Conidial germination in Cercospora arachidicola hori

169

Notes and Brief Articles

The function of these thick walls in relation to sclerotial morphogenesis and germination in C. lagopus is currently being investigated. H. W. is in receipt of a S.R.C. Research Studentship. REFERENCES

ARONSON,]. M. (1965)' The cell wall. In The Fungi, vol. I (ed. by G. C. Ainsworth and A. S. Sussman), ch. 3, pp. 49-76. New York: Academic Press. BARTNICKI-GARCIA, S. (1968). Cell wall chemistry, morphogenesis and taxonomy of fungi. Annual Review ofMicrobiology 22, 87-ro8. CHET, I., HENIS, Y. & KISLEV, N. (1969). Ultrastructure of sclerotia and hyphae of Sclerotium rolfsii Sacco Journal of General Microbiology 57, 143- 147. GOODAY, G. W. & HUNSLEY, D. (1971). Cellulose wall ingrowths in Phytophthora parasitica. Transactions of the British Mycological Society 57, 178- 1 79. HUNSLEY, D. & BURNETT,]. H. (1968). Dimensions of microfibrillar elements in fungal walls. Nature, London 218, 462-463. HUNSLEY, D. & BURNETT,]. H. (1970). The ultrastructural architecture of the walls of some fungi. Journal of General Microbiology 62, 203-218. SCHMID, R. & LIESE, W. (1968). Structural and histochemical observations on fungal fibre hyphae. In Electron Microscopy, vol. 2 (ed. D. S. Bocciarelli), p. 415. Roma: Tipographia Pologlotta Vaticana. SCURTI,]. C. & CONVERSO, L. (1965). Sulla struttura microscopica e ultromicroscopica degli sclerozi di Typhula sp. Caryologia 18, 263-283. EXPLANATION OF PLATE

25

Electron micrographs prepared from material fixed in glutaraldehyde and osmic acid, stained with uranyl acetate and lead citrate. Figs. I, 2. Coprinus lagopus. Longitudinal and transverse sections of sclerotial medullary cells. Note original hyphal wall (0), fibrils (F) in the less compact inner zone (Z) the large interfibrillar spaces (S) and the single fibril profiles (P). Fig. 3. Coprinus lagopus. Section through vegetative hyphal wall. Note laminated substructure. Fig. 4. Coprinus lagopus. Section through sclerotial rind cell wall showing thick inner layer. Fig. 5. Corelius versicolor. Section through thick-walled cell from the subhyrnenial layer of the carpophore. Note uniformity of thickening and the striations due to the fibrillar substructure. Fig. 6. Typhula variabilis. Transverse section through thick-walled medullary cell of the sclerotium. Note uniformity of thickening and the fibrillar substructure. In Figs. 3-6 the scale is equivalent to 250 nm.

CONIDIAL GERMINATION IN CERCOSPORA ARACHIDICOLA HORI oso Department of Botany, University B. A.

ofIbadan,

Nigeria

Cercospora spp., and in particular C. arachidicola, are the causal agents of the 'brown spot' ofgroundnut leaves (Chupp, 1953). This report concerns the conditions and mode ofgermination of conidia ofC. arachidicola. Abundant spores were obtained from infected leaves of groundnut on the Faculty of Agriculture farm, University of Ibadan. Freshly picked leaves were washed in tap-water and rinsed thoroughly in sterile distilled water before being incubated in a desiccator at 100 % r.h. and room Trans. Br. mycol. Soc. 59 (I), (1972). Printed in Great Britain

