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
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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
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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). Several species of Penicillium, Aspergillus, Cladosporium, Alternaria and Fusarium are implicated in some health problems and may
be especially pathogenic to immunocompromised people (Burge, 1989; Schober, 1991). Aspergillus and Penicillium populations detected in greenhouse air may
become critical in the event of significant fungal colony
proliferation.
In conclusion, it is imperative to highlight the value
of a controlled ex situ conservation, with the view of
improving traditional horticultural practices. A correct mycological monitoring may directly support the
protection of both plant collections and workers
health.
Acknowledgements
We thank Dr Andrew Sabuneti for English language revision. This
work was supported by FAR, University of Pavia, Italy.
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