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Conidium ontogeny in the xerophilic fungus Wallemia sebi
J. stored Prod. Res., 1974,
Vol. IO,209-215. Pergamon Press. Printed in Great Britain.
CONIDIUM ONTOGENY IN THE XEROPHILIC FUNGUS WALLEMIA SZBZ S. T. HILL Pest Infestation
Control Laboratory,
Slough, Berks., England
(First received 7 March 1974, and in @aI form 16 April 1974) Abstract-A simple method of slide culture is described which allowed a sequence of photomicrographs to be taken of WaNemia sebi (Fries) von Arx, showing the development of conidia on an individual fertile branch. A cylindrical structure develops above a constriction in the fertile branch. A cross wall divides the cylinder into an upper and lower cell. The upper cell is not meristematic but a cross wall forms in it, cutting it into two equal parts. Each part divides again so that a row of four cells is formed, the length of each cell being equal to its width. These cells round off to produce a row of four conidia. The lower cell of the cylinder is meristematic. It elongates and then divides into an upper and a lower cell, in the same manner as the cylinder, simultaneously with the maturation of the first four conidia. The new upper cell produces four more conidia. This cycle of conidium production in groups of four is repeated many times so that a long chain of conidia is produced. Explanations of conidium ontogeny in W. sebi by various authors since VUJLLEMIN (1906) are discussedin the light of these observations. It is concluded that W. sebi shows characters of both Section IV (conidia produced on
phialides) and Section VIIA (thallic-arthric conidia) and some unique features.
INTRODUCTION
Wallemia sebi (Fries) von Arx is a xerophilic hyphomycete which has been described under
many synonyms. CIFERRI (1958) lists 27. Commonly used names for the fungus have been Sporendonema sebi Fries, S. epizoum (Corda) Ciferri and Redaelli, Hemispora stellata Vuillemin and Wailemia ichthyophaga Johan-Olsen. It is found on a wide variety of products which are relatively dry or have a high sugar or sah concentration. At this laboratory we have isolated it from hay, lucerne pellets, hazel nuts, wheat germ, salted anchovy paste, salted sliced runner beans and lemon curd. Other authors have reported it on cake (SEILER, 1965), dates (CIFERRIand REDAELLI, 1940), dried salt fish (JOHAN-OLSEN, 1887; FRANK and HESS, J941), dried sea weed (SIEBURTHand JENSEN,1967), paper (WANG, 1965) and hay (GREGORYand LACEY,1963). It has been isolated from external lesions of man (GOUGEROT and CARAVEN,1909) but it is considered to be a primary agent of human disease in only a very few cases (CIFERRIand REDAELLI,1934). The fungus is antagonistic to the flour mite (SOLOMON et al., 1964). On agar media it forms small, light to dark brown colonies of a fine, velvety texture resembling actinomycete colonies. It grows relatively rapidly and sporulates heavily on a medium described by HARROLD(1950), consisting of 2 % malt agar with 0.5 % yeast extract and 400 g sucrose/l water. Since the fungus is xerophilic, it will not be detected in a product by culturing methods except by the use of media of low water activity, such as malt agar + 15% sodium chloride, which slow down or inhibit the growth of most fungi but encourage the growth of xerophils. Even then the fungus is easily overlooked because of the small size of its colonies, compared with those of other xerophilic fungi, such as members of the Aspergillus glaucus group, on these media. Conidia are formed in long chains on simple branches. There has been controversy for 209
210
S. T.
HILL
many years over the exact method of conidium formation and the status of the conidia. Since modern systems of hyphomycete taxonomy are based on the manner in which the spores develop (VIJILLEMIN,1910,1910a, 1911; MASON,1933,1937; HUGHES,1953; TUBAKI, 1958, 1963; SUBRAMANIAN, 1962), observations were made on the early stages of conidium development and a series of photomicrographs were taken showing the development of conidia on a single fertile branch (Fig. 3). These observations were summarised in a short report (HILL, 1965). The present paper presents the photomicrographs and in addition scanning and transmission micrographs taken more recently. MATERIALS AND METHODS Ten isolates were studied (Table 1). The five cultures with IMI numbers were kindly supplied by the Commonwealth Mycological Institute; the others were isolated at this laboratory. When growing the fungus to obtain a preparation suitable for mounting on a slide, a drop of a conidial suspension in Oxoid malt extract broth was put on a sterile coverslip; alternatively a drop of Oxoid malt extract agar on the coverslip was inoculated. The coverslip was inverted on to a glass ring 15 mm in diameter and 10 mm deep and which was resting in a hole in a pad of thick, absorbent paper (Whatman Seed Test paper) lining a petri dish. The paper had been previously flooded with 7.5 or 15 % sodium chloride. After several days of incubation at 25°C there was a thin growth of the fungus over the coverslip and the coverslip was removed, excess medium scraped away and the fungus mounted in lactophenol + 0.2 per cent trypan blue (Fig. 4(B)-(I), (M)). Sometimes a drop of 5 % nigrosin in glycerol was mixed with the lactophenol to show the almost hyaline cell walls in greater contrast with the mountant (Fig. 4(L)). In order to observe stages in development of the fungus as they occurred under the compound microscope, two techniques were used. In the first a drop of 7.5 % sodium chloride solution was pipetted into a glass ring, 15 mm in diameter and 3 mm deep, cemented to a microscope slide, The edge of the ring was smeared with petroleum jelly and a coverslip with an inoculated drop of malt extract agar inverted on it. The slide was incubated at TABLE 1. ISOLATES OF
PIL no.* 104 149 150 152 153 155 156 170 171 173
IMI no.?
