I N H I B I T I O N OF CONIDIAL G E R M I N A T I O N IN C O L L E T O T R I C H U M F A L C A T U M W E N T 1)

by

PRITAM SINGH

Division o/ Mycology & Plant Pathology, Indian Agricultural Re- search Institute, New Delhi-12, India

(15.IX. 1964)

Some chemicals are known to stimulate while others inhibit germination of fungal spores (COCHRANE, 1958). The degree of inhibition, however, varies with the nature of the chemical sub- stance employed. Also, spores of different species of fungi respond differently to the same chemical. The inhibitory type of substances have been shown to react by interfering with respiratory fnnctions, with permeability of the cell or spore wall. They may also be effec- tive in the denaturization of proteins or may affect carbohydrate metabolism.

The effect of dyes, like neutral red, methylene blue, dichloro- indophenol and malachite green, and non-nutrient chemicals, like sodium azide, sodium bisxlphite, 2 : 4-dinitrophenol, 5-nitrophenol, copper sulphate, mercuric chloride, potassium permanganate and furfural was studied on the germinability of conidia of C./alcatum [isolate Nos 2) 244, 301,304, 382 and strain ' T ' as light type highly virulent isolates, and isolate Nos 2) 7, 78 and 300 as dark type weakly pathogenic ones~. These dyes and non-nutrient chemicals are known to interfere in the energy reactions, such as respiration and carbohydrate metabolism.

The harvested conidia were freed of adhering external nutrients and mycelial fragments through washing them twice in sterile distilled water by centrifugation (2,800 rpm for 5 minutes, each). Conidia which were not treated with any physical or chemical agents before germination have beert designated as "unactivated". To obtain quantitatively well marked inhibitory effects, it was considered imperative to "activate" the spores, inasmuch as the percentage of germination of the "unactivated" spores is rather

1) This work is a part of the thesis submitted by the author for P h . D . degree I.A.R.I., New Delhi.

2) Nos. of the Indian Type Culture Collection, I.A.R.I., New Delhi.

108 e. sINaH

low (14--18% in the light type isolates and 35--40% in the dark type ones). Initial studies on stimulation showed that conidia of C./alcatum can be activated (as reflected in marked improvement in the germination percentage) either by subjecting them in sus- pension to a temperature of 45 ° C for 5 minutes or presoaking them in 10 ppm EDTA (ethylene diamine tetra-acetic acid) solntion for 8 hours. The germination percentage with heat - - treated spores rose to 65--70% while in E D T A - - treated spores, it was as high as 950/0 .

To determine whether the effects of the dyes and the non- nutrient chemicals are of a permanent nature or they are only transient, the conidia were first soaked in these dyes and chemi- cals individually, for 8 hours and then tested for germination in EDTA. These conidia were considered as "inactivated".

Conidia subjected to various "activation" or "inactivation" treatments were invariably washed first in M/30 phosphate buffer and then in distilled water. They were then tested for germination in various concentrations of the dyes and the chemicals as well as in EDTA at 27 ° C for 18 hours a). Concurrently, with each treat- ment, a 'Control' of "unactivated" conidia was run in distilled water. The concentrations of the dyes and the chemicals used were 0.5, 0.25, 0.125 and 0.0625 ppm, except for furfural which was tried only in 10 ppm solutionS).

The effect observed was remarkable in that, irrespective of the physiologic state of the conidia ("uaactivated" or "act ivated") used and irrespective of the concentration of the dyes and chemicals tried herein, uniformly a total inhibition of the germination oc- curred in all treatments. Moreover, this effect was of a permanent nature and even after treatment with a strong activator like EDTA, germination could not be restored. Only in the case of furhlral- treated spores, their germinability was regained.

Dyes are known to be either st imulatory or inhibitory to the redox system (oxidation reduction process) depending upon their positive or negative role, respectively. Since the dyes produced inhibitory reaction with regard to C. ]alcatum conidia, it appears possible that they acted in a negative fashion and interfered detri- mentally in the energy reactions. The observable effect was total inhibition of germination. Malachite green, being a narcotic as well as a dye belonging to triphenyl methane dye series, is known to inhibit dehydrogenases (QuESTAL & WHEATLEY 3). I t appears that the conidia were either Idiled or irreversibly "inactivated". Sodium azide, sodinm bisulphite, 2:4-dinitrophengl, 5-nitrophenot and potassium permanganate are known to be strong inhibitors of respiration and break off the chain reactions involving cyto- chromes, ATP and DPN (OPPENHEIMER & STERNd; JAMESS; WEST & TODD6). It appears that a similar mechanism is involved with

3) Optimum temperature and incubation period (SING~ 3). 4) Lower concentrations were stimulatory (SINGH 3).

GERMINATION OF COLLETOTRICHUM FALCATUM 109

regard to the inhibition of the germination of C./alcatum conidia. GOLDSWORTHY (~: GREENE 7, DAVSON • DANIELLI s a n d TROGER 9 showed that the heavy metals, like copper and mercury, being di- and tri-valent cations, do not enter the spores, but simply get ad- sorbed on the spore wall and reduce its permeability. The lasting effect of copper sulphate and mercuric chloride in the present in- vestigation shows that either the adsorption as such is permanent and causes immediate killing or the copper and mercury salts form a complex with the proteins present on the spore wall. The hypoth- esis seems plausible in view of the fact that, even in EDTA, the germination of inactivated spores could not be restored. It indicates that a complex of copper and/or mercury with proteins present in the spore wall was formed which could not be chelated by EDTA. Had it not been for the complex, EDTA would have chelated the simple copper and mercury salts (DIxoN & WEBB10). The temporary effect of furfural and that too in higher concentration (10 ppm) definitely shows that its toxic effect is due to higher concentration alone.

All the chemicals, except sodium azide and mercuric chloride, are known to be non-phytotoxic to plants in general including sugarcane, at least in concentrations tried in this study. It suggests a novel possibility of their utilization in experiments to control the secondary spread of the red rot fungus.

Acknowledgement

Gratefulness is acknowledged to Dr. V. V. CHENULU for critically reviewing the manuscript.

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