Microconidium Formation in Magnaporthe grisea

Transcription

1 Microconidium Formation in Magnaporthe grisea Hajime KATO*, Shigeyuki MAYAMA*, Rie SEKINE*, Eiji KANAZAWA*, Yuka IZUTANI*, Alfred S. URASHIMA* and Hitoshi KUNOH** Abstract Magnaporthe grisea produced phialides and microconidia. Some of isolates, which possesed mating ability, from various gramineous plants; finger millet, rice, wheat, Oryza longistaminata, Eriochloa villosa, Panicum bisulcatum, Digitaria sanguinalis, D. smutsii, D. horizontalis and Brachiaria plantaginea had phialide and microconidium production potential. The phialide is darkly pigmented, vase-shaped, spherical to obclavate, with tapered apex, terminal collarette, basal septum, thick-walled, (mean 8.9) ƒêm long, (4.5) ƒêm thick in the broadest part, arising solitarily or sympodially from aerial hyphae. Microconidia are hyaline, cylindrical, rounded at the end first, then lunate with thin cell wall, no septum, 5-8 (mean 6) ƒêm long, (0.7) ƒêm wide and with one nucleus. The first rod-shaped microconidium is formed blastically from the conidiogenous cell apex within the collarette. Successive rod-shaped microconidium is formed blastically from alternate sides of the conidiogenous cell. Subsequent microconidia are produced from the same locus. Accumulated curved microconidia form a globose mass, rarely with slime, at the tip of a phialide. Each conidium continues to grow and then secedes. The shape of the microconidia becomes crescent. Light does not affect microconidium formation. Oatmeal agar and potato dextrose agar are suitable for microconidium production. (Received September 9, 1993) Key words: Magnaporthe grisea, phialide, microconidia. INTRODUCTION Magnaporthe grisea (Hebert) Barr produces pyriform conidia as an anamorph12,17) and spindleshaped ascospores as a teleomorph1,6,17) An additional conidium state was found for the first time. Hermaphroditic behavior is known in this species, but nucleus migration has not been detected at the phase before the crozier formation7,10,14,16). Therefore the observation of nuclear behavior between mycelia of two mating types was proposed by staining the nuclei. As a result, we noticed the different types of conidium development. At first, induction to produce phialides and microconidia was suspected. However, it later became clear that the formation of these structures was limited to individual isolate which had a different genetic background. In this report, morphology of conidiogenous cells and microconidia, conidiogenesis and some conditions affecting microconidium formation will be mentioned. MATERIALS AND METHODS Stock cultures (Table 2) were maintained in barley grain medium at 5 Ž under dry conditions using silica gel5). A mycelium block of 5mm in diameter from PDA culture was transferred to the center of a glass slide covered with oatmeal extract agar about 2mm deep and placed in a 9cm petri dish. Five dishes for each plot were prepared. The lid was sealed with ParafilmTM. A set of dishes was kept continuously under the fluorescent lamp of 15ƒÊE m-2 sec-1 and another set was maintained in a dark