31338 44741 76802 74824 76803
Wallemia sebi
STUDIED
Original substrate Wheat germ Hay Hay Jam Dates Mite (Tyrophugus sp.) from haystack Salted fish Cake Salted fish Lemon curd
* Accession number in the Pest Infestation Control Laboratory culture collection. t Accession number in the Commonwealth Mycological Institute culture collection.
FIG. 3. Time-lapse sequence of conidium formation in Wallemia sebi PIL 170 at 2 5 C over 7.5 ~ sodium chloride using the slide culture on net technique. The sequence starts 21½ hr after inoculation and follows on after 1, 2, 3, 3.~-, 4, 421, 5, 5~, 6, 6~, 6~, 7, 7~ and 8½ hr. Scale line represents 10 t~m.
[facin,~ pa,ee 210
FIG. 4. Wallemiasebi.(A),PILlO4germinatingconidium;(B)-(l),PIL156earlystagesinconidiophore development ; (J), PIL 171 showing a long undivided port ion above the constriction ; (K), PIL 170 detached fertile branch; (L), PIL 156 mature fertile branches; (M), PIL 104 showingaseries of constrictions, sympodial branching of the undivided portion above a constriction and reduced conidial chains. Scale lines repre3ents 10/~m. (B)-(M), all at approximately the same magnification.
FIG. 5. Wallem& sebi. (A), (left) PIL b r a n c h in which the undivided portion Scale line represents 5 ~m. (B), (right), undivided section with two conidia
170 scanning electron m i c r o g r a p h of a m a t u r e fertile above the constriction has been converted to conidia. PIL 170 t r a n s m i s s i o n electron m i c r o g r a p h o f part of an from a m o r e m a t u r e section attached. Scale as A.
Conidium Ontogeny in the Xerophilic Fungus Wdemia se&
211
FIG. 1. Technique of slide culture on net used to obtain a sequence of photomicrographs showing conidium formation in Wallemiu sebi. a seed-test paper; b microscope slide; c gap in cement seal.
25°C and growth of the fungus started in less than 24 hr. The slide was removed periodically for examination then returned to the incubator. This technique was satisfactory for examining germination of conidia (Fig. 4(A)), but unsatisfactory for observing the development of fertile branches because they grew perpendicularly to the coverslip surface. Another disadvantage was that satisfactory illumination of the specimen could not be achieved. The second technique avoided these disadvantages. A 12 mm square of nylon net (2 mm mesh) was soaked in malt extract broth, blotted dry, laid on a sterile slide and inoculated. A 19 mm square sterile coverslip was placed on the net and sealed to the slide with beeswaxgum dammar cement (DADE and WALLER, 1949) leaving a small gap at one corner for aeration (Fig. 1). The slide was rested on a bent glass rod in a petri dish with a seed-test paper soaked in 7.5 % sodium chloride and was incubated at 25°C. Spore bearing branches developed parallel to the coverslip surface into the small cells formed by the meshes of the net, the slide and coverslip. Development of individual branches was recorded by carefully removing the slide from the incubator and taking photomicrographs with a x 45 brightfield objective at approximately hourly intervals (Fig. 3 ; Fig. 4(K)). In order to obtain a preparation suitable for examination in the scanning electron microscope, a small ring, formed from a single strand of wire of 190 pm dia taken from electrical flex, was dipped in double strength malt extract broth. After allowing the broth to dry, the ring was stuck to a specimen stub with a cement consisting of high conductivity paint (Acheson Colloids, Plymouth) and cellulose cement (Rawlplug Co. Ltd, London). The ring was inoculated and incubated in a chamber at 90% r.h. and 30°C. When suitable growth of the fungus had occurred it was coated with gold under vacuum, and examined in a Cambridge Stereoscan S2A. Material to be examined under the transmission electron microscope was fixed in 3 per cent glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1, for 2 hr at room temperature followed by 1% osmium tetroxide in the buffer for 2 hr at 4°C. It was dehydrated in a graded series of ethanol-water mixtures and embedded in Spurr’s resin. Sections were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop IA electron microscope at 80 kV. OBSERVATIONS
Before germinating, conidia increase in diameter by two to three times. Sipgle, or two opposed, germ tubes are produced (Fig. 4(A)). Fertile branches originate froti terminal or
212
S. T. HILL
FIG. 2. Early stages in conidium formation in Wallemia sebi. Each dashed line connects the same point on the fungus. a hyphal swelling; b peg: c darkened part of the wall of the constriction; d meristematic zone; e dumb-bell stage. a + b + c + d = conidiophore.
