I N D U C I N G S P O R U L A T I O N IN D I F F E R E N T S T R A I N S OF ALTERNARIA SOLANI

I. E F F E C T OF V I S I B L E L I G H T

by

B R I J MOHAN SINGH1)

Department o/ Botany and Plant Pathology, Punjab Agricultural University, Hissar, Punjab, India

(with 1 fig.)

(31.VIII.1965)

INTRODUCTION

Spores of Alternaria solani have been recommended for the bio- assay of fungicides in the laboratory by the Committee on the Stan- dardisation of Fungicidal Tests of the American Phytopathological Society (1943). The obvious advantage of the spores of A. solani in preference to other test fungi is their consistency as regards the reproducibility of results, ease of counting because of their large size and their capability to rate specific compounds of a fungicide differently (MCCALLAN et al., 1941).

However, under controlled conditions in the laboratory A. solani fails to sporulate, or if at all, it does so very poorly. The first suc- cessful at tempt at inducing sporulation was that of t~AZcDS (1917), followed immediately by KUNI~EL (1018). Both obtained abundant sporulation by shredding and wounding the mycelium grown on different media. BEECHER (1945) induced sporulation in Alternaria solani by growing it on bran extract medium and subsequently ir- radiating it under a sun lamp. The beneficial effect of light in in- ducing sporulation was also demonstrated by DILLON \¥ESTON (1936), JOHNSON ~: HALPIN (1954) and LUKENS (1960). More recently GUPTA & PUSHKARNATH (1962) reported successful results by irradiating the cultures of Alternaria solani to sun rays and in- frared rays alternately.

The present investigations were undertaken to study the effect of light in inducing sporulation in three physiologically distinct strains of Atternaria solani.

1) Present address: -- Plant Pathology Laboratory, Kulu, Punjab, India

S P O R U L A T I O N OF A L T ~ R N A R I A S O L A N I 145

MATERIAL AND METHODS

Of the three strains of Alternaria solani used in these studies, two were isolated from potato and tomato leaves affected with early blight and designated as strains A and B respectively. The third strain was obtained from the Division of Mycology and Plant Pathology, I.A.R.I., New Delhi and designated as strain C. The three strains were purified by single hyphal tip method and in pre- liminary studies were found to differ in such physiological charac- ters as their comparative growth rates, chromogenic behaviour and type of mycelial growth on potato dextrose agar. (Fig. 1).

Fig. 1. Five day old cultures of the three strains of Alternaria solani on potato dextrose agar.

The experiments were run, in duplicate, in Petri dishes of 9 cm diameter, each containing 25 ml of potato dextrose agar and were incubated at 20+1 ° C for 10 days in the dark after which various treatments were given. The cultures after treatment were returned to incubation at 2 0 ± 1 ° C for 48 hours before making spore counts.

To s tudy the effect of fluorescent light, cultures were exposed to a fluorescent tube of 40 watts hung horizontally over the Petri dishes. Spore counts were made in a haemocytometer cell by making spore suspensoin in 5 % glycerine. An average of 20 counts for each treatment was made and spore yield per Petri dish was calculated. For the biometry of spores, 20 readings at random were taken for every treatment and averaged.

EXPERIMENTAL

Effect of alternate light and darkness

For this experiment, Petri dishes after inoculation were wrapped in colourless cellophane paper and divided into three sets; one set

146 B.M. SINGH

was placed outdoors where it was subject to bright sun light during day time and almost complete darkness during night (temperature, 3--30 ° C). The second set was placed on window sill receiving full natural light during day time but not direct sunlight (temperature, 12--20 ° C) and the third set was placed on laboratory benches re- ceiving diffuse light during the day (temperature, 14--20 ° C). Controls for each treatment were wrapped in thick black paper. After 10 days of incubation, Petri dishes were examined for sporulation. Strain B failed to sporulate whereas strains A and C produced spores in Petri dishes placed out-doors and on window sill and covered with cellophane paper. The results (Table I) clearly show that the intensity of light and a wide fluctuation in temperature had a stimulatory effect on sporulation of strains A and C. The intensity of light had also a marked effect on the length of spores of each strain.

TABLE I .

E//ect o/alternate light and darkness on the spore yield and spore size o/strains A and C

T r e a t m e n t Spore yield in t h o u s a n d s Spore size in microns

S t r a in A St ra in C S t ra in A S t ra in C

Placed 6.50 10.50 ou tdoor s

Placed on 3.00 3.50 W i n d o w sill

Placed on No No l abo ra to ry sporu la t ion sporu la t ion benches

Control No No sporu la t ion sporu la t ion

256.00 × 20.80 159.00 × 16.40

192.96 × 19.20 132.10 × 17.00

The two strains differed considerably from each other in the de- gree of sporulation and spore dimensions.

