Notes and Brief Articles 153LUCAS, R. L. (1971). Autoradiographic techniques in mycology. In Methods in Micro­

biology,vol. 4 (ed. by C. Booth), pp. 501-508. New York: Academic Press.McDOUGALL, B. M. (1970). Movement of 14C-photosynthate into the roots of wheat

seedlings and exudation of 14Cfrom intact roots. The New Phytologist 69, 37-46.PELC, S. R. (1956). Stripping-film technique of autoradiography. The International

Journal of Applied Radiation and Isotopes I, I 72-177.REID, C. P. P. & WOODS, F. W. (1967). Atmospheric transfer of carbon 14: A problem

in fungus translocation studies. Science, New York 157, 712-713.STUMPF, W. E. (1969). Too much noise in the autoradiogram? Science, New York 163,

958-959.WAID,]. S., PRESTON, K.]. & HARRIS, P.]. (1971). A method to detect metabolically­

active micro-organisms in soil and litter habitats. Soil Biology and Biochemistry 3,235-241.

CHANGES IN VOLUME OF REPRODUCTIVESTRUCTURES DURING SPORE PRODUCTION BY

ALTERNARIA BRASSICICOLA

R. CAMPBELL

Department of Botany, University of Bristol

During a study of conidium ontogeny in Alternaria brassicicola (Schw.)Wiltshire (Campbell, 1968, 1969) use was made of time-lapse cinemato­graphy to record the development of the acropetal chains of melanizeddictyospores. Casual observation of the film suggested that the rate ofenlargement of the spores was not uniform. The film was therefore analysedto obtain an estimate of the rates of volume changes of spores in the de­veloping chains. Many authors have used time-lapse cinematography forstudying conidium ontogeny, but very few detailed analyses of volumechanges have been done apart from that of Davison (1968) who studiedsporangium production in Peronospora.

A. brassicicola was grown beneath a thin layer of liquid paraffin in slidecultures (Madelin, 1969) on potato dextrose agar (Oxoid CM 139) atapproximately 25°C. It was filmed with a 16 mm cine camera, theshutter being operated at 30 sec intervals by a solenoid linked to anelectronic timing device. Infrared filters prevented overheating of theculture cell by the microscope lamp. The film was analysed by projectingit, frame by frame, on to the reverse side of a piece of paper resting on aglass plate. The outline of the spores and conidiophores was traced on tothe paper at a known magnification. The diameter of the spores wasmeasured at 2 pm intervals along their long axis and the volume calculatedon the assumption that the spores had a circular cross-section.

Sources of error in this method included inaccuracies in tracing thespore outline, deviations from a truly circular cross-section and changesin the plane offocus as a chain developed. Measurements were taken onlyfrom chains viewed with the plane of focus approximately along the longaxis of the spores. Several chains of spores have been analysed in this wayand the measured volume fluctuated slightly in a random manner, pre­sumably owing to these errors. The general trends in volume changes

Trans. Br. mycol. Soc.59 (I), (1972). Printedin GreatBritain

154 Transactions British Mycological Society

Spore I

Conidiophore

400200

Spore 2

LSpore 3

Diagram of theL..-_._--..JL--....l----I_..I-_II'!_..I---l._.l...--L_ ---I spore chain at

600 600 min

'"a;:l 400"0>

'"...00-

200til

0

800

h' 6008-5

Time from start (min)

Fig. I. The volume changes in spores during formation. Spore I, produced from aprimary conidiophore, increases in volume until the rapid decrease as spore 2 isproduced at its apex. Spore 2 follows a similar pattern and both spore 1 and spore 2decrease in volume as spore 3 is produced from spore 2.

could, however, be seen and were consistent in the chains studied. It willtherefore suffice to examine data from one chain to illustrate the volumechanges during spore production.

The conidiophore studied in this particular film sequence producedthree spores in an unbranched chain during the period of observation(Fig. I). The volume changes during the production of the third spore areshown in more detail in Fig. 2.

Each spore was first visible at the tip of the conidiophore or of thepreceding spore as a spherical extrusion which became 1-2 pm in dia­meter in 3-5 min. During the following 5-10 min, when little increase insize took place (Fig. 2, spore 3), the young spore remained more or lessspherical. The apical portion of the spore then extended very rapidly,giving the main volume increase (Fig. I) and changing the shape fromspherical to ovate. The diameter of the spore also increased at this time,though much less rapidly than the length, and the spore reached its finalshape in about ISO min. The spores now had approximately their finalvolume (Fig. I, spore I); any further increase took place very slowly overthe next hours or days as they matured. After the spores formed their firsttransverse septum they produced a new spore at their apex. As a new sporewent through its phase of rapid enlargement the volume of the motherspore decreased (Fig. I, volume of spore I decreases as that of spore 2

increases). The volume of existing spores decreased again when the thirdspore was produced at the tip of the second (Figs. I, 2). The decrease involume started in the lowest spore of the chain and travelled acropetally.Thus spore I decreased in volume just before spore 2 as spore 3 wasproduced (Fig. 2). This conforms with the view that apical extension iscaused by pressure from behind the apex; if the new wall synthesis at thetip was increasing the chain volume and 'sucking' cytoplasm up the chain