intercalary, deeply-staining swellings of the mycelium. Intercalary swellings may be closely delimited by one or two cross wails (Fig. 4(C),(E)) but usualfy the nearest cross watts are further along the mycelium, as they are when the swelling is terminal. The swelling produces a peg which tapers towards its distal end. The tip of the peg blows out and elongates to produce a cylindrical structure. A cross wall is formed in the cylinder, dividing it into two portions, the upper portion being somewhat longer than the lower (Fig. 2; Fig. 3(A)-(C); Fig. 4 (C)-(I)). Soon after cross wall formation, the outer wall is constricted at this point. The upper portion does not elongate any more from this time (other than a slight elongation due to rounding off during conidium maturation) but a cross wall and constriction form in it, quickly followed by cross walls and constrictions in the sub-sections. At this stage the two pairs of immature conidia look like two dumb-bells placed end to end(Fig. 2e; Fig. 3(D),(H), (I).). Rounding off completes the formation of a line of four conidia derived from one cell. The conidia do not increase in volume as they mature; there is a slight increase in diameter due to rounding off alone. Meanwhile the lower portion of the cylinder has elongated and when it has reached the length of the original undivided cylinder, a cross wall forms in it (Fig. 2; Fig.3 (D)). The upper part again gives rise to four more conidia and the lower part increases in length (Fig. 2 ; Fig. 3 (D)-(I)). This cycle is repeated many times to produce a long chain of conidia. There may be variations from this pattern. Sometimes the undivided portion above the neck becomes very long so that it resembles a hypha(Fig. 4(J)). In fertile branches which have ceased extending, the undivided portion may be converted to conidia (Fig. 5(A)). Under the light microscope, the conidia seem to have two walls: a thin, dark, finelyroughened outer wall and a thick, hyaline to pale yellow inner wall. The young cylindrical structure has a single, thin wall continuous with the wall of the peg and the rest of the mycelium. During active conidial production, the wall of the undivided portion above the neck remains single layered and continuous with the wall of the peg. This was clearly seen in preparations in which the fertile branch was broken from the mycelium just below the neck and the cell contents plasmolysed (Fig. 4(K)). The wall of the upper shoulders of the neck is darker and thicker than elsewhere, creating the impression of a cup or collarette, but there is no break in the wall at this point. Examination of preparations under the scanning electron microscope confirmed outer wall continuity and showed that wall roughening is
Conidium Ontogeny in the Xerophilic Fungus Wallemia sebi
213
coarser just above the neck, reinforcing the impression of a cup (Fig. 5(A)). The wall of the peg is smooth. The meristem giving rise to the extension of the undivided portion seems to be located just above the darkened area. The inner wall develops as the conidia mature. In fertile branches which have ceased growing all walls, including that of the undivided portion, may become double with a thick hyaline inner wall (Fig. 4(L). Transverse septa in fertile hyphae at first consist of a thin, single-layered wall but as the conidia mature, the wall between the segments becomes thick and hyaline with a dark middle-layer along which the conidia separate from each other (Fig. 4(L)). Examination of sections of the fungus under the transmission electron microscope (Fig. 5(B)) showed that the wall of the undivided portion appears to be double with an outer electron dense layer and an inner less dense layer. The outer layer may be an artefact. The outer layers of the conidial walls and the septa between the conidia have the same appearance as the wall of the undivided portion but the conidia have an additional conspicuous, thick, inner layer. There is an abscission layer between conidia. Each conidium has a large lipid droplet. DISCUSSION VUILLEMIN(1906) was the first to consider spore formation in W. se&. He observed the thickened constriction and the cylindrical structure above it, which he called the protoconidium. He introduced the concept of hemispores in which all of the fungus above the lowermost constriction was considered to be a single conidium which may form further constrictions, branches and become partially or totally divided into physiological spores or deuteroconidia. Branching above the first constriction was observed only once in the present work (Fig. 4(M)) and is considered abnormal. Vuillemin did not observe long chains of spores whereas GUBGUEN(19 12) did. GuCguen considered that the conidia were endogenous but they retained part of the protoconidial wall as their outer wall. FONSECAand AREA LBAO (1.927) were the first to note that the distal tip of the protoconidium was the seat of cell division giving rise to the chain of conidia. REDAELLIand CIFERRI(1934) did not observe protoconidia and they found a constriction in the fertile branch in only one strain and then very rarely. However, they consistently found a septum at this point. They accepted GuCguen’s view and referred to this method of spore formation as pseudo-endogenous. More recently several authors (BARRON, 1968 ; ARX, 1970; ELLIS, 1971; KENDRICKand