DD265639A1 - METHOD FOR TESTING ANTIFUNGAL COMPOUNDS - Google Patents

Abstract

A simple, material-economical method (microculture) for the broad-based testing of antifungal agents against different developmental stages (mycelium, sporangium, zoospore) of fungi, in particular of the Oomycete Phytophthora infestans, is described. In him the test organism, z. As Phytophthora infestans, cultured on a single, swollen, sterile, wet rye grain in the Boettcherkammer or on a microplate under standard conditions with and without the addition of the test substance to be examined. By means of a transmitted light or stereomicroscope, mycelial growth and sporangiogenesis are assessed semiquantitatively after an appropriate culture period. The application of the test substance to sporangia, which were formed on untreated mycelium (direct Sporangienkeimung), allows conclusions on the influence of the test substance on the germination of sporangia and zoosporogenesis (indirect Sporangienkeimung). The application of the test substance to released zoospores gives indications of possibilities for influencing the zoospore motility and thus for influencing the spread of the parasite on the host plant by means of these mobile propagation cells.

Description

Field of application of the invention

The invention relates to an effective method for the broad-based assay of antitumor and qualitative scavenging of potential fungicides, preferably to oomycetes, e.g. Phytophthora infestans.

In addition, this method can also be used in the corresponding testing of potential active substances against other, economically relevant parasitic or sapophytic fungi.

The field of application of the invention is in the research as in the chemical industry at dec Sucho for new pesticides.

-2- 265 Characteristic of the known state of the art

For the search for fungicidal agents and for the investigation of the influence of fungicidal agents on various types of fungi, the techniques customary in microbiology are generally used, e.g. The Agarblöckchenmethoden known from antibiotic research, perforated plate tests or Schrägagarkulturen. Many large-scale or fully automatic screoning systems are also based on these principles.

For laboratory investigations, these automated systems do not pay for themselves; one attacks on o.g. Methodologies back and adapting them to the respective study objective. Examples of such general microbiological assay methods are described in detail in the standard literature, et al. in: Booth, C. (ed.). Methods in Microbiology vol.4, Academic Press London, New York 1971, Norris, J.R. and Ribbons, D.W. (ed.). Methods in Microbiology, vol. 1, Academic Press London, New York 1969; Spinning top, H. and Schauer. F. Methods of the Mycological Laboratory ", VEB Gustav Fischer Verlag, Jena 1987. Particularly difficult are investigations in which agents are to be found against low-growing, zoospoian-forming fungi, for example against potato parasites economically important, Tomatoes and many other crops, the Oomycetes Phytophthora infestans, have to be cultivated and maintained in order to maintain and maintain these (optional or obligate) plant parasites, for example by culture in liquid droplets, on parts of plants, on the host plant Methods for this are given in: Lange, L., Olson, W. "Some Methods for studying Zoosporic Plant Pathogenesis Fungi", Buczacki, ST (ed.) "Zoosporic Plant Pathogens - A Modern Perspective", Academic Press London, New York, Paris, Sand Diego, San Francisco, Sao Paulo, Sydney, Tokyo, Toronto 1983.

For the cultivation of Phytophthora infestans, especially for obtaining larger quantities of the asexual stages of propagation (sporangia, zoospores), the use of a rye culture medium according to Tverskoij et al. (Tverskoij, J., Filippova G.G., and Bobkova, Z.S., Substrates for culturing conidia of Phytophthora infestans (MONT.) DE BARY, Mikol.Fitopatol., 15, 1981, 247-250), and Jelke et al. (Jelke, E. Oortel, B., Bohm, K.J., Unger, E., J. Basic Microbiol., 27, 1987 [1], 11-21). However, for a broad-based testing or search of or after antifungal compounds, the cultivation methods mentioned prove to be too material- and labor-intensive. Furthermore, the space required for the incubation of the cultures is too large.

Object of the invention

The invention aims to provide a time and material saving method for testing potential fungicides against different life cycle stages of Oomycetes, especially Phytophthora infestans.

Explanation of the essence of the invention

The invention has for its object to describe a semiquantitativea microbiological process for testing potential fungicides against different life cycle stages of Oomycetes, especially the difficult tu cultured fungus Phytophthora infestans, which allows a high sample throughput with minimal material and time and a broites spectrum of activity recorded ,

According to the invention the problem is solved as follows:

From a preculture of the fungal test organism, an inoculum is initially provided in a manner known per se, by first distilling the first with 10 ml to 30 ml of sterile water. is added, mycelium pieces and sporangia are brought into suspension by stirring. As a fungal test organism, for example, an oumycete such as Phytophthora infestans can be used. Portioned subsets of this approach, ie the mycelial sporangia suspension, are then to be added with defined amounts of a solution or suspension of the respective substance of the substance (batch); At least one portion of the batch is daboi provided for the Konotrolluntersuchung without test substance addition.