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

Notes and Brief Articles 155800 [

600~ Spore I

l~f&2:L~

500 550 600

Time from start (min)

Fig. 2. Detail of the production of spore 3 from spore 2. The volume of both pre-existingspores (I and 2) decreases just before the new spore (3) is produced at the apex of 2.The oscillation after the first rapid flow is noticeable.

the shrinkage of the existing spores would have started from the top of thechain.

Similar volume changes occurred, in other chains, during the produc­tion of conidiophore branches, or secondary conidiophores from a spore,which led to the formation of branched chains.

During spore production the whole chain was therefore pulsating; thespores swelled, then shrank as each new spore was produced at the chaintip. As well as these major changes there were minor fluctuations in volume(Fig. 1, after production of spore 2; Fig. 2, after production of spore 3).The fluctuations may represent the surges in cytoplasm that can beobserved by means of phase-contrast microscopy in the conidiophores andgrowing chains. Though an increase in mass obviously took place in thechain, there were also volume fluctuations which were not true growthbut rather a redistribution of existing material.

Spore enlargement had several phases: of interest from the point of viewof conidium ontogeny was the pause at the spherical stage followed by therapid apical extension of the spore initial. This discontinuity between theinitial and the subsequent extension suggests that separate processes wereinvolved. The first could have been a stretching of a pre-existing wall,while the pause was the period during which the vesicular system for apicalextension (Campbell, 1968) was being organized within the spore initial.

This sort of analysis produces information from time-lapse films whichmay not be obvious in ordinary viewing. It affords a useful introduction tothe spore production in a species, emphasizing the dynamic aspect whichis often lost sight of in subsequent detailed electron microscope examina­tions. It is one of many means which might be used to elucidate conidiumontogeny, the basis of the modern speculations in Deuteromycete taxo­nomy.

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

Transactions British Mycological Society

REFERENCES

CAMPBELL, R. (1968). An electron microscope study of spore structure and developmentin Alternaria brassicicola. Journal of General Microbiology 54, 381-392.

CAMPBELL, R. (1969). Further electron microscope studies of the conidium of Alternariabrassicicola. Archivfur Mikrobiologie fig, 60-68.

DAVISON, ELAINE M. (1968). Development of sporangiophores of Peronospora parasitica(Pers. ex Fr.) Fr. Annals of Botany 32, 623-631.

MADELIN, M. F. (1969). Conidium production by higher fungi within thin layers ofliquid paraffin: a slide culture technique. Journal ofGeneral Microbiology 55,319-324.

INDUCTION OF PHYTOPHTHORA CINNAMOMI OOSPORESIN SOIL BY TRICHODERMA VIR/DE

ROSEMARY J. REEVES* AND R. M. JACKSON

Department ofBiological Sciences, University ofSurrey,Guildford, Surrey

Phytophthora cinnamomi Rands is a heterothallic species that does notnormally produce oospores in cultures of one compatibility type. In recentyears, several studies have been made on the behaviour of this species insoiL These studies have shown that P. cinnamomi is capable of survivingfor long periods (up to 6 years) in soil without a host (Zentmeyer &Mircetich, 1966), but survival of this fungus in soil probably depends uponthe production of resistant spores during or immediately following periodsof active parasitism.

In 1966 Mircetich & Zentmeyer reported the production of oosporesby P. cinnamomi following burial in soil of fibreglass pieces covered withmycelium and chlamydospores. They also observed oospores in naturallyinfected host tissue. The production of oospores in soil by some other speciesof Phytophthora has also been observed. (Legge, 1953; VujiCic & Park, 1964).However, little is known of the factors influencing the development ofoospores of any species of Phytophthora in soil. This paper reports observa­tions on the production of oospores by P. cinnamomi which were madeduring a wider study of the influence of various factors on the behaviourof this fungus in soil.

Spore-free mycelial inocula of an Az isolate of P. cinnamomi, grown for36 h on nylon mesh disks in sterile carrot juice, were thoroughly washedin ten changes of sterile distilled water and blotted dry between glass filter­paper. Two mm slices of Castanea sativa radicle or fleshy feeder root werethen added to some of the disks to provide a nutrient source, as the isolateused was obtained from diseased C. sativa. Other disks had no addednutrient source. The disks were buried in five different soils, the moisturecontents of which had been adjusted to give equal suction pressures

... Part of this work was done during the tenure of a NATO studentship at the SwissFederal Research Station, Wadenswil, and part was supported by a NERC studentshipat the University of Surrey.

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