J. Phytopathology 151, 591–599 (2003)
2003 Blackwell Verlag, Berlin ISSN 0931-1785 Department of Land Ecology and Terrestrial Environments, Section of Mycology, University of Pavia, Italy Study of the Occurrence of Greenhouse Microfungi in a Botanical Garden M. Rodolfi Rodolfi,, E. Lorenzi and A. M. Picco AuthorsÕ address: Università degli Studi di Pavia, Dip. Ecologia del Territorio e degli Ambienti Terrestri, Sezione di Micologia, Via San Epifanio 14, I-27100 Pavia, Italy (correspondence to A. M. Picco. E-mail: apicco@et.unipv.it) With one figure Received March 14, 2003; accepted August 4, 2003 Keywords: greenhouse, botanical garden, phylloplane fungi, airborne spores Abstract Three greenhouses and the Central Garden of The Botanical Garden of Pavia were monitored for 1 year with the objective of investigating the occurrence of both airborne fungal spores and phylloplane fungi. By using an SAS air sampler, the higher fungal spore concentrations were detected in tropical and Mediterranean greenhouses. A total of 72 species belonging to 42 genera, some of which are related to the presence of plants ex situ, were isolated from Petri plates after exposure. Some airborne fungi, such as Aspergillus spp., Penicillium spp. and Conidiobolus spp., which are responsible for human allergies and respiratory problems, were also detected. Forty-four genera of phylloplane fungi were identified from leaves randomly collected from greenhouse plants. Most of the aerial fungal taxa isolated were also detected from the phylloplane. Some phytopathogenic taxa, as exemplified by Gliocladium vermoeseni, Graphium sp., Peronospora sp., and Zygosporium oscheoides, were isolated only from the phylloplane. The information obtained from qualitative and quantitative analysis of fungi can be a useful tool in the control of indoor air quality, thereby guaranteeing ex situ plant conservation and occupational health safety. Introduction The destruction of tropical and temperate habitats because of human activity and the consequent impoverishment of the natural genetic wealth make conservation a key issue for scientists. Because of the role of plants in the maintenance of biodiversity, their conservation continuously receives attention in most countries. Conservation can be effected by using two complementary approaches, in situ and ex situ, the latter of which is mainly represented by plant collections located in botanical gardens and seed banks (Hurka, 1994). The latter is represented by indoor greenhouses designed to protect ornamental plants from pathogens and adverse environmental conditions such as low U. S. Copyright Clearance Centre Code Statement: temperature and precipitation (Jarvis, 1992). The indoor environment is therefore generally warm, humid and wind-free, conditions which permit not only excellent plant growth, but also bacterial and fungal proliferation, and eventually the development of disease (Baker and Linderman, 1979; Cline et al., 1988). Many factors including optimum temperature and the availability or presence of water (in the form of free water, droplets, films and humidity), can determine whether disease will develop or not (Gareth Jones, 1998). Moreover, transmission of fungal spores or other propagules within and between plant populations may be an important process in the development of plant disease. Investigations of the fungal populations usually involve the direct examination of leaf surface on artificial media for subsequent growth, enumeration and identification (Harris and Maramorosch, 1980). As the dispersion and subsequent