Mycol. Res. 103 (7) : 865–872 (1999) 865 Printed in the United Kingdom Observations on the biology and ultrastructure of the asci and ascospores of Julella avicenniae from Malaysia D. W. T. A U, E. B. G. J O N E S A N D L. L. P. V R I J M O ED* Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Ave., Kowloon, Hong Kong Ultrastructure of the marine, bitunicate, ascomycete Julella avicenniae is presented and compared with the marine Pleospora gaudefroyi. Asci of J. avicenniae possess an ocular chamber, a thick endoascus, and a thinner ectoascus. Pseudoparaphyses are enveloped by mucilage (hyphal sheath) which stains with ruthenium red. The mucilage appears to be an extension of the pseudoparaphysis cell wall and internally these cells contain an array of vesicles. Muriform ascospores are surrounded by an exosporial sheath, an electrondense episporium and a bilamellate mesosporium. Optimum conditions for growth are 25–30 °C in 100 % artificial seawater glucoseyeast extract-tryptone media, but the fungus also is able to grow at 35° and at higher salinities. The ability of the fungus to withstand extremes of environmental conditions is discussed. Julella avicenniae (Borse) K. D. Hyde (Thelenellaceae, Ascomycota, Incertae sedis) is common on twigs of Avicennia spp. collected at Morib mangrove, Peninsula Malaysia. It was described first from Avicennia alba Blume collected in India by Borse (1987) and referred to Pleospora. Subsequently, Hyde (1992) transferred the species to Julella because it develops on woody substrata, ascomata are immersed beneath a clypeus and the peridial wall is composed of a single cell type with pseudoparaphyses that are narrow, anastomosing and brightly refractive in a gelatinous matrix. Previous collections have been on submerged roots (Borse, 1987) and driftwood (Hyde, 1992). Neither author referred to the occurrence of this fungus on living trees. At Morib mangrove, Malaysia, the fungus colonizes the tips of twigs}branches and may be involved in their die back. How the fungus enters the host has not been determined, but the Avicennia trees are growing actively, do not appear to be stressed and are free from mechanical damage (e.g. insect attack). Approximately 10–15 % of the Avicennia alba trees are affected (Jones, unpublished observations). The aims of this investigation of J. avicenniae were to examine the ultrastructure of asci and ascospores, and to study the effects of varying temperature and salinity levels on the growth and germination of ascospores. MATERIALS AND METHODS Material of Julella avicenniae on twigs of Avicennia trees was collected from Morib mangrove, Malaysia, 12 April 1995, Ascospores for germination studies were obtained from fresh J. avicenniae ascomata on these twigs. Isolation methods as * Corresponding author. described by Jones & Hyde (1988) were used to obtain a single spore isolate (CY109) which was used for the growth studies. Transmission electron microscopy (TEM) Fresh material was embedded in 2 % Ion agar No. 2 (Oxoid) and fixed in 2 % aqueous potassium permanganate for 15 min. To stain for polysaccharides, material was fixed in 2 % (w}v) paraformaldehyde mixed with 2±5 % (v}v) glutaraldehyde in 0±1  cacodylate buffer (pH 7±2) for 3 h and postfixed in 1 : 1 ratio of 2 % osmium tetroxide and 0±07 % ruthenium red in buffer for 3 h. Fixed material was dehydrated through a graded ethanol series, transferred to propylene oxide and embedded in Spurr’s resin. Ultrathin sections were stained in lead citrate (Reynolds 1963) followed by uranyl acetate and examined with a Philips CM20 transmission electron microscope at 80 kV. The Masson Fontana silver staining method modified by Mckeown (1994) was employed to detect melanin in the ascospore wall. Fresh ascospores were settled onto balsa wood for 2 h before silver staining, dehydration and resin embedment as described above for TEM. Vegetative growth Salinity experiments were carried out with artificial seawater (ASW) prepared according to Lyman & Fleming (1940) buffered with 0±1  Tris at pH 7±5. Different salinity levels (20, 40, 60, 80, 125, 150, 175, 200 %) were obtained either by