Research Article
Aquatic Pycnidial and Hyphomycetes Fungi from Macrophytes and Riparian Plants in the River Nile
Department of Botany and Microbiology, Faculty of Science, Helwan University Cairo, Egypt
Aquatic pycnidial fungi, have been reported from submerged shoots of aquatic macrophytes including Phragmites, Carex and Schoenoplectus (Webster and Descals, 1981; Soars and Barneto, 2006; Kobayashi et al., 2005; Collade et al., 2006).
Macrophytes have several intrinsic properties that make them an indispensable component of constructed wetlands. The most important functions of the macrophytes in relation to the treatment of wastewater are the physical effects brought about by the presence of the plants. The macrophytes stabilize the surface of the beds, provide good conditions for physical filtrations, prevent vertical flow systems from clanging, insulate against frost during winter and provide a huge surface aero for attached microbial growth (Brix, 1994).
Anthony (1999) examined the occurrence of macro fungi and the decay of roofs thatched with water reed, Phragmites australis. Sampling from 20 north- and 20 south-facing roof sides showed that several ascomycetes usually associated with reed in situ are common on thatch. The only basidiomycetes recorded were Mycena species.
Aquatic Hyphomycetes occur commonly on a wide variety of decaying submerged plant substrates in fresh water (Barlocher and Kendrick, 1981; Chauvet, 1992; Gessner and Chauvet, 1994; Descals et al., 1995; Faber, 1998a, b). Many of these spore found in terrestrial habitat (Bandoni, 1981; Fisher et al., 1991; Shearer, 1993, 2001). With the exception of studies by Yuen et al. (1998) and Abdel-Raheem and Shearer (2002) little is known about the enzymatic capabilities and decomposition activities of these fungi. These fungi produce a wide range of plant cell wall degrading enzymes (Chamier, 1985; Abdel Rheem and Shearer, 2002).
In order to decompose plant litter, saprotrophic microbes produce extracellular enzymes. The most relevant enzymes from this aspect involve those that break down the plant fibers (cellulases, hemicellulases, pectinases, phenoloxidases, chitinases) as well as enzymes important for microbial acquisition of nitrogen and phosphorous (peptidases, ureases and phosphatases) (Sinsabaugh et al., 2002). Fungi in general have a more forceful enzymatic capacity than other microbes.
Several of the fungal species generally considered to be terrestrial can also be found in aquatic systems, but also truly aquatic species have been shown to produce a wide range of enzymes (Zemek et al., 1985; Abdel-Raheem and Shearer, 2002).
Decomposition of Phragmites has been found to relate to activity of cellulolytic and xylanolytic enzymes, which can be produced by fungi and other microbes (Tanaka, 1991, 1993; Boschker et al., 1995).
Present study was carried out to inveswtigate Aquatic pycnidial and Hyphomycates fungi occurring on root of plant species near to fresh water of River Nile at Qanatir city and determined the kinds of enzymes produced by pycnidial and Hyphomycetes fungi that could play a role in plant decomposition.
Description of Study Sites
Twigs and branches of aquatic macrophytes and aquatic different plants that grown on wet slopes and water edges were collected at El-Qanatir city front of the bridge about 1 km at sandy area flooded with water frequently. The vegetation in the mire in which the river rises is dominated by Cyperus papyrus, Phragmites australis, Persicaria senegalensis, Persicaria lapathifolia, Eclipta prostrata, Rorripa palustris, Rumex dentatus, Cyperus articulatus, Cyperus alopecroides and Pseudognaphalium luteo-album and Persicaria salicifolia.
Trapped twigs and branches were collected in clean blastic bags and returned to the laboratory for examination and incubation. The twigs were examined under a dissecting microscope at a magnification of x 50. Spores that were found on the wood were removed by a dissecting needle and fungal species were recorded immediately. After that, the twigs were put in a plastic lunch box lined with moist tissue paper. They were examined within 1 week of incubation and at regular intervals for up to 3 months at 27°C. Aquatic pycnidia present were recorded. Isolations were made for some of the fungi on malt and patato, dextrose agar media. Identification of pycnidial fungi were carried out according to Sutton (1980).
The physico-chemical characteristics of water samples during the investigation were as follows: the temperature ranged from 18°C (winter) to 34°C (summer), the pH from 9.5-10.4 and the dissolved oxygen from 3.3-11.4 (ppm).
Culture Media
Malt Extract Agar Medium
This medium was used for isolation of aquatic pycmidia and it has the following composition (g L-1): malt extract, 1; agar, 15; distilled water, 1000 mL. For control of bacterial growth, 0.1% crystamycin was added to the warm agar medium (Descals et al., 1977). Crystamycin is mixture of penicillin and streptomycin. When required, 1 mL of the antibiotic was pipetted into the bottom of the petri-dish before the warm medium was poured. Single conidia were located and cultures were prepared by transferring single germinated spores to 2% Malt extract agar media.
