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VOL. 12

RESEARCH NOTES c

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DECEMBER 1983

NUMBER 4

A Simple Technique for Inducing Sporulation by Alternaria carthaml on Artificial Media C.F. McRae and A.D. Heritage CSIRO Centre for Irrigation Research Griffith, NSW, 2680, and J.F. Brown Department of Botany Univers ity of New England, Arm idale, NSW, 2351 Abstract A simpl e method of inducing abundant spor ulatio n by A. csrthsmi on synthetic media is described. The fungus was grown on potato carr ot agar for 7 to 10 days in darkness. The mycelium was then extensively wounded and sub jec ted to irr adi ati o n (6.5- 9.0 Wm- 2 ) : 12h photoper iod ) fo r 48 h. Abundant sporulation occurred within 24 h. Introduction Many species of Atteme rie , Including the ubiquitous A. solani (Ell. and Mar t.) Jon es and Gro ut, spo rulate reluctantly if at all on synthe tic media (4, 6, 7, 10, 12, 13, 14). We foun d this to be the case with A. carthami Chowd hu ry which ca uses leaf bl ig ht In safflower . Moreover, no satisfactory method of Inducing A. certnemt to sporulate on art ificial media has been descr ibed . Despite the appa rent reluctance of A. cart hami to produce conidia, an abun dant and reliable supp ly of spo res is required by plant pathologists and breeders If they are to screen safflower plants for resistance to the fungus under glasshouse co nditions. The effect of light on the prod uct ion of conidia by a limited num ber of spec ies of Alternaria Is well doc umented (4, 6, 11, 12, 13). In some Instances exposure to light stimulated sporulatio n while In othe rs it did not. Mycelial wounding has been found to Induce spor ulation in some species of Alternaria (4, 7, 10, 13,14). The manipulation of the fungal growt h medium has also proved to be effective In promo ting spor ulation of some spec ies of Alternaria.

This has been ac hieved either by add ing spec ific chemicals to the medium (4, 8, 15) or by causing the medium to dehydrate (4,1 2.1 4). The work report ed in this paper was undertaken to develo p a reliable method of obta ining abundant conidia of A. certnemt on synthetic media In a relatively short time. Materials and Methods An isolate of Alternaria eartham l (DAR 28810) was obtained from the NSW Department of Agriculture. Rydalmere (courtesy J. Walker). The fungus was cultured in 9 em diameter petri dishes containing 15 mls of medium. The effect of four culture media on grow th and sporu lation was tested. These were Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA), Cornmea l Agar (CMA: Oxoid brand ), and V8 vegetable juice Agar (V8A) (6). The agar was Inoculated In the centre with a small piece of agar and myceliu m. Four Inoculated plates of each medium were incubated in darkness at 15, 25, 30 and 35°C. The radial gro wth of colonies was assessed at 3 and 5 days after inoculation by measuring the radial length of colonies at five different positions. The light source used was similar to that descr ibed by the Commonwealth Mycolog ical Institute (6) and consisted of a bank of 3 fluorescent tu bes, each 2.5 m long. The central tube was a Philips black light tub e (T1 40W/ 08 RS F40 BLB). On each side of this was a white tube (Osram 40 WRS). The light bank was controlled by a tim e clock set at 12 h on/off cycle. The incident intensity on the cultures was co ntrolled by varyin g the distance between the light source and the fungal cultures. Distances of 12.5, 22.5, 32.5, 42.5 and 53 em from the light pro duced light intensit ies of 15.5, 12, 9, 6.5 and 4.5 Watts m- 2 measured by a Licor Model No. LI 185A light meter fitted with a Pryo meter head No. pyl 645.7904. The temperature of colonies exposed to each light intensity was monitor ed with a digital thermom eter to see whether the temp eratur e was affected by the differe nt light tr eatments. To study the effect of light on spo rulation, five plates of wounded myceliu m of A. earthami were placed at each light intensity for 48 hours. The con id ia produced on each plate were then washed off the wounded myceliu m with 50 ml of water. The rinse solutions from colonies exposed to each intensity were poo led and a haemocytom eter was used to estimate the co ncentration of co nldla/rn l' of solution . Eight estimates were taken for each solution . 53 '. f

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To study the effect of mycelial wounding on sporulation, A. carthami was grown on PCA and kept at 25°C until the fungus has covered a 9 em diameter petri dish. The mycelium was wounded to different degrees by cutting the agar and mycelium into squares with a sterilized scalpel blade. Vertical and horizontal cuts were made through the agar and mycelium in the petri dishes to produce squares having sides of 1 em, 0.5-0.7, 0.3-0.5 em and 0.2-0.3 em. Some plates were left intact (unwounded). The test plates were then placed under the bank of lights for 2 days (6.5 Wm- 2 ) light intensity; 12 h photoperiod). An additional set of plates with 0.2-0.3 em squares was kept in darkness while another was exposed to continuous light. There were 5 replicates of each treatment. The amount of sporulation was determined by rinsing each plate with 100 ml of water. The number of spores in each rinse solution was estimated by the haemocytometer method described previously. Four readings were taken of each solution.

