Laboratory Protocols in Fungal Biology

Fungal Biology
Vijai Kumar Gupta

Maria Tuohy Editors
Laboratory
Protocols in
Fungal Biology
Current Methods in Fungal Biology
Second Edition
Fungal Biology
Series Editors
Vijai Kumar Gupta
Center for Safe and Improved Food &
Biorefining and Advanced Materials Research Center
SRUC
Edinburgh, Scotland, UK
Maria G. Tuohy
School of Natural Sciences
National University of Ireland Galway
Galway, Ireland
About the Series
Fungal biology has an integral role to play in the development of the biotechnology
and biomedical sectors. It has become a subject of increasing importance as new
fungi and their associated biomolecules are identified. The interaction between fungi
and their environment is central to many natural processes that occur in the
biosphere. The hosts and habitats of these eukaryotic microorganisms are very
diverse; fungi are present in every ecosystem on Earth. The fungal kingdom is
equally diverse, consisting of seven different known phyla. Yet detailed knowledge
is limited to relatively few species. The relationship between fungi and humans
has been characterized by the juxtaposed viewpoints of fungi as infectious agents of
much dread and their exploitation as highly versatile systems for a range of
economically important biotechnological applications. Understanding the biology
of different fungi in diverse ecosystems as well as their interactions with living and
non-living is essential to underpin effective and innovative technological
developments. This series will provide a detailed compendium of methods and
information used to investigate different aspects of mycology, including fungal
biology and biochemistry, genetics, phylogenetics, genomics, proteomics, molecular
enzymology, and biotechnological applications in a manner that reflects the many
recent developments of relevance to researchers and scientists investigating the
Kingdom Fungi. Rapid screening techniques based on screening specific regions in
the DNA of fungi have been used in species comparison and identification, and are
now being extended across fungal phyla. The majorities of fungi are multicellular
eukaryotic systems and therefore may be excellent model systems by which to
answer fundamental biological questions. A greater understanding of the cell
biology of these versatile eukaryotes will underpin efforts to engineer certain fungal
species to provide novel cell factories for production of proteins for pharmaceutical
applications. Renewed interest in all aspects of the biology and biotechnology of fungi
may also enable the development of “one pot” microbial cell factories to meet
consumer energy needs in the 21st century. To realize this potential and to truly
understand the diversity and biology of these eukaryotes, continued development of
scientific tools and techniques is essential. As a professional reference, this series
will be very helpful to all people who work with fungi and should be useful both to
academic institutions and research teams, as well as to teachers, and graduate and
postgraduate students with its information on the continuous developments in
fungal biology with the publication of each volume.
More information about this series at http://www.springer.com/series/11224

Vijai Kumar Gupta • Maria Tuohy
Editors
Laboratory Protocols
in Fungal Biology
Current Methods in Fungal Biology
Second Edition
Editors
Vijai Kumar Gupta Maria Tuohy
Center for Safe and Improved Food & Department of Biochemistry
Biorefining and Advanced Materials National University of Ireland Galway
Research Center Galway, Ireland
SRUC
Edinburgh, Scotland, UK
ISSN 2198-7777 ISSN 2198-7785 (electronic)

Fungal Biology
ISBN 978-3-030-83748-8 ISBN 978-3-030-83749-5 (eBook)
https://doi.org/10.1007/978-3-030-83749-5
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland
AG 2013, 2022
Originally published under Vijai Kumar Gupta, Maria G. Tuohy, Manimaran Ayyachamy, Kevin
M. Turner, and Anthonia O’Donovan, Laboratory Protocols in Fungal Biology: Current Methods in
Fungal Biology
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Contents
1 Various Methods of Long-Term Preservation of Fungal

Cultures in All-Russian Collection of Microorganisms (VKM) . . . . 1
Svetlana M. Ozerskaya, Nataliya E. Ivanushkina,
Galina A. Kochkina, Anastasya A. Danilogorskaya,
Irina P. Pinchuk, and Alexander N. Vasilenko
2 Sabouraud Agar and Other Fungal Growth Media . . . . . . . . . . . . . 69
Tankeshwar Acharya and Janelle Hare
3 Fluorescence In Situ Hybridization of Uncultured Zoosporic
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Télesphore Sime-Ngando, Marlène Jobard, and Serena Rasconi
4 Technique for Identifying and Counting Infective Chytrid
Sporangia Using the Chitinaceous Fluorochrome
Calcofluor White . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Télesphore Sime-Ngando, Serena Rasconi, and Mélanie Gerphagnon
5 Assessment of Host Immune Responses to Fungal Pathogens . . . . . 103
Huilin Su, Chunxiao Li, Jiande Han, Clement K. M. Tsui,
and Min Zhu
6 Recent Advances in Applications of Support Vector Machines
in Fungal Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Sonal Modak, Ashwin Lahorkar, and Jayaraman Valadi
7 Real-Time Quantitative PCR Assay for the Assessment
of Uncultured Zoosporic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Télesphore Sime-Ngando and Marlène Jobard
v
vi Contents
8 Assays for the Quantification of Antioxidant Enzymes

in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Konstantinos Grintzalis, Ioannis Papapostolou,
and Christos D. Georgiou
9 Cellulomics of Live Yeast by Advanced and Correlative
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Zinnat Shahina, Supriya V. Bhat, Easter Ndlovu,
Taranum Sultana, André Körnig, Étienne Dague,
and Tanya E. S. Dahms
10 Molecular Taxonomy and Multigene Phylogeny
of Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Nikita Mehta, Reshma Jadhav, and Abhishek Baghela
11 Fluorochrome-Based Methods for Fungal Sample
Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Silvino Intra Moreira, Lucas Fidelis Pereira,
Elaine Aparecida de Souza, and Eduardo Alves
12 Yeast Isolation Methods from Specialized Habitats . . . . . . . . . . . . . 235
Rameshwar Avchar, Snigdha Tiwari, and Abhishek Baghela
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Chapter 1
Various Methods of Long-Term
Preservation of Fungal Cultures in
All-Russian Collection of Microorganisms
(VKM)
Svetlana M. Ozerskaya, Nataliya E. Ivanushkina, Galina A. Kochkina,