inoculation of pathogenic fungi are facilitated by air currents, their detection may be potentially useful in understanding disease development and management systems (McCartney and Schmechel, 2000). The presence, concentration and vitality of aerial spores and conidia of phytopathogenic fungal genera represent very important information in agriculture and plant conservation (Magyar et al., 2000). Many studies on the monitoring of aerodispersed pathogenic fungi have been carried out for predictive purposes (Aylor, 1998; Bacon et al., 2001; Picco and Rodolfi, 2002), and some of these can be applied in greenhouses (Lacey, 1996). Greenhouses have received little attention, despite the fact that crop plants and workers therein, may be exposed to high fungal concentrations. In our opinion, both qualitative and quantitative information on greenhouse airborne spores may be particularly useful for the control of the greenhouse indoor air quality. The consequences of continued exposure of man to potentially pathogenic fungal species should not be underestimated, especially in terms of occupation 0931–1785/2003/15112–0591 $ 15.00/0 www.blackwell.de/synergy Rodolfi et al. 592 safety in greenhouses. The fact that exposure to airborne fungal particles can cause a variety of respiratory disorders such as allergic rhinitis, asthma, hypersensitivity pneumonitis and infection diseases (Lacey, 1991; Husman, 1996) is very well known. The protection of plants outside their natural habitat is a historical tradition and an important goal of The Botanical Garden of Pavia. This paper reports a 1-year survey on the occurrence of airborne and phylloplane microfungi collected from the three historical greenhouses and from the Central Garden of The Botanical Garden of Pavia. Airborne spore recurrence and host colonization are provided. Materials and Methods The city of Pavia is located in northern Italy. Broadly, it has four distinct seasons (summer, autumn, winter and spring). The samples were collected every 15 days for a period of 1 year. Monitoring was performed between 10 am and 12 am throughout the sampling period. The greenhouses In 1774, Brusati began the construction of greenhouses in the Pavia Botanical Garden. They were built in brickwork and iron (Nocca, 1818). In 1777 Scopoli succeeded Brusati and proceeded with his predecessor’s work by increasing the gardensÕ floral heritage; the greenhouses are now coined ÔScopoli GreenhousesÕ (Fig. 1). Presently, they conserve the original external structure which was maintained throughout the different restorations effected during the years, and approximately 120, 50 and 550 species of Mediterranean, tropical and desert plants, respectively. The progressive switching on and off of the artificial lighting system regulates the photo- and shadow-periods in the greenhouses. Because of the historical infrastructure of the greenhouses, temperature excursions comparable with those of the external environment (Mediterranean greenhouse: 19.5 ± 3.1C during winter and 22.5 ± 3.5C during summer; desert plants greenhouse: 18.5 ± 2C during winter and 20.2 ± 2C during summer; tropical greenhouse: 22.5 ± 2.1C during winter and 25.7 ± 1.4C during summer) are witnessed, regardless of the daily control of aerial temperature and heating inside the greenhouses. On the contrary, the values of relative humidity are almost constant throughout the year (Mediterranean greenhouse: 71.2 ± 4.3%; desert plants greenhouse: 64.3 ± 2.8%; tropical greenhouse: 89.15 ± 4.6%). An artificial rain irrigation system was installed in the