pigmentation. In a culture plate, this is a zone of color tones surrounding the test sample. In a culture tube series, this is color tone differences observed visually or spectrophotometrically.Generally, the ability of this mixture of enzymes to sample is compared with pigmentation in the culture or melanin content of the walls. Melanins in cell walls of Basidiomycotina, on the other hydrolyze fungal cell walls is inversely related to the culture area containing no test sample. Chet, I.
On the basis of malondialdehyde content, culturing of 3 fungi in presence of Zineb led to an increase in their lipid peroxide contents as compared to that in the absence of the fungicide. Soil Biol. Description Technical Field This invention relates to a fungal screen for inhibitors of melanin biosynthesis, particularly for fungicides that inhibit the biosynthesis of 1,8-dihydroxynaphthalene melanin. Observation of altered pigmentation in the culture or culture area containing test sample indicates inhibition of melanin biosynthesis. The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.
The fungus pigments in the light or in the dark. Components of the test media are first prepared using analytical grade or cell culture tested reagents obtained from the sources indicated. D with reference to comparable culture without melanin precursors or inhibitors.
Tricyclazole is employed as a control. Isolation and characterization of melanin: Twenty air dried mycelial mats were used for melanin extraction. D of 1 were diluted 10 folds and measured against a similarly dilute reference culture Nicolaus et al.
Agents that are positive in the test produce altered pigmentation in the fungal growth. In this study, melanization of the tested pathogens was suppressed in a trial to pave the way in front of dicarboximide to show its antifungal effect. Carboximides produce antimicrobial oxidants by introducing oxygen free radical s, however many microorganisms have evolved defenses against oxidants Christman et al. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella lyphimurium.
Such treated fungi produce extra melanin and acquire resistance from prolonged growth in dopamine medium.
Success of this aim will be a solid proof revealing the role of melanin as an antioxidant. A block in melanin biosynthesis is demonstrated by distinctive color differences from wild-type pigmentation. Soil Biol. The first is the capability of the antioxidant to scavenge the toxic free radical s, generated by Zineb in aqueous medium, thus leading the elimination of the inhibitory effect of the fungicide.