Anastasya A. Danilogorskaya, Irina P. Pinchuk, and Alexander N. Vasilenko
Contents
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Cryopreservation of Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Freeze-Drying of Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Drying in Sterile Soil of Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5 Drying of Filamentous Fungi on Silica Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7 Protocol of Drying on Silica Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7.1 Preparation of Sterile Silica Gel and Ampoules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7.3 Preparation of Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7.4 Silica Gel Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.7.5 Filling of Vials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.7.6 Control of Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Annexies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Annex 1: Fields Attributes in the Table «Database Preservation Methods» . . . . . . . . . . . . . . 22
Annex 2: Maximal Preservation Times for VKM Fungal Species . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
S. M. Ozerskaya (*) · N. E. Ivanushkina · G. A. Kochkina · A. A. Danilogorskaya ·

I. P. Pinchuk · A. N. Vasilenko
All-Russian Collection of Microorganisms (VKM), G.K. Skryabin Institute of Biochemistry
and Physiology of Microorganisms, Russian Academy of Science, Pushchino,
Moscow Region, Russia
e-mail: smo@dol.ru
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

V. K. Gupta, M. Tuohy (eds.), Laboratory Protocols in Fungal Biology, Fungal
Biology, https://doi.org/10.1007/978-3-030-83749-5_1
2 S. M. Ozerskaya et al.
1.1 Introduction
Microorganisms are fundamental materials for scientific and practical studies. Cul-
ture collections (biological resource centers) play a primary role in the stable
preservation and long-term storage of microbial resources and ensure regular access
to well-documented strains after a long time from their isolation for scientific or
biotechnological use [32, 33].
Various methods of preservation of fungal cultures have been reported [13, 25,
29]. Freeze-drying (lyophilization) and cryopreservation methods are utilized for
thousands of fungal strains in microbial collections all over the world [7, 12, 27].
Nevertheless, it is clear that the fungal strains of different species vary in the ability
to survive after long-time storage preservation under laboratory conditions. Some of
them are very difficult to maintain ex situ, whereas others could be easily and
successfully preserved alive by using almost any conservation technique.
Storage methods for filamentous fungi result from the type and degree of spor-
ulation. Spore-forming strains (as opposed to nonsporulating strains) can be
effectively freeze-dried. Both types can be frozen and stored for long periods in
liquid nitrogen or in a low-temperature refrigerator. The experience of long-term
preservation of fungal strains shows that the duration of storage directly depends not
only on the choice of the method but also on the laboratory protocol and temperature
of subsequent cultures storage.
This chapter presents the methods of cryopreservation, freeze-drying, drying on
silica gel, and preservation in sterile soil that are utilized in VKM fungal collection,
accompanied by data on maximal storage time registered. The methods take into
consideration the special features of cultures preserved as well as the equipment
used.
VKM fungal collection (All-Russian Collection of Microorganisms, Russia) was
established in 1955 and has a long-term experience in the preservation and storage of
fungal cultures. Collection of filamentous fungi is currently composed of approxi-
mately 7000 strains (590 genera, 1600 species) belonging to species of the kingdoms
Chromista (Oomycota) and Fungi (zygomycetous, ascomycetous and basidiomyce-
tous fungi).
All the information on preservation methods for each VKM fungal strain is
presented in the MS Access database. It keeps curated data on the strain numbers,
preservation dates as well as inspection dates in various methods, and other technical
information. Fields in the database table are presented in Annex 1. For operational
analysis of these data, we use MS Access requests – «FunPreservEnd»,
«FunPreserv_Times», «FunPreserv_MaxTimes». The maximal preservation time
is calculated automatically; the latest results (25.11.2019) are presented in Annex 2.
Preserved for many years fungi of various taxa retain their ability to produce
different substances suitable as a material for industry and medicine. For instance,
the zygomycetous fungus Cunninghamella japonica VKM F-1204D was found to
be a promising lipid producer for biodiesel production [22]. Fungi of the genus
Penicillium, which are supported in the collection for more than 40 years (VKM
1 Various Methods of Long-erm Preservation of Fungal Cultures 3
F-325, VKM F-691, VKM F-1823), are able to synthesize active compounds with
diverse structures [10]. Aspergillus brasiliensis VKM F-1119, which was accepted
by VKM 52 years ago, engaged in the vital process of biotransformation of
artemisinin, uncial medicine for the treatment of tropical malaria [34]. Recently
published data on the assessment of the effect of freeze-drying and long-term storage
on the biotechnological potential of Aspergillus section Nigri strains show
maintaining of biotechnological properties after preservation [19].
1.2 Cryopreservation of Filamentous Fungi
According to published data, the fast cooling rates followed by storage in liquid
nitrogen at -196 C allow secure and long-term preservation of some fungal cultures
[21]. However, the ability to resist damage by freezing and warming differs consid-
erably among genera/species and depends on their particular features (presence and
type of sporulation, chemical composition of cytoplasmic membrane and cell wall,
physiological state, etc.). Selection of optimal cryoprotectants, rates of cooling, and
warming has enabled increasing the number and diversity of taxa preserved by this
method [24, 28].
More than 75% filamentous fungi of VKM are stored using various cryopreser-
vation protocols. Cultures with abundant sexual and nonsexual sporulation usually
were preserved by using fast cooling rates followed by storage either in liquid
nitrogen or in ultralow temperature freezers at 70 C.
It was noticed that some cultures of zygomycetous fungi belonging to the genera
Mortierella, Basidiobolus, Coemansia, and Entomophthora do not survive the
ultrarapid freezing procedure even if they have abundant sporulation. Successful
preservation of such strains was achieved by modification of the cryopreservation
regime, for example, using slow programmed freezing. The same method was used
either for nonsporulating fungi (basidiomycetous fungi) or zoosporeforming former
fungi (Chromista, Oomycota).
According to our data, some parts of strains of Oomycota (20%), basidiomyce-
tous fungi (4%), zygomycetous fungi (1%), and ascomycetous fungi (1%) did not
survive cryopreservation at all freezing regimes and modification applied [9]. The
strains most difficult to maintain belong to genera Dictyuchus and Phytophthora and
to some species of Achlya and Saprolegnia. Similar situations have also been seen
with some species of basidiomycetous fungi (Suillus, Amanita, Dictyophora,
Mutinus, etc.). They are usually maintained by subculturing and preservation
under mineral oil.
It has been suggested that those microbial cultures that are able to survive the
freezing and a short storage will permanently stay in the vital state after any length of
storage [20]. According to our data, this is not quite true: some strains of Achlya
colorata, Achlya intricata, Clitocybe odora, Choanephora conjuncta, Conidiobolus
thromboides, Kickxella alabastrina, Phanerochaete sanguinea, Rhodocollybia
butyracea, and Saprolegnia terrestris have lost their ability to grow after 5–7
years of storage in liquid nitrogen, although they were in the viable state after 24 h of
storage. The reason is not yet known. Nevertheless, the viability test showed that
representatives of 311 species of fungi remain alive after 20–30 years of storage
(Annex 2).
The cooling equipment being used in VKM is storage tanks “Bioproducts-0.5”
with a capacity of 500 liters of liquid nitrogen and ultralow temperature freezers
( 80 C, Sanyo, Japan).
1.3 Freeze-Drying of Filamentous Fungi
Currently, freeze-drying is used to preserve approximately 85% of filamentous fungi