tropical greenhouse. Quantitative samplings Quantitative data were collected in duplicate using a SAS air sampler (PBI, Milan, Italy), held at 1.5 m above the soil level. Samplings, carried out at 200 l/min, were achieved through the use of sabouraud dextrose agar (SAB; Oxoid) medium, which permits the growth of aero-dispersed fungi, and dichloran glicerol agar (DG18; Oxoid, Basingstoke, UK) medium, which is specific for xerophilic fungi. The plates were incubated at 20C for 4–6 days and fungal counts were expressed as colony-forming units per cubic metre of air sampled (CFU/m3). Air samples from outside the Central Garden were cultured on both media to provide an external air spora reference count (ACGIH, 1989). Qualitative samplings Using the gravity settling culture method, the Petri plates containing PDA (potato dextrose agar; Oxoid) medium, were exposed in duplicate for 10 min at soil level. The exposed agar plates were incubated at 25C in natural day/night conditions and examined for 2 weeks. Fungal mycelia were counted and pure cultures were made from all the morphologically different mycelia. Fungal isolates were transferred to culture media suitable for classification and identification based on morphological and physiological characteristics, and following standardized procedures. The counts were expressed as colony forming units per Petri plate area (CFU/154 cm2). Qualitative analysis of the air collected outside Central Garden was also performed as an external air spora reference. Annual and seasonal means were calculated for all the fungal taxa, and monthly percentages were evaluated for each fungal taxon. Isolation of phylloplane fungi Fig. 1 External structure of the ÔScopoli GreenhouseÕ Leaves were randomly collected from some greenhouses plants. The leaves were placed in separate sterile bags and deposited in our laboratory so as to avoid contamination. They were washed several times with sterile water to remove extraneous spores (Dickinson, 1971), dry-blotted with sterile filter paper and aseptically cut into 10 mm · 10 mm fragments. Ten fragments of each leaf were placed onto 90 mm tap-water agar containing Petri dishes which provide the necessary humidity and favour the growth of both pathogens and saprophytes which feed on plants (Harris, 1986; Waller, 2002). The dishes were maintained at room temperature in natural day/night conditions for 30 days. All resulting fungal colonies were directly Study of the Occurrence of Greenhouse Microfungi in a Botanical Garden identified or transferred to an appropriate medium for identification. Results Quantitative data The colony forming units, monitored by quantitative sampling method, are listed in Table 1. Data are expressed as seasonal mean values. Totals of 4102.32 CFU/m3, 899.66 CFU/m3, and 5192.65 CFU/m3 were detected in Mediterranean, desert plants and tropical greenhouses respectively, while a total of 2443.32 CFU/m3 was isolated from Central Garden air. Results showed seasonal variation in the mean spore concentration values especially in the Mediterranean greenhouse and Central Garden air. Highest fungal concentrations were recorded in the tropical greenhouse during summer and autumn. Qualitative data A total monthly mean of 1522.4 colonies was obtained from the Petri plates exposed: 476.34 colonies from the Mediterranean greenhouse, 422.93 from the Central Garden, 348.14 from the tropical greenhouse and 280.18 from the desert plants greenhouse. Table 2 presents a list of the isolated fungal taxa (42 genera and 72 species) and their seasonal mean variations. The