Fungi and cellulolytic activity associated with decomposition of Bauhinia purpurea leaf litter in a polluted and unpolluted Hong Kong waterway

D. W. T. Au' AND I. J. HODGKISS Departrnerzr of Botany, The University of Hong Kong, Pokficlam Roacl, Hong Kong

AND

L. L. P. VRIJMOED Departrnent of Applied Science, City Polyrechr~ic of Hong Korzg, Tat Chee Ave., Kowloon, Horlg Kong

Received October 26, 1991

Au, D. W. T., HODGKISS, I. J., and VRIJMOED, L. L. P. 1992. Fungi and cellulolytic activity associated with decomposition of Bauhinia purpurea leaf litter in a polluted and unpolluted Hong Kong waterway. Can. J. Bot. 70: 1071 - 1079.

A survey of fungal succession on decomposing Bauhinia purprcrea L. leaves in the unpolluted Tai Po Kau Forest Stream (TPKFS) and the animal waste polluted Lam Tsuen River (LTR) was carried out during the winter of 1988 and the summer of 1989. In situ cellulolytic activity of the TPKFS leaf litter was also investigated. Most of the 28 aquatic hyphomycete species found were cosmopolitan or frequently reported in temperate regions. Clavariopsis aqlcatica De Wildeman, L~tnulospora cymbiformis Miura, and Flagellospora penicillioides Ingold were the dominant species at both sites. Among the 49 geofungi species recorded, lymaphilic species were commonly observed in the polluted LTR (e.g., Geotrichlcm candidurn-c ink ex Leman, Fusarircrn oxysporurn Schlecht, and Mucor racernosus Fres.) and lymaxenes in the TPKFS (e.g., Hurrlicola spp., Trichoderma spp. and Gliocladium roseurn Bain.). Species richness of aquatic hyphomycetes was higher in the TPKFS (27 species) than in the polluted LTR (14 species), whereas for the associated geofungi, it was higher in the LTR (35 species) than in the TPKFS (28 species). Conidial production was also higher in the TPKFS. Aquatic hyphomycetes and geofungi showed a complementary sequence of dominance in winter and summer, respectively, in the clean TPKFS. Higher cellulolytic activity occurred in the winter than the summer leaf litter.

Key words: aquatic hyphomycetes, geofungi, leaf litter, pollution, cellulolytic activity.

Au, D. W. T., HODGKISS, I. J., et VRIJMOED, L. L. P. 1992. Fungi and cellulolytic activity associated with decomposition of Ba~chinia purpurea leaf litter in a polluted and unpolluted Hong Kong waterway. Can. J. Bot. 70 : 1071 - 1079.

Au cours de l'hiver 1988 et de l'Ct6 1989, les auteurs ont CtudiC la succession fongique sur des feuilles de Bauhirliapurpurea L. en dCcomposition dans la rivikre Lam Tsuen (LTR), polluCe par des dCchets d'origine animale, et le ruisseau non polluC de la for&t Tai Po Kau (TPKFS). 11s ont mesurk I'activitC cellulolytique in situ, dans la litibre du TPKFS. La plupart des 28 hyphomycbtes rencontrts sont cosmopolites et sont frkquemment rapportis pour les rigions tempCrCes. Le Clavariopsis aqua- tics De Wildeman, le Lunulospora cyrrlbiforrnis Miura, et le Flagellospora penicillioides Ingold sont les espbces dominantes sur les deux sites. Parmi les 49 espbces de giofungi observCes, des espkces lymaphiles sont frkquemment observCes dans le LTR polluC (e.g., Geotrichurn candidum Link ex Leman, Fusarium oxysporurn Schlecht et Mucor racemosus Fres.) alors que dans le TPKFS on retrouve des espkces lymaxbnes (e.g., Hurnicola spp., Trichoderrna spp. et Gliocladilcrn roseurn Bain.). La richesse en espbces de champignons aquatiques est plus ClevCe dans le LTR (35 espkces) que dans le TPKFS (28 espbces). La production de conidies est Cgalement plus ClevCe dans le TPKFS. Les hyphomycbtes aquatiques et les gCofun,' 0 1 montrent une sCquence complCmentaire de dominance, en hiver et en CtC respectivement, dans les eaux propres du TPKFS. L'activitC cellulolytique est plus ClevCe dans la litikre d'hiver que dans celle d'CtC.

