Filamentous fungi isolated from sand and water of “ Bairro Novo ” and “ Casa Caiada ” beaches , Olinda , Pernambuco , Brazil

Fungi comprehend a heterogeneous group of heterotrophic microorganisms that act as saprobes or parasites or, less frequently, as symbionts living in association with other organisms. They are cosmopolitan and important components of ecosystems. Considering the small number of Brazilian papers on the filamentous mycota in marine environments, and the need to improve knowledge of the diversity of these microfungi in “Casa Caiada” and “Bairro Novo” beaches, Olinda, PE, the aim of this work was to isolate and identify the fungi from sand and water samples of these ecosystems. Thirty two samplings of sand (surface and 20 cm deep) and water (surface and 1 m deep) were carried out during the dry and rainy seasons, in low and high tide. From each sand sample, a suspension was made with 50 g of sand diluted in 90 mL of sterilized distilled water. From each sand suspension and water sample, 0.5 mL was spread, in triplicate, onto Petri dishes containing Sabouraud Agar added of chloramphenicol. The plates were incubated at 28 °C ( 2 °C). Fifty seven species were isolated, identified, and classified in 20 genera. Aspergillus and Penicillium were the most frequent genera in both sand and water, with a total of 11 and 19 species, respectively.


Introduction
"Bairro Novo" beach is approximately 2 km long, with 50 m long coast protection walls built perpendicularly to the beach at intervals of 50 m.There are also man-made reef rocks along the sand for breaking the waves.There are many environmental problems like marine erosion, pluvial water and domestic waste disposal on the beach, causing the water to be inappropriate for bathing and contact sports in some areas."Casa Caiada" is an urban beach with calm waters and artificial reefs.It is approximately 4.5 km long, with a hotel on the shore.The beach is used for bathing, soccer and contact sports, as well as fishing and mollusk catching.Its main environmental problems are disposal of pluvial waters, domestic waste and solid residues (Beltrão et al., 1995).Fungi are important components of ecosystems as they are cosmopolitan and usually isolated from tropical, subtropical and temperate countries (Smith and Berry, 1975).They are considered the most active microorganisms in the decomposition of organic compounds both in sand and water (Harley, 1971;Moore-Landecker, 1996).When compared to the thousands of fungal species known from terrestrial environments, only 500 have been described for oceans and estuaries which comprehend the largest part of the Earth's surface (Kohlmeyer and Kohlmeyer, 1979;Kohlmeyer and Volkmann-Kohlmeyer, 1991).The described species are mainly the anamorphous of Ascomycota and Basidiomycota, including some marine yeasts.Some of these marine filamentous fungi are parasites of marine algae or marine angiosperms, or they grow symbiotically with brown algae (Kingham and Evans, 1986).Most of the papers referring to filamentous fungi in marine environments are centered on Europe and North America (Dabrowa et al., 1964, Kishimoto andBaker, 1969;Berner and Wagner-Merner, 1977;Kirk, 1983;Udagawa and Ueda, 1985;Tan, 1985).In Brazil, studies such as Faraco and Faraco (1974), Mattede et al. (1986), Purchio et al. (1988), Pinto et al. (1992) and Sarquis and Oliveira (1996) may be highlighted."Casa Caiada" and "Bairro Novo" are the main beaches in Olinda.However, they present serious environmental problems as they are intensively visited by tourists and locals.The mycota of these beaches is practically unknown, especially with respect to human pathogens.Therefore, considering the lack of studies on the diversity of filamentous fungi in Olinda, the aim of this work was to isolate and identify filamentous fungi from the beach sand and water samples from "Casa Caiada" and "Bairro Novo", in the dry and rainy seasons.