spore concentration was constant throughout the year; a slight decrease in March and an increase in June, September and November were observed. An unusual increase of airspore concentration was identified in the Mediterranean greenhouse in November. Cladosporium cladosporioides was dominant both in the greenhouses and in the Central Garden, reaching the monthly mean presence of 59.31%. Other dominant fungal taxa were Penicillium fellutanum (7.28%), P. olsonii (6.38%), Epicoccum nigrum (6.29%), Alternaria alternata (2.97%) and Conidiobolus major (2.91%). Fungal distribution in the monitored sites was uneven. Cladosporium cladosporioides was mainly present in the Central Garden (68.84%). Low values of P. fellutanum (5.8%) and P. olsonii (4.02%) were observed in the desert plants greenhouse, while they were of considerable importance in tropical (11.99%) and Mediterranean greenhouses (8.15%). E. nigrum was present in very low concentrations in the tropical greenhouse (1.94%) while presenting a 10-fold increase in the Central Garden (10.67%). The genus Conidiobolus was represented by C. major, C. obscurus, C. apiculatus and C. coronatus; high C. major concentrations were observed in the Mediterranean greenhouse (5.37%). While Botryosporium longibrachiatum was only observed in autumn and Table 1 Seasonal values of CFU/m3 monitored by SAS air sampler Sampling sites Winter Spring Summer Autumn Mediterranean greenhouse Desert plants greenhouse Tropical greenhouse Central garden 572.33 207.33 1157.66 190.33 1070.00 262.33 1195.66 810.66 847.66 220.00 1337.33 357.00 1612.33 210.00 1502.00 1085.33 593 in the tropical greenhouse, it was present throughout the year in the desert plants greenhouse. Beltrania rhombica was isolated only in summer in the Mediterranean greenhouse, and all throughout the year in the Central Garden. Phylloplane fungi A total of 324 fungal isolates including 45 genera and 60 species were identified from the phylloplane of 13 Mediterranean plants, 13 desert plants and 18 tropical plants. All fungi are listed in Table 3. Some taxa, such as A. alternata, Cladosporium cladosporioides and Penicillium spp., were mainly present on all the leaves. Alhough not dominant, Fusarium sp. and Gliocladium sp. were isolated. Aspergillus spp. was isolated from desert and tropical plants but never from the Mediterranean. Colletotrichum sp., Nigrospora sp. and Zygosporium spp. seemed to be more associated with tropical plants. Significant quantities of Acremonium butyri (on Caryota urens), Aureobasidium pullulans (on Coffea sp. and Eugenia caryophyllata), Chaetomium globosum (on Coffea sp.), Clonostachys rosea (on Anthurium scherzerianum), Cunninghamella sp. (on Opuntia inamoena), Curvularia affinis (on Persea gratissima), Didymostilbe sp. (on Bombax palmeri), Melanospora sp. (on Alluaudia dumosa), Mortierella sp. (on Latania borbonica), Mycosphaerella sp. (on Monstera deliciosa), Peronospora sp. (on Bahuinia aculeata), Sphaeropsis sp. (on Dieffenbachia sp.), Stemphylium sp. (on Chamaedorea oblungata), Gliocladium vermoeseni (on Chamaedorea spp.) and Truncatella sp. (on Chamaedorea stolonifera) were observed. Discussion This paper is a result of the necessity to control indoor air quality in three historical greenhouses of The Botanical Garden of Pavia, and the need to verify the phytopathological state of plants thereof since correct ex situ conservation of plants is dependent on controlled and secure habitat areas (Sinclair et al., 1995). Quantitative studies revealed that the number of greenhouse airborne fungi is generally higher