2 box at 17 Ž, 20 Ž and 25 Ž, respectively. Light intensity was measured by Quantum Sensor, Li-Cor Inc. The slide with mycelia was stained by HCl-Giemsa method after fixation with Carnoy solution. The number of phialides with microconidia per microscopic field, 0.25mm2, of each of the 5 slides, was counted at a distance of 0.79mm, 1.0mm and 1.2mm from the center, respectively, every day for 4 days. As growth medium, oatmeal agar, oatmeal extract agar, potato dextrose agar, Sachs' agar and Czapek agar were used. To make oatmeal extract, oatmeal with triple amount of water was shaken at 25 Ž for 24hr. Then 2% agar and 2% sucrose were added to the extract. Mycelium fragments of 5mm in diameter were transferred to the center of each medium and cultured at 25 Ž for 4 days. The number of phialides bearing microconidia was counted in 20 microscopic fields, 0.08mm2 each, at the place of 1.2mm far from the center. For observation by scanning electron microscopy, mycelium segments were mounted at different growth stages on aluminum stubs and immediately dipped into liquid nitrogen. Specimens were placed on the cold stage of the scanning electron microscope (SEM, Hitachi S4000). Ice was sublimed by conductive heating at -80 Ž and samples were sputter-coated with gold. Temperature was maintained at -120 Ž. SEM observation was made at an accelerating voltage of 2kV and recorded by photography. RESULTS Morphology of conidiogenous cell and microconidium This species demonstrates considerable variation in the morphology of conidiogenous cells, that is, phialides (Plate I). The phialide is darkly pigmented, thick-walled, vase-shaped, spherical and obclavate, with tapered apex, terminal collarette, basal septum, 5.9 to 12.5 (mean 8.9) ƒêm long, 3.3 to 7.2 (mean 4.5) ƒêm thick in the broadest part, arising solitarily or sympodially from aerial hyphae. Microconidia are hyaline, cylindrical, rounded at the ends first, then lunate with thin cell wall, no septum, 5 to 8 (mean 6) ƒêm long, 0.5 to 0.8 (mean 0.7) ƒêm wide and with one nucleus. Conidiogenesis The first rod-shaped microconidium is formed blastically from the conidiogenous cell apex within the collarette. At least two successive rod-shaped microconidia are formed blastically from alternate sides of the conidiogenous cell apex. Subsequent microconidia are produced from the same locus. Accumulated curved microconidia form a globose mass, rarely with slime, at the tip of a phialide. Each microconidium continues to grow and then secedes. The shape of microconidia becomes crescent. Effect of temperature and light The rate of growth was 1.5mm at 17 Ž, 2mm at 20 Ž and 2.4mm at 25 Ž a day. The production of phialides and microconidia started on the first day after the initiation of mycelium growth and gradually increased for 4 days at 25 Ž under both light and darkness. A few phialides and microconidia were formed on the first day at 20 Ž and the second day at 17 Ž under both light and darkness. The number of these organs increased during the period of observation. There was no effect of light on the Fig. 1. Effect of temperature and light on the formation of phialides with microconidia. Solid line shows the effect of darkness and dotted line shows the effect of light.

3 Ann. Phytopath. Soc. Japan 60 (2). April, Table 1. Number of phialides with microconidia formed on various media at 25 Ž a) Mean number of phialides with microconidia per microscopic field, 0.08mm2 and standard deviation. 20 replicates. b) Isolates7) from Eleusine coracana. production of phialides and microconidia (Fig. 1). Effect of nutrients The production of microconidia was easy to observe on oatmeal extract agar, although microconidia were produced on oatmeal agar and PDA. However, there was no formation of phialides and microconidia on Sachs' agar and Czapek agar (Table 1). Relations between isolates and microconidium formation Some isolates from Eleusine coracana of Japan, India, Nepal and Uganda produced phialides and microconidia (Table 2). Ten isolates out of eleven from Triticum aestivum in different locations of Brazil produced phialides and microconidia of the same shape. The isolate CD 141 from Oryza longistaminata of Ivory Coast, the isolate IN from rice of India and the isolate Guy 6 from rice of Guyana produced phialides and microconidia. However, in the rice strain, 22 isolates from Japan, 14 isolates from Brazil, 3 isolates from India and one isolate from Indonesia tested did not produce these organs. Among strains from other gramineous plants, there was formation of these organs on some isolates from Digitaria horizontalis and Brachiaria plantaginea of Brazil, Eriochloa villosa, Digitaria sanguinalis, D. smutsii, Eragrostis lehmanniana and Panicum bisulcatum of Japan. No phialide and microconidium formation was observed in 6 isolates from Panicum miliaceum and 3 isolates from Setaria italica of Japan and one isolate each from Festuca elatior, Leersia oryzoides, Phalaris arundinacea, Setaria viridis, Anthoxantum odoratum and Pennisetum ciliare of Japan. These results show that both mating type MATT-1 (formerly A) and MAT1-2 (formerly a) isolates have the ability of microconidium formation. Among the hermaphroditic, male- and female-fertile isolates, there were isolates which had the ability to produce microconidia. DISCUSSION In conidiogenesis of Magnaporthe grisea, the collarette of phialide surrounds the base of a young microconidial initial which has begun to grow from below and to one side of the previously formed microconidium. It is unlike the typical basipetal phialoconidium formation2,3). Successive microconidia are farmed from alternative sides of conidiogenous cell apex, as in the case of Chloridium which constitutes a sympodulo-phialide3,4). The staining of nuclei with Giemsa solution was tried in the region of mycelium confluence of both mating types to detect the process of nucleus migration. These organs had been difficult to identify without staining. It was concluded that microconidia were formed in each isolate without regard to mating type. Hermaphroditic isolates forming perithecia tend to produce microconidia. The role of microconidia as spermatia was expected. However, there is no definite tendency among male- and female-fertile isolates. Two manuscripts indicated phialide and phialoconidium formation in Magnaporthe species; both M. rhizophila Scott et Deacon13) and M. poae Landschoot et Jackson9). In both cases, the size of the phialide is equivalent to that of M. grisea, but being hyaline. The size of conidia are larger in M. rhizophila than in M. grisea. M. poae produces the wider conidium. The shape of conidium in both M. rhizophila and M.