Effect of d i f f erent d u r a t i o n s of s u n l i g h t Cultures of the three strains were exposed to morning sunlight

for 30, 60 and 90 minutes in two lots. Petri dishes of the first lot were covered with lids whereas those of the second lot had their covers removed prior to exposure. Controls were covered with thick black paper. Strains A and B failed to sporulate and only strain C responded favourably to the treatments. I t may be seen that both the time of exposm-e and presence or absence of covers markedly influenced spore yield. Although spore yield increased with a cor- responding increase in the time of exposure, the dishes without cov- ers far outyielded those with covers on the Petri dishes. Mean spore length increased with an increase in the time of exposure ir- respective of whether the covers of Petri dishes were removed or not (Table II).

SPORULATION OF ALTERNARIA SOLANI 147

TABLE I I .

E/[ect o/ time O] exposure to sunlight on the spore yield and spore size o/ strain C.

Time of exposure

in m inu t e s

Spore y ie ld in t h o u s a n d s Spore size in microns

P la t e s w i t h P l a t e s w i t h P la t e s w i t h P la t e s w i t h covers covers Covers covers

r emoved r emoved

Contro l No No -- - - spo ru l a t i on sporu la t ion

30 21.50 24.50 142.70 × 17.18 140.80 × 17.35 60 48.00 159.00 156.20 × 16.36 162.60 × 16.26 90 97.00 296.00 181.44 × 18.72 177.28 × 17.60

Effect of expos ing scraped and unscraped cultures

Scraped and unscraped cultures of the three strains were placed on the window sill for 48 hours (temperature, 31---35 ° C). Scraped and unscraped controls were covered with thick black paper, as usual. Spores were produced by Strain B only (Table III) . I t is of interest to note that in the first experiment strain B had failed to sporulate whereas sporulation was observed in strains A and C. Scraped cultures sporulated more profusely than the unscraped ones. Mean length of the spores in scraped cultures was slightly more than that of the unscraped ones.

TABLE I I I .

Effect of exposing scraped and unscraped cultures on the window sill on spore yield and spore size o/ strain B.

T r e a t m e n t Spore y ie ld in t h o u s a n d s

Spore size in microns

Scraped 394.00 251.67 × 13.12 Unsc raped 127.50 233,42 × 13.60 Control No spo ru l a t i on --

Effect of f luorescent l ight

Scraped cultures of the three strains were exposed under the fluorescent tube for 10, 20 and 40 minutes at a distance of 45 cm by removing Petri dish covers prior to irradiation. Non irradiated scra- ped cultures were kept as control. Only strain C sporulated.

I t is evident from Table IV that 20 minutes exposure was opti- mum for sporulation, further irradiation causing considerable in- hibition. The time of exposure, however, did not exercise any in- fluence on spore size.

148 ~. M. SINGH

TAI3L]~ IV.

E//ect o/fluorescent light on the spore yield and spore size o/ strain C.

Time of exposure Spore y ie ld in in m inu t e s t h o u s a n d s

Spore size in microns

Control No sporu la t ion -- 10 34.00 153.60 × 17.92 20 128.00 157.44 x 17.28 40 48.00 158.72 X 17.28

E f f e c t o f d i f f e r e n t c o l o u r s o f l i g h t

In order to determine the effective region of visible spectrum of light for inducing sporulation, the cultures of the three strains were scraped and with their covers removed, were wrapped in cellophane papers of blue, green, yellow and red colours, so as to allow the transmission of rays of respective wavelengths. The cultures were irradiated under the fluorescent tube at 45 cm distance for 10, 20 and 40 minutes. Only strain C sporulated for which the results are given (Table V). It is evident that exposure to green light for 40 minutes was effective in inducing maximum sporulation in strain C and exposure to red light resulted in minimum spore yield. Where- as exposure to red light for 10 and 20 minutes was not effective in inducing sporulation, the exposure to blue light for 40 minutes resulted in inhibition of sporulation due to irradiation for a longer period. Not much variation was observed in spore dimensions under different treatments.

TABLE V.

E/leer o/di//erent eolours of light on the spore yield and spore size o/strain C.

