Decomposition
of Deciduous
in a Woodland I. A S c a n n i n g
Electron
Leaf
Litter
Stream Microscopic
Study
K. F. SUBERKROPP AND M. J. KLUG
w. K. Kellogg Biological Station, Michigan State University Hickory Corners, Michigan 49060 Abstract Microorganisms associated with decomposing deciduous leaf litter in a woodland stream were examined by scanning electron microscopy. The use of a critical point drying method allowed the preservation of a wide variety of microorganisms as well as the decomposing litter with a minimum of distortion. The micrographs provide evidence that the aquatic hyphomycetes are the major fungal flora present during decomposition. Two distinct groups of these fungi were found during the seasonal cycle with one group occurring only in the summer while the other occurred throughout the rest of the year. The presence of all developmental stages of these organisms in the environment is considered further evidence of their active role in the decomposition of litter. Scanning electron microscopy (SEM) has been used to examine the microflora associated with detritus in a variety o f environments [2, 3, 8, 9 ] . As a part of a comprehensive study o f deciduous leaf decomposition in streams, we have employed SEM to examine the in situ morphological and physical relationships among the microorganisms associated with leaf litter. One group o f fungi, the aquatic hyphomycetes, has been found to be ubiquitous invaders o f this leaf material in streams [4, 7]. These fungi produce specialized tetraradiate or sigmoid spores which appear to provide them with an ecological advantage [5, 10]. This paper presents representative scanning electron micrographs of these fungi and associated microorganisms on decomposing leaf litter during a seasonal study. Materials and Methods Autumn shed white oak (Quercus alba) and pignut hickory (Carya glabra) leaves were collected and air dried. In the fall and summer, leaves were fastened to a masonry brick and placed perpendicular to the current in Augusta Creek, a small hardwater stream in Southwestern Michigan [6]. Leaves were regularly removed for light microscopic and cultural studies, and relative frequencies of fungi sporulating were determined. Based on light micro96 MICROBIAL ECOLOGY, Vol. 1, 96-103 (1974) 9 1974 by Springer-Verlag New York Inc.
Decomposition of Deciduous Leaf Litter I
97
scopic observations, leaves with representative microflora were periodically removed and examined with SEM. Leaf disks (1 cm diameter) were cut from the leaves, and disks were either immediately fixed for 1 hr in 2% osmium tetroxide buffered with 0.05 M sodium phosphate, pH 6.8, or were directly started through the dehydration series (see below) without fixation. Except where noted, materials were examined without incubation in the laboratory. All samples were passed through a dehydration series of ethanol in water (10, 40, 70, and 100%) followed by a series of isoamyl acetate in ethanol (10, 40, 70, and 100%) for 10 min each. Liquid carbon dioxide was flushed through the sample to replace the isoamyl acetate and the sample was dried with a critical point drying method [1]. The dried samples were mounted on stubs, vacuum coated with gold, and examined with an AMR, model 900, scanning electron microscope. Identification of the aquatic hyphomycetes sporulating on the leaf material was routinely made from observations with the light microscope. However, the presence of all stages of sporulation and the distinctive spores of these fungi simplified their identification in the scanning electron micrographs.
Results and Discussion
Preparation for SEM. The critical point drying method reduces or eliminates the causes of distortion which can occur during air drying or freeze drying; i.e., it reduces surface tension effects during evaporation and eliminates the formation of solid-phase boundaries [1], and therefore allows examination of natural materials with a minimum of distortion. Fungal structures, e.g., hyphae and thin-walled spores, remained distinct from each other and from the leaf substratum whether the samples were previously fixed with osmium (Figs. 1A, 2, and 3) or not (Fig. 1B). Bacterial cells either singly (Fig. 4C) or in microcolonies (Fig. 4B) remained intact and were easily distinguishable from detritus. Protozoa, such as the group o f Vorticella sp. in Fig. 5, and diatoms (Fig. 2) which were occasionally seen on this material also retained their shape and structural integrity. Thus a wide range of types of microorganisms as well as the leaf substratum have been prepared for SEM with a minimum of distortion by the use of this critical point drying technique.