maintained in VKM. Fungi from different taxonomical groups (zygomycetous fungi,
ascomycetous fungi – both teleo- and anamorph) able to produce dormant structures
(spores, sclerotia, etc.) usually survive freeze-drying [11]. According to our data,
about 90% of strains of these fungal groups remain alive in this method. We noticed
that the freeze-dried strains of 817 species stored at 5 C for more than 20 years were
in a viable state, and cultures of 289 species have been sustained for even 40–50
years of storage. Some species did not survive freeze-drying even when the sporu-
lation is abundant, those are Conidiobolus coronatus, C. thromboides,
Entomophthora thaxteriana, E. conica, E. dipterigena, Cunninghamella
homothallica, and C. vesiculosa. Species of genus Botrytis (B. fabae and B.
squamosa), forming only sclerotia as a dormant structure, remain in a vital state in
freeze-drying only for rather a short time – less than 10 years [9].
Nonsporulating microorganisms from Oomycota and basidiomycetous fungi are
not stored in VKM by freeze-drying, since sterile mycelia generally do not remain
viable.
The equipment used in VKM for freeze-drying is the centrifugal freeze-dryer
system Micromodulyo (Edwards, UK).
1.4 Drying in Sterile Soil of Filamentous Fungi
This simple and popular method for the preservation of fungi was applied at the
beginning of the twentieth century [18]. Species of Aspergillus and Penicillium can
be maintained by this way more effectively than other micromycetes. According to
T.P. Suprun [31] who investigated the preservation of 78 Penicillium species (more
than 1000 strains) in sterile soil for 7–10 years, the best preserved strains were
representatives of Assymmetrica section. Less effectively preserved species were
Biverticillata-Symmetrica, and the lowest effectiveness was observed with strains of
the section Monoverticillata.
This method is also efficient for preservation of some human, animal, and plant
pathogens with retaining their virulence [21]. For example, Alternaria japonica (syn.
A. raphani), Fusarium oxysporum, and the species of Septoria (S. avenae, S.

nodorum, S. passerinii, S. tritici) have retained their ability to infect a plant host
after 2–5 years of storage [2, 8, 23]. Some degraded strains of micromycetes partly
recuperated their lost qualities after preservation in soil [30].
According to our data, fungal strains of 167 species stored by this method are able
to maintain viability for more than 30 years, and cultures of 87 species have been
sustained for even 40–55 years of storage.
1.5 Drying of Filamentous Fungi on Silica Gel
Immobilized cells of microorganisms retain viability and biological activity at action

of different stressors, as a rule, better than free ones [4]. Therefore, the preliminary
drying of the cells on the adsorbent allows the microorganisms to remain viable for a
longer time. As an adsorbent on which a suspension of microorganisms is applied for
subsequent drying, silica gel (a dried gel of polysilicic acid with numerous pores) is
most often used [5]. Silica gel promotes the dehydration of microorganisms and
helps them to survive a thermal stress [24]. Since the silica gel can prevent all fungal
growth and metabolism, the risk of any morphological, physiological, and genetic
changes could be minimized [1].
Using of anhydrous silica gel particles for maintaining stock cultures of Neuros-
pora crassa was suggested by D. Perkins in 1962 [17]. This new method has proved
consistently useful and effective over several years.
At present, this method is widely used in relation to different taxa and ecological
groups of fungi. So, the method was effective for the storage of entomopathogenic
fungi of the order of Hypocreales for 2 years [3] and, in particular, Metarhizium
anisopliae [6], as well as for fungi of many other taxa, including the spores of
obligate biotrophic parasite Podosphaera fusca [16] and rust fungi, which cannot be
grown on agar media [1].
As a disadvantage of the silica gel preservation method, researchers note that the
time of storage is quite short (between 2 and 4 years) [14, 26]. But it is clear that the
features of the methodological protocols can be crucial for the fungi preservation by
this method, wherein the temperature at which frozen fungi are stored affects how
long they could be preserved while remaining viable.
The method of storage on silica gel was introduced in VKM in the middle of the
1980s [24]. Our experience has shown that several groups of fungi can be preserved
by this method without losing vitality for many years (Table 1.1).
The viability of more than 300 strains of zygomycetous fungi with various types
of sporogenous structures (6 classes, 6 orders, 15 families, 35 genera, and 118
species) and near 300 strains of dark-colored anamorphic ascomycetous fungi with
different types of conidiogenesis (7 classes, 18 orders, 34 families, 79 genera, and
164 species) (Table 1.2) was assessed from 1 to near 30 years of preservation (Figs.
1.1 and 1.2).
Table 1.1 Drying of VKM fungal cultures on silica gel (storage time)
Storage time at
different temperature
(years)
Number 12 70
No. Name of species of strains 5 С C
C
1 Absidia caerulea Bainier 1889 4 13,98 8,84 29,95
2 Absidia cuneospora G.F. Orr et Plunkett 1959 1 1,21 1,21 30,57
3 Absidia cylindrospora Hagem 1908 2 16,15 11,79 30,02
4 Absidia glauca Hagem 1908 4 10,77 7,78 29,95
5 Absidia repens van Tieghem 1878 1 1,07 1,07 29,44
6 Absidia spinosa Lendner 1907 1 1,66 1,09 30,00
7 Acrophialophora fusispora (S.B. Saksena 1953) 1 32,24 22,34 32,24
Samson 1970
8 Actinomucor elegans (Eidam 1884) C.R. Benjamin 7 15,52 11,34 30,04
et Hesseltine 1957
9 Albifimbria verrucaria (Albertini et Schweinitz 1 11,05 11,05 32,41
1805) L. Lombard et Crous 2016
10 Alternaria alternata (Fries 1832) Keissler 1912 5 10,98 8,66 31,61
11 Alternaria atra (Preuss 1852) Woudenberg et Crous 4 31,81 13,31 31,87
2013
12 Alternaria botrytis (Preuss 1851) Woudenberg et 9 25,40 11,32 32,11
Crous 2013
13 Alternaria brassicicola (Schweinitz 1832) Wiltshire 1 21,75 10,36 31,68
1947
14 Alternaria chartarum Preuss 1848 4 26,64 10,07 31,97
15 Alternaria consortialis (Thuemen 1876) Groves et 3 9,63 8,25 32,25
Hughes 1953
16 Alternaria japonica Yoshii 1941 1 6,96 3,04 32,26
17 Alternaria macrospora Zimmermann 1904 2 5,47 1,63 31,97
18 Alternaria multirostrata E.G. Simmons et C.R. 1 1,30 6,18 31,56
Jackson 1968
19 Alternaria oudemansii (E.G. Simmons 1967) 1 1,00 4,89 31,26
Woudenberg et Crous 2013
20 Alternaria radicina Meier et al. 1922 1 5,88 2,84 32,32
21 Alternaria solani Sorauer 1896 1 1,19 3,21 31,91
22 Alternaria tenuissima (Kunze 1818) Wiltshire 1933 1 22,35 10,91 32,26
23 Amerosporium concinnum Petrak 1953 1 31,26 9,91 31,26
24 Ampelomyces artemisiae (Voglino 1905) Rudakov 1 10,61 6,64 31,95
1979
25 Ampelomyces heraclei (Dejeva 1967) Rudakov 1979 1 10,61 10,61 31,97
26 Ampelomyces humuli (Fautrey 1890) Rudakov 1979 1 31,99 10,61 31,95
27 Ampelomyces polygoni (Potebnia 1907) Rudakov 1 0,72 2,51 22,56
1979
28 Ampelomyces ulicis (Adams 1907) Rudakov 1979 1 22,03 10,61 31,95
29 Ampelomyces uncinulae (Fautrey 1893) Rudakov 1 21,69 1,35 31,62
1979
(continued)
Table 1.1 (continued)