CARMICHAEL,1973) have considered that Wallemia shows a combination of two methods of spore formation: the part of the fertile branch up to the constriction is phialide-like and from the mouth of the collarette of this ‘phialide’ a sporogenous hypha grows out which septates basipetally or simultaneously or randomly into arthroconidia. Barron suggested that the tip of the ‘phialide’ acts as a meristem and the present work confirms this. However, the sporogenous hypha does not, arise within the ‘phialide’ tip and if the ‘phialides’ proliferate (Fig. 4(M)) they do not do so percurrently through collarettes. If the conidiophore is a phialide then it is Madelin’s type C, which produce conidia by transverse septation of the conidiophore tip without any longitudinal splitting of the wall of the phialide tip (MADELIN, 1966). Pirozynski (p. 234 in KENDRICK, 1971) suggested that WalIemia has a phialide in which the delimitation of conidia is delayed. Delayed maturation of conidia produced from phialides is seen in some other fungi such as Aspergillus restrictus Smith and Penicillium digitatum Saccardo. As Madelin pointed out, there is little basic diRerence between the. mechanics of the type C phialide and those of meristem-arthrospores, that is thalllic-meristem S.P.R.---P
214
S. T. HILL
conidia following the recommendations of the First International Workshop-Conference on the Classification of Fungi Imperfecti (KENDRICK, 1971). However in Wullemia there is no increase in volume of the sections cut off from the tip of the conidiophore whereas thallicmeristem conidia swell as they mature. The conidia of Wallemia differ from the arthroconidia produced by other fungi in that the hypha producing the conidia continues to grow from its base and the conidia are formed in groups of four derived from one cell. Although each conidium is totally enclosed by a thick inner secondary wall, the conidia are not endogenous because they are not released from the outer wall that encloses them. It is concluded that W. sebi cannot be placed in any one of the existing sections of the hyphomycetes. Its method of conidium ontogeny seems to be a combination of that found in Section IV (conidia produced from phialides) and VIIA (thallic-arthric conidia) with some unique features. Acknow/edgements-The author is indebted to the Commonwealth Mycological Institute, Kew, which supplied some of the isolates. to Mr. M. LAMBOURNE of this Laboratory for taking the scanning electron micrograph, to Mr. R. CROSSEand Mr. P. MASON of Glaxo Research Ltd, Stoke Poges, for taking the transmission electron micrograph and to Mr. G. AYERSTwho encouraged him in this work.
REFERENCES Aux, J. A. VON (1970) The Genera of Fungi Sporulating in Pure Culture. Cramer, Lehre. BARRON,G. L. (1968) The Genera of Hyphomycetes from Soil. Williams & Wilkins, Baltimore. CIFERRI,R. (1958) Mauginiella a synonim of Sporendonema. Atti Ist. bot. Univ. Lab. crittogam. Pavia Series 5, 15,126-133. CIFERRI, R. and REDAELLI,P. (1934) Sporendonema epizoum (Corda) Cif. et Red.; an entity including Hemispora stellata and Oospora d’Agatae. J. trop. Med. Hyg. 36, 167-170. CIFERRI,R. and REDAELLI,P. (1940) Segnalazione dell0 Sporendonema epizoum (Cda.) Cif. et Red. su frutti di dattero in Libia. Mycopath. Mycoi. appl. 2, 162-163: DADE. H. A. and WALLER.S. (1949) New techniaues for mounting fungi. Mvcol. Putt. 27. l-5. Eurs:M. B. (1971) Dematiaceous Z?yphomycetes. Commonwealth Mycological Institute, Kew. FONSECA,0. da, and AREA LEAo, A.-E. DE, (1927) Contribution a l’btude d’Hemispora stellata. C.r. Seanc. Sot. Biol. 97, 1790-1792. FRANK, M. and HESS, E. (1941) Studies on salt fish. V. Studies on Sporendonema epizoum from dun salt fish J. Fish. Res. Bd Can. 5,276-286. GOUGEROT,H. and CARAVEN,(1909) Mycose nouvelle: 1’Htmisporose. Osteite humaine primitive du tibia due k I’Hemispora stellata. Cr. Seance. Sot. Biol. 26,474-476. GREGORY, P. H. and LACEY,M. E. (1963) Mycological examination of dust from mouldy hay associated with farmer’s lung disease. J. gen. Microbial. 30,75-88. GU~GUEN,F. (1912) Developpment de l’appareil conidien et synonymie de I’Hemispora stellata Vuillemin.. C.r. Seance. Sot. Biol. 64, 32-34. HARROLD,C. E. (1950) Studies in the genus Eremascus-I. The rediscovery of Eremascus albus Eidam and some new observations concerning its life-history and cytology. Annls. Bot. 14,127-148. HILL, S. T. (1965) Spore formation in Sporendonema sebi. Pest Infest. Res. 1%4,17. HUGHES,S. J. (1953) Conidiophores, conidia and classification. Can. J. Bot. 31, 577-659. JOHAN-OLSEN(1887) Om sop pa klipfisk, den sakaldte mid. Forh. Vidensk. S&k. Krist., 12,1-20. KENDRICK,W. B. (Editor) (1971) Taxonomy of Fungi Zmperfecti. University of Toronto Press, Toronto and Buffalo. KENDRICK,W. B. and CARMICHAEL, J. W. (1973) Hyphomycetes in The Fungi, An Advanced Treatise (Edited bv AINSWORTH.G. C.. SPARROW.F. C. and SUSSMAN,A. S.), 4A, 323-509. Academic Press, New York. MA;ELIN, M. F. (1966) The genesis’of spores of higher fungi, in The Fungus Spore (Edited by MADELIN,M. F.), 15-37. Butterworths, London. MASON. E. W. (1933) Annotated account of fungi received at the Imperial Mycological Institute, List II (fasckle 2). Mycol. Pap. 3, l-67. MASON, E. W. (1937) Annotated account of fungi received at the Imperial Mycological Institute. List II (fascicle 3-General Part). Mycol. Pap. 4, 68-99.
