Microfungal diversity in the swash zone interstitial water (SZIW) of three Ligurian urban beaches (NW, Italy)
10.6092/issn.2531-7342/6778
È stata studiata l’abbondanza e la composizione specifica della comunità microfungina presente nell’acqua interstiziale di battigia in tre spiagge urbane ubicate in prossimità di Genova caratterizzate da una forte pressione antropica. In particolare si è voluto: 1) caratterizzare la diversità microfungina nell’acqua interstiziale di battigia; 2) verificare la presenza di funghi potenzialmente patogeni e/o allergenici per l’uomo; 3) studiare l’influenza dei parametri biotici e abiotici sulla distribuzione delle specie fungine. A tal fine sono state effettuate tre campagne di campionamento (aprile, luglio e settembre 2013) nelle quali sono stati raccolti dati qualitativi e quantitativi. Complessivamente sono stati isolati e identificati 1157 morfotipi (MTU) appartenenti a 52 taxa. Tra i 52 taxa identificati, il 45% era comune a tutte e tre le spiagge. Sono state isolate diverse specie opportuniste (principalmente appartenenti al genere Aspergillus) e allergeniche (come Aspergillus, Cladosporium, Mucor, ecc). Inoltre, sono state individuate diverse colonie della specie patogena Aspergillus niger. Per quanto riguarda i dati quantitativi solo il mese di campionamento sembra influenzare il numero di MTU presenti.
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Mirca Zotti
Italian Journal of Mycology
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Microfungal diversity in the swash zone interstitial water (SZIW) of three Ligurian urban beaches (NW, Italy)
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2017-03-16
2017-01-24
2018-08-20
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Ital J Mycol
Mirca Zotti
2017/03/16
10.6092/issn.2531-7342/6778
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2531-7342
anthropic impact
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Microfungal diversity in the swash zone interstitial water (SZIW) of three Ligurian urban beaches (NW, Italy)
https://italianmycology.unibo.it/article/download/6778/6579
46
S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,               Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778




  Microfungal diversity in the swash zone interstitial
  water (SZIW) of three Ligurian urban beaches
  (NW, Italy)
  ________________________________________________________________________________

        Simone Di Piazza 1*, Susanna Baiardo 2, Grazia Cecchi 1, Elia Ambrosio 1,
                             Chiara Paoli 2, Paolo Vassallo 2, Mirca Zotti 1
  1
   Laboratorio di Micologia, 2Laboratorio di Ecologia
  Dipartimento di Scienze della Terra dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso
  Europa 26, 16126 Genova, Italy.

  Correspondig Author e-mail: simone.dipiazza@unige.it; Telephone: +39010352169 Fax: +39010352169

  Abstract

      Microfungal species abundance and specific composition of three urban beaches near Genoa (Liguria, NW
      Italy) in the swash zone interstitial water (SZIW) were evaluated. These beaches are characterized by a high
      presence of tourists. The investigation was carried out in order to: i) characterize the beach SZIWs microfungal
      diversity; ii) evaluate the presence of potential pathogenic and/or allergenic fungi for human; iii) investigate the
      influence of biotic and abiotic parameters on fungal species distribution. Qualitative and quantitative data were
      collected during April, July and September 2013.
      On the whole, 1,157 Morphological Taxonomic Units (MTUs), belonging to 52 taxa, were identified. Among
      these 52 taxa, 45% were common to all three beaches. The majority of them were saprotrophic species. Several
      opportunistic (mainly belonging to genus Aspergillus) and allergenic (such as Aspergillus, Cladosporium,
      Mucor, etc) fungi were found. Moreover several colonies of pathogenic species Aspergillus niger were found.
      Concerning the quantitative results, only the month (sampling period) seems to affect the amount of MTUs.

      Keywords: filamentous microfungi; fungal community; environmental stress; seashore; anthropic impact.


