Pycnidium production by Aureobasidium pullulans type-cultures

Pycnidium production by Aureobasidium pullulans type-cultures


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Notes and brief articles



J. M.


Scottish Horticultural Research Institute, Invergowrie, Dundee DDz 5DA

While re-examining the statistics given by Duncan (1976), it was found that they had been processed wrongly. The author had assumed, incorrectly, that the Rothamsted Maximum Likelihood computer programme allowed data from samples ofundiluted field soil to be processed in the same manner as those from diluted samples. In consequence, all MPNs and associated 95 % Confidence Limits were too high. In Tables 1,3 and 4 the MPNs should be one-fifth (the dilution factor) and in Table 2 onequarter of the given values. The number of plants infected at a given dilution and the associated chisquare test data remain unaltered. Fortunately, the error does not affect all the conclusions. Thus comparisons made in Table 1 are still valid because all the figures in that table are altered proportionately. In contrast, the numbers of oospores or zoospores calculated to give detectable infection (Tables 3, 4) must be increased by a

factor of five and the test therefore is not as sensitive as claimed, an infective unit being equivalent to 27 not 5'4 zoospores. Data from undiluted soil samples can be included in the computation using the Rothamsted ML programme providing the volume of material used in each pot is multiplied by the reciprocal of the dilution factor and the resultant larger figure then entered as the volume of soil used in each case. The undiluted sample should then be treated as the first level of dilution in a dilution series. The author accepts full responsibility for the error and apologizes for any inconvenience caused. REFERENCE


J. M. (1976). The use of bait plants to detect

Phytophthora fragariae in soil. Transactions of the British Mycological Society 66, 85-89.


Aberdeen University, Forestry Department, Aberdeen AB9 2UU, Scotland Aureobasidium pullulans (De Bary) Arnaud has

always been considered as a fungus which does not produce pycnidia except where it has been confused with Dothichiza (Sclerophoma) pityophila (Corda) Petro (Haddow, 1941; Robak, 1952; Batko, Murray & Peace, 1958). As such it has been placed in the Moniliales rather than the Sphaeropsidales. In 1966, Hudson described Guignardia jagi Hudson which produced A. pullulans-type cultures from single ascospores. Several other ascomycetes have been claimed to produce this type of conidial state but these have not been checked by single ascospore isolations. On fallen leaves of broad-leaved trees bearing ascocarps of G. fagi, Hudson also found microsclerotia which produced A. pullulans-type conidia

* Presentaddress: Forest ResearchInstitute, Terma Alkmanos, Athens 615, Greece. Trans. Br, mycol, Soc. 68 (1), (1977).

after a period in a damp chamber. Pugh & Buckley (1971) recorded ' furnagoid structures' on the surface of leaves of Acer pseudoplatanus L. which were clustered to give the appearance of microsclerotia. We have seen similar microsclerotia on the surface of pine needles sprayed previously with spores of A. pullulans (Xenopoulos, 1974). During a study ofthe relation of D. pityophila to A. pullulans on Scots pine (Pinus sylvestris L.) the possible production of pycnidia by these two fungi was examined with the results recorded here. The method of culture was similar to that desscribed by Batko, Murray & Peace (1958). Healthy Scots pine needles were first washed in running tap water for 15-20 min and then sterilized by autoclaving for 15 min at 1 bar gauge pressure. The autoclaved needles were laid in plastic or glass dishes on sterilized moist cotton wool and inoculated either with mycelium or with a suspension

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Notes and brief articles


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1. Imperfectly formed pycnidium of A. pullulans on an autoclaved needle of Pinus syluestris, Inoculum of A. pullulans obtained from a needle of P. syluestris,

Fig. 2. Pycnidium of A. pullulans immersed in an autoclaved needle of P. sylvestris. Note the stomatal guard cells at the base of the pycnidium. Inoculum of A. pullulans obtained from leaf of Acer pseudoplatanus, Fig. 3. Pycnidium of A. pullulans immersed in an autoc1aved needle of P. syluestris, Note the stomatal guard cells at the top of the pycnidium. Inoculum of A. pullulans obtained from single ascopore of Guignardia fagi. Fig. 4. Pycnidium of D. pityophila immersed in an autoc1avedneedle of P. syluestris. Inoculum of D. pityophila obtained from needle of P. sylvestris. Scale = 10 microns. Transverse sections of pine needles e = epidermis, g = guard cells, h = hypodermis, m = mesophyll.

Trans Br. mycol. Soc. 68 (1), (1977).