Mots c l b : hyphomycktes aquatiques, gCofungi, litikres foliaires, pollution, activitC cellulolytique. [Traduit par la redaction]

Introduction

Two ecological groups of Fungi play a major role in the decomposition of organic materials in freshwater ecosystems. These include the obligate aquatic fungi, the most conspicuous of which are the aquatic hyphomycetes that have striking adap- tations for aquatic dispersal, and the facultative aquatic fungi or geofungi that are regular inhabitants of soil and organic substrates in terrestrial habitats but that are able to grow on submerged organic matter (Cooke 1961, 1976). The abundance, distribution, and role of these aquatic fungi have been studied in various regions of the world i n ~ l u d i n ~ - ~ a r t s of Asia, e.g., Suzuki and Nimura (1960a, 1960b) for Japan, Nawawi (1985, 1987) and Kuthubutheen (1987) for Malaysia, and Sridhar and Kaveriappa (1988a, 1988b, 1989) for India. No equivalent data is available for the Asian subtropics in the region of Hong Kong. In this study, diversity and abundance of aquatic hyphomycetes and geofungi on Bauhinia pulpurea L. leaves are compared

'Author to whom all correspondence should be addressed Primed in Canada I Irnprirnd au Canada

between a polluted and unpolluted waterway in Hong Kong. The cellulolytic activity (Sinsabaugh et al. 1981; Hodgkiss and Leung 1986) of leaves during different stages of decomposi- tion and at two seasons of contrasting temperature was examined in an attempt to correlate the patterns of enzymatic activities with the associated microbial community.

Materials and methods

Description of sites Two sites in the New Territories of Hong Kong were selected, one

being the clean Tai Po Kau Forest Stream (TPKFS) and the other the animal waste polluted Lam Tsuen River (LTR) near Tai Po. The site descriptions and the preparation of B. purpurea L. leaf litter bags for both decomposition and mycological studies are described by AU et al. (1992). Litter bags were sampled six times, once every 2-3 weeks from 4 November 1988 to 24 February 1989 (winter series) and from 6 June 1989 to 11 July 1989 in the LTR but to 8 August 1989 in the TPKFS (summer series). Collections were more frequent in summer because of the rapid decomposition.

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CAN. J . BOT. VOL. 70. 1992

TABLE 1. Ranking of aquatic hyphomycetes associated with Bauhinia purpurea L. leaf litter in the Tai Po Kau Forest Stream (TPKFS) and the Lam Tsuen River (LTR) during winter

1988 and summer 1989

TPKFS LTR

Species Winter Summer Winter Summer

Alatospora pulchella Marvanova 8.7 6.8 Articulospora moniliforma Ranzoni 12.2 Articulospora tetracladia Ingold 13.3 7.6 Calcarispora hiemalis Marvanova & Marvan 13.2 Campylospora chaetoclades Ranzoni 15.1 12.2 8.0 Centrospora aquatica Iqbal 12.4 6.7 Clavariopsis aquatica De Wildeman 3.6 9.6 4.1 2.7 Clavariopsis sp. 10.3 Dendrospora fisca Descals & Webster 13.6 Flagellospora curvula Ingold 11.3 6.6 5.7 Flagellospora pet~icillioides Ingold 4.5 6.8 5.2 4.2 Lemonniera sp. 6.5 10.6 Lunulospora curvula Ingold 6.8 10.0 5.9 6.5 Lunulospora cymbifonnis Miura 4.5 3.8 4.4 2.3 Mycocentrospora filiformis Iqbal 11.3 Pseudoanguillospora stricta Iqbal 9.6 3.2 Pyratnidospora flumit~ea Miura & Kudo 5.0 6.8 Sigmoidea aurantiaca Descals 12.6 7.4 6.0 Tetrachaetum elegans Ingold 11.6 12.3 Tricladiutn attenuatum Iqbal 13.2 Tricladium chaetocladium Ingold 8.2 Triscelophorus acuminatus Nawawi 6.5 Triscelophorus monosporus Ingold 8.2 4.2 Triscelophorus sp. 14.4 Varicosporium delicatum Ingold 13.5 2.0 Hyphomycetes sp. 1 (sigmoid) 14.0 7.6 Hyphomycetes sp. 2 (tetraradiate) 14.8

Total no. of genera found 15 14 8 9 Total no. of species found 20 18 10 10

NOTE: Values are the average of the rank numbers from the six collections in each series (based on thc percent occur- rence of fungi in each collection). Boldface indicates the five top-ranked species in each series (six in the case of the TPKFS summer series).