  Riassunto

      È stata studiata l’abbondanza e la composizione specifica della comunità microfungina presente nell’acqua
      interstiziale di battigia in tre spiagge urbane ubicate in prossimità di Genova caratterizzate da una forte
      pressione antropica. In particolare si è voluto: 1) caratterizzare la diversità microfungina nell’acqua interstiziale
      di battigia; 2) verificare la presenza di funghi potenzialmente patogeni e/o allergenici per l’uomo; 3) studiare
      l’influenza dei parametri biotici e abiotici sulla distribuzione delle specie fungine. A tal fine sono state
      effettuate tre campagne di campionamento (aprile, luglio e settembre 2013) nelle quali sono stati raccolti dati
      qualitativi e quantitativi.
      Complessivamente sono stati isolati e identificati 1157 morfotipi (MTU) appartenenti a 52 taxa. Tra i 52 taxa
      identificati, il 45% era comune a tutte e tre le spiagge. Sono state isolate diverse specie opportuniste
      (principalmente appartenenti al genere Aspergillus) e allergeniche (come Aspergillus, Cladosporium, Mucor,
  8
S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,            Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778



    ecc). Inoltre, sono state individuate diverse colonie della specie patogena Aspergillus niger. Per quanto riguarda
    i dati quantitativi solo il mese di campionamento sembra influenzare il numero di MTU presenti.

    Parole chiave: funghi filamentosi; comunità fungina; stress ambientale; costa; impatto antropico.

  Introduction

  Marine coastal environments are complex and extreme ecosystems characterized by a high level of
  biodiversity (Hyde 1989; Gonzalez et al. 1998; Migahed 2003; Babu et al. 2010; Gonçalves de Oliveira et al.
  2011). The human exploitation of beaches often alters ecological balance, and causes the deterioration of the
  environment itself (Lucrezi et al. 2009; Montefalcone et al. 2009). In this scenario, urban beach represents an
  interesting and multidimensional environment subjected to the marine, terrestrial and human influence
  (Salvo et al. 2005; Roca and Villares 2008). Several studies have shown that urban beach soils host a great
  number of organisms, such as small invertebrates, bacteria, fungi, virus, algae, and diatoms which can adapt
  in constantly changing environments (Mancini et al., 2004; Goncalves de Oliveira et al., 2011; Zakaria et al.,
  2011). Specifically, Moore-Landecker (1996) has demonstrated that microfungi are important components of
  beach ecosystems, in both soil and water. Indeed, they play a key role in nutrient cycle decomposing
  complex organic substrates such as cellulose, and animal products, biodegrading hydrocarbons, or living as
  opportunistic parasite on marine organisms (Gonzales et al., 1998; Kohlmeyer and Volkmann-Kohlmeyer,
  2003; Vogel et al., 2007; Gomes et al., 2008; Babu et al., 2010; Matallah-Boutiba et al., 2011; Zakaria et al.,
  2011).
  However, the microfungal communities of urban beach swash zones are poorly investigated and only few
  studies are available till now (Vezzulli et al., 2009; Onofri et al., 2011; Vassallo et al., 2012). These zones
  represent a dynamic environment, characterized by large sediment transport rates, rapid morphological
  changes, and high turbulence levels (Masselink and Puleo, 2006). Some authors compared several beach
  habitats (e.g. backshore, foreshore) and found a microbial reservoir and a high level of species richness in
  swash zones (Vassallo and Fabiano, 2005; Vezzulli et al., 2009; Onofri et al., 2011; Vassallo et al., 2012).
  Owing to their wide ecological range (plasticity), several urban beach microfungi may also behave as
  opportunistic or pathogenic (Hyde, 1989; de Hoog et al., 2000; Heaney et al., 2009; Lee et al., 2011),
  becoming a human health potential risk. Nevertheless, urban beach microorganism communities have been
  often neglected and real health risks have not been assessed (Hyde, 1989; Salvo and Fabiano, 2007; Vezzulli
  et al., 2009; Onofri et al., 2011). Recently, the study of such communities has received a greater attention in
  view of the safety of beach environments since they are very attractive and intensely visited recreational
  areas (Guidelines for recreational water environments – Volume 1 Coastal and Fresh Waters – World Health
  Organization, 2003). Therefore, the investigation of urban beaches swash zone interstitial water (SZIW)
  mycobiota results essential to know the related mycological biodiversity, to prevent potential sources of
  pollution, and possible contaminations of adjacent waters. In addition, the study of SZIW mycobiota permits
  to estimate the quality and health of urban beaches, and to assess the risks of potential contaminations by
  opportunistic, pathogenic or allergenic fungi.
  The paper deals with the microfungal species abundance and diversity of three urban beaches SZIWs in
  Liguria (NW Italy) and presents the results of some statistical tests on the potential influence of biotic and
  abiotic parameters on microfungal communities.
  The main goals are: i) characterize the beaches SZIWs microfungal diversity; ii) evaluate the presence of
  potential pathogenic or allergenic fungal species to humans; and iii) investigate the influence of biotic (in