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Notes and brief articles Table 1, Source of isolates of Aureobasidium pullulans Host Acer platanoides L. Garrya elliptica Dougl. Fagus syluatica L. Fraxinus ornus L. Ilex aquifolium L. Pinus sylvestris L. Quercus coccinea Muenchh, Rhododendron viscosum Torr. Skimmia [aponica Thunb. Sorbus aria (L.) Crantz Ulmus glabra Huds. Verbascum nigrum L.

Plant part Leaf litter Petiole Leaf litter Bud scale Fruit Needle Bark Bud sclae Male flower Bud scale Bud scale Leaf

of blastospores from 2 % malt agar cultures of A. pullulans or D. pityophila. The inoculated needles were incubated at room temperature (22-24°) in natural light. Many isolates of A. pullulans, derived from surface-sterilized material from a variety of hosts (Table 1), were tested for pycnidium production. In addition, four isolates of the A. pullulans state of G. fagi on poplar each derived from a single ascospore, and sent to us by Dr H. J. Hudson, were tested. Two strains of D. pityophila (Xenopoulos, 1974) which were known to produce pycnidia under these conditions were included for comparison. All the attempts to produce pycnidia on autoclaved pine needles from the isolates of A. pullulans including those derived from G .fagi were successful after about two weeks incubation. Re-isolation from the pycnidiospores confirmed that the pycnidia belonged to A. pullulans, Most of the pycnidia were superficial on the needle (Fig. 1) but few were immersed totally (Figs. 2, 3). In both cases the pycnidia were usually imperfectly formed. Uninoculated autoclaved needles remained sterile. In the early stages of development the structures were sclerotial-like with a wall of pseudoparenchymatous tissue of dark-walled cells. With the immersed pycnidia, which usually formed below stomata, in some cases the stomatal guard cells were below the pycnidium (Fig. 2) whilst in other cases they remained in their original position (Fig. 3). The pycnida produced pycnidiospores endogenously without forming conidiophores and the spores were embedded in mucilage, When the pycnidia were kept for a few days in very humid conditions at room temperature they usually developed aerial hyphae. The pycnidia measured 60-200 pm and the pycnidiospores 5-16 x 3'5-5.6 pm (mean 8'3 x 4'3 pm) and corresponded morphologically in all respects to Trans. Br. mycol. Soc. 68 (1), (1977). 5


pycnidia and spores of D. pityophila produced under the same conditions (Fig. 4). The two fungi were tested also for production of pycnidia on lacquered pine boards since Butin (1963) had used this method to distinguish them. In Burin's tests, D. pityophila produced pycnidia after 6-8 weeks whereas A. pullulans did not. In our tests D. pityophila produced pycnidia after only 3 weeks at room temperature in sealed humid chamber but after 8 weeks A. pullulans had produced only pycnidial initials under a mass of spores in mucilage. This is the first published account of pycnidium production by A. pullulans, although, at our suggestion, Lehmann (1974) produced pycnidia on autoclaved pine needles from isolates of A. pullulans from pine. Of particular interest is the fact that cultures of A. pullulans derived from single ascospores of G. jagi also produced pycnidia on pine needles. It is noteworthy that D. pityophila, which is found frequently on conifer needles, has never been isolated from angiosperm leaves (Hudson, 1966) and this agrees with OUI own observations. A. pullulans-type cultures are obtained most readily from angiosperm material but have been isolated from gymnosperms. On the whole the two fungi occupy the same ecological niche but on widely different hosts. We have often seen microsclerotia or imperfectly formed natural pycnidia on brown pine needles, similar to those of A. pullulans described by Hudson (1966) and Pugh & Buckley (1971) on broad leaves. So far these have always proved, on examination, to be D. pityophila but it is reasonable to assume that a pycnidial state of A. pullulans might exist in nature. There is no doubt that at least two genetically different species are involved, as genetic connections have been demonstrated between A. pullulans and G. fagi (Hudson, 1966) and D. pityophila and Sydotoia polyspora (Bref. et Tavel) Muller (Butin, 1964). Although genetically different they show many morphological similarities and our demonstration of"the production of Dothichiza-type pycnidia by A. pullulans adds further to these. We wish to thank Professor H. Butin who examined cultures for us and Dr H. J. Hudson who supplied the cultures of Guignardia jagi, read the manuscript and made helpful suggestions. Cultures have been deposited at IMI as follows: A. pullulans (1M I 189459), G. jagi (IMI 189460), D. pityophila (IMI 189457 and IMI 189458).

