Mode of parasitism of a mycoparasite, Cladosporium gallicola, on western gall rust, Endocronartium harknessii

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1 CANADIAN JOlJRNAL OF PLANT PATHOLOGY I: 31-36,1979 Mode of parasitism of a mycoparasite, Cladosporium gallicola, on western gall rust, Endocronartium harknessii A. Tsuneda and Y. Hiratsuka Canadian Forestry Service. Northern Forest Research Centre, Edmonton. Alberta T6H 3S5. First author is a National Research Council of Canada Visiting Fellow. Accepted for publication Cladosporium gallicola is a destructive mycoparasite of western gall rust. Endocronartium harkness;i. Parasitism occurs either by means of contact without penetration or by penetration of the rust spores. The warty surface layer ofthe fungal host cell wall often disintegrates after being contacted by the parasite hyphae: appressoria are frequently formed. Penetration of the rust spores occurs with or without formation of appressoria. Host cytoplasm coagulates underneath the penetrated parasite cell and eventually disappears. I n the late stages of parasitism. the parasite hyphae usually form conidiophores and conidia directly on the host spores. Cladosporium gallicola est un mycoparasite destructeur de la rouille-tumeur. Endocornartium harknessii. Le parasitisme a lieu soit par voie de contact. sans penetration ou encore par penetration des spores de la rouille. La surface verruqueuse de la paroi cellulaire de l'hote se desintegre souvent apres avoir ete en contact avec les hyphes parasitaires; il se forme frequemment des appressorium. La penetration des spores de la rouille se produit avec ou sans formation d'appressorium. Le cytoplasme de la cellule hote se coagule en presence de la cellule du pathogene et finit par disparaitre. Dans les dernieres etapes de I'infection, les hypes parasites habituellement des conidiophores et des conidies directement sur les spores hotes. Western gall rust caused by Endocronartium harknessii (J.P. Moore) Y. Hiratsuka is the most common, most conspicuous, and probably the most important rust of hard pines in western Canada (Ziller 1974, Hiratsuka & Powell 1976). The pathogen causes globose galls on branches or stems that grow larger every year and sporulate each spring. The disease usually does not cause tree mortality except when a main stem infection occurs on young trees (Hiratsuka & Powell 1976). The rust is autoecious, requiring no alternate host; infection occurs directly from pine to pine (Hiratsuka 1969). Thus, this fungus cannot be controlled by removal of alternate hosts; its inoculum potential is directly proportional to the amount of viable spores produced on pine. A biological control measure is therefore worthy of serious consideration. Our recent field surveys in western Canada revealed that E. harknessii galls on Pinus eontorta Dougl. var. latifolia Engelm. and P. banksiana Lamb. were often covered by secondary fungi, and some of these fungi appeared to destroy the sporulating surface of the rust sori. One of these fungi, Cladosporium gallieola Sutton, was previously reported to form a close association with E. harknessii by Sutton (1973), who noted the penetration of aeciospores (= peridermioid teliospores (Hiratsuka 1973)) by Cladosporium hyphae. Powell (1971) reported a Cladosporium species, later confirmed as C. gallieola, to be one of the most common fungi associated with cankers caused by Cronartium eomandrae Pk. and suggested that C. gallieola may be parasitic to aeciospores of the rust. Byler et al. (1972) similarly reported that C. aecidiicola Thurn. attacked spores of E. harknessil These authors, however, did not provide detailed information on the nature of these associations. This study was conducted to determine the mode of parasitism of C. gallicola on E. harknessii. Materials and methods Galls on P. contorta or P. banksiana caused by E. harkness;; were collected at several locations in Alberta. Cladosporium gallicola used in this study was isolated from a gall collected near Smoky Lake. Galls that were either naturally infected or artificially inoculated with C. gallicola were used for