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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,             Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778



  particular human frequentation, organic matter content) and abiotic (seasonality, pH, temperature of air and
  water, relative humidity) parameters on fungal species temporal distribution.

  Materials and methods

  Study area
  The study focuses on three intensely visited urban beaches located within the boundaries of the Municipality
  of Genoa (Italy, Liguria, NW Mediterranean Sea), from west to east: Voltri, Multedo and Quinto, hereinafter
  referred as VB, MB and QB respectively (Fig. 1).




  Fig. 1. Localization of studied beaches (red circles) within Genoa municipality, Voltri (VB), Multedo (MB) and Quinto
  (QB).
  Fig. 1. Localizzazione delle spiagge oggetto di studio (cerchi rossi) all’interno del comune di Genova, Voltri (VB),
  Multedo (MB) and Quinto (QB).


  VB is located on the western boundary of Genoa, and limited westward by the presence of a freshwater
  outlet. It is characterized by a great confluence of urban activities located in the immediate nearby of the
  beach: residential and municipal buildings, commercial activities, sports facilities, railway, and beach resorts
  (Fig. 2). MB is located within the Genoa harbor, between the mouth of a stream (west) and the Genoa oil
  terminal (east). For this reason bathing activities are forbidden all-year-round. It undergoes several pressures
  by sports facilities, residential buildings, petrol stations, and some sailing and fishing associations (Fig. 2).
  QB is a typical gravely cobble pocket beach, which is nested within one of the most valuable stretches of the
  urban coast. The residential context is characterized by an alternation of ancient seafaring centers, historical
  mansion houses with wide gardens, and more recent buildings. QB hosts a freshwater discharge that may
  cause an increase of nutrients due to the organic matter and fecal pollution contamination in the adjacent
  coastal waters (Fig. 2).




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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,              Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778




  Fig. 2. Stellite view of the analyzed beaches. Red dots identify sampling stations distribution. White arrows show main
  freshwater outlet (images from www.regione.liguria.it)
  Fig. 2. Immagini satellitari delle spiagge analizzate. I punti rossi indicano la distribuzione delle stazioni di
  campionamento. Le frecce bianche indicano le pricipali foci di torrenti (immagini prese da www.regione.liguria.it)


  Sampling and samples processing
  The three beaches were sampled during April, July, and September 2013. For each beach, three sampling
  areas were chosen along the swash zone equally spaced along the beach extent. In each sampling area three
  samples were collected. As a consequence, 27 samples of water and sediment were collected overall in each
  month. On the whole 81 water and sediment samples were collected during this study.
  Sampling activities were always carried out with dry weather conditions (from at least three days before
  sampling) and calm sea (Beaufort scale 2 or lower). Sample areas were determined by first locating the
  landward edge of the swash zone, which is the zone alternatively covered and exposed by waves, and then
  chosen 1 m inland from the outer edge of the swash zone. For the interstitial water, the depth of dry sand was
  measured and the samples were harvested with a shovel. Each hole was dug deep 20 cm enough to allow free
  interstitial water to accumulate (Fig. 3).
  Interstitial water was collected by means of sterile Falcons (45 ml) and conserved at 4°C until the arrival at
  the laboratory. Additional samples of interstitial water were also collected for the pH measurement, and
  sediment samples were collected for the quantification of the total organic matter present (% Total Organic
  Matter; TOM) in the sediments of the swash zone.
  In addition, data about air temperature (°C), humidity (%) and seawater temperature (°C) were acquired from
  www.dicat.unige.it and www.ambienteinliguria.it for every sampling month.
  Microfungi were isolated by a dilution soil-plate method (Gams 1987) that permits to isolate culturable
  fungal strains. The final dilution (1:100000) was obtained mixing 1 ml of interstitial water with sterile water.
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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,         Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778