Storage time at
(years)
Number 12 70
C
30 Apenidiella strumelloidea (Milko et Dunaev 1986) 1 0,96 3,09 32,48
W. Quaedvlieg et P.W. Crous 2014
31 Aposphaeria caespitosa (Fuckel 1869) Jaczewski 1 10,64 3,30 32,05
1917
32 Arthrinium arundinis (Corda 1838) Dyko et Sutton 1 10,28 10,30 31,62
1981
33 Arthrinium sphaerospermum Fuckel 1874 1 21,99 10,57 31,91
34 Ascochyta malvicola Saccardo 1878 1 2,17 2,17 31,62
35 Aureobasidium melanogenum (Hermanides-Nijhof 6 28,72 8,05 32,33
1977) Zalar et al. 2014
36 Aureobasidium microstictum (Bubak 1907) W.B. 1 31,99 6,68 31,99
Cooke 1962
37 Aureobasidium pullulans (de Bary 1866) G. Arnaud 7 24,50 9,05 32,09
1918
38 Backusella circina J.J. Ellis et Hesseltine 1969 1 4,30 7,25 29,98
39 Backusella indica (Baijal et B.S.Mehrotra 1965) G. 1 4,42 4,42 30,49
Walther et de Hoog 2013
40 Backusella lamprospora (Lendner 1908) Benny et 3 6,55 4,25 29,93
R.K. Benjamin 1975
41 Backusella oblongielliptica (H. Naganishi et al. ex 1 1,33 1,33 29,67
Pidoplichko et Milko 1971) G. Walther et de Hoog
2013
42 Backusella recurva (E.E. Butler 1952) G. Walther et 1 4,45 7,42 29,84
de Hoog 2013
43 Backusella tuberculispora (Schipper 1978) G. 1 4,11 6,66 28,58
44 Backusella variabilis (A.K. Sarbhoy 1965) G. 1 7,42 4,45 29,84
45 Beauveria brongniartii (Saccardo 1892) Petch 1926 1 2,98 6,02 32,37
46 Benjaminiella poitrasii (R.K. Benjamin 1960) Arx 1 16,01 7,33 29,84
1981
47 Berkeleyomyces basicola (Berkeley et Broome 1850) 1 3,24 3,24 31,99
W.J. Nel et al. 2017
48 Bipolaris australiensis (M.B. Ellis 1971) Tsuda et 4 19,07 5,70 31,58
Ueyama 1981
49 Bipolaris cynodontis (Marignoni 1909) Shoemaker 1 21,62 10,21 31,56
1959
50 Bipolaris sorokiniana (Saccardo 1890) Shoemaker 2 8,51 8,45 31,82
1959
51 Bipolaris victoriae (F. Meehan et H.C. Murphy 1 0,96 0,96 21,23
1946) Shoemaker 1959
52 Bispora antennata (Persoon 1801) E.W. Mason 1953 1 4,79 1,70 31,19
(continued)