Production of extracellular enzymes was determinel by incorporation of test substrates into a basic medium, inoculating the medium with discs of fungal hyphae, allowing the fungi to grow out on the medium and adding reagents to the plates to detect the test substrate remaining. Colony radial growth rates and substrate clearing zones were measured for each fungal species on each substrate. Three replicates of each treatment were assayed and non-inoculated plates with substrates served as negative controls. Inoculated plates were checked at 5-7 days depending on the growth rates of the individual species.
Amylase
Amylase activity was assayed by growing the fungi on starch medium (starch, 2 g; peptone, 1 g; agar, 20 g; distilled water, 1 L). After 5-10 days, the plates were flooded with a 1% aqueous IKI solution. A yellow one around the colony in an otherwise blue medium was considered a positive test for starch hydrolysis (Gessner, 1980).
Proteolytic Enzyme
The medium used to detect proteolytic enzyme activity contained gelatin as the protein substrate (Hankin and Anagnostakis, 1975).
Pectolytic Enzyme
To detect pectolytic activity, we used the medium described by Liao and Wells (1987).
Cellulase
The basal media were supplemented with carboxymethyl cellulose, were proved to be best sources for the production of cellulase, according to Bland and Douglas (1977) and Datta et al. (1989).
Effect of Ground Twigs of Rorripa palustris and Persicaria salicifolia on Growth
Samples were dried separately at 50°C for 2 days, after which time they were finely milled. The mill (2g L-1) was added to 2% MEA medium. These samples were thoroughly mixed and poured into Petri-dishes aseptically. Cultures were cut into 1 cm disks using a flamed cork borer and transferred to the petri-dishes that contained the twigs powders and the plates were incubated at 25°C for 10 days. Three plates were prepared for each organism and the increase in colony diameter was measured as usual.
Results shown in Table 1 showed 10 aquatic pycnidial fungi, 2 Hyphomycetes and one Ascomycetes isolated from submerged plant substrates were selected for study: five species have been reported only from Phragmites australis (Dinemasporium cytosporoides, Sordaria sp. Cylindrocarpon sp. Camarosporula sp. and Cheilaria agrostis. Two species have been reported from Cyperus articulatus (Cystotricha striola and Leptomelanconium australiense. Four species have been recorded on Cyperus papyrus (Cylidrocarpon sp. Clypeopycnis aeruginascens, Fusarium sp. and Pleurothyrium sp.).
Table 1: | Fungal strains and substrate isolated from which they were isolated |
1 = Cyperus articulatus, 2 = Cyperus alopecroides, 3 = Phragmitesaustralis, 4 = Persicaria salicifolia, 5 = Pseudognaphalium luteo-album, 6 = Persicaria lapathifolia, 7 = Rumex dentatus, 8 = Rorripa palustris, 9 = Cyperus papyrus, 10 = Eclipta prostrate, 11 = Persicaria senegalensis, 12 = % = Frequency of occurrence of fungus, + = Fungus recorded, - = Fungus not recorded |
Clyeopycnis aeruginascens was found on most plants and it was only found on Rorripa palustris, although other species were not faund on it. Sordaria sp. and Cammarosporula sp. were only found on Phragmites australis.
Koriniak and Belomesytseva (2005) were identified 45 fungus species occurring in confierous forests of the Minsk elevation 8 genera living on 26 plant species from 18 families. Two new fungal were recorded from aquatic weeds native to Barzil and were described Passalora barretoana stat-et comb-nov. and Paraphaeosphaeria michotii. The latter was described in association with its anamorph, which belong to the genus Microsphaeropsis (Soares and Barreto, 2006). Two new pycnidial members of the Atractiollapes: Basidiopycnis hyallina and Proceropycnis pinicola (Oberwinkler et al., 2006).
Among plant-inhabiting fungi, Kobayashi et al. (2005) were described four fungi that found among plant-inhabiting collected in June 2001 and in September 2002 on Hachijo Island, Tokgo They consist of two new species, namaly Stagonospora hachijoensis on Miscanthus sinensis var. Condensatus and Ascochyta ixorae on Ixora Chinensis and two fungi newly added to the Japanese mycoflora, namely Discosiella cylindrospora on Callistemon speciosum and Robillarda sessilis on Parthenocissus tricuspidatus. Van Ryckegen and Verbeken (2000) were discovered a new Rosellinia species on dead culms of Phragmites australis.