Each subset of the thus prepared batch portions is then transferred to a microculture. The microculture used is in each case a single sterile, moist, unheated cereal grain, preferably a pre-swollen rye or barley grain, which is arranged in a humid, subjective observation by means of a coagulating chamber permitting reflected light microscopy or in the sample chambers of a microtest plate.

Another variant of the test procedure is carried out so that the microculture immediately inoculates with the pure inoculum (without test substance) and only after mycelium formation, i. after 3 days to 5 days of incubation, the solution or suspension dor test substance feeds and is further incubated.

The microculture thus prepared, in which the cereal grain acts as a qualitatively and quantitatively well standardized nutrient substrate, is incubated at a temperature of 16 ° C to 20X, preferably 19 ° C, for the total duration of 6 days to 12 days (first incubation ).

After completion of the first incubation, the microculture, if appropriate still in the coker chamber or in the microtest plate, is first evaluated according to criteria such as mycelial growth and the presence of sporangia.

Subsequently, mycelial spores are sporanged from the test substance-treated microcultures

- After short-incubation (about 2 hours) at temperatures of 3 ° C to 10 ° C, with Phytophthora infestans preferably bsi a temperature of 5 * C, followed by another short-term incubation (about 15 minutes) at temperatures of 20 ° C to 28 ° C, preferably of 25 ° C, on zoospores Lildung and mobility of the zoospores (test step: with Zweitinkubation) and

- On the other hand after placing in sterile bidistilled water. and renewed incubation (about 24 hours) at a temperature of 2O ⁰ C to 28 ⁰ C, voizug-, from 25 ⁰ C to germ tube formation (likewise test step with Zweitinkubation)

examined.

In parallel, mycelium flakes from the control cultures (without test substance) are treated and examined in the same way. However, the respective second incubation of the untreated mycelium is formed here. Sporangia in a solution or suspension of the test substance. In this way the influence of the test substance on the zoosporon formation of sporangia, which was formed on ungelucted mycelium, is examined. Furthermore, the influence of the test substance on the mobility of the zoospores released from the sporangia treated with the test substance is analyzed. For the assessment of mycelium growth, a magnifying glass or a stereo optical microscope, for the study of zoospores use a transmitted light microscope with 100- to 400-fold magnification. The following advantages are associated with the use of this test method: First, only a very small amount of material and personnel is required.

Secondly, the influence of the test substances on different stages of differentiation can be examined in the same research approach (mycelial growth, germination of active ingredient-treated sporangia after removal of the active substance, germination of untreated sporangia in the presence of the active substance, zoosporemnotility and morphology). Third, the method requires only minimal application rates for the test substances (pg quantities).

Ausfuhrungsbelsplel

The effectiveness of the described method will be demonstrated by the flowchart of three preferred embodiments.

example 1

1.1. Vorkulur

Phytophtho'a infestans as a test organism is cultured for 14 days at 19 ⁰ C in Eprouvetten on 17 ml slants of the following composition: 17g bean flour, 34g oatmeal, 8g sucrose, 20g agar, 11 distilled water (ZOLLFRANK).

1.2. Production of the Approach to Microculture Inoculation

The pre-culture (14 days grown) of the mushroom is bidest with 30ml of sterile aqua. added. Mycol pieces and sporangia are distilled by vigorous stirring of the water. with a glass rod on the surface of mycelium cover the preculture in suspension (sterile). This suspension should be portioned according to the number of substances to be tested; the corresponding portions are to be mixed with the suspension or solution of the Teitsubstanzen. As a test substance, alkaloids, other natural substances or synthetic substances can be used. The Testsubstanzon be added in such a concentration the inoculum that, in the approach (inoculum and the test substance) is present the test substance in 10 ^"J mol to 10 '" mol dilution. Part of the mycelial sporangia suspension should be used for the inoculation of microcultures without addition of the test substances.

1.3. microculture

Each sterile, moist rye grain (sterilization for 20 minutes in an autoclave) is allowed in a coop chamber of the following structure

incubate:

Place 2 slices of filter paper (moist, sterile, 1 cm in diameter) on a slide (side by side). About these Filter paper slices each a 1 cm high glass hollow cylinder is provided with an inner diameter of 1 cm. The upper edge

of each glass hollow cylinder is equipped with a filter paper ring (dimensions corresponding to the wall thickness of the

Glass hollow cylinder) provided and covered with a cover slip 18mm χ 18mm. Each rye grain contained in these chambers is spiked with 0.05 ml of mycelia-sporgenia-test substance suspension

applied and is to be subjected to a cultivation of 10 days at 19 ° C in a chamber.