Storage time at
(years)
Number 12 70
C
53 Bispora betulina (Corda 1838) S. Hughes 1958 1 3,38 3,38 3,38
54 Bispora effusa Peck 1891 1 6,24 2,17 10,30
55 Blakeslea trispora Thaxter 1914 8 16,69 12,33 29,84
56 Botryotrichum piluliferum Saccardo et Marchal 1885 4 17,10 6,78 25,82
57 Botrytis aclada Fresenius 1850 2 10,87 6,84 32,22
58 Botrytis anthophila Bondartsev 1913 1 5,82 2,72 32,20
59 Botrytis cinerea Persoon 1794 8 1,30 1,65 15,34
60 Botrytis convoluta Whetzel et Drayton 1932 2 5,23 6,26 32,11
61 Botrytis elliptica (Berkeley 1881) Cooke 1901 1 1,02 1,02 1,02
62 Botrytis galanthina (Berkeley et Broome 1873) 1 1,02 1,02 1,02
Saccardo 1886
63 Botrytis gladiolorum Timmermans 1941 2 2,11 4,48 17,66
64 Botrytis tulipae (Libert 1830) Lind 1913 1 0,08 0,08 2,90
65 Cadophora fastigiata Lagerberg et Melin 1928 1 10,76 3,35 32,14
66 Cadophora malorum (Kidd et Beaumont 1924) W. 4 8,82 6,40 32,16
Gams 2000
67 Cadophora melinii Nannfeldt 1934 1 3,31 8,34 32,07
68 Cephalotrichum gorgonifer (Bainier 1907) 1 3,02 7,00 32,24
Sandoval-Denis et al. 2016
69 Cephalotrichum purpureofuscum (Schweinitz 1832) 1 0,98 0,98 31,24
S. Hughes
70 Cephalotrichum stemonitis (Persoon 1801) Nees 3 3,21 4,22 23,24
1812
71 Chaetocladium brefeldii van Tieghem et G. Le 2 23,8 12,77 30,4
Monnier 1873
72 Chaetocytostroma sp. 1 0,01 1,02 21,34
73 Chloridium caesium (Nees et T. Nees 1818) Réblová 1 1,32 1,32 31,58
et Seifert 2016
74 Chloridium virescens (Persoon 1797) W. Gams et 1 1,13 1,13 31,39
Holubova-Jechova 1976 var. caudigerum (Hoehnel
1903) W. Gams et Holubova-Jechova 1976
75 Choanephora infundibulifera (Currey 1873) 1 17,56 7,35 29,88
Saccardo 1891
76 Circinella muscae (Sorokin 1870) Berlese et de Toni 3 21,11 5,88 30,11
1888
77 Circinella umbellata van Tieghem et G. Le Monnier 1 15,27 6,34 28,33
1873
78 Cladophialophora chaetospira (Grove 1886) Crous 1 10,57 3,21 31,91
et Arzanlou 2007
79 Cladosporium aecidiicola Thuemen 1876 1 1,01 4,82 31,24
(continued)

Storage time at
(years)
Number 12 70
C
80 Cladosporium brevicompactum Pidoplichko et 2 6,23 6,21 31,65
Deniak 1941
81 Cladosporium cladosporioides (Fresenius 1850) 2 9,08 5,15 32,41
G.A. de Vries 1952
82 Cladosporium colocasiae Sawada 1916 1 1,04 2,94 32,15
83 Cladosporium cucumerinum Ellis et Arthur 1889 1 1,07 1,07 32,07
84 Cladosporium elegantulum Pidoplichko et Deniak 2 10,51 10,53 31,86
1938
85 Cladosporium gossypiicola Pidoplichko et Deniak 2 16,32 6,72 32,08
1941
86 Cladosporium herbarum (Persoon 1794) Link 1816 15 9,14 7,05 29,98
87 Cladosporium lycoperdinum Cooke 1883 1 4,82 4,82 31,24
88 Cladosporium macrocarpum Preuss 1848 3 3,32 4,61 31,68
89 Cladosporium pseudocladosporioides Bensch et al. 1 31,22 9,87 31,22
2010
90 Cladosporium sphaerospermum Penzig 1882 5 10,02 7,61 30,28
91 Cladosporium straminicola Pidoplichko et Deniak 1 3,09 3,09 32,48
1938
92 Cladosporium transchelii Pidoplichko et Deniak 1 11,13 3,09 32,48
1938
93 Cokeromyces recurvatus Poitras 1950 2 16,73 7,31 29,45
94 Colletotrichum gloeosporioides (Penzig 1882) 2 13,94 2,12 20,95
Penzig et Saccardo 1884
95 Colletotrichum musae (Berkeley et M.A. Curtis 1 9,94 1,00 31,26
1874) Arx 1957
96 Conidiobolus coronatus (Costantin 1897) Batko 1 1,74 1,74 30,37
1964
97 Coniothyrium concentricum (Desmazieres 1840) 1 31,62 21,69 31,62
Saccardo 1878
98 Coniothyrium hellebori Cooke et Massee 1886 1 10,96 1,05 10,96
99 Coniothyrium rosarum Cooke et Harkness 1882 2 10,49 6,52 31,83
100 Coniothyrium wernsdorffiae Laubert 1905 1 1,28 2,15 2,15
101 Cunninghamella blakesleeana Lendner 1927 1 7,80 7,80 30,16
102 Cunninghamella echinulata (Thaxter 1891) Thaxter 11 17,54 6,51 30,00
ex Blakeslee1905
103 Cunninghamella japonica (Saito 1905) Pidoplichko 7 10,65 7,76 29,56
et Milko 1971
104 Curvularia comoriensis Bouriquet et Jauffret 1955 1 3,17 10,59 31,93
ex M.B. Ellis 1966
105 Curvularia geniculata (Tracy et Earle 1896) Boedijn 2 5,44 3,45 16,72
1933
(continued)

Storage time at
(years)
Number 12 70
C
106 Curvularia inaequalis (Shear 1907) Boedijn 1933 1 31,91 6,96 32,01
107 Curvularia lunata (Wakker 1898) Boedijn 1933 2 5,93 3,35 32,11
108 Dematioscypha delicata (Berkeley et Broome 1859) 1 0,00 1,94 4,99
Hosoya 2014
109 Dicyma ampullifera Boulanger 1897 1 6,20 6,20 6,20
110 Dicyma olivacea (Emoto et Tubaki 1970) Arx 1982 1 0,80 5,90 5,90
111 Didymella glomerata (Corda 1840) Q. Chen et L. Cai 6 32,92 8,97 31,92
2015
112 Didymella pomorum (Thümen 1879) Q. Chen et L. 2 16,31 6,67 31,95
Cai 2015
113 Dinemasporium strigosum (Persoon 1801) Saccardo 1 1,30 1,30 31,56
1881
114 Discula brunneotingens E.I. Meyer 1953 1 0,00 0,96 0,96
115 Discula pinicola (Naumov 1926) Petrak 1927 var. 1 5,83 5,83 31,28
mammosa Lagerberg et al. 1927
116 Dothiora prunorum (Dennis et Buhagiar 1973) 1 31,97 10,62 31,97
Crous 2016
117 Entomophthora conica Nowakowski 1883 1 1,33 6,86 29,67
118 Entomophthora thaxteriana I.M. Hall et J. Bell 1963 1 0,10 0,10 30,7
119 Epicoccum nigrum Link 1815 2 2,20 2,20 31,98
120 Exophiala castellanii Iwatsu et al. 1984 1 0,00 2,92 32,33
121 Exophiala salmonis J.W. Carmichael 1966 1 5,99 5,99 31,42
122 Fennellomyces linderi (Hesseltine et Fennell 1955) 1 16,94 7,34 29,39
Benny et R.K. Benjamin 1975
123 Fonsecaea pedrosoi (Brumpt 1922) Negroni 1936 1 1,07 1,07 29,34
124 Fulvia fulva (Cooke 1883) Ciferri 1954 1 0,00 0,00 1,32
125 Geomyces pannorum (Link 1824) Sigler et J.W. 1 5,82 5,82 10,79
Carmichael 1976
126 Gilbertella persicaria (E.D. Eddy 1925) Hesseltine 1 16,72 16,72 30,37
1960
127 Gliocephalotrichum bulbilium J.J. Ellis et Hesseltine 1 1,04 1,04 32,24
1962
128 Gliomastix murorum (Corda 1838) S. Hughes 1958 1 6,23 6,23 31,66
var. murorum
129 Gongronella butleri (Lendner 1926) Peyronel et Dal 5 0,90 3,34 24,40
Vesko 1955
130 Gonytrichum macrocladum (Saccardo 1880) S. 1 22,34 22,34 32,24
Hughes 1951
131 Hansfordia pulvinata (Berkeley et M.A. Curtis 1875) 1 0,98 5,83 31,24
S. Hughes 1958
132 Harzia acremonioides (Harz 1871) Costantin 1888 3 6,29 6,31 31,46
(continued)