dilution of 100 % ASW with Tris buffer or by increasing the concentrations of various salts of ASW accordingly. A medium lacking ASW salts also was included. The salinity of Biology and ultrastructure of Julella avicenniae 866 Figs 1–4. Julella avicenniae. TEM. Aldehydes, osmium tetroxide and ruthenium red fixation. Fig. 1. Longitudinal median section of ascus apex. The ascus is apically rounded (arrowed), the ocular chamber (arrowed OC) is formed by the subapical thickening of the endoascus (En). Bar ¯ 4 µm. Fig. 2. Transverse section of an ascus. The electron density of the ascus wall layers increases from the endoascus (arrowed En) to ectoascus (arrowed Ec). Lipid bodies, vacuoles and vesicles are present in the epiplasm (E). Bar ¯ 4 µm. Fig. 3. Longitudinal section of pseudoparaphyses. The wall (CW) is multilayered with electron density increasing from the inner layer to the outer layer, and surrounded by the hyphal sheath which is fibrillar and electron-dense. Mucilage (*) arises as fine fibrils from the cell wall (arrows). Numerous vesicles (Ve) and vacuoles (Va) are associated with or in close vicinity to the highly convoluted plasma membrane (arrowed pm). My ¯ Myelin figures. Bar ¯ 1 µm. Fig. 4. Ruptured ascus. Extension of the endoascus (arrowed En) rupturing the ectoascus (arrowed Ec). Bar ¯ 5 µm. 100 % ASW was at 30 parts per thousand. The basic medium consisted of 10 g glucose, 1 g yeast extract, 2 g tryptone, with or without 18 g agar in 11 of buffered ASW (GYT}ASW). Agar plates (in triplicate) were inoculated with agar discs (5 mm diam) obtained from the edge of an active growing colony of J. avicenniae on GYT}ASW. Agar plates then were sealed with parafilm and incubated at 15, 20, 25, 30, 35 and 40° for 25–35 d. Colony diameter was measured at 2–3 d intervals. Liquid cultures (in triplicate) were inoculated with five agar discs from an actively growing culture and incubated at 25° for 4 wk without shaking. Growth was monitored by measuring the dry weight biomass at weekly intervals. Mycelium was filtered through a pre-dried and pre-weighed filter paper followed by drying and weighing under similar conditions (dried at 80° for 48 h and cooled overnight in a desiccator). Initial dry weight biomass of the inoculum was D. W. T. Au, E. B. G. Jones and L. L. P. Vrijmoed estimated by melting four sets of five agar discs in Tris buffer in a boiling water bath, filtering, drying and weighing as described. 867 Potassium permanganate fixed material shows no lamellation of the endoascus (Fig. 8). At maturity, the endoascus extends and ruptures the ectoascus (Figs 4, 6). The discharge of ascospores follows (Fig. 9). Germination of ascospores Ascospores were prepared in a similar method as for the single spore isolation procedure described above. The agar disc method, adapted from Byrne & Jones (1975) was used. Agar discs (5 mm diam.) cut rom 100 % ASW agar plates were transferred to a Petri dish lined with moistened filter paper. An ascospore suspension in 100 % ASW (in adjusted spore concentration to approximate 50–60 spores per drop) was dropped onto each disc and the plates sealed with parafilm and incubated at 25°. Spore germination was determined at regular intervals by transferring four discs onto a glass slide and fixing the spores with lactophenol (with or without cotton blue). Forty spores were examined under a microscope and percent germination, number of germ tubes produced and their corresponding linear extension rates were determined. RESULTS Ultrastructure of asci Asci of Julella avicenniae are fissitunicate, 130–200¬26–31 µm long, clavate, with a thick endoascus and a thinner ectoascus (Fig. 2). At the apex the ectoascus is thin-walled, while the endoascus is thickened subapically with an ocular chamber (Fig. 1). The thick ascus wall appears to be multilayered when fixed by aldehydes, osmium tetroxide and ruthenium red (Figs 2, 5–7). Pseudoparaphyses (1–2 µm) are surrounded by mucilage (hyphal sheath) which stains with ruthenium red (Fig. 3). The mucilage appears to be an extension of the cell wall while internally pseudoparaphyses contained an array of vesicles (Fig. 3). The