Aquatic hyphomycetes Methods used for the study of aquatic hyphomycetes on leaf samples

were modified from Wood-Eggenschwiler and Barlocher (1983). Two leaf samples from each site (with their surface areas measured by means of grating paper) were incubated separately in 250 mL of distilled water in a 500-mL flask and aerated for 3 days with com- pressed air at 20°C. The resulting conidial suspension was then filtered (0.45 pm membrane filter) and stained (with 0.1 % lactophenol cotton blue) for identification and counting under the microscope (Sherry and Qureshi 1981). The key references used for identification were Descals and Webster (1977, 1980, 1982), Ingold (1975), Nawawi (1985), and Nilsson (1964). The density of conidia was expressed in terms of numbers of conidia per leaf area in cm2. The overall value of dominance for each aquatic hyphomycete was expressed by calcu- lating the percent frequency of its conidia in each of the six samples, ranking each species in each sample according to its frequency value and then averaging the six rank values obtained for each species.

Geofingi To determine numbers and species of associated geofungi, both

disc plating and moist chamber incubation techniques were used. Two leaves from each site were randomly selected, and two leaf discs (1 cm diameter) from each leaf sample were incubated at 20°C on (1.2%) plain water agar plates (containing 0.05 % penicillin and 0.05 % strep- tomycin) (Chamier et al. 1984). The remainder of each leaf was incubated in a Petri dish lined with glass filter papers soaked with water containing the same concentration of antibiotics. Fungal

TABLE 2. Comparison of the conidial production (conidia/cm2 leaf) of the three dominant aquatic hyphomycete species from decompos- ing Bauhinia p u p r e a L. leaves in the Tai Po Kau Forest Stream

(TPKFS) and the Lam Tsuen River (LTR)

TPKFS LTR

Clavariopsis aquatica 0.7 - 154.8 0 - 13.6 Flagellospora penicillioides 0 - 127.0 0 - 17.5 Lunulospora cymbifonnis 0 - 154.6 0 - 11.2

NOTE: Results are ranges obtained from six samples collected in winter 1988 and summer 1989.

hyphae - conidiophores growing out from the four leaf discs and from 10 random spots on the leaf fragments in the moist chambers were transferred to cornmeal agar or malt extract agar (and other growth media if required) for identification. The key references for identification were Barron (1968), Domsch et al. (1980), Ellis (1971, 1976), and Onions et al. (1981). The number of occurrences of each fungus was recorded. The frequency of occurrence of each fungus in each series at each site was determined for each method by summing the number of occurrences obtained from the six samples and expressing this as a percentage of the total number of leaf discs or spots inves- tigated (24 for the leaf disc method and 60 for the moist chamber method). These percent occurrences were ranked for each method and

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Lam Tsuen R i v e r T a i Po Kau F o r e s t Stream

d . . , . . . , , . . , , 0 4 , . . , . . , , , , , , 0 10 20 30 40 50 60 70 00 90 100110120 0 10 20 30 40 50 60 70 80 90 100110120

Time (days) Time (days)

FIG. 1. Number of aquatic hyphomycete species associated with decomposing B. purpuren L. leaves during winter 1988 (solid line) and summer 1989 (broken line).

the sum of these ranks indicates the overall dominance for each fungus. Details of the crude enzyme extract preparation for the cellulolytic

activity estimation of TPKFS leaves can be found in Leung (1986). Ash-free dry weights (AFDW) of leaves were also determined (ashing at 550°C in a muffle furnace for 10 h). Cellulolytic activity in the extract was analysed by the loss in viscosity of carboxymethyl cellu- lose (CMC). Six millilitres of the crude enzyme was pipetted into 3 mL of 2 % CMC solution (Sigma Chemical Co., St. Louis, Mo.; low viscosity) in 0.2 M citrate buffer at pH 5.5 (27"C), with 0.02% sodium azide added as a preservative. An appropriate amount of thoroughly mixed (by vortex mixer) mixture was pipetted into a buffer calibrated viscometer that was secured in a constant tempera- ture (27°C) water tank. Viscosity was measured at the beginning of the experiment and subsequently every 5 min for 1 h. Each enzyme extract sample was measured in triplicate. Cellullolytic activity was defined as the specific activity on the basis of ash-free dry weight (Sinsabaugh et nl. 1981).

