The pathogenic yeasts of Cryptococcus neoformans species complex have been isolated in the environment from bird
excreta, soil, trees, and decaying wood. Although previous studies showed the ability of C. neoformans species complex to
grow and mate on bird excreta and plant materials, there are no studies on soil. Therefore, the aim of the present study is to
verify if C. neoformans species complex is able to grow on media containing soil and if it is able to sexually reproduce on
this substrate. Soil media were prepared using four different soil samples collected in four different Italian regions, and the
two reference strains JEC20 and JEC21 were tested to verify their ability to mate on these media. All the media supported the
growth and sexual reproduction confirming that soil represents a realized niche for C. neoformans species complex

M. Cogliati

AIR Universita degli studi di Milano

International Journal of Fungal Genetics and 
Biology 
BMRJ, 1(1): 1-4 
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Original Case Report: Open Access 
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Int J Fungal Genet Biol (IJFGB) 1 
Soil Enables Mating and Sporulation of Cryptococcus Neoformans Species 
Complex 
Massimo Cogliati* 
*Lab. Medical Mycology, Dept. Biomedical Sciences for Health, Università degli Studi di Milano, Milano, Italy
Received December 24, 2017; Accepted March 23, 2018; Published May 10, 2018
ABSTRACT 
The pathogenic yeasts of Cryptococcus neoformans species complex have been isolated in the environment from bird 
excreta, soil, trees, and decaying wood. Although previous studies showed the ability of C. neoformans species complex to 
grow and mate on bird excreta and plant materials, there are no studies on soil. Therefore, the aim of the present study is to 
verify if C. neoformans species complex is able to grow on media containing soil and if it is able to sexually reproduce on 
this substrate. Soil media were prepared using four different soil samples collected in four different Italian regions, and the 
two reference strains JEC20 and JEC21 were tested to verify their ability to mate on these media. All the media supported the 
growth and sexual reproduction confirming that soil represents a realized niche for C. neoformans species complex. 
INTRODUCTION
Encapsulated yeasts belonging to Cryptococcus neoformans 
and Cryptococcus gattii species complexes are the main 
fungal infectious agents of cryptococcosis, a life-threatening 
fungal infection occurring in both immunocompromised and 
immunocompetent hosts causing meningitis and meningo-
encephalitis as the most frequent symptom [1]. The infection 
is acquired by inhalation of small cells or basidiospores 
present in the environment, which are able to reach 
pulmonary alveoli and to cause the onset of the disease [2]. 
Understanding the ecology of these fungal pathogens is a 
crucial step to elucidate which are their ecological niches 
and therefore the major sources and reservoir in the 
environment in order to prevent infection of hosts included 
in high risk categories [3]. In the environment, C. 
neoformans species complex was isolated from bird excreta, 
soil, trees, and decaying wood [4-8]. A recent environmental 
survey carried out in Europe showed also that some trees, 
such as olive trees and carob trees, present a higher rate of 
colonization than others. In addition, soil collected near the 
colonized trees was often contaminated with the fungus 
suggesting that trees are a potential reservoir and soil is a 
secondary niche [6]. Although previous studies showed the 
ability of C. neoformans species complex to grow and mate 
on bird excreta [9] and plant materials [10], there are no 
studies on soil. Therefore, the aim of the present study is to 
verify if C. neoformans species complex is able to grow on 
media containing soil and if it is able to sexually reproduce 
on this substrate. 
MATERIALS AND METHODS 
Sampling and soil medium preparation 
Soil samples were collected in four different regions of Italy: 
Lombardia, Toscana, Marche, and Puglia (Figure 1). Soil 
samples from Marche and Toscana were collected from 
vineyards whereas those from Lombardia and Puglia were 
from olive groves (Table 1). Soil samples were not 
contaminated with bird excreta. 
Soil medium (SM) was prepared dissolving 50 g of soil in 
500 ml of distilled water. The suspension was boiled for at 
least 30 min and then filtered throughout some overlapping 
pieces of gauze. Volume was adjusted with further distilled 
water and 20 g of agar were added. The medium was 
sterilized by autoclave and then poured in 90 mm Petri 
dishes. For sugar soil medium (SSM), 2.5 g of glucose were 
added. Medium pH was measured before storage at 4°C. In 
addition, Murashige Skoog medium (Sigma-Aldrich, 
Milano, Italy) was prepared as control mating medium. 
*Corresponding Author: Massimo Cogliati, Lab. Medical Mycology, 
Dept. Biomedical Sciences for Health Università degli Studi di Milano, Via 
Pascal 36, 20133 Milano, Italy, Tel: +39 02 50315144; Fax: +39 02 
50315146; E-mail: massimo.cogliati@unimi.it 
Citation: Cogliati M. (2018) Soil Enables Mating and Sporulation of 
Cryptococcus Neoformans Species Complex Int J Fungal Genet Biol, 1(1): 
1-4. 
Copyright: ©2018 Cogliati M. This is an open-access article distributed 
under the terms of the Creative Commons Attribution License, which 
permits unrestricted use, distribution, and reproduction in any medium, 
provided the original author and source are credited. 
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Int J Fungal Genet Biol (IJFGB) 2 
International Journal of Fungal Genetics and Biology, 1(1): 1-4    Cogliati M 
Table 1. Incubation time to observe growth, filaments and basidiospores on the different soil media and on control medium. 
Sugar soil mediuma (days) Soil medium (days) 
Soil 
geographical 
origin 
Soil 
source 
Medium 