ectoascus is 500–600 nm thick comprising two (or more) layers (Fig. 5), while the endoascus is 1500–1700 nm thick comprising six (or more) layers consisting of alternating layers of different electron-density (Figs 6, 7). Ultrastructure of ascospores Mature ascospores of J. avicenniae are yellow to pale brown, ellipsoidal, 26–36¬12–16 µm with up to three longitudinal septa ; six to seven transverse septa, and constricted at the central septum (Figs 6, 9, 10, 12). Ascospores are surrounded by a prominent exosporial sheath, 60–100 nm thick, comprising whorls of mucilaginous material of different electrondensity (Figs 5–7). When released from the ascus, the mucilaginous sheath becomes more diffuse (Figs 9, 10). The spore wall consists of an outer mucilaginous exosporium, a central electron-dense episporium (30–50 nm thick) and a bilamellate mesosporium (400–500 nm) (Figs 1, 6, 8, 10–12). The inner mesosporial layer is electron-transparent, while the outer mesosporial layer is electron-dense, with electron-dense inclusions, and forms the septum between individual cells (Figs 1, 7, 11, 12). Silver nitrate solution staining of ascospores results in the formation of silver which appear as electron-dense particles over the electron-dense region of the mesosporium (Figs 13, 14) confirming the presence of melanin in the cell wall. Multivesicular bodies in the cytoplasm also stain with silver particles (Fig. 14). Effect of varying temperature and salinity levels on vegetative growth Fig. 15 shows the linear extension of J. avicenniae on GYT agar medium of various salinities for an incubation period of 30 d at 25°. The linear extension rate for each salinity level then was computed along the linear phase of the growth curve and plotted against temperature (Fig. 16). One way ANOVA of results for each test temperature indicates salinity had a significant effect on the linear extension rate at the various Fig. 5. High magnification TEM of Julella avicenniae ascus and ascospore walls. The ectoascus consists of two layers (arrowed Ec and " Ec ) and the endoascus consists of three layers (arrowed En , En , En ). The exosporial sheath (arrowed Ex) of the ascospore is well # " # $ developed comprising whorls of mucilaginous material of different electron-density. Bounding the exosporium is the delimiting membrane (arrowed D) and this is continued by observation of the whole ascospore. Strands of electro-dense material (arrowed) are associated with the episporium. Bar ¯ 1 µm. Biology and ultrastructure of Julella avicenniae 868 Figs 6–9. Julella avicenniae. TEM. Figs 6, 7. Aldehydes, osmium tetroxide and ruthenium red fixation. Figs 8, 9. KMnO fixation. Fig. % 6. Mature ascus. The ascus is clavate with a narrow stalk. Ectoascus (arrowed Ec) ruptured near the base of the ascus, the endoascus (arrowed En) is multilayered. Mature ellipsoidal ascospores (arrowed Asp) have both longitudinal and transverse septa, enclosed by the mucilaginous exosporial sheath (arrowed Ex). Bar ¯ 10 µm. Fig. 7. Higher magnification of the ascus wall in Fig. 6. Remnant of the ectoascus (arrowed) remains on the endoascus (arrowed En) which comprises five lamellae of differing electron-density. The mucilaginous material within the exosporial sheath (Ex), is heterogeneous in texture with an electron-dense fibrillar network embedded in a less electron-dense matrix and bounded by the delimiting membrane (arrowed D). E ¯ epiplasm. Bar ¯ 2 µm. Fig. 8. Higher magnification of the ascus wall. The endoascus (arrowed En) is a single layer without lamellation. The mature ascospore (Asp) is delimited from the ascus wall by a discontinuous delimiting membrane (arrowed D) or remnants of the ascus plasma membrane. Mucilaginous exosporium ¯ Ex. Bar ¯ 2 µm. Fig. 9. Dehiscent ascus, endoascus (arrowed En) with released ascospores (Asp) enrobed in a mucilaginous sheath (boundary arrowed). Bar ¯ 1 µm. D. W. T. Au, E. B. G. Jones and L. L. P. Vrijmoed 869 Figs 10–14. Julella avicenniae. TEM. Figs 10–11. KMnO fixation. Figs 12–14. Silver staining. Fig. 10. Released ascospore % surrounded by a mucilaginous sheath (boundary arrowed), has two longitudinal