than that of outdoor environments. High RH and temperature values required for the maintenance of the natural status of the habitat may be responsible for high fungal counts observed in indoor ambient. This was confirmed by the high counts observed in the tropical greenhouse, which is characterized by high temperature and humidity values. Although the microclimate of the greenhouses is controlled, it is interesting to note the seasonal variation of fungal counts which may probably be related to the life cycles of plants and the entrance of spores from the external environment. Recent studies have shown that the concentration of fungal propagules in indoor environments is mainly dependent on their outdoor concentration and the activities and/or substrates in the indoor environment (Blomquist and Andersson, 1994). Regarding indoor air quality, the general perception of a ÔhealthyÕ indoor air spora is similar to that of the 594 Table 2 Fungal taxa isolated using the gravity settling method Mediterranean greenhouse Fungal taxa W Sp Su A Desert plants greenhouse AM W Sp Su A Tropical greenhouse AM W Sp Su A Central Garden AM W Sp Su A AM Rodolfi et al. Acremonium fusidioides (Nicot) Gams 6.33 1.58 0.33 0.08 Acremonium sp. 0.33 0.08 Alternaria alternata (Fr.) Keissl. 1.00 2.33 22.00 22.33 11.92 2.00 2.00 20.66 19.33 11.00 1.33 2.33 10.00 11.00 6.17 A. longipes (Ellis & Everth.) Mason 0.33 0.08 6.33 2.66 19.33 39.00 16.83 A. tenuissima (Kunze) Wiltshire 0.33 0.08 Arthrinium phaeospermum (Corda) Ellis 0.33 0.08 0.66 0.17 1.33 0.33 0.42 Arthrobotrys sp. 0.33 0.08 Aschersonia sp. 0.33 0.08 Aspergillus flavus Link 0.33 0.08 0.33 0.08 A. fumigatus Fresen. 2.33 1.00 0.33 0.92 0.33 2.00 0.58 1.33 6.66 2.00 A. giganteus Wehmer 0.66 0.17 0.66 0.17 0.33 0.08 A. niger Van Tieghem 3.00 3.00 26.66 8.17 1.00 1.33 5.33 1.92 0.33 2.00 2.00 4.33 2.17 0.33 1.00 1.33 0.67 A. nidulans Wint. 0.33 0.08 A. ochraceus Wilh. 1.33 0.66 3.33 1.33 0.33 1.66 2.66 4.00 2.16 5.33 0.33 17.33 2.33 6.33 Aspergillus sp. 0.66 0.17 1.00 0.33 0.33 1.66 0.33 0.50 2.66 0.33 0.75 Aureobasidium pullulans 1.66 0.66 0.66 0.66 0.91 1.33 2.00 0.66 1.00 3.33 0.66 0.66 1.16 0.33 0.66 0.25 (De Bary) Arnaud Beltrania rhombica Penz. 0.33 0.08 5.00 2.00 1.66 0.66 2.33 Bipolaris cynodontis (Marign.) Shoem. 0.66 0.17 Botryosporium longibrachiatum 1.33 0.33 0.33 0.66 0.66 2.00 0.50 (Oudem.) Maire Botrytis cinerea Pers. 1.33 4.33 0.33 2.00 2.00 1.00 0.33 0.33 2.00 0.66 0.66 0.66 1.00 Chaetomium bostrychodes Zopf 0.33 0.66 1.33 0.58 Cladosporium cladosporioides 152.33 165.00 208.33 555.66 270.33 44.66 100.00 218.66 289.66 163.25 151.00 91.00 186.66 285.66 178.58 1.00 0.25 (Fres.) De Vries C. herbarum (Pers.) Link 0.33 0.08 128.00 215.00 315.33 504.66 290.75 C. macrocarpum Preuss 0.66 0.17 C. oxysporum Berk & Curtis 0.33 2.00 0.58 0.33 0.33 1.00 0.42 2.00 0.50 C. sphaerospermum Penz. 0.33 0.33 0.17 0.66 0.17 0.33 0.33 0.33 0.25 Clonostachys rosea 0.33 0.08 0.33 0.08 0.33 0.08 (Link) Schroers, Samules, Seifert & Gams Cochliobolus australiensis 0.66 0.17 (Tsuda & Ueyama) Alcorn Colletotrichum gleosporioides 0.66 14.33 3.75 (Penz.) Penz. & Sacc. Conidiobolus apiculatus Rem. & Kell. 40.00 10.00 C. coronatus (Cost.) Batko 0.33 21.00 5.33 C. major Rem. & Kell. 0.66 20.66 81.00 25.58 1.00 0.25 4.33 0.66 32.33 20.00 14.33 0.33 20.33 0.66 5.33 C. obscurus Rem. & Kell. 7.00 42.00 0.66 12.42 5.33 20.00 20.33 11.42 Cylindrocarpon sp. 0.66 0.33 0.25 Curvularia affinis Boedijn 0.33 0.08 Epicoccum nigrum Link 1.33 3.33 20.00 61.00 21.42 2.33 2.00 19.66 66.66 22.66 2.33 0.33 5.66 18.66 6.75 20.00 6.66 28.00 125.66 45.08 Erynia sp. 0.33 0.08 0.33 0.08 Fusarium acuminatum Ellis & Everth. 