Microorganisms Involved in Leaf Decomposition. During the seasonal study of the decomposition o f leaf litter in Augusta Creek it became evident that two species of aquatic hyphomycetes were observed on fresh and incubated samples much more frequently than other fungi. Alatospora acuminata (Fig. 1) was the dominant species during the fall, winter, and spring when the mean water temperature was below about 15~ and Lunulospora curvula (Fig. 2) was dominant in the summer when the mean temperature exceeded about 18~ Figures 1A and 2A show typical densities o f spore production on litter taken directly from the environment during the winter and summer, respectively. The tetraradiate spores of A. acuminata and several stages of development of these phialospores were
98
K.F. Suberkropp and M. J. Klug
generally found in each cluster of sporophores (Fig. 1B). Although the sigmoid spores of L. curvula are produced singly, several stages of development were often observed in close proximity to one another (Fig. 2B). The occurrence of all stages of spore development of these two fungi at the observed densities during decomposition of the litter indicates active growth and completion of their life cycle.
Fig. 1. Alatospora acuminata on hickory leaves. Leaves were placed in the stream for 7 weeks (Jan-Feb). (A, 180X ; B, 1800•
Decomposition of Deciduous Leaf Litter I
99
It should be noted that most of the fungal biomass visible in these micrographs consists of sporulating structures and that the vegetative hyphae (presumably the majority of the biomass) must be, therefore, almost entirely within the leaf tissue itself.
Fig. 2. Lunulospora curvula on oak leaves. Leaves were placed in the stream for 1 week (Aug). Diatoms of the genus Cymbella can also be seen in A (A,180X; B, 900X ).
I00
K . F . Suberkropp and M. J. Klug
Ten species o f aquatic hyphomycetes were found to be o f common occurrence on the oak and hickory leaves examined in this study. Besides the two dominant species (Figs. 1 and 2), four additional species are illustrated in Fig. 3.
Fig. 3. (A) Clavariopsis aquatica on oak (900X). Leaves were placed in the stream for 6 months (Nov-May). (B) Flagellospora curvula on hickory (450X). Leaves were placed in the stream for 7 weeks (Jan-Feb). (C) Clavatospora tentacula on oak (900X). Leaves were placed in the stream for 1 week (Aug). (D) Tetracladium marchalianum on hickory (900X). Leaves were placed in the stream for 6 weeks (Jan-Feb) and incubated for 5 days at 5~ in aerated stream water in the laboratory.
Decomposition of Deciduous Leaf Litter I
1 O1
Clavariopsis aquatica (Fig. 3A), ~Tagellospora curvula (Fig. 3B), Lemonniera aquatica, and species of Anguillospora were present with A. acuminata when the temperature was below 15~ Clavatospora tentacula (Fig. 3C), Flagellospora penicilliodes, and Triscelophorus monosporus occurred in the summer while
Fig. 4. (A) Hickory leaves placed in the stream for 2 weeks (Aug) (450X). Arrow indicates cuticle peeling. (B) Bacterial microcolony on oak leaves placed in the stream for 1 week (Aug)(1800• (C) Stomate and associated bacteria on oak leaf (4500X), Arrows indicate bacterial ceils. Placed in the stream for 6 months (Nov-May).
102
K.F. Suberkropp and M. J. Klug
Fig. 5. Vorticella sp. on oak leaves placed in the stream for 6 months (Nov-~vlay) (500X).
Tetracladium marchalianum (Fig. 3D) was present throughout the year. On hickory leaves, T. marchalianurn was nearly as prevalent as the two dominant species.
In the inital stages of decomposition it is thought that the fungi penetrate the interior of the leaf whereas the bacteria are primarily limited to the surface. As the cuticle of the leaf is removed (Fig. 4A, arrow), bacterial cells may begin acting on the leaf tissue underneath. Bacteria may also enter initially through the leaf stomates (Fig. 4C) and thereby gain access to the interior of the leaf. From these observations it can be concluded that the aquatic hyphomycetes are the major fungal flora on decomposing leaves in this stream. The vegetative hyphae of these fungi presumably grow chiefly within the leaf tissue producing the characteristic tetraradiate or sigmoid spores at the surface. They can be found in all stages of development in high densities, emphasizing that they are actively growing in this environment. Further studies are now being undertaken to determine the hydrolytic and metabolic capabilities of these fungi and selected bacterial isolates so that their role in the decomposition of leaf litter might be better understood.