17°

Transactions British Mycological Society

temperature (28°) for 2 days, when conidia developed on the lesions. These were scraped into a drop of sterile distilled water on a glass slide which bore a I cm 2 grid (subdivided into millimetre squares) to facilitate the counting during subsequent observations. The drop of water, together with the conidia, was spread out with a needle to cover the entire grid and then left to dry. For each experiment each I ern" grid contained about 600-800 conidia and all these were counted during the observations. The slides were inverted over staining blocks employed as humidity chambers, and distilled water and saturated solutions of appropriate inorganic salts were used to obtain the various relative humidities at different temperatures (Wexler & Hasegawa, 1954). Each block with its slide was then placed in a separate container with either distilled water or the same conditioning solution as in the staining block. The container was in turn kept in an incubator at a constant temperature. Germination of conidia was observed microscopically. In experiments at 20° and 100 % r.h. the conidia were observed, after 24 h. incubation, to germinate either by giving rise to germ-tubes which later developed into vegetative hyphae, or by producing short, stout projections (incipient conidiophores) on which conidia were later borne. Sometimes germination was by both methods from different cells of the same conidium. In further experiments conidia were incubated for 24 h (except where otherwise stated) at temperatures of 1°,5°, 10°, 15°,20°,25°,30°,33°,35° and 37°, and relative humidities up to 100 %. It was found that in saturated air no germ-tubes were produced at 1°, 5°, 10°, 35° and 37° after 24 h incubation. Short germ-tubes were, however, formed at IS° and 33°, while much longer ones were obtained at 20°,25° and 30°. Hence the optimum temperature for germ-tube formation at 100 % r.h. was in the range 20-30°. Conidia that had failed to germinate after 24 h at 1°, 5°, 10° and 35° produced germ-tubes when transferred to 20° for 24 h. A temperature of 37° for 24 h at 100 % r.h. appeared to be fatal to the conidia since a subsequent transfer to 20° for up to 72 h failed to induce germ-tube production. Table I shows the result of the combined effect of different temperatures and relative humidities on germ-tube production. There was no germination at 1°, 5°, 10° and 35° at any of the humidities, nor below 96'5 % r.h. at any temperature. These results show that the conidia of C. arachidicola require a saturated or near-saturated atmosphere to germinate at optimum temperature (20-30°). Conidia from groundnut leaves were incubated for a few days with daily observations. It was found that a secondary conidiophore originates from the primary conidium as a slight protuberance of one of its cells (PI. 26, fig. I). From the first this can be recognized as distinct from a vegetative hypha by its greater width. This protuberance elongates (PI. 26, fig. 2), and after attaining its full length, which varies considerably, the secondary conidium is initiated as a terminal bud (PI. 26, fig. 3). This elongates and is Trans. Br. mycol, Soc. 59 (I), (1972). Printed in Great Britain

Notes and Brief Articles Table

I.

Percentage germination ofconidia of Cercospora arachidicola in relation to temperature andrelative humidity Mean (range) of three experiments) r.h, \

Temp.

100%

96'5%

88%

10 15 20 25 3° 33 35

° 27'3 (II) 73'3 72·6 5) 70·6 (2) 14.6 (I)

° ° ° 17'3 (I) 16·6 (7) °

° ° ° ° ° °

15)

0

0

0

soon cut off from the conidiophore by a transverse septum (PI. 26, figs.4, 5). This occurs during the second 24 h of incubation; and during the third 24 h period, mature, septate secondary conidia are observed on the conidiophores (PI. 26, fig. 6). A secondary conidium therefore takes about 2 days to develop to maturity. Any cell of the primary conidium can apparently form a secondary conidiophore, which sometimes, as well as producing a secondary conidium, also gives rise to a vegetative hypha. One or two septa can be observed on some of the secondary conidiophores (PI. 26, fig. 6). A primary conidium does not produce more than one secondary conidiophore, which, however, can bear up to two or three conidia (PI. 26, fig. 8). The conidiophore, after producing the first conidium, continues to elongate and subsequently gives rise to another one (PI. 26, figs.!6, 8). A secondary conidium is lighter in colour than a primary one, and there is some variation in its mode of attachment to the conidiophore (PI. 26, figs. 6, 7). About 24 h after maturation some of the secondary conidia give rise to germ-tubes even while still attached. These germ-tubes arise mainly at the tips of the conidia (PI. 26, fig. 9). Mostly these conidial tips become swollen prior to the germ-tube formation. Sometimes, however, these germ-tubes are formed by the intercalary cells of the secondary conidium (PI. 26, fig. 10). No tertiary conidia have been observed. All these observations were made on conidia in saturated air at 20-30°. At lower humidities, secondary conidia were not formed at any temperature. Conidial initiation and maturation took a longer time (48-72 h) at 15° and 100 % r.h, and at no other temperatures were secondary conidia produced. The primary conidia, when incubated on nutrient agar or on fresh groundnut leaves at 100 % r.h. and optimum temperatures, produced only vegetative hyphae. Observations made on the infection of groundnut leaves in the field show that leaves are more heavily infected during the rainy than in the dry season. This can reasonably be correlated with the fact that the air is very moist during the rainy season, thus providing the optimum relative humidity for conidial germination. This is quite unlike the dry season, when Trans. Br. mycol. Soc. 59 (I), (1972). Printedin Great Britain