Results Aquatic hyphomycetes

The species of aquatic hyphomycetes found in this study are listed in Table 1. A total of 28 species belonging to 20 genera were found. Among the 13 species found at both sites, Clavari- opsis aquatica, Flagellospora penicillioides, and Lun~ilospora cymbiformis were among the top five dominant species in both the TPKFS and the LTR. However, their conidial production on the LTR leaves was on average 10 times lower compared with the TPKFS leaves (Table 2). In fact, the TPKFS samples always yielded a higher number of species (Fig. 1) and conidial abun- dance (Fig. 2) when compared with those of the polluted LTR.

In the winter series, a total of 20 species from 15 genera were recorded on the TPKFS leaf litter (Table 1). The five most com- mon hyphomycetes were Clavariopsis aquatica, Lunulospora cymbiformis, Flagellospora penicillioides, Lemonniera sp., and Lunulospora curvula (Table I), and their frequency ofoccur- rence is shown in Table 3. In the polluted LTR, a total of 10 species from eight genera were found (Table 1). Pyramid-

osporafluminea and Variosporium delicatum together with the three dominant species previously mentioned made up the five top ranked species and their frequency of occurrence is shown in Table 3. The drop in water temperature at both sites (from 15 to 5°C) 47 days after submersion of leaf litter bags (details concerning the hydrological data of the two waterways were given in Au et al. 1992) appeared to have inactivated most of the fungi, except C. aquatica whose frequency of occurrence at this time in the TPKFS was 72% and in the LTR was 54.6% (Table 3). This water temperature effect is also reflected by a decrease in the total conidial count per leaf area, which is best shown in the TPKFS winter series (Fig. 2).

In the summer series at TPKFS, a slightly smaller number (18) of species (Table 1) were recorded. Moreover, the concen- tration of conidia also fell markedly (Fig. 2). With regard to ranking the five most prevalent fungi, six species were included (since two were equally ranked). They were Triscelophorus monosporus, Flagellospora curvula, Alatospora pulchella (all three were sporadic in the winter series), Triscelophorus acuminatus (not present in the winter series), Lunulospora cymbiformis and Flagellospora penicillioides (which were dominant species in winter). Clavariopsis aquatica, though still present in summer, was very low in abundance. In the LTR, the loss of leaf material from litter bags in summer was very rapid, and quantitative studies of the conidial number were terminated after the 3rd week, thus species succession was difficult to observe (also true for the study of geofungi).

Geofung i A list of the geofungi recorded on decomposing B. purpurea

L. leaves is presented in Table 4. The leaf disc on agar culture method yielded either the same or more frequently higher numbers of species than the moist chamber incubation method. The similarity of the geofungi community recorded by these two methods ranged from 41.6 to 66.7% (Sgrensen Index) (Table 4). A total of 49 species belonging to 41 genera was

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CAN. 1. BOT. VOL. 70, 1992

4 0 0 ~ Lam Tsuen R i v e r 4001 T a i Po Kau F o r e s t Stream

Time (days) Time (days)

FIG. 2. Conidial production by aquatic hyphomycetes associated with decomposing B. purpurea L. leaves during winter 1988 (solid line) and summer 1989 (broken line).

TABLE 3. Percent frequency of conidia of the top-ranked aquatic hyphomycetes on Bauhinia purpurea L. leaf litter after different submersion periods (days)

(A) Winter 1988

14 28 47 70 9 1 112

Tai Po Kau Forest Stream Clavariopsis aquatica 6.6 5.2 5.3 72 0.4 77.7 Flagellospora penicillioides 0 17.5 34.8 15 22.9 1.6 Letnmoniera sp. 57.1 12.5 1.5 0.6 0.3 0 Lunulospora curvula 4.1 2.9 7.5 0.4 4 0 Lunulospora cymbifartnis 0 11.9 42.4 4.9 69.8 4.5

Lam Tsuen River Clavariopsis aquatica 0.3 35.9 1.8 54.6 0 95.4 Flagellospora penicillioides 69.1 16.8 0 0 0.8 0.3 Lunulospora cytnbifortnis 1.9 8.2 3.6 2.5 36.6 0.3 Pyratnidospora jlutninea 0 1.3 14.3 0 1.6 2.4 Variosporium delicatunl 10 30.5 50 12.6 61 0.5