septa, six transverse septa and constricted at the central septum. Bar ¯ 5 µm. Fig. 11. Higher magnification of ascospore wall. The episporium (arrowed Ep) is an electron-dense layer. The mesosporium is multilayered : inner mesosporium (arrowed Me ) and outer mesosporium, electron-dense (Me ) which divides the spore # " into individual cells. Bar ¯ 1 µm. Fig. 12. Released ascospore. The silver particles are electron-opaque and distributed evenly on the multilayered mesosporium (arrowed Me). Bar ¯ 5 µm. Fig. 13. Ascospore wall with electron-opaque silver particles which concentrate on the electron-dense region of the mesosporium (arrowed Me ). Bar ¯ 0±5 µm. Fig. 14. Ascospore wall. Electron-opaque silver particles " are present in both the outer mesosporium (arrowed Me ) and in the multivesicular bodies (arrowed MVB) in the cytoplasm. The " electron-dense particles on the cell wall are artifacts of lead staining. L ¯ lipid bodies. Bar ¯ 0±5 µm. temperatures (P ! 0±05). The combined effect of temperature and salinity on vegetative growth was analysed by two way ANOVA which revealed significant interaction between the two parameters (P ! 0±01) with temperature being the more important factor. With the exception of the 15° and 35° data, the linear extension rates increased with increasing salinity levels. Maximum growth was at 20 to 100 % ASW at 25° but at 60 to 100 % ASW at 30°, reaching a linear extension rate 0±75–0±95 mm d−". Linear extension rates at 15° and 35° were between 15 % and 25 % of the rates at the intermediate Biology and ultrastructure of Julella avicenniae 15 % ASW 70 100 0 20 40 60 80 100 50 40 17 80 Germination (%) 60 Colony diam. (mm) 870 30 60 40 20 20 10 0 0 0 5 10 15 20 Time (d) 25 30 1 35 0 20 40 60 80 100 20 24 15 Germ tube length (ím) Linear extension rate (mm d–1) 12 16 % ASW 0·8 8 18 20 1·0 4 0·6 0·4 10 5 0·2 0 15 20 25 Temperature (°) 30 0 35 Figs 15, 16. Effect of varying temperature and salinity levels on linear growth of Julella avicenniae in glucose yeast extract tryptone ASW media buffered with Tris at pH 7±5. Fig. 15. Radial growth (mm³...) at varying salinities at 25°. Fig. 16. Linear extension rate (mm d−") at varying temperatures and salinities. Germination Ascospores germinated rapidly, each producing a germ tube from each cell of the multi-celled ascospore. Germination occurred within the first hour, with a small percentage of spores producing four to six germ tubes. At 24 h, over 90 % of the ascospores had germinated (Fig. 17) with half of these spores having more than seven germ tubes while approximately 20 % had more than nine germ tubes per ascospore (Fig. 19). Germ tube growth increased linearly with time (Fig. 18). 4 8 4 8 12 20 24 19 100 75 Spores (%) temperatures. No growth was recorded at 40° after 30 d. Dry weight biomass of J. avicenniae, harvested from stationary growth in liquid GYT medium after 28 d at 25 °, revealed the following salinity preference : 150 " 125 " 100 " 75 " 175 " 200 " 50 " 25 % ASW with the biomass ranging between 150 mg (25 % ASW) and 410 mg (150 % ASW). The anamorphic state of J. avicenniae, a Phoma sp., was observed in both agar and liquid cultures. A total of 10 strains was isolated but only some developed the anamorphic stage. 1 50 25 0 1 12 Time (h) 20 24 Figs 17–19. Profile of germination of Julella avicenniae on 100 % ASW in 24 h at 25°. Four replicates of 40 spores each were used for determination of each data point. Fig. 17. Germination (³...). Fig. 18. Linear growth of germ tubes (³...). Fig. 19. Percentage of germinating spores with different numbers of germ tubes : 7, no germination ; :, 1–3 germ tubes, *, 4–6 ; 8, 7–9 ; 9, " 9. DISCUSSION Julella avicenniae asci and ascospores are superficially similar to those of Pleospora gaudefroyi Pat. at the light microscope and ultrastructural levels (Yusoff, Moss & Jones, 1994). Both D. W. T. Au, E. B. G. Jones and L. L. P. Vrijmoed species have bitunicate asci with a thick-walled endoascus and a distinct ocular chamber. Ascospores are brown to dark brown, muriform, with thick cell walls and an outer mucilaginous sheath. Ultrastructurally the ascospore wall