Mycelial wounding, although not necessary for sporulation, enhanced sporulation of A. carthami (Table 1). Sporulation, did not occur unless the mycelium was subjected to a period of light and a period of darkness (Table 1). Sporulation was not observed in colonies that were kept in either continuous light or darkness. This result is consistent with the findings of other workers (1, 3, 5, 9) for other species of Alternaria. The sporulation process in many fungi is divided into two processes; an assimilation process in which light is necessary for conidiophore production and a dark period necessary for subsequent conidial formation (2). Under favourable conditions sporulation occurred within 24 hours after wounding and exposure to the light/dark regime. A second crop of spores could be obtained by rinsing off the spores and replacing the plates of wounded mycelium under the lights. The light intensity to which colonies were exposed had a marked effect on sporulation. An intensity of between 6.4 and 9.0 Wm- 2 produced significantly more sporulation (P<0.5) than higher or lower light intensities. The different light intensities had little effect on the temperature of the colonies (Table 2). This research was funded in part by the Oilseeds Research Committee of Australia.

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Table 1. Effect of mycelium wounding on sporulation by Alternaria carthami under different light regimes.

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Sporulation (conidia/rnl)

0 1 0.5-0.7 0.3-0.5 0.2-0.3 0.1-0.3 0.1-0.3

12 h light: 12 h dark 12 h light: 12 h dark 12 h light: 12 h dark 12 h light: 12 h dark 12 h light: 12 h dark Continuous darkness Continuous light

0.80 ab 1.25 be 1.80cd 2.00 d None None

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Table 2. Sporulation of A. carthami on artificial media after exposure to light at varying intensities.

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Values followed by the same lower case letter do not differ significantly (p<0.5 = 0.58).

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TEMPERATURE I'CI

Fig. 1. Effect of temperature on culture medium on growth of Alternaria earthami. PCA A = radial growth after 3 days PDA 8 = radial growth after 5 days CMA V8A

Distance from light source (em)

Temperature DC

Light Watts/m 2

Sporulation (conldla/rnl) x 10-'

12.5 22.5 32.5 42.5 53

22.8 22.6 22.2 22.0 21.7

15.5 12.0 9.0 6.5 4.5

8Ab* 9.3b 10.5c 10.9c 6.1a

'Values followed by the same lower case letter do not differ significantly (P<0.5 = 0.94).

REFERENCES

Result. and Discussions In general, A. Carthami grown on PCA at 25°C produced significantly more growth (P
(1) Aragaki, M. (1961) - Radiation and temperature interaction on the sporulation of Alternaria. Phytopathology. 51: 803804. (2) Aragaki, M. (1964) - Relation of radiation and temperature to the sporulation of Alternaria tomato and other fungi. Phytopathology 54: 565-569.

54

zoospores are considered to be the most important infective stage of the fungal life cycle (14). In 1935, Mehrlich (10) found that P. cinnamomi could readily be induced to form zoosporangia by incubating young mycelia in non-sterile soil leachate, but not in sterilized leachate from the same soil. This was confirmed in the course of subsequent investigations, the results of which indicated that the sporangium-inducing properties of soil solutions were due to the activity of bacteria (3,6, 9, 13), or to unidentified substances produced by bacteria (1, 11). In two studies (3, 6) fungi were also tested for effects on sporangium formation but no evidence was found to suggest a stimulatory role for these organisms. The purpose of the present paper is to report stimulation of sporangium production in P. cinnamomi by species of Penicillium Link ex Fr. from the rhizosphere of plants native to the jarrah forest of Western Australia. Isolates of Pe. verruculosum Peyronel (I.M.1.268173), Pe. janthinellum Biourge (I.M.1.270808), Pe. citrinum Thom (I.M.1.270809) and Pe. melinii Thom (I.M.1.270811) were studied. The first two species were isolated from Banksia grandis Willd. and the latter two from Acacia pulchella R.Br. The fungi were recovered from rhizosphere soil using methods described by Malajczuk and McComb (5) except that the isolation medium (peptone dextrose agar (7)) was modified by substituting 2 p.p.m. aureomycin hydrochloride for rose bengal and altering the cone. of streptomycin sulphate to 100 p.p.m. An isolate of the A2 mating strain of P. cinnamomi (I.M.1.264384) was used. Media were prepared using distilled deionised water (DDW) of pH7 and dispensed at the rate of 20ml/90mm Petri dish. Inoculum of Penicillium spp. was grown for 21 days on potato dextrose agar supplemented with bacto agar (Difco Laboratories, Detroit, U.S.A.) at 5 gl-'. (PDA). Pea agar (PA), containing 400g homogenized peas and bacto agar at 15 gl-', was used to culture inoculum of P. cinnamomi. Polycarbonate membranes (13mm diam., 8/lm pore) (Nuclepore Corp., Pleasanton, Ca., U.S.A.) were placed on the surface of the PA (10/dish) which was centrally inoculated with P. cinnamomi and incubated for 7 days to allow formation of a dense mycelial mat over the membranes. The mycelium was then peeled from the agar to expose membranes on which hyphae had penetrated the pores and become attached to the surface.