1.4. Appraisal and follow-up

1.4.1. The microculture is first to be assessed according to the following criteria (using a stereomicroscope):

• Mycelium growth equals unrestricted control + + +

good, + +

very little mycelium +

no mycelium

Presence of sporengia (yes / no)

1.4.2. Subsequently, in order to remove a mycelium flake with sporangia from the microcultures treated with test substance, it is distilled for 2 hours in 0.1 ml of double-distilled water. at 5 ° C and 1S minutes at 25 ⁰ C to incubate. After applying a drop of the incubation liquid on a slide, it is to be checked by means of a light microscope at a magnification of 100 to 400 times whether the sporangia formed on the test substance-treated mycelium released zoospores in the absence of the test substance. The morphology of these zoospores is to be judged in comparison to the control (changes of the form etc.). Also the zoosporen mobility is to beurtoilen.

1.4.3. Another mycelium flake with sporangia from these test substance-treated cultures is redistilled in 0.1 ml of aqua. Incubate for 24 hours at 25 * C:

It is to be checked by means of light / microscope at 100- to 400-fold magnification, whether these Sporai formed on Wirkstoffstoffbehandoltem mycelium glei are able to germinate after removal of the test substance with germ tube.

1.4.4. With mycelium flakes from the non-bottomed microcultures to proceed as in 1.4.2. or 1.4.3. described. However, the second incubation of the sporangia formed on untreated mycelium is carried out in suspension or solution of the corresponding test substance.

Example 2

The procedure is as in Example 1, but the microculture with mycelial sporangia suspension without test substance addition to inoculate and vorzukultivieren 3 to 5 days to well-recognizable mycelial growth (19 ⁰ C). Then 0.05 ml of the test substance solution or suspension should be dripped onto each rye grain. The further cultivation is carried out as described in Example 1.

Example 3

Ec is to proceed as in Example 1, however, is to be used as the inoculum provided with a test substance Sporangiensusponsion.

Claims (11)

1. A method for testing antifungal compounds in microculture and recourse to a fungal test organism, characterized in that - On a single, sterile, moist, ungated cereal grain as substrate of the microculture a portion of an inoculum consisting of a staggered with a defined amount of the respective test substance mycelium Sporangien- or sporangium suspensions of the fungal test organism used, transmits and thus prepared microculture , as well as at least one control microculture without test substance then incubated (initial incubation) or a portion of an inocuium consisting of mycelial Spo ^r angien- or sporangium suspension of the fungal test organism used, transmits the microculture thus prepared in a first phase incubated, after the formation of mycelium performs an application of the test substance and incubation in a second phase of Erstinkubation continues, while also at least one control microculture without test substance remains, Finally, mycelium flakes with sporangia taken from each incubated microculture were finally examined with regard to the formation and mobility of zoospores as well as germ tube formation. 2. The method according to claim 1, characterized in that arranging the cereal grain in a humid, a subjective observation by means of incident light microscope permitting cooper chamber 3. The method according to claim 1, characterized in that arranging the cereal grain in a moist, a subjective observation by means of incident light microscope permitting chamber of a micro test plate. 4. The method according to claim 1 to 3, characterized in that one uses as the substrate of the microculture in each case a pre-swollen rye grain. 5. The method according to claim 1 to 3, characterized in that one uses as the substrate of the microculture in each case a pre-swollen barley grain. 6. The method according to claim 1 to 5, characterized in that one carries out the initial incubation at a temperature of 16 ° C to 2O ⁰ C for a period of 6 days to 12 days. Process according to claim 6, characterized in that the initial incubation in the case of the test organism Phytophthora infestans is carried out at a temperature of 19 ° C for a period of 10 days. 8. The method according to claim 6, characterized in that in the two-phase first incubation for each of the two phases provides a duration of 3 days to 6 days. 9. The method according to claim 1 to 8, characterized in that for the investigation of the microculture taken from the microcracked spores with sporangia in terms of formation and mobility of the zoospores about a 2-hour short-term incubation at temperatures of 3 ⁰ C to 10 ⁰ C, followed by an approximately 15-minute short-term incubation at temperatures of 20 ⁰ C to 28 ⁰ C performs (two-minute incubation). 10. The method according to claim 1 to 8, characterized in that for examining the extracted from the incubated microcracked mycelium spores with respect to germ tube formation about a 24-hour incubation at temperatures of 20 ⁰ C to 28 ° C durchfuhrt (second incubation). 11. The method according to claim 1 to 10, characterized in that one Retrospectively admixed with test substance solution or suspensions mycelium flakes with sporangia taken from the incubated control microculture, and - also carries out the studies on the formation and motility of zoospores and germ tube formation.