Storage time at
(years)
Number 12 70
C
133 Helicostylum elegans Corda 1842 1 18,23 18,23 30,33
134 Helicostylum pulchrum (Preuss 1851) Pidoplichko et 2 10,5 10,5 30,36
Milko 1971
135 Hesseltinella vesiculosa H.P. Upadhyay 1970 1 0,10 0,10 0,98
136 Hormoconis resinae (Lindau 1906) Arx et G.A. de 8 17,31 13,39 32,08
Vries 1973
137 Hormonema macrosporum L. Voronin 1986 1 3,21 10,60 31,97
138 Humicola fuscoatra Traaen 1914 2 16,11 2,02 31,73
139 Hyphopichia burtonii (Boidin et al. 1964) Arx et Van 1 10,98 3,11 32,33
der Walt 1976
140 Kickxella alabastrina Coemans 1862 1 8,30 8,30 17,34
141 Lecythophora decumbens (J.F.H. Beyma 1942) E. 1 11,05 11,05 32,41
Weber et al. 2002
142 Lecythophora fasciculata (J.F.H. Beyma 1939) E. 1 31,99 10,62 31,99
Weber et al. 2002
143 Lecythophora hoffmannii (J.F.H. Beyma 1939) W. 2 27,12 10,72 32,08
Gams et McGinnis 1983
144 Lecythophora mutabilis (J.F.H. Beyma 1944) W. 1 32,18 2,98 32,18
Gams et McGinnis 1983
145 Lichtheimia blakesleeana (Lendner 1924) Kerst. 3 16,79 11,03 30,38
Hoffmann et al. 2009
146 Lichtheimia corymbifera (Cohn 1884) Vuillemin 11 15,65 7,93 29,86
1903
147 Lichtheimia hyalospora (Saito 1906) Kerst. 1 16,72 11,88 30,37
Hoffmann et al. 2009
148 Linderina pennispora Raper et Fennell 1952 1 1,00 1,00 30,16
149 Macrophoma mantegazziana (Penzig 1882) Berlese 1 1,19 1,19 31,91
et Voglino 1886
150 Memnoniella echinata (Rivolta 1884) Galloway 2 12,21 6,52 31,57
1933
151 Menispora ciliata Corda 1837 1 0,96 0,96 31,15
152 Microsphaeropsis olivacea (Bonorden 1869) 1 22,13 10,73 32,03
Höhnell 1917
153 Monodictys paradoxa (Corda 1938) S. Hughes 1958 1 32,47 11,05 32,41
154 Mortierella alpina Peyronel 1913 1 7,10 4,51 28,99
155 Mortierella beljakovae Milko 1973 1 0,10 0,10 29,63
156 Mortierella capitata Marchal 1891 1 22,67 7,03 29,8
157 Mortierella dichotoma Linnemann 1936 ex W. Gams 1 1,33 4,35 29,67
1977
158 Mortierella exigua Linnemann 1941 1 6,87 1,29 29,63
159 Mortierella gemmifera M. Ellis 1940 1 6,90 4,34 29,63
(continued)

Storage time at
(years)
Number 12 70
C
160 Mortierella globulifera O. Rostrup 1916 1 1,33 1,33 29,67
161 Mortierella hyalina (Harz 1871) W. Gams 1970 var. 3 2,24 3,24 29,47
hyalina
162 Mortierella jenkinii (A.L. Smith 1898) Naumov 1 1,15 1,15 7,06
1935
163 Mortierella lignicola (G.W. Martin 1937) W. Gams 1 15,82 7,06 28,99
et R. Moreau 1959
164 Mortierella mutabilis Linnemann 1941 1 0,10 15,44 28,38
165 Mortierella parvispora Linnemann 1941 4 2,43 2,43 18,19
166 Mortierella polycephala Coemans 1863 1 2,12 6,20 28,16
167 Mortierella pusilla Oudemans 1902 1 4,78 1,29 29,63
168 Mortierella reticulata van Tieghem et G. Le Monnier 1 0,42 0,42 1,29
1873
169 Mortierella stylospora Dixon-Stewart 1932 1 7,16 7,16 7,16
170 Mortierella verticillata Linnemann 1941 5 2,12 3,32 29,52
171 Mortierella zychae Linnemann 1941 1 0,10 0,75 3,05
172 Mucor aligarensis B.S. Mehrotra et B.R. Mehrotra 1 1,24 2,40 29,27
1969
173 Mucor bainieri B.S. Mehrotra et Baijal 1963 1 6,27 6,27 29,10
174 Mucor circinelloides van Tieghem 1875 var. 9 20,75 6,18 29,34
circinelloides
janssenii (Lendner 1907) Schipper 1976
lusitanicus (Bruderlein 1916) Schipper 1976
177 Mucor durus G. Walther et de Hoog 2013 1 15,4 6,43 29,27
178 Mucor exponens (Burgeff 1924) G. Walther et de 4 7,54 2,87 29,95
Hoog 2013
179 Mucor flavus Bainier 1903 13 10,27 7,5 30,04
180 Mucor fuscus Bainier 1903 3 5,72 4,15 30,19
181 Mucor genevensis Lendner 1908 3 3,02 2,95 28,98
182 Mucor griseocyanus Hagem 1908 2 16,08 4,08 29,88
183 Mucor guilliermondii Nadson et Philippow 1925 1 7,27 4,39 29,78
184 Mucor heterogamus Vuillemin 1903 1 4,62 4,62 30,16
185 Mucor hiemalis Wehmer 1903 var. corticolus 2 12,46 12,2 30,24
(Hagem 1910) Schipper 1973
186 Mucor hiemalis Wehmer 1903 var. hiemalis 13 10,78 7,06 29,94
187 Mucor hiemalis Wehmer 1903 var. silvaticus 3 2,30 2,30 29,78
(Hagem 1908) Schipper 1973
188 Mucor indicus Lendner 1930 2 16,86 12,54 30,29
189 Mucor laxorrhizus Y. Ling 1930 5 4,24 5,36 25,11
(continued)