(3) Basnl, E. and Rotem. J. (1975) - The effect of light on sporulation of Alternaria potri 1. sp. solanl and Stemphyllum betryosum f. sp, Iycoperslcl In vitro: Phytoparasltlca 3: 63-67. (4) Charlton, K.M. (1953) - The sporulation of Alternaria soteni in culture. Transactions of the British Mycological Society, 38: 349-355.

(5) Cohen, Y., Levi. Y. and Eyal. H. (1978) -Sporogenesis of some fungal plant pathogens under intermittent light conditions. Canadian Journal of Botany, 58: 2578-2543.

Mycological Institute (1968) Plant Pathologist's Pocketbook - Commonwealth Mycological Institute. Kew.

(6) Commonwealth

(7) Douglas. D.R. and Pavek. J.J. (1971) - An efficient method of inducing sporulation of Alternaria solanl in pure culture. Phytopathology, 81: 239. (8) Ellers, K.L. and Baxters, LW. (1974) - Induced conidial formation in Alternaria zinntee on media amended with Morestan (Abstract). Proceedings of the American Phytopathological Society, 1: 59. (9) Gupta, R.B.L., Desai. B.G. and Pathak, V.M. (1972) - Effect of light on growth and sporulation of Alternaria brassicae (Berk.) Sacco Phytopathologica mediterranea, 11: 61-62. (10) Kundel, L.a. (1918) - A method of obtaining abundant sporulation in culture of Macrosporium solani E & M. Members of Brooklyn Institute of Arts and Sciences Botanic Gardens, 1: 306-312. (11) Leach, C.M. (1962) - The sporulation of diverse species of fungi under near ultraviolet radiation. Canadian Journal of Botany, 40: 151-161. (12) Lukens, R.J. (1963) - Conidial production from filter paper cultures of Helminthosporium vegans and Alternaria solani. Phytopathology, 50: 867-868. (13) McCallen, S.E.A. and Chan. S.Y. (1944) - Inducing sporulation of Alternaria soieni in pure culture. Contributions from the Boyce Thompson Institute for Plant Research. 13: 323-325. (14) Rand, R.D. (1917) - Production of spores by Alternaria soleni in pure culture. Phythopathology. 7: 316-317.

To examine effects on sporangium production, 20ml of sterile DDW was inoculated concurrently with one membrane supporting hyphae of P. cinnamomi and two discs of PDA (5mm diam.) bearing mycelium and conidia of a Penicillium sp, Control treatments were inoculated only with P. cinnamomi, or with two blank PDA discs instead of Penicillium. After 8 day's incubation at 26°C, the membranes were recovered and stained with 0.5% trypan blue in polyvinyl alcohol before mounting on microscope slides. Total numbers of sporangia were estimated by inspection of the entire membrane at x 200 magnification. Each treatment was replicated five times in randomized blocks. The experiment, which was repeated three times, was maintained in an incubator illuminated by a 15W white fluorescent tube. P. cinnamomi produced sporangia in sterile DDW inoculated with Penicillium spp., but not in non-inoculated controls or in controls receiving PDA discs instead of Penicillium. Significantly greater (p 0.001) numbers of sporangia formed in DDW inoculated with Pe. janthinellum, compared to Pe. verruculosum or Pe. melinii, whilst sporulation was poor in the treatment with Pe. citrinum (Table 1). Zoospores were released by some

(15) Shahin. E.A. and Shepard, J.F. (1979) - An efficient technique for inducing profuse sporulation of Alternaria species. Phytopathology. 89: 618-620.

Stimulation of Sporangium Production in Phytophthora clnnamoml by Penicillium spp. from the Jarrah Forest of Western Australia D.I.L. Murray Division of Forest Research, CSIRO, P.O. Box 4008, Canberra, A.C.T. 2600 Phytophthora cinnamomi Rands, a soil-borne plant pathogen of ubiquitous distribution, has been reported to parasitise a wide range of hosts including many species of economic significance (14). Although plants, primarily their roots, may be infected by vegetative hyphae (8), 55