4 Table 2. Microconidium formation of Magnaporthe grisea from various gramineous plants

5 a) Isolates designated BR were collected by S. Igarashi and A.S. Urashima15), NI by N. Nishihara11), G 10-1 and Z 2.1 by S. Itoi et al.7), Guy 6 by J.L. Notteghem, CD 141 by J.M. Bidaux and the others by H. Kato (UG was sent from J. Mukiibi as specimens)8). b) H indicates hermaphroditic, M male-fertile and F female-fertile. poae is different from that of microconidia of M. grisea, and pyrif orm conidium was not documented. However, coincidence of formation of phialiditic conidia suggests the phylogenic similarity among these species. We thank Hideo Koike and Peter H. Tsao, who critically reviewed the manuscript. Literature 1. Barr, M.E. (1977). Magnaporthe, Telimenella, and Hyponectria (Physosporellaceae). Mycologia 69: Cole, G.T. and Kendrick, W.B. (1973). Taxonomic studies of Phialophora. Mycologia 65: Cole, G.T. and Samson, R.A. (1979). Patterns of Development in Conidial Fungi. Pitman, London, pp Hammill, T.M. (1972). Electron microscopy of conidiogenesis in Chloridium chlamydosporis. Mycologia 64: Hayashi, N. and Kato, H. (1988). Viability and aggressiveness of Pyricularia cultures preserved by silica gel-drying grain method. Proc. Kanto-Tosan Plant Protec. Soc. 35: (in Japanese). 6. Hebert, T.T. (1971). The perfect stage of Pyricularia grisea. Phytopathology 61: Itoi, S., Yamamoto, J., Karino, S., Arase, S. and Kato, H. (1980). Hermaphroditic isolates of Pyricularia isolated from Ragi, Eleusine coracana (L.) Gaertn. Ann. Phytopath. Soc. Japan 46: (in Japanese). 8. Kato, H. and Yamaguchi, T. (1980). Host ranges and interrelations of Pyricularia spp. from various cereals and grasses. Proc. Kanto-Tosan Plant Protec. Soc. 27: (in Japanese). 9. Landschoot, P.J. and Jackson, N. (1989). Magnaporthe poae sp. nov., a hyphopodiate fungus with a Phialophora anamorph from grass roots in the USA. Mycol. Res. 93: Leung, H. and Williams, P.H. (1987). Nuclear division and chromosome behavior during meiosis and ascosporogenesis in Pyricularia oryzae. Can. J. Bot. 65: Matsuyama, N., Kato, H. and Yamaguchi, T. (1977). Comparison of the isozyme patterns of extracellular enzymes in Pyricularia strains from gramineous and zingiberaceous plants. Ann. Phytopath. Soc. Japan 43 : Rossman, A.Y., Howard, R.J. and Valent, B. (1990). Pyricularia grisea, the correct name for the rice blast disease fungus. Mycologia 82: Scott, D.B. and Deacon, J.W. (1983). Magnaporthe rhizophila sp. nov., a dark mycelial fungus with a Phialophora conidial state, from cereal roots in South Africa. Trans. Br. mycol. Soc. 81: Tanaka, Y., Murata, N. and Kato, H. (1979). Behavior of nuclei and chromosomes during ascus development in the mating between either rice-strain or weeping lovegrass-strain and ragi-strain of Pyricularia. Ann. Phytopath. Soc. Japan 45: Urashima, A.S., Igarashi, S. and Kato, H. (1993). Host range, mating type, and fertility of Pyricularia grisea from wheat in Brazil. Plant Dis. 77: Yaegashi, H. and Hebert, T.T. (1973). Perithecial development and nuclear behavior in Pyricularia. Phytopathology 66: Yaegashi, H. and Udagawa, S. (1978). The taxonomical identity of perfect state of Pyricularia grisea and its allies. Can. J. Bot. 56: cited