pH 
Growth Filaments Basidiospores Growth Filaments Basidiospores 
Lombardia Olive 
grove 
7.6 2 7 15 2 16 30 
Marche Vineyards 7.7 2 7 15 2 16 30 
Toscana Vineyards 7.8 2 7 15 2 A A 
Puglia Olive 
grove 
7.9 2 7 15 2 A A 
Control 
medium 
Murashige and 
Skoog 
5.5 2 7 10 
a Soil medium additioned with glucose; A=Absent 
Reference strains 
Two  reference  strains  were  used  in  mating  assays: 
JEC20  (Cryptococcus  neoformans  var. neoformans, 
mating type a) and JEC21 (Cryptococcus neoformans var. 
neoformans, mating type ). The strains were obtained by 
the National Institutes of Health (NIH), Bethesda, USA. 
Mating assays 
SM and SSM were prepared for each of the four soil 
samples. Each plate was inoculated with three streaks: one 
with JEC20 strain alone, one with JEC21 strain alone, and 
the third with the mix of the two strains. Plates were then 
incubated for four weeks at 25°C in the dark. Plates were 
observed periodically for the presence of filaments by a 
stereomicroscope (Zeiss, Oberkochen, Germany). Once the 
filaments appeared the culture was let grow for a further 
week and then a piece of agar containing hyphae was 
collected and observed by an optical microscope (Zeiss) in 
order to check and photograph the presence of clamp 
connections, basidia, and basidiospores. One plate 
containing Murashige Skoog medium was also inoculated as 
described above in order to test if the two tester strains were 
actually able to mate. 
RESULTS 
The four media prepared with the four soil samples 
presented different pH values ranging from 7.6 to 7.9. Soil 
from Lombardia presented the lowest pH and that from 
Puglia the highest one (Table 1). The two reference strains 
were able to grow on all soil media with a visible growth 
after 48 h of incubation. On SSM agar, filaments become 
macroscopically visible at the edge of the streak of all mixed 
cultures at days 7 of incubation. The same streaks produced 
basidia and basidiospores at day 15. On SM, filaments and 
basidiospores were produced only on two mixed culture 
streaks (soil samples from Lombardia and Marche) at day 16 
and 30, respectively (Table 1, Figure 1). As expected 
streaks inoculated with JEC20 or JEC21 strains alone did not 
produce any filament in all plates. The two tester strains 
confirmed their ability to mate on Murashige Skoog 
medium. 
DISCUSSION 
This study shows that C. neoformans species complex is 
able to grow on media containing soil material either with or 
without addition of glucose. This means that soil is a suitable 
substrate for the growth and survival of this fungus in 
agreement with several studies reporting isolation of C. 
neoformans species complex from soil samples [6,7,11,12] 
Cryptococcus neoformans species complex has been shown 
to grow and to mate on medium containing pigeon excreta 
[9] as well as on medium containing plant materials [10] but,
at present, similar studies on media containing soil has not
yet been carried out. Our results show that soil represents a
realized niche for C. neoformans species complex where it
can grow and sexually reproduce. In addition, we showed
that soil enables growth and mating of this yeast also
without bird excreta contamination and confirming that
Cryptococcus neoformans species complex is able to
colonize a wide range of microhabitats. Potentially, each
kind of soil is able to support sporulation of this pathogen
especially if it is rich of carbohydrates and it is sufficiently
acidic. This is also suggested by our results which showed
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Int J Fungal Genet Biol (IJFGB) 4 
International Journal of Fungal Genetics and Biology, 1(1): 1-4    Cogliati M 
that media prepared with soil from Puglia and Toscana, 
which were the least acidic media, enabled sporulation only 
in presence of glucose, whereas on the other two more acidic 
media (Lombardia and Marche) sporulation occurred also 
without glucose addition. 
Figure 1. Filaments produced on sugar soil medium prepared from four different soil samples and on the control medium, 
Murashige Skoog agar. Figure insets show basidia and basidiospores (magnification 1000X, bar 5 µm). At the bottom-left 
side of each figure the location of the Italian regions of sampling sites is shown.
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Int J Fungal Genet Biol (IJFGB) 4 
International Journal of Fungal Genetics and Biology, 1(1): 1-4    Cogliati M 
Basidiospores are the most resistant propagules of C. 
neoformans species complex and can be easily dispersed in 
the environment by wind, water, insects, birds, and other 
animals, as well as by human activities [13]. 
Therefore, the ability to produce basidiospores in soil 
represents an important step in Cryptococcus life cycle. In 
addition, presence of the fungus in the soil as yeast or 
basidiospores represents a potential source of infection 
because soil aerosols could transfer small cells and spores in 
pulmonary alveoli of humans and animals causing the onset 
of the infection as shown in previous studies [14,15]. At 
present, little is known about interaction between 
Cryptococcus neoformans and soil microhabitat, and many 
questions remain opened: how long can survive cryptococcal 
yeasts or basidiospores in soil? How do they interact with 
other organisms in this microhabitat? What is the abundance 
of this yeast in soil, and which kind of soil does better 
support their growth and mating? Further studies and efforts 
addressed to understand these and other questions about the 
ecology of this important fungal pathogen are needed in 
future. 
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Soil enables mating and sporulation of Cryptococcus neoformans species complex

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Soil enables mating and sporulation of Cryptococcus neoformans species complex

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