We have summarized the occurrence and frequency values for the pycnidial fungi and Hyphomycetes for Nile River at El Qanatir as colonizers of submerged of riparian plant roots (Table 1). The maximum occurance was on Cyperus alopecroides, Phragmites australis and Cyperus papyrus. Clypeopycnis aeruginascens showed maximum frequency of occurance (60%) followed by Cylindrocarpon sp. and Fusarium sp. (50%) Pleurothyrium sp. had 14% frequency of occurrence. The minimum frequency of occurrence was recorded for the remaining species (4.5%). Rorripa palustris (Brassicaceae) and Persicaria salicifolia were not colonized by pycnidial fungi and Hyphomycetes only one or two species were found after long incubation time at 25°C. This results might be due to the presence of some substance (allelopathic compound) inhibited the growth of these fungi. McCarthy and Hanson, (1998) recorded that the production of potentially allelopathic compounds in members of the Brassicaceae did not have to mean that allelopathy was involved in the success of members of this family invading woodlands as exotic weeds.
Anthony (1999) were recorded eleven species (Ascomycetes and Basidiomycetes) in total on Phragmites australis and the average of 2.4 species per roof did not increase with the age of thatch or degree of decay.
Generally, species from macrophytes substrates and woody riparian substrates had the fastest growth rates on PCA and most of the test substrates (Table 2). The least growth generally occurred on 2% MEA which added to it ground Rorripa twigs and 2% MEA which added to it ground Persicaria twigs media. Otherwise, all species grew to some degree on most test substrates.
Table 2: | Colony diam (cm) after growth for seven days on the different test media |
MR = Malt extract agar media + ground Rorripa palustris twigs, MP = Malt extract agar media + ground Persicaria salicifolia twigs, AM = Amylase; Pr = Proteolytic; PEC = Pectinase; CL = Cellulase |
Table 3: | Production of extracellular enzymes by Aquatic pycnidial and Hyphomycetes fungi |
++ = Strong reaction; + = Weak reaction; - = No reaction; AM = Amylase; Pr = Proteolytic enzyme; PEC = Pectinase; CL = Cellulase |
Table 4: | Production of extracellular enzymes by pycnidial and Hyphomycetes fungi; as measured by width of clearing zone (cm) |
AM = Amylase; Pr = Protease; PEC = Pectinase; CL = Cellulase, - = No reaction |
Most of species were positive for cellulase and pectinase (Table 3). Cystotricha striola was able to degrade all substrates tested. Libertella faginea and Fusarium sp. and Sordaria sp. Fusamen amenamentorum were postive for all enzymes except amylase. One species (Cystotricha striola) was positive for amylase. Six species were positive for proteolytic enzyme while 12 species were positive for pectinase and cellulase. Danninger et al. (1979) were investigated the ability of five aquatic Hyphomycetes to produce amylase, pectinase and cellulose. They found that all the tested strains were week productions of amylase and good producers of pectinase, whereas degradation of cellulose was only found with two strains. Boschker et al. (1995) suggested that enzymatic hydrolysis of polysaccharides in common reed litter (Phargmites australis) which contains cellulose and arabinose as its main polysaccharides, was the main source of glucose, xylose, arabinose and galactose accumulation which was probably caused by lyses of the microbial population in toluene-treated reed litter.
Cylindrocarpon sp., Cystotricha striola, Sordaria sp. were able to degrade to some degree, starch, protein, pectin and cellulose (Table 4). Fusamen amenamentorum and Clypeopycnis aeruginascens were only decomposed protein, pectin and cellulose, but Fusarium sp. degraded only pectin and cellulose. Although all species grow on test media but not all produce enzymes (Table 3 and 4). Positive growth but negative enzymes results could be due to the ability of the fungus to use other materials in the medium rather than the test substrate. These results were similar to those recorded for tropical freshwater fungi (Yuen et al., 1998).
Pettersen (1984) found that, the most abundant polymer in wood, cellulose, may account for about 40-50% of dry weight of temperate woods. Native cellulose requires three hydrolytic enzymes acting synergistically for its complete degradation (Kirk and Cowling, 1984). All species in this study tested positive for cellulose. Rohrmann and Molitoris (1992) recorded cellulose activity on acid-swollen avicel for marine Ascomycetes and Raghukumar et al. (1994) recorded about 80% of the marine species they tested were positive for cellulotic enzymes.
Au et al. (1992) reported that tested aquatic hyphomycetes showed higher cellulolytic activity in the winter than summer leaf litter. On the other hand, Parado and Forchiassim (1999) found that temperature between 50-55°C was the optimal temperature for cellulose system in Nectria catalinensis.
This study demonstrates that aquatic pycnidial and Hyphomycetes which found on submerged substrates of macrophytes are able to produce many extracellular enzymes in the decomposition of these substrates and species specificities in types of substrates decomposed.
Author is very grateful to D. Emad M. Farahat for collecting plants and for their identification.