0.33 3.00 1.33 1.17 0.33 1.00 1.33 2.00 1.17 2.66 1.00 1.00 1.17 0.66 0.66 2.00 0.83 F. avenaceum Sacc. 0.33 0.08 F. equiseti (Corda) Sacc. 0.33 0.08 Colony mean number 1.33 1.00 0.25 0.66 1.33 1.00 0.33 0.33 0.33 0.66 0.42 0.08 0.42 1.00 0.33 0.33 0.08 0.33 0.33 1.33 2.00 0.33 0.33 0.66 0.33 1.00 0.33 0.17 0.33 0.08 0.67 0.17 0.58 0.25 0.66 0.33 0.33 0.08 0.33 0.17 0.08 0.33 0.33 0.08 0.75 3.00 0.33 0.08 0.08 0.08 0.33 0.33 0.33 0.66 0.66 0.33 0.33 0.08 0.66 3.00 0.33 0.08 0.33 2.33 0.08 16.00 83.66 31.66 20.00 31.33 41.66 7.00 11.08 0.08 9.08 25.66 0.33 1.33 46.66 17.00 12.00 0.66 25.33 0.25 0.42 18.33 0.33 0.33 0.08 0.08 0.17 0.75 0.66 50.00 79.00 9.66 3.33 40.33 44.75 0.83 79.66 9.33 2.33 52.00 4.33 4.66 0.66 19.33 6.00 1.00 26.66 38.83 3.83 2.16 6.67 5.00 0.33 2.33 0.33 3.00 0.08 0.25 0.75 0.08 8.66 1.00 0.33 0.66 0.33 0.33 0.08 0.33 2.33 4.33 0.08 1.92 1.17 0.17 1.33 0.08 8.75 0.08 1.33 19.00 36.00 6.66 22.00 2.00 15.33 32.00 0.33 1.66 0.17 0.33 1.33 0.33 0.66 0.33 0.33 1.00 2.00 3.00 0.33 0.66 1.33 34.33 0.33 1.66 1.00 1.00 0.66 0.33 0.66 0.33 0.33 19.33 4.00 3.33 8.42 40.00 2.00 26.33 5.33 1.33 0.33 0.33 1.00 2.17 0.25 0.08 0.25 0.33 1.00 0.33 1.00 0.33 0.33 0.66 1.00 2.33 0.66 0.66 0.33 1.33 0.33 8.66 1.66 3.00 34.33 1.33 3.00 0.33 0.33 1.33 2.91 0.33 0.33 0.41 0.66 1.00 3.66 0.33 0.33 0.33 4.00 10.75 1.33 4.66 0.33 0.33 0.50 2.66 1.66 16.83 0.66 1.33 0.66 0.66 0.33 23.33 1.33 24.33 0.66 1.00 0.33 0.33 0.17 0.33 0.42 0.25 0.33 0.33 0.33 1.00 0.33 0.50 22.50 0.42 0.17 0.83 0.17 0.08 0.33 0.17 0.08 0.25 0.17 7.25 0.50 0.66 2.00 2.33 1.66 1.00 1.00 2.00 0.50 0.66 7.58 0.17 0.50 0.08 0.33 1.00 0.58 1.00 0.25 1.33 6.33 12.66 45.00 0.66 16.16 0.33 1.66 0.33 1.00 0.83 0.66 8.00 2.00 1.00 2.92 0.08 2.50 10.00 1.00 1.00 3.33 0.66 0.33 2.33 0.33 0.17 Study of the Occurrence of Greenhouse Microfungi in a Botanical Garden F. graminearum Schwabe F. oxysporum (Fr.) Schlechtend. F. proliferatum (Matsushima) Nirenberg F. solani (Mart.) Sacc. Fusarium sp. Gleosporium sp. Gliocladium sp. Gliomastix luzulae (Fuckel) Mason Gonatobotrys simplex Corda Monodictys sp. Mucor sp. Neozygites fresenii Witlaczil Nigrospora oryzae (Berk. & Br.) Petch N. sphaerica (Sacc.) Mason Oidiodendron sp. Paecilomyces lilacinus (Thom) Samson Penicillium citrinum Thom P. claviforme Bain. P. duclauxii Delacr. P. fellutanum Biourge P. griseofulvum Dierckx P. janthinellum Biourge P. olsonii Bainier & Sartory P. oxalicum Currie & Thom P. purpurescens (Sopp) Biourge P. purpurogenum Stoll P. restrictum Gilman & Abbott P. variabile Sopp P. verrucosum Dierckx P. waksmanii Zaleski Penicillium sp. Pestaloptiopsis guepini (Desmaz.) Steyaert Phoma betae Frank P. eupyrena Sacc. P. exigua Desm. P. lingam (Tode) Schw. P. medicaginis Malbr. & Roum. Phoma sp. Pyricularia grisea (Cooke) Sacc. Rhizopus stolonifer (Ehrenb.) Vuill. Scopulariopsis brumptii Salvanet-Duval Sporobolomyces roseus Kluiver & Niel Stemphylium vesicarium (Wallr.) Simmons Trichoderma viride (Fr.) Pers. Truncatella sp. Verticillium dahliae Kleb. V. tenerum (Pers.) Link Verticillium sp. Non-sporing isolates 0.33 0.50 60.00 0.66 0.33 2.00 22.33 1.66 0.08 6.66 327.59 367.26 355.56 854.93 476.34 138.58 197.95 348.91 435.26 280.18 338.23 219.20 377.88 457.23 348.14 198.93 324.92 477.60 690.26 422.93 W, winter; Sp, spring; Su, summer; A, autumn; AM, annual mean. 595 596 Table 3 Fungal taxa isolated from the phylloplane Mediterranean greenhouse Fungal taxa Tropical greenhouse 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d Rodolfi