(B) Summer 1989

Tai Po Kau Forest Stream Alatospora pulchella 0.1 1.4 5.6 0 23.3 16.9 Flagellospora curvula 0 10 1.3 7.6 5.1 0.7 Flagellospora penicillioides 70.9 0.5 4.8 59.9 4.2 0 Lunulospora cymbiformis 14.1 68.6 22.1 14.4 0.6 0.7 Triscelospora monosporus 0 17.3 29.9 5.5 23.6 27.7 Triscelospora acuminatus 0 0 22.9 3 34.8 51.4

found. There were 14 species common to both sites and six of In the winter series, the polluted LTR yielded the highest them, Acremonium kiliense, Gliocladium roseum, Humicola (28) species richness of geofungi on leaf litter among the four grisea, Monilia sp., Trichoderma lignorum, and unknown series (Table 4). The five prevalent species, Gliocladium sp. 2 were found regardless of season. In all, a total of 28 and roseum, Fusarium oxysporum, Geotrichum candidum, Mucor 35 species of geofungi were reported in the TPKFS and LTR, racemosus, and Penicillium canescens are very common soil respectively, during the entire study period using both methods fungi, but only Gliocladium roseum was recorded in the (Table 4). TPKFS. These dominant species were detected during almost

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TABLE 4. Ranking of geofungi associated with Bauhinia purpurea L. leaf litter in the Tai Po Kau Forest Stream (TPKFS) and Lam Tsuen River (LTR) during winter 1988 and summer 1989

TPKFS LTR

Winter Summer Winter Summer

Species D MC D MC D MC D MC

Acremotzium kiliense Griitz 5.0 15.0 Acremonium strictum W. Gams Aspergillus flavipes Bain. & Sart. Aspergillus flavus Link ex Gray Aspergilhs niger van Tieghem Cylitldrocarpon mangnusianum (Sacc.) Wollenw. 17.0 6.0 Fusarium oxysporum Schlecht. Geotrichum candidurn Link ex Leman Geniculifera sp. Gleosporum fructigenum Berk. Gliocladium roseum Bain. 6.5 9.0 Gonatobotrys simplex Corda 4.0 15.0 Graphium sp. Humicola grisea Traaen 17.0 7.0 Humicola insolenus Cooney & Emerson 1.0 4.0 Monilia sp. 12.5 15.0 Monodictys levis Hughes Mucor racemosus Fres. Nectria gliocladioides Smalley & Hansen Paecilomyces variotii Bain. Papulaspora sp. Penicillium canescens Sopp. Penicillium funiculosum Thom. 6.5 15.0 Penicillium simpicissimum Thom. Pestalotia sp. Phialophora sp. Phoma sp. 12.5 1.0 Phythium sp. Sagenomella striatispora W. Gams Sclerotium sp. 17.0 2.5 Sporendonema purpurascens Mann & Hughes Sporothrix schenkii Hektoen & Perkins Sympodiella acicola Kendrick Synnematium jonesii Speare 12.5 15.0 Trichoderma glaucum Abbott 8.5 15.0 Trichoderma harziatlum Rifai 3.0 15.0 Trichodema lignorum Tode ex Harz 2.0 10.0 Trichosporon cutaneum De Beurm 10.0 15.0 Trichothecium sp. Tritntnatostroma betulinum Corda Ulocladium chartarum (Preuss) Simon 12.5 15.0 Verticillium atro-album Reinke & Berth. 8.5 2.5 Volutella ciliata (Alb. & Schw.) Fr. Sterile mycelia

sp. 1 (red-brown chlamydospore) 17.0 8.0 sp. 2 (light brown) 17.0 5.0 sp. 3 (dark green) sp. 4 (hyaline) sp. 5 (dark brown)

Yeast-like organism

Total no. of genera Total no. of species Total no. of species from D Total no. of species from MC Number of common species Similarity index (%)?

NOTE: D is the number of records from leaf discs on 1.2% plain agar (total of 24 discs) and MC is the number of records from leaf fragment incubated in moist chamber (total of 60 spots). Boldface indicates the five top-ranked species in each series.

*Results from four samplings only. ' ~ ~ r e n s e n index = (2 x S) X 100 divided by (S, + S2) where S is the number of common species, S, and Sz are the number of species from

D & MC, respectively.