Storage time at
(years)
Number 12 70
C
190 Mucor luteus Linnemann 1936 2 0,70 0,70 30,14
191 Mucor microsporus Namyslowski 1910 1 0,71 0,71 28,16
192 Mucor moelleri (Vuillemin 1903) Lendner 1908 4 12,54 9,00 29,84
193 Mucor mousanensis Baijal et B.S. Mehrotra 1966 1 30,38 8,33 30,38
194 Mucor mucedo Linnaeus 1753 6 6,99 6,70 29,71
195 Mucor odoratus Treschew 1940 2 1,46 3,22 29,02
196 Mucor piriformis A. Fischer 1892 3 8,27 5,37 29,02
197 Mucor plasmaticus van Tieghem 1875 1 0,99 0,99 29,88
198 Mucor plumbeus Bonorden 1864 10 16,68 9,50 28,71
199 Mucor psychrophilus Milko 1971 1 17,19 7,02 29,75
200 Mucor racemosus Fresenius 1850 var. racemosus 17 17,53 10,28 30,10
201 Mucor racemosus Fresenius 1850 var. 1 16,79 30,07 30,07
sphaerosporus (Hagem 1908) Schipper 1970
202 Mucor ramosissimus Samoutsevitch 1927 1 15,23 6,27 29,10
203 Mucor saturninus Hagem 1910 1 4,47 17,10 30,55
204 Mucor sinensis Milko et Beliakova 1971 1 16,57 7,95 30,29
205 Mucor strictus Hagem 1908 1 7,32 7,32 29,84
206 Mucor zonatus Milko 1967 2 16,42 7,75 29,97
207 Mucor zychae Baijal et B.S. Mehrotra 1965 var. 1 0,10 0,10 30,49
zychae
208 Mycogone cervina Ditmar 1817 1 31,58 5,33 31,58
209 Mycogone nigra (Morgan 1895) C.N. Jensen 1912 3 15,45 7,84 31,88
210 Mycogone rosea Link 1809 4 0,87 1,23 31,30
211 Mycosticta cytosporicola Frolov 1968 2 5,76 2,06 21,25
212 Mycotypha africana R.O. Novak et Backus 1963 1 18,05 18,05 30,51
213 Myrothecium sp. 2 0,94 0,94 31,12
214 Neocamarosporium betae (Berlese 1888) 1 11,01 11,01 32,37
Ariyawansa et K.D. Hyde 2015
215 Neottiospora caricina (Desmazieres 1836) Hoehnel 1 4,76 4,76 31,17
1924
216 Nigrospora gorlenkoana Novobranova 1972 2 6,00 6,00 31,78
217 Nigrospora gossypii Jaczewski 1929 1 5,91 5,96 31,24
218 Nigrospora oryzae (Berkeley et Broome 1873) Petch 2 10,55 4,13 31,90
1924
219 Nodulisporium verrucosum (J.F.H. Beyma 1929) G. 1 5,82 2,94 29,15
Smith 1954
220 Ochrocladosporium elatum (Harz 1871) Crous et U. 1 22,58 11,13 32,48
Braun 2007
221 Oidiodendron cereale (Thuemen 1880) G.L. Barron 1 9,92 9,92 31,24
1962
(continued)

Storage time at
(years)
Number 12 70
C
222 Paraconiothyrium fuckelii (Saccardo 1878) Verkley 1 10,28 21,69 31,62
et Gruyter 2012
223 Paraconiothyrium sporulosum (W. Gams et Domsch 2 7,00 6,78 32,05
1969) Verkley 2004
224 Paramyrothecium roridum (Tode 1790) L. Lombard 1 31,81 10,46 31,81
et Crous 2016
225 Parasitella parasitica (Bainier 1884) Sydow 1903 1 6,31 6,31 28,33
226 Pestalotia pezizoides de Notaris 1841 1 21,69 9,27 30,59
227 Phialophora atrovirens (J.F.H. Beyma 1935) Schol- 1 2,94 1,04 32,15
Schwarz 1970
228 Phialophora bubakii (Laxa 1930) Schol-Schwarz 1 10,83 22,82 32,18
1970
229 Phialophora lagerbergii (Melin et Nannfeldt 1934) 1 0,98 3,22 31,99
Conant 1937
230 Phialophora verrucosa Medlar 1915 1 8,27 8,27 31,99
231 Phycomyces blakesleeanus Burgeff 1925 4 4,75 5,50 23,91
232 Phycomyces nitens (C. Agardh 1823) Kunze 1823 2 7,47 5,91 29,44
233 Phyllosticta pucciniospila C. Massalongo 1900 1 3,19 3,19 31,95
234 Pilaira anomala (Cesati 1851) J. Schroeter 1886 1 4,39 4,39 29,78
235 Pilaira caucasica Milko 1970 1 6,92 17,12 29,75
236 Pirella circinans Bainier 1882 var. volgogradensis 1 6,29 6,29 29,1
(Milko 1974) Benny et Schipper 1988
237 Pirella naumovii (Milko 1970) Benny et Schipper 1 8,05 8,05 30,33
1992
238 Pleotrichocladium opacum (Corda 1837) 1 22,09 10,64 31,99
Hernández-Restrepo et al. 2017
239 Pleurophoma cava (Schulzer 1871) Boerema 1996 3 18,57 7,63 21,54
240 Pyrenophora biseptata (Saccardo et Roumeguere 1 9,89 0,98 31,24
1881) Crous 2013
241 Radiomyces spectabilis Embree 1959 1 15,95 7,16 29,98
242 Rhinocladiella atrovirens Nannfeldt 1934 1 22,34 6,95 32,24
243 Rhizomucor miehei (Cooney et R. Emerson 1964) 1 17,04 8,54 30,49
Schipper 1978
244 Rhizomucor pusillus (Lindt 1886) Schipper 1978 3 16,98 16,65 30,10
245 Rhizomucor tauricus (Milko et Schkurenko 1970) 1 16,52 7,92 30,24
Schipper 1978
246 Rhizopus arrhizus A. Fischer 1892 8 17,71 8,60 29,74
247 Rhizopus microsporus van Tieghem 1875 var. 2 16,67 10,37 30,17
chinensis (Saito 1904) Schipper et Stalpers 1984
248 Rhizopus microsporus van Tieghem 1875 var. 4 15,88 6,76 29,38
microsporus
(continued)