Conidium Ontogeny in the Xerophilic Fungus Wallemia sebi
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REDAELLI,P. and CIPERRI,R. (1934) Studio comparative di ventun ceppi di Sporendonema epiroum (Corda Nobis (= Hemispora stellata Vuillemin; = Oospora d’dgatae Saccardo; etc.). Atti ht. bot. Univ. Lab. crittogam. Pavia, Series 4, 5, 145-198. SEILER.D. A. L. (1965) Factors a&tine the mould-free shelf life of cake with uarticular reference to the use of ahtimould agents: British Baking IIndustries Research Association, Rept. ‘No. 18. SIEBURTH,J. McN. and JENSEN,A. (1967) Effect of processing on the microflora of Norwegian seaweed-meal with observations on Sporendonema minutum Heye) Frank & Hess. Appl. Microbial. 15,830-838. SOLOMON,M. E., HILL, S. T., CUNNINGTON,A. M. and AYERST,G. (1964) Storage fungi antagonistic to the flour mite (Acarus siro L.). J. appl. Ecol. 1, 119-125. SUBRAMANIAN, C. V. (1962) The classification of the Hyphomycetes. Bull. bot. Surv. India, 4,249-259. TUBAKI, K. (1958) Studies on Japanese Hyphomycetes. V. Leaf and stem group with a discussion of the classification of Hyphomycetes and their perfect stages. J. Hattori bot. Lab. 20,142-244. TUBAKI, K. (1963) Taxonomic study of Hyphomycetes. A. Rep. Inst. Fermentation, Osaka (1961-1962) 1, 25-54. VunLahrtN, P. (1906) Un nouveau genre de Mr.&din&: Hemispora stellata. Bull. trimest. Sot. mycol Fr. 22, 125-129. VUILLEMM,P. (1910) Materiaux pour une classification rationelle des Fungi Imperfecti. C.r. hebd. Shmc. Acad. Sci., Paris, 150, 882-884. VUILLEMIN,P. (1910a) Les conidiospores. Bull. SPanc. Sot. Sci. Nancy, Series III, 1,129-172. VUILLEMIN,P. (1911) Les aleuriospores. Bull. She. Sot. Sci. Nancy, Series III, 12,151-175. WANG, C. J. K. (1965) Fungi of pulp andpaper. New York State Univ. Coil. Forestry Tech. Publ. No. 87.
Vol. IO,209-215. Pergamon Press. Printed in Great Britain.
CONIDIUM ONTOGENY IN THE XEROPHILIC FUNGUS WALLEMIA SZBZ S. T. HILL Pest Infestation
Control Laboratory,
Slough, Berks., England
(First received 7 March 1974, and in @aI form 16 April 1974) Abstract-A simple method of slide culture is described which allowed a sequence of photomicrographs to be taken of WaNemia sebi (Fries) von Arx, showing the development of conidia on an individual fertile branch. A cylindrical structure develops above a constriction in the fertile branch. A cross wall divides the cylinder into an upper and lower cell. The upper cell is not meristematic but a cross wall forms in it, cutting it into two equal parts. Each part divides again so that a row of four cells is formed, the length of each cell being equal to its width. These cells round off to produce a row of four conidia. The lower cell of the cylinder is meristematic. It elongates and then divides into an upper and a lower cell, in the same manner as the cylinder, simultaneously with the maturation of the first four conidia. The new upper cell produces four more conidia. This cycle of conidium production in groups of four is repeated many times so that a long chain of conidia is produced. Explanations of conidium ontogeny in W. sebi by various authors since VUJLLEMIN (1906) are discussedin the light of these observations. It is concluded that W. sebi shows characters of both Section IV (conidia produced on
phialides) and Section VIIA (thallic-arthric conidia) and some unique features.