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1076 CAN. J. BOT. VOL. 70, 1992

0 0 10 20 30 40 50 60 70 00 90 100 110 120

Time (days)

FIG. 3. Cellulolytic activity of enzyme extracts from decomposing B. pulpurea L. leaves in the Tai Po Kau Forest Stream during winter 1988 (solid line) and summer 1989 (broken line). Each bar represents the standard error of the mean.

the entire period of sampling. Nineteen species belonging to 16 genera were found in the TPKFS (Table 4). Of these, Humicola insolenus, Trichoderma lignorum, and Phoma sp. were constantly high in occurrence. Both Gliocladium roseum and Verticillium atro-album were among the top five ranked species but appeared during the latter stages of leaf decompo- sition.

Although the total number of species (20) found on the TPKFS leaves in summer was almost the same as that in winter, the individual numbers in each leaf sample were found to be on average five species more on the summer leaves than on the winter leaves. The similarity between species recorded by the two methods was higher (Sgrensen index = 66.7%) than in winter (41.6%). Fifteen species were detected by each of the two methods (Table 4). The five top-ranked species of the summer series (in ascending order), i .e., Sclerotium sp., Penicil- lium funiculosum, Gliocladium roseum, unknown sp. 2 , and Humicola grisea, were present in the winter leaf litter samples as well, but only Gliocladium roseum was highly ranked. Although the LTR summer study involved only four litter bag samples (due to the insufficient leaf material remaining), there were still 19 species reported. The three highest ranked winter species, Gliocladium roseum, Geotrichum candidum, and Fusarium oxysporum, were also recorded on leaves over almost the entire period of study.

Cellulolytic activity studies Figure 3 shows the cellulolytic activity in the enzyme extracts

of B. purpurea L. leaf litter in the TPKFS. The Kruskal -Wallis test indicated a significant difference ( p < 0.01) in cellulo-

lytic activities in the two seasonal studies with a higher level in the winter series. The patterns of change in enzyme level differed greatly. During the winter period, enzyme activity increased in the first 4 weeks to reach its peak and then dropped immediately from the 4th week to the 6th week. It kept declining, at a moderate pace, until the 16th week and then remained at a level similar to the initial value. During the summer period, the enzyme level in the leaf extracts was always lower than in winter. It was initially low but gradually increased from the 3rd to the 6th week and dropped again (Fig. 3).

Discussion

Aquatic hyphomycetes Conway (1970) found that the frequencies and numbers of

aquatic hypomycete species were lowest at a site downstream from sewage inputs. Results of the present study agree with this finding in that both greater species richness and conidial abun- dance of aquatic hyphomycetes was noted on B. purpurea L. leaves in the clean TPKFS than on those in the polluted LTR. This does not, however, agree with the findings of Sladeckova (1963), who found a higher species richness in polluted areas of the River Zelivka (Bohemia), or with those of Suberkropp et al. (1988), who found no difference between species rich- ness in polluted and unpolluted regions of the River Erms, Germany.

The study of Wood-Eggenschwiler and Barlocher (1983) in 16 streams in Europe showed no significant relationship between the species richness of aquatic hyphomycetes and adjacent riparian vegetation. Nutritional resources appear not to be the major factor in determining the species richness of aquatic hyphomycetes. Therefore, the difference in the sur- rounding vegetation, rich in the TPKFS and poor in the LTR, could not explain the low species diversity found in the pol- luted LTR. In addition, the lower conidial production of the three dominant fungi, Clavariopsis aquatica, Flagellosporum penicillioides, and Lunulospora cymbiformis, on LTR leaf litter compared with that on the TPKFS litter suggests that sporulation on the LTR leaves was inhibited. However, it may be that the conditions in the polluted LTR were not favourable for colonization by these fungi.

Among the physical-chemical factors studied (water temper- ature, dissolved oxygen, biological oxygen demand, pH, tur- bidity), oxygen availability would probably determine the ability of aquatic hyphomycetes to compete with other micro- organisms (mainly bacteria, algae, and other groups of fungi), since well-oxygenated water is required for growth and sporu- lation of aquatic hyphomycetes (Nilsson 1964; Webster 1975). Deposition of layers of organic material from the nearby poultry farms on the submerged B. purpurea L. leaves in the LTR created an anaerobic microclimate around the leaf litter.