Decomposition of Deciduous Leaf Litter I
103
Acknowledgments This investigation was supported by Grant GB-36069X from the National Science Foundation. We thank W. S. McAfee and V. E. ShuU for technical assistance with the electron microscopy and P. Gerhardt for support during part of this work. This article is Kellogg Biological Station Publication No. 255 and Michigan Agricultural Experiment Station Journal Article No. 6661.
References l.
Anderson, T. F. 1956. Electron microscopy of microorganisms, p. 17%240. In: G. Oster and A. W. PoUister, editors. Physical Techniques of Biological Research. VoL Ill. Academic Press, New York.
2;
Gessner, R. V., Goos, R. D., and Sieburth, J. ,'r 1972. The fungal microcosm of the internodes of Spartina aherniflora. Mar. Biol. 16: 269-273.
3.
Gray, T. R. G. 1967. Stereoscan electron microscopy of soil microorganisms. Science 155: 1668-1670.
4.
Ingold, C. T. 1942. Aquatic hyphomycetes of decaying alder leaves. Trans. Brit. Mycol. Soc. 25: 339-417.
5.
Ingold, C. T. 1966. The tetraradiate aquatic fungal spore. Mycologia 58: 43-56.
6.
Manny, B. A., and Wetzel, R. G. 1973. Diurnal changes in dissolved organic and inorganic carbon and nitrogen in a hardwater stream. Freshwater Biol. 3: 31-43.
7.
Nilsson, S. 1964. Freshwater hyphomycetes. Symb. Bot. Upsal. 18 (2): 1-130.
8.
Paerl, H. W. 1973. Detritus in Lake Tahoe: Structural modification by attached microflora. Science 180: 496-498.
9.
Todd, R. L., Cromack, K., Jr., and Knutson, R. M. 1973. Scanning electron microscopy in the study of terrestrial microbial ecology. Bull. Ecol. Res. Comm. (Stockholm) 17: 109-118.
10.
Webster, J. 1959. Experiments with spores of aquatic hyphomycetes, I. Sedimentation, and impaction on smooth surfaces. Ann. Bot. N. S. 2 3 : 5 95-611.
of Deciduous
in a Woodland I. A S c a n n i n g
Electron
Leaf
Litter
Stream Microscopic
Study
K. F. SUBERKROPP AND M. J. KLUG
w. K. Kellogg Biological Station, Michigan State University Hickory Corners, Michigan 49060 Abstract Microorganisms associated with decomposing deciduous leaf litter in a woodland stream were examined by scanning electron microscopy. The use of a critical point drying method allowed the preservation of a wide variety of microorganisms as well as the decomposing litter with a minimum of distortion. The micrographs provide evidence that the aquatic hyphomycetes are the major fungal flora present during decomposition. Two distinct groups of these fungi were found during the seasonal cycle with one group occurring only in the summer while the other occurred throughout the rest of the year. The presence of all developmental stages of these organisms in the environment is considered further evidence of their active role in the decomposition of litter. Scanning electron microscopy (SEM) has been used to examine the microflora associated with detritus in a variety o f environments [2, 3, 8, 9 ] . As a part of a comprehensive study o f deciduous leaf decomposition in streams, we have employed SEM to examine the in situ morphological and physical relationships among the microorganisms associated with leaf litter. One group o f fungi, the aquatic hyphomycetes, has been found to be ubiquitous invaders o f this leaf material in streams [4, 7]. These fungi produce specialized tetraradiate or sigmoid spores which appear to provide them with an ecological advantage [5, 10]. This paper presents representative scanning electron micrographs of these fungi and associated microorganisms on decomposing leaf litter during a seasonal study. Materials and Methods Autumn shed white oak (Quercus alba) and pignut hickory (Carya glabra) leaves were collected and air dried. In the fall and summer, leaves were fastened to a masonry brick and placed perpendicular to the current in Augusta Creek, a small hardwater stream in Southwestern Michigan [6]. Leaves were regularly removed for light microscopic and cultural studies, and relative frequencies of fungi sporulating were determined. Based on light micro96 MICROBIAL ECOLOGY, Vol. 1, 96-103 (1974) 9 1974 by Springer-Verlag New York Inc.