et al. Acremonium butyri (van Beyma) Gams Acremonium sp. Alternaria alternata (Fr.) Keissl. Arthrobotrys sp. Aspergillus flavus Link A. niger Van Tieghem Aspergillus sp. Aureobasidium pullulans (De Bary) Arnaud Botrytis cinerea Pers. Chaetomium globosum Kunze Chaetomium sp. Cladosporium cladosporioides (Fres.) De Vries Clonostachys rosea (Link) Schroers, Samules, Seifert & Gams Colletotrichum sp. Coniella musaiensis Sutton var. hibisci var. nov. Cunninghamella sp. Curvularia affinis Boedijn Didymostilbe sp. Doratomyces sp. Epicoccum nigrum Link Fusarium sp. Gliocladium catenulatum Gilman & Abbott Gliocladium vermoeseni (Biourge) Thom Gliocladium sp. Gonatobotrys simplex Corda Graphium sp. Melanospora sp. Mortierella sp. Mycosphaerella sp. Myrothecium roridum Tode Myrothecium sp. Nectria sp. Nigrospora sp. Paecilomyces farinosus (Holmsk.) Brown & Sm. Paecilomyces lilacinus (Thom) Samson Penicillium sp. Periconia byssoides Pers. Periconia sp. Peronospora sp. Phoma sp. Pithomyces chartarum (Berk & Curtis) Ellis Pithomyces sp. Rhinocladiella sp. Rhizopus stolonifer (Ehrenb.) Vuill. Sphaeropsis sp. Stachybotrys atra Corda Desert plants greenhouse d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d d Stachybotrys sp. Stemphylium sp. Torula herbarum (Pers.) Link Torula sp. Trichoderma viride (Fr.) Pers. Trullula sp. Truncatella sp. Ulocladium sp. Verticillium tenerum (Pers.) Link Verticillium sp. Volutella ciliata (Fr.) Alb. & Schw. Volutella sp. Zygosporium oscheoides Mont. Zygosporium sp. 1: Bauhinia aculeata; 2: Chamaerops humilis; 3: Citrus limon; 4: Cordia superba; 5: Cynnamomum nitidum; 6: cyphomandra betacea; 7: Eugenia uniflora; 8: Iasminum sp.; 9: Persea gratissima; 10: Picraena excelsa; 11: Pimenta acris; 12: Sapindus australis; 13: Spidium friedium; 14: Alluaudia comosa; 15: Aloe massawana; 16: Ananas comosus; 17: Bombax palmeri; 18: Caralluma dummeri; 19: Cereus sp.; 20: Crassula arborescens; 21: Crassula capitello; 22: Crassula lactea; 23: Euphorbia grandicornis; 24: Opunzia cilindrica; 25: Opunzia inamoena; 26: Peperomia dolabriformis; 27: Aglaonema marantifolium; 28: Anthurium scherzerianum; 29: Asplenium nidum; 30: Coffea sp.; 31: Caryota urens; 32: Chamaedorea oblungata; 33: Chamaedorea stolonifera; 34: Croton sp.; 35: Dieffenbachia sp.; 36: Dizygotheca elegantissima; 37: Dracaena meremensis; 38: Eugenia caryophyllata; 39: Latania borbonica; 40: Monstera deliciosa; 41: Philodendron bipinnarifidum; 42: Platycerium willinkii; 43: Puya sp.; 44: Spatiphyllum wallisi regei. d d d d d d d d d d d d Study of the Occurrence of Greenhouse Microfungi in a Botanical Garden 597 outdoor environment (Flannigan and Miller, 1994). Qualitative monitoring showed that the same taxa (Cladosporium cladosporioides, P. fellutanum, P. olsonii, E. nigrum, A. alternata) are dominant in both greenhouses and the external environment. However, the availability of colonizable substrate and the favourable microclimatic conditions facilitated the enrichment and the diversification of the indoor aeromycological pattern: 58 taxa were isolated from the tropical greenhouse, 49 from the Mediterranean greenhouse, 43 from the desert plants greenhouse and 41 from the outdoor environment. The use of the gravity settling culture method resulted in the identification of fungal taxa commonly occurring in greenhouses (B. longibrachiatum) (Barron, 1968), and taxa typical of tropical areas but also present in Mediterranean areas (Beltrania rhombica) (Rambelli and Pasqualetti, 1990) as confirmed in this study from air from the Mediterranean greenhouses and