  Fig. 3. Plan for the SZIW collection
  Fig. 3. Schema di campionamento della SZIW.


  Each sample was inoculated in Petri dishes (12 cm diameter) on modified Sabouraud (15% agar, 10%
  glucose, 5% neopepton) and Malt Extract Agar at 24 °C in the dark (Hyde and Pointing, 2000). The Petri
  dishes were inspected daily for 20 days. Single colonies of fungi and yeasts were counted and isolated. The
  number of morphological taxonomic units (MTU) per 100 ml was calculated in order to quantify the total
  amount of colonies for each station.
  The colonies were identified by polyphasic approach. In order to obtain a clear identification, in
  particular for the most critical strains, the results of morphological and physiological identification
  (Rifai 1969; Ellis 1971; Raper and Fennel 1977; Schipper 1978; Kohlmeyer and Kohlmeyer 1979;
  Bisset 1984, 1991a, 1991b, 1991c; Kiffer et al. 1999; Klich 2002) were compared with the results of
  sequence of the b-tubulin and ITS gene (Glass N.L., Donaldson 1995). The sequence data were
  searched by means of sequence databases (NCBI/BLASTN algorithm) to confirm the identification.
  Interstitial water pH was measured with a digital pH-Meter, and the TOM was determined through mass
  loss-on-ignition (LOI) method that involves the heated destruction of all organic matter in sediments. A
  known quantity of sample oven-dried at 60 °C for 24 h, was washed at 450 °C for 4 hours. The sample was
  then cooled and weighed. The TOM is confuted as the difference between the initial and final sample
  weights divided by the initial sample weight.
  Statistical data analyses
  The species accumulation curves were plotted (number of samples vs number of identified species) and the
  estimation of species richness was computed to interpret the effect of sampling effort on survey efficiency.
  A Principal Component Analysis (PCA) was performed in order to analyze the effect of environmental
  variables (organic matter, pH, humidity, water and air temperature) on the mycobiota. Since the
  environmental variables are expressed in different measurement scales, we computed a PCA on the
  correlation matrix. The Euclidian distances were used to jointly display sites and variables in the PCA plot.
  Vector fitting was executed to interpret environmental variables onto ordination. A two-way ANOVA test
  was performed to analyze the fungal abundance dependence on beach and season, respectively (Beach: three
  fixed levels; Month: three fixed levels). All the statistical analyses were computed by using Vegan package
  in R system (version 3.1.0; 2014).

  Results

  From the SZIW samples were isolated 1,157 MTUs belonging to 52 taxa (Table 1). 37 species of
  filamentous fungi were identified and 15 taxa were unidentified. Among the latter taxa, seven fungi were

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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,               Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778



  unable to complete their lifecycle and grew as sterile mycelia. In spite of the filamentous fungi that were the
  main target of our survey, 8 different morphotypes of yeasts were distinguished on the basis of the macro-
  morphological features of the colony. The number of MTUs per 1 ml is summarized in Table 2 and the data
  collected in each sampling month about pH, TOM, air and water temperature and humidity values are
  displayed in Table 3.
  In table 4 the results of a TWO WAY ANOVA revealed significant differences among sampling month (0.1
  confidence level) whereas differences among beaches did not result statistically significant. Figure 4 shows
  the accumulation curve approach asymptote for all the three areas, both plotted as total and separated per
  sites. The PCA plots are shown in Figure 5 and Table 5. The cumulative percentage variance explained along
  the first two axes amounts to 84% (50% on the first axis; 34% on the second axis). The eigenvalues
  associated to the first and second axes are 2.50 and 1.72, respectively (Tab. 5). The gradient along the first
  axis appears to be correlated with a seasonality trend; whereas, air and water temperatures are significantly
  correlated with the different sites (= beaches) composition. This trend is further confirmed by the results of
  vector fitting test (Tab. 6). The most significant variables (see r2 and Pr(>r)) in fact, were the water and air
  temperatures which depend on the season. Relevant for the communities species composition it was also the
  value of pH.