Decomposition of Deciduous Leaf Litter I
97
scopic observations, leaves with representative microflora were periodically removed and examined with SEM. Leaf disks (1 cm diameter) were cut from the leaves, and disks were either immediately fixed for 1 hr in 2% osmium tetroxide buffered with 0.05 M sodium phosphate, pH 6.8, or were directly started through the dehydration series (see below) without fixation. Except where noted, materials were examined without incubation in the laboratory. All samples were passed through a dehydration series of ethanol in water (10, 40, 70, and 100%) followed by a series of isoamyl acetate in ethanol (10, 40, 70, and 100%) for 10 min each. Liquid carbon dioxide was flushed through the sample to replace the isoamyl acetate and the sample was dried with a critical point drying method [1]. The dried samples were mounted on stubs, vacuum coated with gold, and examined with an AMR, model 900, scanning electron microscope. Identification of the aquatic hyphomycetes sporulating on the leaf material was routinely made from observations with the light microscope. However, the presence of all stages of sporulation and the distinctive spores of these fungi simplified their identification in the scanning electron micrographs.
Results and Discussion
Preparation for SEM. The critical point drying method reduces or eliminates the causes of distortion which can occur during air drying or freeze drying; i.e., it reduces surface tension effects during evaporation and eliminates the formation of solid-phase boundaries [1], and therefore allows examination of natural materials with a minimum of distortion. Fungal structures, e.g., hyphae and thin-walled spores, remained distinct from each other and from the leaf substratum whether the samples were previously fixed with osmium (Figs. 1A, 2, and 3) or not (Fig. 1B). Bacterial cells either singly (Fig. 4C) or in microcolonies (Fig. 4B) remained intact and were easily distinguishable from detritus. Protozoa, such as the group o f Vorticella sp. in Fig. 5, and diatoms (Fig. 2) which were occasionally seen on this material also retained their shape and structural integrity. Thus a wide range of types of microorganisms as well as the leaf substratum have been prepared for SEM with a minimum of distortion by the use of this critical point drying technique.
Microorganisms Involved in Leaf Decomposition. During the seasonal study of the decomposition o f leaf litter in Augusta Creek it became evident that two species of aquatic hyphomycetes were observed on fresh and incubated samples much more frequently than other fungi. Alatospora acuminata (Fig. 1) was the dominant species during the fall, winter, and spring when the mean water temperature was below about 15~ and Lunulospora curvula (Fig. 2) was dominant in the summer when the mean temperature exceeded about 18~ Figures 1A and 2A show typical densities o f spore production on litter taken directly from the environment during the winter and summer, respectively. The tetraradiate spores of A. acuminata and several stages of development of these phialospores were
98
K.F. Suberkropp and M. J. Klug
generally found in each cluster of sporophores (Fig. 1B). Although the sigmoid spores of L. curvula are produced singly, several stages of development were often observed in close proximity to one another (Fig. 2B). The occurrence of all stages of spore development of these two fungi at the observed densities during decomposition of the litter indicates active growth and completion of their life cycle.
Fig. 1. Alatospora acuminata on hickory leaves. Leaves were placed in the stream for 7 weeks (Jan-Feb). (A, 180X ; B, 1800•
Decomposition of Deciduous Leaf Litter I
99
It should be noted that most of the fungal biomass visible in these micrographs consists of sporulating structures and that the vegetative hyphae (presumably the majority of the biomass) must be, therefore, almost entirely within the leaf tissue itself.
Fig. 2. Lunulospora curvula on oak leaves. Leaves were placed in the stream for 1 week (Aug). Diatoms of the genus Cymbella can also be seen in A (A,180X; B, 900X ).
I00
K . F . Suberkropp and M. J. Klug
Ten species o f aquatic hyphomycetes were found to be o f common occurrence on the oak and hickory leaves examined in this study. Besides the two dominant species (Figs. 1 and 2), four additional species are illustrated in Fig. 3.
Fig. 3. (A) Clavariopsis aquatica on oak (900X). Leaves were placed in the stream for 6 months (Nov-May). (B) Flagellospora curvula on hickory (450X). Leaves were placed in the stream for 7 weeks (Jan-Feb). (C) Clavatospora tentacula on oak (900X). Leaves were placed in the stream for 1 week (Aug). (D) Tetracladium marchalianum on hickory (900X). Leaves were placed in the stream for 6 weeks (Jan-Feb) and incubated for 5 days at 5~ in aerated stream water in the laboratory.