consists of an outer mucilaginous exosporium (sheath) which swells in water, an electron-dense episporium and a multilamellate mesosporium. In J. avicenniae, the mesosporium is bilamellate, while in P. gaudefroyi three layers are discernible (Yusoff et al., 1994). The outermost mesosporial layer of both species contains electron-dense particles and divides the spores into a number of individual cells. In J. avicenniae, the electron-dense particles are not as prominent as those of P. gaudefroyi. Using the staining technique developed by Mckeown, Moss & Jones (1996), these particles have been confirmed as melanin. Ascospore walls of marine fungi generally comprise three layers, the exosporium (often elaborated into appendages, but lacking in some species), the episporium (the electron-dense middle layer, except when the exosporium is absent, it becomes the outer layer) and the inner electron-transparent mesosporium. As we have examined more species, it becomes evident that the ascospore walls of some species are more complex, in particular the mesosporium. In the Loculoascomycetes P. gaudefroyi, Massarina thalassiae, Paraliomyces lentiferus and Dactylospora haliotrepha, all have an exosporium and an episporium (Yusoff et al., 1994 ; Read et al., 1995 ; Au et al., 1996), as in the unitunicate Halosphaeriales e.g. Corollospora maritima, Carbosphaerella leptosphaerioides and Bovicornua intricata (Jones et al., 1983 ; Johnson et al. 1984 ; Yusoff et al., 1993). The episporium is, however, lacking in Lignincola laevia, Kohlmeyeriella tubulata (Halosphaeriales : Yusoff et al., 1991 ; Jones et al., 1983) and Mycosphaerella ascophylli (Loculoascomycetes : Stanley, 1991). It is the mesosporium that is most variable : one layer, C. maritima, L. laevis, B. intricata, M. thalassiae, Mycosphaerella ascophylli, P. lentiferus (Jones et al., 1983 ; Read et al., 1992, 1994 ; Stanley, 1991) ; two-layered, C. leptosphaerioides, K. tubulata (Jones et al., 1983 ; Johnson et al., 1984), while in P. gaudefroyi and D. haliotrepha it is many layered (Yusoff et al., 1994 ; Au et al., 1996). The mesosporial layer in J. avicenniae is more complex than that of many marine fungi and further developmental studies of ascosporogenesis in these fungi are warranted. Little is known of the physiology of mangrove fungi, in particular, those exposed to extreme environmental conditions. J. avicenniae has been collected on submerged wood previously, but at Morib mangrove, Malaysia, it was growing on twigs of young shrubs of Avicennia alba, branches that are not submerged even at the high tide level. The fungus has been found growing on Avicennia species in collections from Oman, Egypt, Thailand and Hong Kong (Jones, unpublished observations). Is it, therefore, a true mangrove}marine species ? The salinity data demonstrate that its optimum for growth in liquid culture is 150 % ASW, although it can tolerate higher salinities (200 % ASW). Optimum temperature for growth was in the range 25–30°, which agrees with data presented by Panebianco (1990) for tropical marine fungi. It is interesting that J. avicenniae also can grow at 35° but its growth is restricted at lower temperatures, a feature also noted for other tropical fungi (Jones, 1993). Data show that J. avicenniae is well 871 adapted for the extreme environmental conditions found in the mangrove ecosystem, namely, wide salinity tolerance and tolerance of higher temperatures, and the production of mucilage from the pseudoparaphyses to protect the developing asci and ascospores. Active ejection of the ascospores results in the good dispersal of the species, while the mucilaginous sheath (exosporium) surrounding the ascospores undoubtedly aids spore adhesion to new substrata (Hyde, Jones & Moss, 1986 ; Rees & Jones, 1984 ; Jones, 1994). Information is available on the effects of varying salinity and temperature levels on the vegetative growth of marine fungi (e.g. Schaumann, 1974 ; Molina & Hughes, 1982 ; Vrijmoed, 1987 ; Lorenz & Molitoris, 1992), but hardly any is available for mangrove fungi. In published works on salinity tolerance of marine fungi, the Phoma-pattern, first