  Tab. 1. List of 52 taxa discerned (37 species of filamentous fungi and 15 unidentified taxa) and their abundance of
  MTU measured during the isolation procedure.
  Tab. 1. Lista dei 52 taxa individuati (37 species di fungi filamentosi e 15 taxa non identificati) e la loro abbondanza di
  MTU misurata durante le procedure di isolamento.
                                                                              April             July             September
 Fungal species                                                          QB MB VB QB MB VB QB MB VB
 Aspergillus flavus var. oryzae (Ahlb.) Kurtzman                           5     4   3     -     22      3      -     -  16
 Aspergillus flavus Link                                                   -     1   -     -      -      -      6    2    -
 Aspergillus niger Tiegh.                                                  1     -   -     2      2      -     20    1    -
 Aspergillus ochraceus Wilh.                                               -     -   2     -      2      2     18     -   -
 Aspergillus parasiticus Speare                                            -     -   -     -      -      -      -     -   4
 Aspergillus pulverulentus (McAlpine) Thom                                 -     -   2     -      -      -      -     -   -
 Aspergillus tamarii Kita                                                  -     -   -     2      1      1      -     -   -
 Aspergillus terreus Thom                                                  -    12 18      -      -      -      9    1    -
 Aspergillus tubingensis Mosseray                                          -     -   -     -      3      -      -     -   -
 Boeremia exigua var. exigua (Desm.) Aveskamp, Gruyter & Verkley 3               -   -     -      3      -      -     -   -
 Circinella minor Lendn.                                                   -     4   1     4      -      -      -    3    1
 Cladosporium cladosporioides (Fresen.) G.A. de Vries                      -     -   -     -      -      -      1     -   1
 Cladosporium herbarum (Pers.) Link                                        -     -   -     -      -      2      -     -   -
 Cladosporium sphaerospermum Penz.                                         -     -   -     -      -      -      -     -   1
 Cladosporium tenuissimum Cooke                                            5     -   -     -      -      -      -     -   -
 Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams            2     2   -     -      -      2      -     -   -
 Mucor hiemalis Wehmer                                                     -     -   -     -      -      3      2    3    2
 Mucor mucedo Fresen.                                                      1     -   3     1      2     10      5     -   -
 Mucor racemosus Bull.                                                     -     -   -     -      -      1      1     -   1
 Penicillium brevicompactum Dierckx                                       26 14      7     7      -      3      7    1    8
 Penicillium chrysogenum var. chrysogenum Thom                             1     -   -     -      6      3      -     -   1
 Penicillium citrinum Thom                                                 -     -   1     -     11      -      -     -   -
 Penicillium decumbens Thom                                                7     -   -     -      4      -      -     -   -
 Penicillium digitatum (Pers.) Sacc.                                       -     2   -     -      -      -      -     -   -
 Penicillium griseofulvum Dierckx                                          -     -   -     -      5      4      -     -   -
 Penicillium italicum Wehmer                                               -     -   2     -      -      -      -     -   -
 Penicillium simplicissimum Thom                                           -     -   -     -      -      7      -     -   -

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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,            Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778