Decomposition of Deciduous Leaf Litter I
1 O1
Clavariopsis aquatica (Fig. 3A), ~Tagellospora curvula (Fig. 3B), Lemonniera aquatica, and species of Anguillospora were present with A. acuminata when the temperature was below 15~ Clavatospora tentacula (Fig. 3C), Flagellospora penicilliodes, and Triscelophorus monosporus occurred in the summer while
Fig. 4. (A) Hickory leaves placed in the stream for 2 weeks (Aug) (450X). Arrow indicates cuticle peeling. (B) Bacterial microcolony on oak leaves placed in the stream for 1 week (Aug)(1800• (C) Stomate and associated bacteria on oak leaf (4500X), Arrows indicate bacterial ceils. Placed in the stream for 6 months (Nov-May).
102
K.F. Suberkropp and M. J. Klug
Fig. 5. Vorticella sp. on oak leaves placed in the stream for 6 months (Nov-~vlay) (500X).
Tetracladium marchalianum (Fig. 3D) was present throughout the year. On hickory leaves, T. marchalianurn was nearly as prevalent as the two dominant species.
In the inital stages of decomposition it is thought that the fungi penetrate the interior of the leaf whereas the bacteria are primarily limited to the surface. As the cuticle of the leaf is removed (Fig. 4A, arrow), bacterial cells may begin acting on the leaf tissue underneath. Bacteria may also enter initially through the leaf stomates (Fig. 4C) and thereby gain access to the interior of the leaf. From these observations it can be concluded that the aquatic hyphomycetes are the major fungal flora on decomposing leaves in this stream. The vegetative hyphae of these fungi presumably grow chiefly within the leaf tissue producing the characteristic tetraradiate or sigmoid spores at the surface. They can be found in all stages of development in high densities, emphasizing that they are actively growing in this environment. Further studies are now being undertaken to determine the hydrolytic and metabolic capabilities of these fungi and selected bacterial isolates so that their role in the decomposition of leaf litter might be better understood.
Decomposition of Deciduous Leaf Litter I
103
Acknowledgments This investigation was supported by Grant GB-36069X from the National Science Foundation. We thank W. S. McAfee and V. E. ShuU for technical assistance with the electron microscopy and P. Gerhardt for support during part of this work. This article is Kellogg Biological Station Publication No. 255 and Michigan Agricultural Experiment Station Journal Article No. 6661.
References l.
Anderson, T. F. 1956. Electron microscopy of microorganisms, p. 17%240. In: G. Oster and A. W. PoUister, editors. Physical Techniques of Biological Research. VoL Ill. Academic Press, New York.
2;
Gessner, R. V., Goos, R. D., and Sieburth, J. ,'r 1972. The fungal microcosm of the internodes of Spartina aherniflora. Mar. Biol. 16: 269-273.
3.
Gray, T. R. G. 1967. Stereoscan electron microscopy of soil microorganisms. Science 155: 1668-1670.
4.
Ingold, C. T. 1942. Aquatic hyphomycetes of decaying alder leaves. Trans. Brit. Mycol. Soc. 25: 339-417.
5.
Ingold, C. T. 1966. The tetraradiate aquatic fungal spore. Mycologia 58: 43-56.
6.
Manny, B. A., and Wetzel, R. G. 1973. Diurnal changes in dissolved organic and inorganic carbon and nitrogen in a hardwater stream. Freshwater Biol. 3: 31-43.
7.
Nilsson, S. 1964. Freshwater hyphomycetes. Symb. Bot. Upsal. 18 (2): 1-130.
8.
Paerl, H. W. 1973. Detritus in Lake Tahoe: Structural modification by attached microflora. Science 180: 496-498.
9.
Todd, R. L., Cromack, K., Jr., and Knutson, R. M. 1973. Scanning electron microscopy in the study of terrestrial microbial ecology. Bull. Ecol. Res. Comm. (Stockholm) 17: 109-118.
10.
Webster, J. 1959. Experiments with spores of aquatic hyphomycetes, I. Sedimentation, and impaction on smooth surfaces. Ann. Bot. N. S. 2 3 : 5 95-611.