Study area
Olinda city is approximately 40.83 km ² located between the parallels 7° 57' 30" S and 8° 02' 30" S and the meridians 39° 49' 41" W and 39° 55' 00" W. It is delimited by the city of Paulista to the North, the Atlantic Ocean to the East, and the city of Recife to the South.The original vegetation covering this area was composed of the Atlantic Forest and its related ecosystems: mangroves and "restinga".According to Köppen's general classification, Olinda's weather is of the type As', hot and humid, with a rainy season during autumn and winter, lasting from March to August, with the coldest temperature of 18 °C and the mean temperature of 27 °C.The months with the greatest amount of rain are May (224.4 mm) and July (453.3mm), and the annual precipitation varies from 1,000 to 2,000 mm with the annual average ranging from 74 to 94%.The beaches of "Casa Caiada" and "Bairro Novo" are the main beaches in Olinda.As urban beaches, they are intensively visited by tourists and locals, and present serious environmental conflicts (Beltrão et al., 1995).The sampling points were chosen in the regions with the greatest access of bathers and release of domestic sewage.

Sand and water samplings
Sand and water samplings were concomitantly collected in "Bairro Novo" and "Casa Caiada" beaches, in December/2000 and February/2001 (dry season) and June and July/2001 (rainy season).Thirty two sand samples and 32 water samples were collected in syzygy's low-tide and high-tide, based on the harbor of Recife tide tables (Brasil, 2000).The sand sampling in each site was carried out using a garden shovel, in mid-seashore at 1 m from the tideline, on the surface and at 20 cm deep.The samples were placed in labeled plastic bags.Surface and 1 m deep water samples were taken in each sampling site using sterile labeled glass tubes.The sand and water samples were kept at room temperature and transferred to the laboratory at the Dept.Mycology (Universidade Federal de Pernambuco).

Isolation, purification and identification of filamentous fungi
Each sand sample (50 g), was diluted in 90 mL of sterilized distilled water; 0.5 mL of this suspension was spread, in triplicate, onto Petri dishes containing Sabouraud Agar (SA) added of chloramphenicol (500 mg.L -1 ).Each water sample (0.5 mL, undiluted) was also spread onto Petri dishes as above.The plates were incubated at 28 °C ( 2 °C).As soon as the first colonies were developed, they were transferred to test tubes containing SA.After the purity of the colonies was confirmed, they were subcultured onto Potato Dextrose Agar, Malt Agar or Czapeck, in glass tubes.Fungal identification was carried out by macroscopic and microscopic observation of colonies and when needed, by microculture on a microscope glass slide (Riddell, 1950).Raper and Thom (1949), Booth (1971), Ellis (1971), Ellis (1976), Raper and Fennell (1977), Domsch et al. (1980), Sutton (1980) and Pitt (1985) were mainly used for species identification.

Sand and water abiotic data
Sand and water pH and temperature were measured with a digital pH-Meter and a digital thermometer (Hanna), respectively.Species frequency.The occurrence of fungal species was calculated according to Dajoz (1983), using the formula Fo = Ta.100/TA,where Ta = number of samples in which a taxon has occurred, TA = total number of samples.Values were considered according to the following classification: <10% = Rare, 10 25% = Low frequency, 25 <35% = Frequent, 35 < 50% = Abundant, and > 50% = Very abundant.