 Penicillium spinulosum Thom                                          3     -    -     -    39     -    -    -    1
 Phoma pomorum Thüm.                                                  3     -    -     1     -     -    -    -     -
 Rhizopus arrhizus A. Fisch.                                          1     3    -     1     1    2     -    -    1
 Rhodotorula Type 1 F.C. Harrison                                    40     2    6     -     1    7    19    -     -
 Rhodotorula Type 2 F.C. Harrison                                     -     -    -     -     1     -    -    -     -
 Sterile mycelia Type 1                                              23     3    9     2     -    10    7    6     -
 Sterile mycelia Type 2                                               4     -    -     -     4    1     -    -     -
 Sterile mycelia Type 3                                               1     -    -     1     6    1    10    3    19
 Sterile mycelia Type 4                                               -     1    7     1     5    3    11    -    5
 Sterile mycelia Type 5                                               -     -    -     -     -    4     -    -     -
 Sterile mycelia Type 6                                               -     -    -     1     -     -    -    -     -
 Sterile mycelia Type 7                                               -     -    -     2     1     -    -    -     -
 Talaromyces purpureogenus Samson, Yilmaz, Houbraken,
 Spierenburg, Seifert, Peterson, Varga & Frisvad                      6     7    3     -     -    -     -     -    -
 Talaromyces ruber (Stoll) Yilmaz, Houbraken, Frisvad & Samson        3     6    6     -    38    5    11    21    6
 Trichoderma asperellum Samuels, Lieckf. & Nirenberg                  -     -     -    4     -    -     4     -    4
 Trichoderma atroviride P. Karst.                                     -     -     -    -     1    -     -     -    -
 Trichoderma harzianum Rifai                                          5     7    15    -     -    -     7     -    1
 Trichoderma koningii Oudem                                           -     -     -    -     1    -     -     -    -
 Trichoderma viride Pers.                                            26     3     -    1     1    -    15     2    1
 Yeast Type 1                                                        19     -    12    6     1    -     2    14    3
 Yeast Type 2                                                        50    28    93   27     3    -     4     -    4
 Yeast Type 3                                                         -     -     -    -     1    3     -     -    -
 Yeast Type 4                                                         6     -     -    2     3    -     -     -    1
 Yeast Type 5                                                         -    17    1     -     1    -     -     -    -
 Yeast Type 6                                                         -     -     -    -     1    -     -     -    -

  Tab. 2. Mean amount of MTUs in 1 ml for each beach and month of sampling: QB = Quinto, MB = Multedo and VB =
  Voltri.
  Tab. 2. Media delle MTUs in 1 ml calcolate in ogni campionamento effettuato su ogni spiaggia: QB = Quinto, MB =
  Multedo e VB = Voltri.
           Site              April              July             September             Total
           QB                8.0 E+03           2.2 E+03         5.3 E+03              5.2 E+03
           MB                3.9 E+03           5.7 E+03         1.9 E+03              3.8 E+03
           VB                6.4 E+03           2.6 E+03         2.7 E+03              3.9 E+03
           Total             6.1 E+03           3.5 E+03         3.3 E+03              4.3 E+03


  Tab. 3. Environmental parameters measured during each sampling month for each station: QB = Quinto, MB =
  Multedo and VB = Voltri.
  Tab. 3. Parametri ambientali misurati durante ogni mese di campionamento nelle rispettive stazioni: QB = Quinto, MB
  = Multedo e VB = Voltri
                              Air               Water            Humidity        Organic matter        pH interstitial
  Sampled beaches
                      temperature (°C) temperature (°C)             (%)                 (%)                water
      QB April               14.6                 15                75.2                0.55                8.2
      MB April               14.6                 15                75.2                0.49                8.3
      VB April               14.6                 15                75.2                0.34                8.3
       QB July               24.3                 26                83.7                0.45                8.2
       MB July               24.3                 26                83.7                0.42                8.2
       VB July               24.3                 26                83.7                0.51                8.2
    QB September             20.5                 24                68.8                0.35                7.7
    MB September             20.5                 24                68.8                0.38                8.2
    VB September             20.5                 24                68.8                0.28                 8



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C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778




  Tab. 4. Two-way ANOVA results.
  Tab. 4. Risultati dell’analisi ANOVA a due vie.
  Source               Sum Sq            d.f.            Mean Sq            F                 Prob > F

  Beach               60.6               2               30.32              0.6               0.5475

  Month               254.1              2               127.05             2.53              0.0809

  Error               23271.9            463             50.26

  Total               23586.6            467




  Fig. 4. Accumulation curves of species richness in the three areas (MB = Multedo, QB = Quinto and VB = Voltri)
  plotted both separately, and as total.
  Fig. 4. Curve di accumulo della ricchezza specifica nelle tre aree analizzate (MB = Multedo, QB = Quinto e VB =
  Voltri) e del loro totale.