INTRODUCTION
Wallemia sebi (Fries) von Arx is a xerophilic hyphomycete which has been described under
many synonyms. CIFERRI (1958) lists 27. Commonly used names for the fungus have been Sporendonema sebi Fries, S. epizoum (Corda) Ciferri and Redaelli, Hemispora stellata Vuillemin and Wailemia ichthyophaga Johan-Olsen. It is found on a wide variety of products which are relatively dry or have a high sugar or sah concentration. At this laboratory we have isolated it from hay, lucerne pellets, hazel nuts, wheat germ, salted anchovy paste, salted sliced runner beans and lemon curd. Other authors have reported it on cake (SEILER, 1965), dates (CIFERRIand REDAELLI, 1940), dried salt fish (JOHAN-OLSEN, 1887; FRANK and HESS, J941), dried sea weed (SIEBURTHand JENSEN,1967), paper (WANG, 1965) and hay (GREGORYand LACEY,1963). It has been isolated from external lesions of man (GOUGEROT and CARAVEN,1909) but it is considered to be a primary agent of human disease in only a very few cases (CIFERRIand REDAELLI,1934). The fungus is antagonistic to the flour mite (SOLOMON et al., 1964). On agar media it forms small, light to dark brown colonies of a fine, velvety texture resembling actinomycete colonies. It grows relatively rapidly and sporulates heavily on a medium described by HARROLD(1950), consisting of 2 % malt agar with 0.5 % yeast extract and 400 g sucrose/l water. Since the fungus is xerophilic, it will not be detected in a product by culturing methods except by the use of media of low water activity, such as malt agar + 15% sodium chloride, which slow down or inhibit the growth of most fungi but encourage the growth of xerophils. Even then the fungus is easily overlooked because of the small size of its colonies, compared with those of other xerophilic fungi, such as members of the Aspergillus glaucus group, on these media. Conidia are formed in long chains on simple branches. There has been controversy for 209
210
S. T.
HILL
many years over the exact method of conidium formation and the status of the conidia. Since modern systems of hyphomycete taxonomy are based on the manner in which the spores develop (VIJILLEMIN,1910,1910a, 1911; MASON,1933,1937; HUGHES,1953; TUBAKI, 1958, 1963; SUBRAMANIAN, 1962), observations were made on the early stages of conidium development and a series of photomicrographs were taken showing the development of conidia on a single fertile branch (Fig. 3). These observations were summarised in a short report (HILL, 1965). The present paper presents the photomicrographs and in addition scanning and transmission micrographs taken more recently. MATERIALS AND METHODS Ten isolates were studied (Table 1). The five cultures with IMI numbers were kindly supplied by the Commonwealth Mycological Institute; the others were isolated at this laboratory. When growing the fungus to obtain a preparation suitable for mounting on a slide, a drop of a conidial suspension in Oxoid malt extract broth was put on a sterile coverslip; alternatively a drop of Oxoid malt extract agar on the coverslip was inoculated. The coverslip was inverted on to a glass ring 15 mm in diameter and 10 mm deep and which was resting in a hole in a pad of thick, absorbent paper (Whatman Seed Test paper) lining a petri dish. The paper had been previously flooded with 7.5 or 15 % sodium chloride. After several days of incubation at 25°C there was a thin growth of the fungus over the coverslip and the coverslip was removed, excess medium scraped away and the fungus mounted in lactophenol + 0.2 per cent trypan blue (Fig. 4(B)-(I), (M)). Sometimes a drop of 5 % nigrosin in glycerol was mixed with the lactophenol to show the almost hyaline cell walls in greater contrast with the mountant (Fig. 4(L)). In order to observe stages in development of the fungus as they occurred under the compound microscope, two techniques were used. In the first a drop of 7.5 % sodium chloride solution was pipetted into a glass ring, 15 mm in diameter and 3 mm deep, cemented to a microscope slide, The edge of the ring was smeared with petroleum jelly and a coverslip with an inoculated drop of malt extract agar inverted on it. The slide was incubated at TABLE 1. ISOLATES OF
PIL no.* 104 149 150 152 153 155 156 170 171 173
IMI no.?
31338 44741 76802 74824 76803
Wallemia sebi
STUDIED
Original substrate Wheat germ Hay Hay Jam Dates Mite (Tyrophugus sp.) from haystack Salted fish Cake Salted fish Lemon curd
* Accession number in the Pest Infestation Control Laboratory culture collection. t Accession number in the Commonwealth Mycological Institute culture collection.
FIG. 3. Time-lapse sequence of conidium formation in Wallemia sebi PIL 170 at 2 5 C over 7.5 ~ sodium chloride using the slide culture on net technique. The sequence starts 21½ hr after inoculation and follows on after 1, 2, 3, 3.~-, 4, 421, 5, 5~, 6, 6~, 6~, 7, 7~ and 8½ hr. Scale line represents 10 t~m.
[facin,~ pa,ee 210
FIG. 4. Wallemiasebi.(A),PILlO4germinatingconidium;(B)-(l),PIL156earlystagesinconidiophore development ; (J), PIL 171 showing a long undivided port ion above the constriction ; (K), PIL 170 detached fertile branch; (L), PIL 156 mature fertile branches; (M), PIL 104 showingaseries of constrictions, sympodial branching of the undivided portion above a constriction and reduced conidial chains. Scale lines repre3ents 10/~m. (B)-(M), all at approximately the same magnification.