suggested by Ritchie (1957), is always discussed and has been confirmed in several fungi e.g. Lignincola laevis (Hughes, 1960 ; Lorenz & Molitoris, 1992), Zalerion maritimum (Ritchie & Jacobsohn, 1963 ; Schaumann, 1974 ; Molina & Hughes, 1982) and Digitatispora marina (Doguet, 1964). For these Phoma-pattern orientated fungi, their maximum salinity for vegetative growth increases with higher temperatures, until the temperature becomes limiting. Intertidal fungi with such a physiology have an obvious advantage in their survival, especially for tropical and subtropical fungi (Lorenz & Molitoris, 1992). The J. aviciennae isolate used in this investigation was collected above the high tide level, rarely inundated but definitely affected by salt spray of tidal waters. Thus it is not surprising that J. avicenniae can grow at a salinity greater than 100 % ASW. Its optimal temperature range (20–30°) also conforms with its growth conditions in nature. The absence of a Phoma-pattern may be due to the relatively constant high saline and temperature regime the fungus is exposed to, as compared with other mangrove fungi, such as Hypoxylon oceanicum Schatz which is truly ‘ intertidal ’ and which exhibits a Phoma response (Vrijmoed & Jones, unpublished). In a number of bitunicate mangrove fungi the asci are embedded in a matrix of mucilage e.g. Pyrenographa xylographoides Aptroot, Melaspilea mangrovei Vrijmoed, K. D. Hyde & E. B. G. Jones, Massarina ramunicola K. D. Hyde, Dactylospora haliotrepha (Kohlm. & E. Kohlm.) Hafellner. These fungi often are exposed to extreme environmental conditions : high air temperature, desiccation during their intertidal exposure, variable salinity (freshwater in the rainy season, normal salinity during inundation, high salinities during the noon time exposure at low tide). Thus, mucilage may aid in the conservation of water and in the protection of the developing asci. The site of mucilage production is unknown, but Kohlmeyer & Kohlmeyer (1979) and Nakagiri (1993) have suggested it is developed from the ascus cap – at least in D. haliotrepha. This is unlikely as no organelles are active in mucilage secretion in the ascus wall or within the ascus (Au, Vrijmoed & Jones, 1996). In J. avicenniae, there is evidence to suggest that the pseudoparaphyses may be implicated in mucilage production, cells possess well preserved organelles, often lacking in other fungi we have studied at the TEM level (Read, Moss & Jones, 1994). Also fibrillar extensions (mucilage) are visible in our electron micrographs of the pseudoparaphyses (Fig. 3). This is a more plausible site for mucilage Biology and ultrastructure of Julella avicenniae production than the asci which are predominantly designed to ensure ascospore development and their release. The multi-germ tube germination of the ascospores appears to ensure the early and rapid colonization of substrata, a key issue in the capture of a resource domain, and the elimination of competition from other fungi}micro-organisms. The role of J. avicenniae in the die back of the apical twigs of A. alba warrants investigation and is in progress. We thank the Research Grant Council of the University Grants Committee, Hong Kong (Grant No. 9040096) for financial support. Prof. E. B. G. Jones and Dr L. L. P. Vrijmoed are grateful to the British Council, Hong Kong, for travel grants for exchange visits under the British Academic Links Scheme and Prof. E. B. G. Jones to the British Council, Malaysia, for a travel grant to visit Malaysia. The technical assistance of Mr Barry Ng and Ms Lau Lai Yip also are gratefully acknowledged. REFERENCES Au, D. W. T., Vrijmoed, L. L. P. & Jones, E. B. G. (1996). Ultrastructure of asci and ascospores of the mangrove ascomycete Dactylospora haliotrepha (Kohlm. et E. Kohlm.) Hafellner. Mycoscience 37, 129–135. Byrne, P. J. & Jones, E. B. G. (1975). Effect of salinity on spore germination of terrestrial and marine fungi. Transactions of the British Mycological Society 64, 497–503. Borse, B. D. (1987). Marine fungi from India-V. Current Science 56, 1109–1111. Doguet, G. (1964). 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