Colour of T ime of Spore y ie ld Spore size in microns l igh t exposure in t h o u s a n d s

in m i n u t e s

Blue

Green

Yel low

Red

10 28.00 149.44 × 17.12 20 222.00 142.40 x 17.28 40 No sporu la t ion --

10 117.00 151.04 × 17.44 20 241.00 151.36 × 17.28 40 393.00 t46.24 x 16.64

10 170.00 166.08 x 17.60 20 220.00 167.04 × 18.08 40 173.00 159.68 × 17.60

10 No sporu la t ion -- 20 No sporu la t ion -- 40 14.00 145.92 × 17.12

SPORULATION OF ALTERNARIA SOLANI 149

DISCUSSION

Alternate light and darkness coupled with a wide variation in temperature had a beneficial effect on the sporulation of strains A and C of Alternaria solani. Increased intensity of light resulted in greater sporulation in both the strains. Sporulation in strain C was affected by the duration of exposure to sunlight and the presence or absence of covers. This indicates the associative effect of a com- paratively more rapid desiccation of the cultures than those covered with lids. The favourable effect of desiccation on sporulation has been reported in the case of Macrosporium carotae by PIRONNE (1935). The effect of sunlight in inducing sporulation has been demonstrat- ed by DILLON WESTOX (1936) and BEECHER (1945). Spore length in both cases increased with an increase in the intensity of light. JOHNSON & HALPIN (1954) also reported similar effects of the in- tensity of light on the spore morphology of Alternaria solani.

The light and temperature interaction on sporulation was re- markable. Strain B which had failed to sporulate at comparatively low temperature at the time of exposure to sunlight sporulated profusely when exposed on the window sill at higher temperature with minimum fluctuation. Such interaction of light and tempera- ture was clearly demonstrated by HOUSTON & OSWALD (1946) in the case of Helminthosporium gramineum.

Fluorescent light induced moderate sporulation in strain C only, the most effective colour of visible spectrum of light being green. Evidently, intermediate wave lengths within the visible spectrum are more stimulatory than the extremes on either side.

Summary

The role of visible light in inducing sporulation in three strains of Alternaria solani has been studied. The three strains respond dif- ferently to sunlight and fluorescent light. The intensity of light, duration of exposure, temperature at the time of exposure and scrap- ing the cultures have a considerable effect on spore yield and spore dimensions. Most effective colour of visible spectrum in inducing sporulation is green.

Acknowledgements

Grateful thanks are due to late Professor C. S. PARACER for his valuable guidance during the course of this investigation.

References BEXCHER, F. S. 1945. Light and composition of media as factors inducing sporulation

in strains of Alternaria solani. Phytopathology 35: 483. Committee on the Standardizat ion of Fungicidal Tests 1943. The slide germinatioI1

method of evaluating protectant fungicides, Phytopathology 33: 627--632.

150 B.M. SI~GH

DILLON ~VESTON, W. A, R, 1936. The sporulation of Helminthosporium avenae and Alternaria solani in artificial culture. Trans, Brit, mycol. Soc. 20: 112--115.

GUPTA S. K. & PUSHKARNATH 1962. Inducing sporulation in artificial culture of Alternaria solani (Ell. & Martin) Jones & Grout. Mycopathol. et Mycol. appl. 17: 82--88.

HOUSTON, B. R. & OSWALD J. W. 1946. The effect of light and temperature on coni- dium production by Helminthosporium gramineum in culture. Phytopathology 36: 1049--1055.

*JOHNSON, T. W. & J. E. HAI.PIN 1954. Environmental effects on conidialvariation in some fungi imperfecti. J. Elisha Mitchell Sci. Soc. 70: 314--326. Original not seen.

KUNKEL, L. O. 1918. A method of obtaining abundant sporulation in cultures of Macrosporium solani E. & M. Brooklyn Bot. Gdn. Mem. 1: 306--312.

LUKENS, R. J. 1960. Conidial production from filter paper cultures of Helmin- thosporium vagans and A lternaria solani. Phytopathology 80: 867--868.

McCALLAN, S. E. A. et al. 1941. An analysis of factors causing variation in spore germination tests of fungicides. III. Slope of toxicity curves, replicate tests and fungi. Contrib. Boyce Thomp. Inst. 12: 49--78.

PIRONNE, P. P. 1935. An improved method for inducing spore fructification ill cer- tain species of 3lracrosporium. Phytopathotogy 25: 31--32.

RANDS, R. D. 1917. The production of spores by Alternaria solani in pure culture. Phytopathology 7: 316--317.