Studies have been carried out worldwide on seasonal changes in conidial concentration of aquatic hyphomycetes in streams. For instance, Iqbal and Webster (1977) and Iqbal et al. (1979) examined the River Exe in England; Haeckel and Marvanova (1979) investigated a subarctic stream in Sweden; Suberkropp (1984) carried out studies in Michigan, U.S.A.; Aimer and Segedin (1985) examined a New Zealand stream; and Sridhar and Kaveriappa (1989) investigated a tropical stream in India. Despite the differences in geographical locations and climatic factors, all the studies recorded a decline in conidial numbers in streams during spring and summer, with higher numbers during autumn and winter. Higher conidial production on the

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AL.: 11 1077

winter leaf litter was indirectly related to the availability in the streams of a fresh supply of autumn-shed leaves. This can also explain the higher conidial abundance found in the vegetation- rich TPKFS. Moreover, the present investigation also revealed a significantly higher bacterial population on leaf litter in sum- mer (Au et al. 1992), so the numbers and activities of the associated bacteria may modify the development of aquatic hyphomycetes on the summer leaf litter.

Most of the aquatic hyphomycetes recorded in the present investigation have been reported frequently in temperate regions, and the three dominant species, Clavariopsis aquatica, Lunulospora cymbijormis, and Flagellospora penicillioides, have been reported to be able to grow well in a wide range of temperatures and stream conditions (Webster and Descals 198 1). C. aquatica has been found frequently in cold regions (Nilsson 1964; Webster and Descals 1981). This may explain its presence when winter water temperature dropped to 5OC in the LTR and TPKFS, at which point the other species were suppressed.

The LTR winter-dominant species Variosporium delicatum, which disappeared during summer, was also frequently found in the Dartmoor streams studied by Iqbal and Webster (1977). The water temperature of their study streams ranged from 4.5 to 20°C, coinciding with the temperature range in the LTR during winter. Pyrarnidospora flitminea, which was found only in the polluted LTR, could be considered a pollution- tolerant species.

~riscelo~hora acunzirzatus and Triscelophora monosporus were among the major colonizers on different types of leaf litter in tropical Indian streams (Sridhar and Kaveriappa 1988a, 1988b, 1989). This could explain why they were the prevalent summer species in the TPKFS. The in vitro studies of hyphal interaction among wood- and leaf-inhabiting freshwater fungi (Shearer and Zare-Maivan 1988) indicated that wood inhabit- ing species such as Clavariopsis aquatica were more inhibi-

t tory to, and more resistant to inhibition by, leaf-inhabiting species such as Triscelophora monosporus. Thus it was not surprising to find that Clavariopsis aquatica and Triscelophora monosporus were never codominants.

Geofingi Incubation of leaf litter in laboratory conditions inevitably

modified the actual living environment for the leaf associated geofungi. To prevent or minimize rapid growth of selected groups of fungi due to the presence of external nutrient sources, nonnutritious plain water agar and glass filter paper were used for the leaf disc and leaf fragment incubation. These conditions would be favourable for the growth of those species that truly rely on leaf litter as their nutrient source.

The two methods used for the detection of geofungi showed a certain disparity in species composition. The transparent nature of the plain water agar medium undoubtedly provides a better background to reveal the presence of inconspicuous fungal hyphae or sporulating structures extending from the leaf tissue when examining the leaf disc cultures under a dis- section microscope. This would lead to a higher rate of isola- tion of sterile hypha or propagules from the leaf discs than from the moist chamber.

According to Cook's checklist (1957) of fungi isolated from polluted water and sewage, among the five prevalent species restricted to the LTR leaves, Aspergillus flavus, F~uarium oxysporum, and Geotrichum candidum are all classified as lymaphiles, i.e., those that grow commonly in the presence of pollutants but may also grow elsewhere. Mucor racemosus as

well as most of the species of geofungi found on the TPKFS leaves (e.g., the prevalent species Humicola spp. and Gliocla- dium roseum) belong to the lymaxene group, i.e., those that on occasion will colonize waste materials. Although Penicillium finiculosum is lymaphilic, it was also common on the TPKFS leaves. This illustrates the difficulty of applying strict defini- tions, since as long as the available substrates and the environ- ment are suitable, lymaphiles also are able to survive in unpolluted waters. Neither Penicillium canescerls nor Tricho- derma lignorum appear on Cook's (1957) list, but they were very often isolated from LTR leaf litter. This implies that they should be included in the lymphophile list.