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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,            Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778




  Fig. 5. Ordination diagrams obtained through Principal Component Analysis (PCA). Sites abbreviations: QBA=site QB
  in April, QBJ= site QB in July; QBS= site QB in September etc. Variable abbreviations: Orgmatt=Organic Matter,
  pH=pH, Humidity= Humidity, Tempwat=water temperature, Tempair=air temperature.
  Fig. 5. Diagramma relative all’analisi delle component principali (PCA). Abbreviazione dei siti: QBA=sito QB ad
  aprile, QBJ= sito QB a luglio; QBS= sito QB a settembre ecc. Abbreviazione delle variabili: Orgmatt=Materia
  Organica, pH=pH, Humidity= Umidità, Tempwat=Temperatura dell’Acqua, Tempair=Temperatura dell’Aria.


  Tab. 5. Summary of PCA: eigenvalues, their contribution to the correlations and variables (Organic Matter, pH,
  Humidity, temperature water, temperature air) scores.
  Tab. 5. Risultato della PCA e valori delle singole variabili (Materia Organica, pH, Umidità, Temperatura dell’Acqua,
  Temperatura dell’Aria) rispetto agli assi (PCA1, PCA2, PCA3, PCA4).
                                            PC1                   PC2                 PC3                PC4
   Eigenvalue                              2.5083                1.7226              0.6018           0.16731
   Proportion Explained                    0.5017                0.3445              0.1204           0.03346
   Cumulative Proportion                   0.5017                0.8462              0.9665           1.00000
   Temperature water                      -1.5081               -0.5811             -0.2116            -0.2579
   Temperature air                        -1.2995               -1.0420             -0.1578            -1.1032
   Organic matter                         -0.2150                1.6067             -1.6221            -1.0323
   pH                                     -0.6458                1.3280              1.8870            -0.7637
   Humidity                               -1.3778                0.7456             -0.2509               1.




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S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,                 Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778


  Tab. 6. Results of vector fitting test of significant variables. The first column shows the environmental variables; the
  second and third column show eigenvalues for Axis 1 (PC1) and Axis2 (PC2). r2 corresponds to correlation values. P
  values, in the last column, are based on 1000 permutations. Significance codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
  Tab. 6. Risultati del test vector fitting per le variabili significative. La prima colonna mostra le variabili ambientali; la
  seconda e la terza colonna mostrano l’eigenvalues per l’Asse 1 (PC1) e Axis2 (PC2). r2 corrisponde al valore di
  correlazione. Il P values, nell’ultima colonna è basato su 1000 permutazioni. Significatività: 0 '***' 0.001 '**' 0.01 '*'
  0.05 '.' 0.1 ' ' 1
                                  PCA 1                    PCA 2                    r2                 Pr(>r)
    Temperature water             -0.93313                 -0.35953                 0.9940             0.002997 **
    Temperature air               -0.78014                 -0.62560                 0.9654             0.002997 **
    Organic matter                -0.13264                 0.99116                  0.7214             0.021978 *
    pH                            -0.43732                 0.89931                  0.6458             0.003996 **
    Humidity                      -0.87947                 0.47596                  0.9043             0.025974 *