FIG. 5. Wallem& sebi. (A), (left) PIL b r a n c h in which the undivided portion Scale line represents 5 ~m. (B), (right), undivided section with two conidia
170 scanning electron m i c r o g r a p h of a m a t u r e fertile above the constriction has been converted to conidia. PIL 170 t r a n s m i s s i o n electron m i c r o g r a p h o f part of an from a m o r e m a t u r e section attached. Scale as A.
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FIG. 1. Technique of slide culture on net used to obtain a sequence of photomicrographs showing conidium formation in Wallemiu sebi. a seed-test paper; b microscope slide; c gap in cement seal.
25°C and growth of the fungus started in less than 24 hr. The slide was removed periodically for examination then returned to the incubator. This technique was satisfactory for examining germination of conidia (Fig. 4(A)), but unsatisfactory for observing the development of fertile branches because they grew perpendicularly to the coverslip surface. Another disadvantage was that satisfactory illumination of the specimen could not be achieved. The second technique avoided these disadvantages. A 12 mm square of nylon net (2 mm mesh) was soaked in malt extract broth, blotted dry, laid on a sterile slide and inoculated. A 19 mm square sterile coverslip was placed on the net and sealed to the slide with beeswaxgum dammar cement (DADE and WALLER, 1949) leaving a small gap at one corner for aeration (Fig. 1). The slide was rested on a bent glass rod in a petri dish with a seed-test paper soaked in 7.5 % sodium chloride and was incubated at 25°C. Spore bearing branches developed parallel to the coverslip surface into the small cells formed by the meshes of the net, the slide and coverslip. Development of individual branches was recorded by carefully removing the slide from the incubator and taking photomicrographs with a x 45 brightfield objective at approximately hourly intervals (Fig. 3 ; Fig. 4(K)). In order to obtain a preparation suitable for examination in the scanning electron microscope, a small ring, formed from a single strand of wire of 190 pm dia taken from electrical flex, was dipped in double strength malt extract broth. After allowing the broth to dry, the ring was stuck to a specimen stub with a cement consisting of high conductivity paint (Acheson Colloids, Plymouth) and cellulose cement (Rawlplug Co. Ltd, London). The ring was inoculated and incubated in a chamber at 90% r.h. and 30°C. When suitable growth of the fungus had occurred it was coated with gold under vacuum, and examined in a Cambridge Stereoscan S2A. Material to be examined under the transmission electron microscope was fixed in 3 per cent glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1, for 2 hr at room temperature followed by 1% osmium tetroxide in the buffer for 2 hr at 4°C. It was dehydrated in a graded series of ethanol-water mixtures and embedded in Spurr’s resin. Sections were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop IA electron microscope at 80 kV. OBSERVATIONS
Before germinating, conidia increase in diameter by two to three times. Sipgle, or two opposed, germ tubes are produced (Fig. 4(A)). Fertile branches originate froti terminal or
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FIG. 2. Early stages in conidium formation in Wallemia sebi. Each dashed line connects the same point on the fungus. a hyphal swelling; b peg: c darkened part of the wall of the constriction; d meristematic zone; e dumb-bell stage. a + b + c + d = conidiophore.
intercalary, deeply-staining swellings of the mycelium. Intercalary swellings may be closely delimited by one or two cross wails (Fig. 4(C),(E)) but usualfy the nearest cross watts are further along the mycelium, as they are when the swelling is terminal. The swelling produces a peg which tapers towards its distal end. The tip of the peg blows out and elongates to produce a cylindrical structure. A cross wall is formed in the cylinder, dividing it into two portions, the upper portion being somewhat longer than the lower (Fig. 2; Fig. 3(A)-(C); Fig. 4 (C)-(I)). Soon after cross wall formation, the outer wall is constricted at this point. The upper portion does not elongate any more from this time (other than a slight elongation due to rounding off during conidium maturation) but a cross wall and constriction form in it, quickly followed by cross walls and constrictions in the sub-sections. At this stage the two pairs of immature conidia look like two dumb-bells placed end to end(Fig. 2e; Fig. 3(D),(H), (I).). Rounding off completes the formation of a line of four conidia derived from one cell. The conidia do not increase in volume as they mature; there is a slight increase in diameter due to rounding off alone. Meanwhile the lower portion of the cylinder has elongated and when it has reached the length of the original undivided cylinder, a cross wall forms in it (Fig. 2; Fig.3 (D)). The upper part again gives rise to four more conidia and the lower part increases in length (Fig. 2 ; Fig. 3 (D)-(I)). This cycle is repeated many times to produce a long chain of conidia. There may be variations from this pattern. Sometimes the undivided portion above the neck becomes very long so that it resembles a hypha(Fig. 4(J)). In fertile branches which have ceased extending, the undivided portion may be converted to conidia (Fig. 5(A)). Under the light microscope, the conidia seem to have two walls: a thin, dark, finelyroughened outer wall and a thick, hyaline to pale yellow inner wall. The young cylindrical structure has a single, thin wall continuous with the wall of the peg and the rest of the mycelium. During active conidial production, the wall of the undivided portion above the neck remains single layered and continuous with the wall of the peg. This was clearly seen in preparations in which the fertile branch was broken from the mycelium just below the neck and the cell contents plasmolysed (Fig. 4(K)). The wall of the upper shoulders of the neck is darker and thicker than elsewhere, creating the impression of a cup or collarette, but there is no break in the wall at this point. Examination of preparations under the scanning electron microscope confirmed outer wall continuity and showed that wall roughening is