Transactions British Mycological Society

172

humidity is low and temperature is usually very high . Both these factors operate against infection by airborne conidia since a conidium, alighting on a leaf, germinates and infects it only if the temperature is nearly optimal and humidity is high as in the rainy season. Although much work has been done on Cercospora spp. (Nagel, 1934; Miller, 1949; Kovachich, 1954; Kilpatrick & Johnson, 1956; Jackson, 1960; Holtzmann & Aragaki, 1966; Calpouzos & Stallknecht, 1967), secondary conidial formation has been reported only in C. bougainvilleae, the causal agent of a leaf spot of Bougainvillea (Sobers & Martinez, 1966). However, this species produces its secondary conidium directly on the primary one without the prior formation of a conidiophore. Observations on C. personata and C. nicotianae during this study showed that they germinate by vegetative germ-tubes only. Since C. arachidicola produces these secondary conidia on a slide and not on nutrient agar, it could be inferred that the phenomenon of secondary conidia production is a means of ensuring the further propagation of the species in an environment where nutrients for mycelial growth are lacking. In nature such environments could be the surface of stones from which the secondary conidia could be carried by rain splash on to leaf surfaces. I wish to thank Dr S. O . Alasoadura for useful discussions and to acknowledge Dr A. O. Fajola's assistance in the photographic aspect of the work. REFERENCES

CALPOUZOS, L. & STALLKNECHT, G. F. (1967). Effects oflight on sporulation ofCercospora beticola. Phytopathology 57, 679-681. CHUPP, C. (1953). A monograph ojthefungus genus Cercospora. Ithaca, New York. HOLTZMANN, O. V. & ARAGAKI, M. (1966). Susceptibility of Acerola to Cercospora leaf spot. PhytopatJwlogy 56 , I I 14-1 I 15. JACKSON, C. R. (1960) . Cercospora blight of snapdragon. Phytopathology 50 , 190-192. KILPATRICK, R. A. & JOHNSO~, H . W. (1956). Sporulation of Cercospora spp. on carrot leaf decoction agar. Phytopathology 46, 180-18 I. KOVACHlCH, W. G. (1954). Cercospora elaeidis leaf spot of the oil palm. Transactions of the British Mycological Society 37, 20g-212. MILLER, L. L (1949). Cultural and parasitic races of Cercospora arachidicola and C. personate. Phytopathology 39, 15· NAGEL, C. M. (1934). Conidial production in species of Cercospora. Phytopathology 24, 1101-1110. SOBERS, E. K. & MARTINEZ, S. P. (1966). A leaf spot of Bougainvillea caused by Cercospora bougainvilleae. Phytopathology 56, 128-130. WEXLER, A. & HASEGAWA, S. (1954)' Relative humidity-temperature relationships of some saturated salt solutions in the temperature range I to 50 Journal ojResearch ofthe National Bureau ofStandards 53, 19-26.

ac.

EXPLANATION OF PLATE

dsc

26

developing secondary conidium germ-tube which later develops into vegetative hyphae pa point of attachment of secondary conidium to conidiophore pc primary conidium sc secondary conidium sca scar left by secondary conidium on conidiophore scp secondary conidiophore scpp secondary conidiophore primordium sp septum gt

Trans. Br. mycol. Soc. 59 (I), (1972). Printed in Great Britain

Trans. Br. mycol. Soc.

V ol. 59.

Plate 26

(Facing p. 172)