the Central Garden. Most of the fungal taxa identified from air samples were also present on the phylloplanes of plants. Aureobasidium, Cladosporium and Sporobolomyces can grow and sporulate in fluctuating climatic and nutritional conditions: Alternaria and Epicoccum are phylloplane invaders normally grow extensively only when conditions are particularly favourable. These taxa were classified by Dickinson (1976) as non-pathogenic epiphytes, and redefined by Dix and Webster (1995) early colonizers, saprophytic or weakly parasitic fungi that are mainly restricted to the leaf surface until leaf senescence. Other taxa including Coniella musaiensis var. hibisci, G. vermoeseni, Graphium sp., Peronospora sp. e Zygosporium oscheoides were exclusively isolated from plant phylloplane. Regarding the control of plant health, we highlight two particular isolations: the ÔtropicalÕ Zygosporium oscheoides, a polyphagous saprophytic pathogen which feeds on plant debris and is the causative agent of leaf spot on Cordyline foliage (Farr et al., 1989), was isolated from the phylloplane of Dracaena meremensis and Puya sp.; G. vermoeseni, the causative agent of Gliocladium Blight (alias ÔPink RotÕ) in ornamental palms, was initially reported by Bliss (1938) and coined P. vermoeseni. The latter is mainly found in southern Italy (Polizzi, 2000) on Chamaedorea elegans, Howeia forsteriana and Washingtonia filifera. The isolation of Conidiobolus spp. using the gravity settling method was particularly significant. The isolation of this entomopathogenic taxon was concomitant to an infestation by greenhouse whiteflies (Trialeurodes vaporarium Westwood) observed in both tropical and Mediterranean greenhouses. This entomopathogen, initially reported in Oahu in 1907 (Lloyd, 1922) is widely distributed throughout the world and occurs in greenhouses of temperate zones. Mound and Halsey (1978) provided an extensive list of greenhouse whitefly hosts. Preventive measures can be effective in delaying whitefly infestation. Actually, several parasite species (Prospaltella transvena Timberlake and Encarsia formosa 598 Gahan) offer biological control in greenhouse situations (Gerling, 1983; van Vianen and van Lenteren, 1986). Recent advances in the production, formulation and application of mycoinsecticides have resulted in dramatic improvements of fungal products against insect pathogens. Fungi pathogenic to insects are also potential biological control agents for selected crop pests (Glare and Milner, 1991; Hemmati et al., 2001). Further work is required to elucidate the natural occurrence of Conidiobolus spp. in greenhouses and the complex interactions between biological and environmental factors. Studies on the occurrence of C. coronatus should be intensified because of its potential human pathogenicity. The species was reported as causative agent for nasal granuloma in humans (Ng et al., 1991), which leads to nasal obstruction and the formation of subcutaneous nodes. This disorder was reported particularly in workers in West African tropical rain forests, where the fungus is normally found in soil and in rotten plant material (Fromentin and Ravisse, 1977). Consequently, the problem of fungi in greenhouses air may be analysed in so far as the biomedical consequences of exposure to fungal propagules is concerned. Fungi may cause several problems when large numbers of conidia are present in indoor environments. Attention has been focused on allergy problems in workers (Miller, 1992; Crooke and Sherwood-Higham, 1997). 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