scanning electron microscopy. Artificial inoculation was performed by spraying young sporulating galls with about I ml of spore suspension of C. gallicola per gall. The galls were incubated at room temperature in sterile petri dishes (15 X 2 cm) lined with moist filter paper. The spore suspension of C. gallicola was obtained by adding sterile distilled water to a 10- to 14-day-old culture on potatodextrose agar and scraping the culture surface with a needle. The conidia were washed in a Buchner funnel and suspended in sterile distilled water. Spore density was adjusted to about 107 sporesl ml. Small pieces (about 7 X 7 mm) of gall surface covered by C. gallicola were cut from the galls and fixed overnight either with osmium tetroxide vapor or in 3% glutaraldehyde in 0.1 M Na2HP04 - NaH2P04 (ph 7.0). The vapor-fixed material was air-dried for at least 24 h. The material fixed in glutaraldehyde was washed in the phosphate buffer 31

2 32 CANADIAN JOURNAL OF PLANT PATHOLOGY VOLUME L 1979 and postfixed in 2% osmium tetroxide in the same buffer for 3-5 h. It was then dehydrated in an ethanol series, taken to amyl acetate, and criticalpoint-dried in a Denton vacuum DCP unit using carbon dioxide. The air-dried and critical-pointdried materials were shadowed with gold and examined with a Cambridge Stereoscan S4 scanning electron microscope. For observation with a light microscope, 0.5 ml of the spore suspension of C. gal/icola, prepared as described above, was spread evenly on a cellophane membrane that was placed on water agar in a plastic petri dish. Mature spores of E. harknessii were then dusted evenly on the membrane. and the petri dish was kept at 20 C in the dark. After incubating for 48 or 72 h. the cellophane membrane was cut into strips of appropriate sizes. mounted in water or in cotton blue-iactophenol on a microscope slide, and examined under a Leitz Orthoplan microscope with phase-contrast and interference-contrast equipment. Results N omarski Peridermia (spore blisters) of E. harknessii were bleached white when invaded by C. gal/icola. The hyperparasite produced abundant conidiophores and conidia on the sori, giving them a brownish green to a dark brown appearance. Growth of C. gal/icola was usually restricted to several layers of rust spores from the exposed surface. Young actively sporulating sori of E. harknessii were rapidly overgrown by C. gal/icola when they were spray-inoculated with the hyperparasite and were completely covered in 2-3 days after the inoculation (Fig. I). Microscopic examination revealed that hyphae of C. ga//icola were attracted to rust spores and became attached to them with or without forming appressoria (Figs. 2-5). In some cases. branching of Cladosporium hypha occurred at the point of contact, forming a hand-shaped appressorium (Figs. 3, 4). The external wall. or warts, of the contacted rust spores became disorganized and eventually disintegrated (Fig. 5). Penetration of rust spores by C. gal/icola was common (Figs. 6-11). The parasite hyphae penetrated the host either with or, more commonly, without forming appressoria prior to the penetration. When an appressorium was involved, a fine penetration peg (Fig. 7) was formed that became swollen after piercing the host cell wall (Fig. 6). This hyphal swelling was not so obvious when penetration occurred without the formation of an appressorium, but several knot-like swellings were often observed in the internal parasite hypha (Fig. 8). Coagulation of the host cytoplasm occurred beneath the parasite hypha in the initial stage of penetration, as revealed by N omarski interferencecontrast microscopy (Fig. 6). The host cells, however. soon became empty, and the warty surface layer of the penetrated rust spore was readily detached (Fig. 8, arrow). Scanning electron microscopy of the penetrated rust spores showed no sign of indentation in the host cell wall at the penetration sites (Figs. 9, 10). At late stages of parasitism, the warts of the infected spores became shriveled and disorganized (Fig. II). Dissolution of the host cell wall was clearly evident where the internal parasitic hyphae reappeared (Fig. 12, arrow). These hyphae usually formed conidia immediately after their emergence (Fig. 13). Conidiophores formed on or in the vicinity of the host spores (Figs. 10, 13) were Jlm long and 3-4 Jlm wide. Although penetration was common. it was not invariable in the parasitism by C. ga//icola: appressoria formed on the host spores often failed to produce a penetration peg, yet resulted in the disintegration of the host spores. A conidiophore often developed directly from the appressoria, and the host cells eventually became empty. None of these changes in the host spore was observed, however, unless there had been physical contact between the host and parasite. Discussion Results presented here clearly indicate that C. ga//icola is a destructive parasite of E. harknessii. Parasitism occurs either by contact without penetration (contact parasitism) or by penetration of the living rust spores. The parasite destroys the host cytoplasm. absorbs nutrients from it. and usually forms conidiophores directly on or adjacent to the host spores. The warty surface layer of the host cell wall often disintegrates when contacted by the parasite hyphae. The dissolved appearance of this surface layer (Fig. 5) may indicate an enzymatic or other chemical breakdown of the host material. Penetration of the fungal host cell wall by mycoparasites has been suggested as primarily due to either a chemical (Carling et al. 1976, Tsuneda & Skoropad 1977) or a mechanical (Manocha & Lee 1971) process. In the E. harknessii C. gal/icola interaction, the process appears to be primarily chemical in nature, because there is no obvious sign of indentation or rupturing in the host cell wall at the penetration sites (Figs. 8-10). Also, in the late stages of parasitism, the host cell wall has a dissolved appearance where internal parasite hyphae reappear (Fig. 12), Wound plug-like structures, such as "infection

3 TSUNEDA, HIRATSUKA: CLADOSPORIUM ON ENDOCRONARTIUM 33 Figure 1. Galls on Pinus conrorta caused by Endocronartium harknessii. showing an uninfected sorus (left) and one infected by Cladosporium gallicola. Peridia (arrows) are clearly seen on the healthy gall. but they are obscured on the other because of the profuse growth of C. gallicola. X 0.5. Figure 2. Hyphae of C. gallicola appressed to a spore of E. harknessii. X Figures 3-4. Hand-shaped appressoria of C. gallicola formed on rust spores. Fig. 3, X 2000; Fig. 4, X Figure 5. Greatly disintegrated surface structure (arrows) of rust spores contacted by Cladosporium hyphae. X 1100.

4 34 CANADIAN JOURNAL OF PLANT PATHOLOGY VOLUME I, 1979 Figures 6-7. Nomarski interference-contrast micrographs showing penetration of spores of Endocronartium harknessii by hyphae of Cladosporium gallicola. Fig. 6. Early stage of penetration from an appressorium (AP). Note coagulation in the host cytoplasm (arrow) beneath a swollen internal cell of the parasite. X Fig. 7. Same spore as Fig. 6 viewed with different focus. A fine penetration peg (arrow) is piercing the host cell wall. X Figure 8. Late stage of parasitism showing an empty host spore. Arrowheads indicate knot-like swellings of the parasite hypha inside the host spore. Remnants of surface wall layer (arrow) of the host spore were removed from the spore during mounting. Compare the surface of infected spore with that of uninfected one. X Figure 9. Scanning electron micrograph showing penetration (arrow) of a rust spore by a short side branch of C. gal/icola. There is no obvious sign of identation in the host cell wall around the penetrating hypha. X 3000.

5 TSUNEDA, HIRATSUKA: CLADOSPORIUM ON ENDOCRONARTIUM 35 Figure 10. Formation of a very short conidiophore (arrowhead) by C. gallicola. which has penetrated (arrow) an adjacent rust spore. X Figure 11. Late stage of parasitism by penetration showing a shriveled and disorganized surface structure. X Figure 12. Emergence of internal parasite hyphae that dissolved host cell wall (arrow). X Figure 13. N omarski interference-contrast micrograph showing conidiophores of C. gallicola formed on the rust spore. Arrows indicate outline of internal parasite hypahe. X 1500.