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Notes and brief articles REFERENCES

BATKO, S., MURRAY, J. S. & PEACE, T. R. (1958). Sclerophoma pityophila associated with needle-cast and its connection with Pullularia pullulans, Transactions of the British Mycological Society 41,

126-128. BUTIN, H. (1963). Uber Sclerophoma pityophila (Corda) v. Hohn. als Blaupilz an verarbeitetemHolz. Phytopathologische Zeitschrift 48, 298-305. BUTIN, H. (1964). Uber zwei Nebenfruchtformen von Sydotoia polyspora (Bref, et v, Tav.) Muller, Sydouna 17,114-118. HADDOW, W. R. (1941). Needle blight and late fall browning of Red pine (Pinus resinosa Ait.) caused by gall midge (Cecidomyiidae) and the fungus Pullularia pullulans Berkhaut, Transactions of the Royal Canadian Institute 23, 161-189.

HUDSON, H. J. (1966). An ascomycete with Aureobasidium pullulans-type conidia. Nova Hedwigia 10, 319-328. LEHMANN, P. (1974). The biology of fungi decomposing pine leaf litter. Ph,D. Thesis, University of Cambridge. PuGH, G. F. J. & BUCKLEY, N. G. (1971). The leaf surface as a substrate for colonization by fungi. In Ecology of leaf-surface micro-organisms (ed. T. F. Preece and C. H. Dickinson),pp. 432-445. London: Academic Press. ROBAK, H. (1952). Dothichiza pithyophila (Cda) Petr., the pycnidial stage of a mycelium of the type Pullularia pullulans (de B.) Berkhout, Sydowia 6, 361-362. XENOPOULOS, S. (1974). Relationship and pathogenicity of Sclerophoma pityophila and Aureobasidium pullulans on Scots pine. Ph.D. Thesis, University of Aberdeen.



Institut fiir Spezielle Botanik der Unioersitdt, 6500 Mains, Germany Cladosporium herbarum is a common member of the Fungi Imperfecti and has received much attention because it can cause serious spoilage of many foodstuffs. However, there is little information on the cytology of this species and especially on nuclear behaviour in hyphae and conidia. De Vries (1967) shows drawings of nuclei in the mycelium of several species of Cladosporium but details are lacking. In his study on the nuclei in vegetative hyphaeof several phytopathogenic fungi, Hoffmann (1968) observed that the average number of nuclei per hyphal cell of C. fulvum was 1'2. There are, however, investigations on genera related to Cladosporium, e.g, Stemphylium (van Warmelo, 1971 a, b) and Alternaria (Stall, 1958; Hartmann, 1964, 1966; Brushaber, Wilson & Aist, 1967). A comparison of these results with the nuclear behaviour in C. herbarum was therefore of interest. Cladosporium herbarum (strain F4) was cultured in Petri dishes on malt extract agar (Difco). Test preparations for the cytological study were made by growing the fungus on cover slips in malt extract solution (Oxoid) in moist chambers. After an incubation period of 24 h the preparations were fixed and stained. The preparations were fixed for 30 min in freshly prepared ethanol-glacial acetic acid (3: 1, vjv), rinsed in water and hydrolysed in 1 N-HCI at 60°C for 8 min. The nuclei were stained for 60 min Trans. Br, mycol. Soc. 68 (1), (1977).

with HCl-Giemsa, prepared according to Hrushovetz (1956): two drops of Giemsa stain (Chroma, Stuttgart, Germany) were added to 1 ml phosphate buffer (pH 7'0). After careful rinsing the stained preparations were mounted in Euparal (Chroma). Most of the hyphal cells contain only one large nucleus of oval to ellipsoid shape. In interphase the darkly stained chromatin surrounds the nucleolus which appears as a lighter centre (Fig. 1). Dikaryotic cells were very rare. A network of strands appears in the prophase nucleus which still seems to be surrounded by an intact membrane (Fig. 2). Discrete chromosomes can be seen during metaphase because of their greater contraction (Fig. 3). Five chromosomes were tentatively counted. In anaphase the groups of chromosomes separate and move away from one another (Figs. 4, 5). A spindle is probably involved in the separation of the daughter chromosomes (Fig. 5). A new cross wall is subsequently formed (Fig. 5) and may even penetrate the spindle elements (Fig. 6). Branching begins with the formation of hyphal proliferations into which one of the daughter nuclei migrates (Figs. 7, 8). Anastomoses often occur and their development is similar (Fig. 9). A nucleus from one of the fused hyphae migrates into the hyphal bridge (Fig. 10). During conidiation short side-hyphae develop buds at their tips into which the nucleus migrates

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