6 Explanation of plate Plate I 1: Comparison of conidiophores with conidia and phialides with microconidia stained by Giemsa solution. Isolate Z2-1 from Eleusine coracana, Japan. Bar shows 75ƒÊm. 2: Phialides on oatmeal extract agar. Isolate G10-1 from Eleusine coracana, Japan. 4 days after inoculation. Bar shows 50ƒÊm. 3: A part of plate 2. Initial stage of microconidium formation. Isolate G10-1. Bar shows 20ƒÊm. 4: Phialides and microconidia without staining on PDA. Isolate G10-1. Bar shows 10ƒÊm for 4 and 5. 5: Phialides and microconidia stained by methylene blue. Isolate G : Phialides formed on a hypha. Isolate Z2-1. 7: Initial stage of microconidium formation, rod-shape. Isolate Z2-1. 8: Initial stage of microconidium formation, rod-shape. Isolate G-10. 9: Globose mass of microconidia elongated and curved. Isolate UG from Eleusine coracana, Uganda. Bar shows 10ƒÊm for 6, 7, 8, 9 and : Young globose mass of rod-shaped microconidia and mass of mature luna-shaped microconidia. Isolate UG from Eleusine coracana, Uganda. 11: Lunate microconidia. Isolate UG : Lunate microconidia. Isolate G : Phialide formation on loop-formed hypha. Isolate Z : Singly formed phialide. Isolate G : Microconidium formation of isolate CD 141 from Oryza longistaminata, Ivory Coast. Bar shows 10ƒÊm. 16: Microconidium formation of isolate IN from Eleusine coracana, India. Bar shows 10ƒÊm for 11, 12, 13, 14 and : Microconidium formation of isolate IN from Oryza sativa, India. 18: Microconidium formation of isolate BR049 from Triticum aestivum, Brazil. 19: Microconidium formation of isolate NI980 from Digitaria smutsii, Japan. 20: Microconidium formation of isolate NI986 from Eragrostis lehmanniana, Japan. 21: Microconidium formation of isolate NI885 from Panicum bisulcatum, Japan. Bar shows 10ƒÊm for 17, 18, 19, 20 and : Collarettes of phialides. Isolate G10-1. Bar shows 3ƒÊm. 23: Side view of phialide with developing microconidium, Isolate G10-1. Bar shows 3ƒÊm. 24: Microconidium formation from phialide. Isolate G10-1. Bar shows 3ƒÊm. 25: Three microconidia produced from the same apex of a phialide. Isolate G10-1. Bar shows 3ƒÊm. 26: A mass of curved microconidia produced from a phialide. Isolate G10-1. Bar shows 3ƒÊm. 27: Mature microconidia with phialides. Isolate G10-1. Bar shows 10ƒÊm.

7

8

9

10

11