  Discussion and Conclusion

  Some researches (Salvo and Fabiano, 2007; Vezzulli et al., 2009) indicated that SZIW is characterized by
  good mycological diversity, and represent a reservoir of fungal micro-organisms due to the optimal
  combination of environmental factors (e.g. high levels of humidity and organic matter, etc…). For this
  reason SZIW environment can be fruitfully taken into account to characterize urban beach fungal diversity
  and composition, and to evaluate the quality of the beach by means of the presence of fungal pathogenic or
  allergenic species.
  Our quantitative results according to the data reported in Vezzulli et al. (2009) reveal a high presence of
  fungi in the SZIW of all beaches. The total number of MTUs from each beach is almost the same probably
  due to the high human frequentation that acts as a vector of fungal propagules. A significant variation of
  MTU amount was detected among months, and the total abundance appear to be higher in April than in July
  and September. In general, organic matter on the beach represents the essential substrate and nutrient source
  for microbial organisms, and surely it can affect the fungal abundance. However, on urban beaches organic
  matter has different origin, such as decayed wood and algae, washed-up leaves, animal products, (e.g. chitin
  and keratin), and sewage discharges from publicly owned treatment works (Hyde, 1989; Babu et al., 2010;
  Gonçalves de Oliveira et al., 2011; Lee et al., 2011, Zakaria et al., 2011).
  PCA results show that environmental factors, mainly water and air temperature, seem to have influenced on
  site fungal composition in April and July, and not in September. This is likely due to the high human
  frequentation, which during the summer increase the number of fungal propagules transported; and as
  suggested by Pereira et al. (2013) the other recreational seashore activities contribute to increase the organic
  matter amount.
  In spite of the high MTUs abundance, there are few differences in fungal species composition among the
  beaches themselves. In fact, we identify 52 taxa: 45% of which are common to all three beaches, and 70%
  are common to at least two beaches This similarity probably is due to the anthropic pressure that often tends
  to support a limited group of widespread species manly related to the human activity.
  As concerns filamentous fungi, the most recurrent genera are: Aspergillus, Mucor, Penicillium, Talaromyces,
  Trichoderma, Circinella and Clonostachys. Concerning the yeasts, four of seven morphotypes discerned
  were isolated from all beaches.
  The most fungal species are really ubiquitous, saprotrophic potential allergenic species (e.g. Cladosporium
  cladosporioides (Fresen.) G.A. de Vries, Mucor mucedo Fresen., Rhizopus arrhizus A. Fisch., Trichoderma
                                                                                                                             17
S. Di Piazza, S. Baiardo, G. Cecchi, E. Ambrosio,         Italian Journal of Mycology vol. 46 (2017) ISSN 2531-7342
C. Paoli, P. Vassallo, M. Zotti
DOI: https://doi.org/10.6092/issn.2531-7342/6778



  harzianum Rifai), and only few species are opportunistic or true pathogens to human (e.g. Aspergillus flavus
  Link, A. ochraceus Wilh., A. niger Tiegh., A. terreus Thom, A. tubingensis Mosseray, Mucor racemosus
  Bull., and Rhodotorula sp.) in accord with previous researches on urban beaches (Salvo and Fabiano, 2007).
  Opportunistic species could be threatening for human immunodepressed individuals infecting by
  conidia/spores inhalation, or by contact with sand causing irritations and/or sensitivity (Matavulj et al.,
  2005). In particular, Aspergillus tubingensis rarely producted osteomyelitis (Bathoorn et al., 2013), while
  Rhodotorula spp. recently caused allergenic reactions on skin (Wirth and Goldani, 2012).
  Our results show that no dermatophitic and keratinophilic fungi were found in urban beach SZIW zones.
  Furthermore, visitors could be possible vectors of opportunistic fungi that can find a suitable habitat in the
  SZIW itself (Sabino et al., 2011). This suggests that human impact in general contribute to affect seasonality
  distribution of fungal species on beaches. In our case, however, these beaches are undergone to human stress
  throughout the year and, as a consequence, the effect caused by summer beach frequentation is hidden by
  background noise.
  Our results show how urban SZIW represents a reservoir of microfungi. Most of the fungal species were
  saprotrophic, some of which were opportunistic. As concerning fungal pathogens for human only A. niger
  were found. Furthermore, a statistical analysis has revealed that the fungal composition in April and July is
  affected by water and air temperature. The human impact, in particular beach recreational frequentation and
  the related activity, seems to affect the fungal communities in September. Further studies are needed in order
  to better characterize the urban beaches mycobiota and to well understand the effect of environmental factors
  on organisms distribution.

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