Concerning the succession pattern of geofungi on B. purpurea L. leaf litter, it was found that most of the prevalent fungi were dominant from the initial stages of decomposition. This differs from the succession pattern described by Hudson (1968) for plant litter in the terrestrial environment.

The species numbers of geofungi in the winter series as well as the overall total species numbers were higher on the pol- luted LTR leaves than on the TPKFS leaves. The opposite was true in the case of the aquatic hyphomycetes. In fact, geofungi can tolerate low levels of oxygen concentration, high levels of organic pollution, or sometimes both (Cooke 1976). They should thus have a competitive advantage over the aquatic hyphomycetes in the anaerobic microclimate on leaves sub- merged in the organically polluted LTR.

With the decline in the aquatic hyphomycete diversity in summer, the species diversity of geofungi on individual TPKFS leaves was higher. No such trend was observed in the LTR leaves, possibly due to the fact that the aquatic hyphomycete communities were less well developed. In addition, data for the LTR summer study were not complete.

Cellulolytic activity studies As pointed out by Barlocher and Kendrick (1976) and Fell

and Newel1 (1981), fungi are superior to bacteria in their ability to degrade cellulose and lignin because of the ramifying nature of the fungal hyphae, whereas bacteria are often restricted to substrate surfaces only. In the present study, no parallel trend was found between the abundance of heterotrophic bacteria and cellulolytic activity in the leaf litter, and the higher bac- terial counts in the summer leaf litter did not accompay a higher specific activity of CMCase (Au et al. 1992). Though the enzymatic activity of microbes may not necessarily be related to their abundance, the result at least revealed that the number of bacteria on the leaf litter has no influence on the overall microbial cellulolytic activity.

Hodgkiss and Leung (1986) claimed that the detection of cellulolytic activity could be used as an indirect biochemical indicator of fungal activity in decomposing mangrove leaves as well as a measure of general microbial activity. Numerous studies have shown that aquatic hyphomycetes are able to assimi- late not only soluble cellulose substrates, e.g., CMC, but also the native cellulose microfibrils of plant cells (Nilsson 1964; Suberkropp and Klug 1980, 1981; Jones 1981; Singh 1982; Chamier et al. 1984). The comparatively low species richness and conidial production of aquatic hyphomycetes on the summer leaf litter was also accompanied by a lowering of cellulolytic activity. The peak cellulolytic activity at day 28 of the TPKFS winter series corresponded to the codominance of Flagellospora penicillioidies, Lemmonniera sp., and Lunulospora cyrnbifomis, whereas Lemtnoniera sp. was replaced by Flagellospora curvula and Triscelosporus monosporus in the summer leaf litter

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(Table 3). These are the aquatic hyphomycete species that had been tested for cellulolytic activities by the authors mentioned above. The prevalent geofungi associated with the TPKFS leaves, e .g. , species of Gliocladium, Humicola, Trichoderma, Penicillium, and Sclerotium sp., are found to be cellulose- producing organisms of established o r potential commercial use and are capable of degrading native cellulose (Bisaria and Ghose 1981; Coughlan 1985; Gomes et al . 1989).

Barlocher and Kendrick (1974) found that decom~osit ion of leaves proceeded faster if the five species of aquatic hypho- mycete species tested were inoculated together onto the leaf discs rather than just a single species. This may be explained by the synergistic interactions among fungi. It is also true that no enzyme complex of any species is as efficient as the range of enzymes expressed by a group of associated fungi (Chamier and Dixon 1982).

In conclusion, it is clear that the organic pollution in the LTR is one of the decisive factors resulting in a comparatively poor species diversity and spomlation of aquatic hyphomycetes and a more diversified geofungi community on the submerged B. purpurea L. leaf litter. he seasonal studies in the clean TPKFS also suggest that aquatic hyphomycetes and geofungi have a complementary sequence of dominance on leaf litter in winter and summer, respectively. The higher aquatic hypho- mycete activity in the winter coincided with the higher cellulo- lytic activity exhibited in the leaf litter. The majority of the dominant fungi have been shown to demonstrate cellulolytic activity. Further studies of the cellulase production of the fungi associated with decomposing leaves in freshwater habitats are essential to a better understanding of the fungal succession pattern in freshwater detritus systems.

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