6 36 CANADIAN JOURNAL OF PLANT PATHOLOGY VOLUME I, 1979 papillae" (Hashioka & Fukita 1969, Hoch & Fuller 1977) and "reaction zones" (Tsuneda et al. 1976), have been reported to occur in the cells of certain fungi parasitized by their mycoparasites. This phenomenon is also common in interactions between fungi and higher plants (Bracker & Littlefield 1973, Aist 1976). Formation of any of these structures, however, was not observed in the cells of E. harknessii parasitized by C. gallieola. It has been suggested that toxic factors are involved in necrotrophic (or destructive) mycoparasitism (Hashioka & Fukita 1969, Barnett & Binder 1973, Traquair & McKeen 1978). However, a culture filtrate of C. gallicola did not cause any visible change in the spores of E. harknessii (Tsuneda & Hiratsuka, unpublished). Furthermore, physical contact between the host spores and the hyphae of C. gallieola seems to be necessary for the parasitic process. Therefore, we suggest that a highly diffusible toxin is not likely involved in this mycoparasitism. In the original account of C. gallieola occurrence, Sutton (1973) described the size of conidiophores as Mm long and 8-9 Mm wide at the bases, becoming 5-6Mm wide towards the apices. However, conidophores are much shorter and thinner, as short as 2 Mm and as thin as 3 Mm (Figs. 10, 13), when produced directly on or in the close vicinity of infected rust spores. Byler et al. (1972) reported that many fungi were associated with E. harknessii galls and that several of these fungi, especially Neetria fuekeliana Booth (probably N. maerospora (Wr.) Ouellette), killed the rusted gall tissues, resulting in the mortality of galls and host branches distal to the galls. Unlike N. [uekeliana, C. gallieola's growth is usually restricted to several spore layers of the rust sori, and it does not affect the gall tissues. Therefore, it is unlikely that C. gallieola will cause gall mortality; however, because the growth of C. gallieola on the peridermia of E. harknessii is rapid and profuse under moist conditions, the inoculum level of the rust may be significantly lowered. More observations and experiments are needed to ascertain the significance of this mycoparasite in nature and to evaluate its potential use as a biological control agent for western gall rust and other pine stem rusts. The authors thank G. Braybrook. Department of Entomology. University of Alberta. for assistance with the scanning electron microscopy. and P. Maruyama of the Northern Forest Research Centre for providing some of the materials used in this study. Aist, J.R Papillae and related wound plugs of plant cells. Annu. Rev. Phytopathol. 14 : Barnett, H.L., and F.L. Binder The fungal host-parasite relationship. Annu. Rev. Phytopathol. II : Bracker, C.E., and L.J. Littlefield Structural concepts of host-pathogen interfaces. Pages in R.J. W. Byrde and C.W. Cutting. eds. response. Academic Press. New York. Fungal pathogenicity and the plant's Byler, J.W., F.W. Cobb, Jr., and J.R. Parmeter, Jr Occurrence and significance of fungi inhabiting galls caused by Peridermium harknessii. Can. J. Bot. 50: Carling, D.K, M.F. Brown, and D.F. Millikan Ultrastructural examination of the Puccinia graminis-darluca filum host-parasite relationship. Phytopathology 66: Hashioka, Y., and T. Fukita Ultrastructural observations on mycoparasitism of Trichoderma, Gliocladium and Acremonium on phytopathogenic fungi. Rep. Tottori Myco!. Inst. 7:8-18. Hiratsuka, Y Endocronartium. a new genus for autoecious pine stem rusts. Can. J. Bot. 47: Hiratsuka, Y The nuclear cycle and the terminology of spore states in Uredinales. Mycologia 65: Hiratsuka, Y., and J.M. Powell Pine stem rusts of Canada. Can. Dep. Environ.. Can. For. Serv.. For. Tech. Rep. No pp. Hoch, H. C., and M.S. Fuller Mycoparasitic relationships. I. Morphological features of interactions between Pythium acanthicum and several fungal hosts. Arch. MicrobioI. 111: Manocha, M.S., and K.Y. Lee Host-parasite relations in mycoparasites. I. Fine structure of host. parasite. and their interface. Can. J. Bot. 49: Powell, J.M Fungi and bacteria associated with Cronartium comandrae on lodgepole pine in Alberta. Phytoprotection 52: Sutton, B.C Hyphomycetes from Manitoba and Saskatchewan. Mycol. Pap. 132: Traquair, J.A., and W.E. McKeen Necrotrophic my coparasitism of Ceratocystis jimbriata by Hirschioporus pargamenus (Polyporaceae). Can. J. Microbiol. 24: Tsuneda, A., and W.P. Skoropad The Alternaria brassicae - Nectria inventa host-parasite interface. Can. J. Bot. 55: Tsuneda, A., W.P. Skoropad, and J.P. Tewari Mode of parasitism of Alternaria brassicae by Nectria inventa. Phytopathology 66: Ziller, W.G The tree rusts of western Canada. Can. Dept. Environ., Can. For. Servo PubI. No. 1329, 272 pp.