Sand and water abiotic data for "Bairro Novo" and "Casa Caiada" beaches
Factors such as water salinity, temperature and pH may influence in the activity, abundance and distribution of marine fungi (Dix and Webster, 1995).The water temperature of "Bairro Novo" and "Casa Caiada" was between 24.3 °C and 29.4 °C in the dry season (December and February).In the rainy season (June and July), the minimum water temperature was 25.3 °C while the maximum temperature was 28.2 °C.The sand temperature in the dry season reached its minimum at 25.7 °C and maximum at 29.4 °C, and in the rainy season, it was between 24.4 and 28.8 °C.In general, marine fungi need high temperatures (usually between 25 and 30 °C), to reproduce (Griffin, 1981).All water and sand samples had a slightly alkaline pH, varying from 7.6 to 8.2.In the "Bairro Novo", the water and sand salinity was of 20 and 40‰ in the rainy and dry seasons, respectively.Water and sand salinity in "Casa Caiada" was of 24 and 39‰ in the dry and rainy seasons, respectively (Table 1).According to Gambale et al. (1977), salinity has a great influence in the microbiota of the estuaries.However, very little is known about the distribution of Ascomycetes and anamorphous fungi related to water temperature.Some observations made by Borut and Jonhnson (1962) on the fungi isolated from estuary sediments on North Carolina, EUA, showed that the germination and development of the fungi was influenced by salinity.
Isolation and identification of filamentous fungi from sand and water, during the dry and rainy seasons, on "Bairro Novo" and "Casa Caiada" beaches.Thirty six species of filamentous fungi were isolated and identified from the sand and water samples taken from "Casa Caiada" during the dry and rainy seasons.Most of the species were of anamorphous fungi.Penicillium and Aspergillus were represented by 11 and 8 species, respectively, followed by Cladosporium (3), Emericella (2), Acremonium, Chaetomium, Cunninghamella, Curvularia, Eurotium, Fusarium, Lasiodiplodia, Monilia, Paecilomyces, Phoma, Tilachlidium and Trichoderma (1) (Figure 1).In the "Bairro Novo", during the dry and rainy seasons, 44 species of filamentous fungi, mostly anamorphous, were isolated and identified.These were mainly represented by Penicillium and Aspergillus, with 14 and 9 species, respectively, Fusarium and Trichoderma (4), Cladosporium (3), Absidia, Acremonium, Colletotrichum, Curvularia, Emericella, Eurotium, Paecilomyces, Phoma, Spegazzinia and Stilbella (1) (Figure 2).Some species were common for both beaches, like Aspergillus flavus Link, A. janus Borut and Johnson (1962) and Stilbella clavispora Seifert is mentioned as present in a marine environment, probably for the first time.Tilachlidium brachiatum Petch is mentioned for the second time in a marine environment; the first occurrence was registered on Boa Viagem beach, Recife, Pernambuco, by Pinto et al. (1992).The main pathogenic fungi to men and animals can be found within the anamorphic fungi.These fungi are saprophytic and occasionally pathogenic, and can be isolated from water, soil, animals and humans.Species of Aspergillus, Cladosporium and Penicillium, found in these beaches, can be a source of infection for superficial and deep mycosis (Sidrim and Moreira, 1999).Most of the filamentous fungi isolated from the beaches of "Casa Caiada" and "Bairro Novo" were anamorphous.The same was observed in researches carried out in beaches of California (Dabrowa et al., 1964), Hawaii (Kishimoto and Baker, 1969), Florida (Bergen and Wagner-Merner, 1977), and Brazil, in Boa Viagem (Pinto et al., 1992) and Ipanema (Sarquis and Oliveira, 1996).The number of genera in "Casa Caiada" was higher than in "Bairro Novo".The reason for this may be the fact that "Casa Caiada" is more frequently visited by tourists and local bathers than "Bairro Novo".This certainly contributes to increasing the pollution.Similar facts were observed in research carried out by Kishimoto andBaker (1969), Bergen andWagner -Merner (1977) and Purchio et al. (1988), who obtained a higher incidence of fungal genera in the beaches of Oahu (Hawai), Tampa (EUA), and São Vicente and Bertioga (São Paulo).In the studies carried out by Mattede et al. (1986), the opposite was found.The authors evaluated dry and wet sand samples from polluted and non-polluted beaches of the city of Vitória, Espírito Santo.The incidence of fungal genera was greater in non-polluted beaches (55%) than in polluted ones (45%).The frequency and incidence of fungi varied and pollution factors inhibited some species.According to Gambale et al. (1983), the differences of fungal genera found in this study may be related to the sampling methods, to the geographical location and to the method of data analysis.

Figure 2 .
Figure 2. Filamentous fungi isolated from "Bairro Novo" beach during the dry and rainy seasons.

Table 1 .
Temperature, pH and salinity of the water and sand from "Bairro Novo" and "Casa Caiada" beaches.