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coarser just above the neck, reinforcing the impression of a cup (Fig. 5(A)). The wall of the peg is smooth. The meristem giving rise to the extension of the undivided portion seems to be located just above the darkened area. The inner wall develops as the conidia mature. In fertile branches which have ceased growing all walls, including that of the undivided portion, may become double with a thick hyaline inner wall (Fig. 4(L). Transverse septa in fertile hyphae at first consist of a thin, single-layered wall but as the conidia mature, the wall between the segments becomes thick and hyaline with a dark middle-layer along which the conidia separate from each other (Fig. 4(L)). Examination of sections of the fungus under the transmission electron microscope (Fig. 5(B)) showed that the wall of the undivided portion appears to be double with an outer electron dense layer and an inner less dense layer. The outer layer may be an artefact. The outer layers of the conidial walls and the septa between the conidia have the same appearance as the wall of the undivided portion but the conidia have an additional conspicuous, thick, inner layer. There is an abscission layer between conidia. Each conidium has a large lipid droplet. DISCUSSION VUILLEMIN(1906) was the first to consider spore formation in W. se&. He observed the thickened constriction and the cylindrical structure above it, which he called the protoconidium. He introduced the concept of hemispores in which all of the fungus above the lowermost constriction was considered to be a single conidium which may form further constrictions, branches and become partially or totally divided into physiological spores or deuteroconidia. Branching above the first constriction was observed only once in the present work (Fig. 4(M)) and is considered abnormal. Vuillemin did not observe long chains of spores whereas GUBGUEN(19 12) did. GuCguen considered that the conidia were endogenous but they retained part of the protoconidial wall as their outer wall. FONSECAand AREA LBAO (1.927) were the first to note that the distal tip of the protoconidium was the seat of cell division giving rise to the chain of conidia. REDAELLIand CIFERRI(1934) did not observe protoconidia and they found a constriction in the fertile branch in only one strain and then very rarely. However, they consistently found a septum at this point. They accepted GuCguen’s view and referred to this method of spore formation as pseudo-endogenous. More recently several authors (BARRON, 1968 ; ARX, 1970; ELLIS, 1971; KENDRICKand CARMICHAEL,1973) have considered that Wallemia shows a combination of two methods of spore formation: the part of the fertile branch up to the constriction is phialide-like and from the mouth of the collarette of this ‘phialide’ a sporogenous hypha grows out which septates basipetally or simultaneously or randomly into arthroconidia. Barron suggested that the tip of the ‘phialide’ acts as a meristem and the present work confirms this. However, the sporogenous hypha does not, arise within the ‘phialide’ tip and if the ‘phialides’ proliferate (Fig. 4(M)) they do not do so percurrently through collarettes. If the conidiophore is a phialide then it is Madelin’s type C, which produce conidia by transverse septation of the conidiophore tip without any longitudinal splitting of the wall of the phialide tip (MADELIN, 1966). Pirozynski (p. 234 in KENDRICK, 1971) suggested that WalIemia has a phialide in which the delimitation of conidia is delayed. Delayed maturation of conidia produced from phialides is seen in some other fungi such as Aspergillus restrictus Smith and Penicillium digitatum Saccardo. As Madelin pointed out, there is little basic diRerence between the. mechanics of the type C phialide and those of meristem-arthrospores, that is thalllic-meristem S.P.R.---P
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conidia following the recommendations of the First International Workshop-Conference on the Classification of Fungi Imperfecti (KENDRICK, 1971). However in Wullemia there is no increase in volume of the sections cut off from the tip of the conidiophore whereas thallicmeristem conidia swell as they mature. The conidia of Wallemia differ from the arthroconidia produced by other fungi in that the hypha producing the conidia continues to grow from its base and the conidia are formed in groups of four derived from one cell. Although each conidium is totally enclosed by a thick inner secondary wall, the conidia are not endogenous because they are not released from the outer wall that encloses them. It is concluded that W. sebi cannot be placed in any one of the existing sections of the hyphomycetes. Its method of conidium ontogeny seems to be a combination of that found in Section IV (conidia produced from phialides) and VIIA (thallic-arthric conidia) with some unique features. Acknow/edgements-The author is indebted to the Commonwealth Mycological Institute, Kew, which supplied some of the isolates. to Mr. M. LAMBOURNE of this Laboratory for taking the scanning electron micrograph, to Mr. R. CROSSEand Mr. P. MASON of Glaxo Research Ltd, Stoke Poges, for taking the transmission electron micrograph and to Mr. G. AYERSTwho encouraged him in this work.
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