Storage time at
(years)
Number 12 70
C
249 Rhizopus stolonifer (Ehrenberg 1818) Vuillemin 13 19,86 9,71 29,17
1902 var. stolonifer
250 Scopulariopsis brevicaulis (Saccardo 1882) Bainier 1 31,22 9,87 31,22
1907
251 Spadicesporium acrosporum V.N. Borisova et 1 21,69 10,32 31,64
Dvoinos 1982
252 Spadicesporium acrosporum-majus V.N. Borisova 1 6,22 6,22 31,64
et Dvoinos 1982
253 Spadicesporium bifurcatum V.N. Borisova et 1 6,22 6,22 31,64
Dvoinos 1982
254 Spadicesporium bifurcatum-majus V.N. Borisova et 1 21,69 6,22 31,64
Dvoinos 1982
255 Spadicesporium copiosum V.N. Borisova et Dvoinos 1 10,32 6,22 31,64
1982
256 Spadicesporium persistens V.N. Borisova et 1 10,28 2,14 31,60
Dvoinos 1982
257 Spadicesporium ramosum V.N. Borisova et Dvoinos 1 21,69 10,30 31,64
1982
258 Sphaerostilbella penicillioides (Corda 1840) 2 9,88 5,80 31,22
Rossman et al. 2015
259 Stachybotrys chartarum (Ehrenberg 1818) S. Hughes 9 18,27 5,33 31,88
1958
260 Stachybotrys cylindrospora C.N. Jensen 1912 1 31,95 10,57 31,91
261 Stemphyliomma sp. 1 10,57 5,52 31,91
262 Stemphylium botryosum Wallroth 1833 1 10,59 10,59 31,93
263 Stemphylium sarciniforme (Cavara 1890) Wiltshire 1 3,17 6,67 31,93
1938
264 Striaticonidium brachysporum (Nicot 1961) L. 1 10,37 6,31 31,70
Lombard et Crous 2016
265 Striaticonidium cinctum (Corda 1842) L. Lombard et 1 0,01 2,35 5,16
Crous 2016
266 Syncephalastrum racemosum Cohn ex J. Schroeter 6 18,97 8,06 30,08
1886
267 Syncephalis cornu van Tieghem et G. Le Monnier 1 18,62 18,62 30,48
1873
268 Thamnidium elegans Link 1809 2 19,05 6,77 29,2
269 Thamnostylum piriforme (Bainier 1880) Arx et H.P. 2 16,7 23,5 30,35
Upadhyay 1970
270 Thysanophora canadensis Stolk et Hennebert 1968 1 0,90 3,50 32,26
271 Thysanophora penicillioides (Roumeguere 1890) 4 0,61 2,13 18,34
W.B. Kendrick 1961
272 Torula ligniperda (Willkomm 1866) Saccardo 1906 1 31,91 1,28 31,91
(continued)

Storage time at
(years)
Number 12 70
C
273 Trichocladium asperum Harz 1871 1 32,13 3,31 32,07
274 Trichocladium griseum (Traaen 1914) X. Wei Wang 2 15,71 1,31 26,17
et Houbraken 2018
275 Trichocladium nigrospermum (Schweinitz 1832) X. 1 10,83 10,83 32,18
Wei Wang et Houbraken 2018
276 Trichoderma deliquescens (Sopp 1912) Jaklitsch 1 31,15 9,79 31,15
2011
277 Truncatella angustata (Persoon 1801) S. Hughes 1 1,05 1,05 31,68
1958
278 Umbelopsis isabellina (Oudemans 1902) W. Gams 6 16,65 6,59 29,24
2003
279 Umbelopsis longicollis (Dixon-Stewart 1932) Y.N. 3 20,11 5,82 28,9
Wang et al. 2015
280 Umbelopsis nana (Linnemann 1941) Arx 1984 2 7,07 4,29 29,78
281 Umbelopsis ramanniana (Moeller 1903) W. Gams 6 14,18 8,67 29,29
2003
282 Umbelopsis vinacea (Dixon-Stewart 1932) Arx 1984 1 4,34 3,34 29,41
The analysis of the results testifies that this method has proved very successful for
the storage of most of the investigated fungi within 3–7 years (Table 1.2).
Where it is desired to keep and constantly to renew cultures within 1–2 years, a
temperature of 5 C is perfectly applicable. More than 97% of the studied
zygomycetous fungi and 94% of ascomycetous fungi were viable after storage.
For long-term (more than 10 years) storage, however, this temperature is not reliable,
since the viability of fungi in both groups is reduced to 57% and 55 % respectively.
A temperature of 12 C is least favorable for long storage. Only 60% of
zygomycetous fungi and 35% of ascomycetous fungi stored at such a temperature
were viable after 10 years. After 17–20 years viability decreased to 10–13% and 4%,
respectively (Fig. 1.1). Among zygomycetous fungi representatives of the classes
Mortierellomycetes, Entomophthoromycetes, and Kickxellomycetes lost their vitality
most rapidly at these temperatures. After 17 years of storage, their viability
decreased to 0–4%. In contrast, the strains from the psychrotolerant species
Helicostylum elegans and Thamnostylum piriforme and thermotolerant species
Rhizomucor pusillus remained steady. Among dark-colored anamorphic ascomyce-
tous fungi the best viability at temperature 12 C after 20 years was found in strains
of the genera Acrophialophora, Alternaria, Coniothyrium, Gonytrichum,
Hormoconis, Paraconiothyrium, and Phialophora. After 30 years, only 1 strain
(Hormoconis resinae) was viable.
Table 1.2 Viability (%) of different taxa of VKM fungi after long-term preservation (30 years) at various temperatures on silica gel
Viability (%) after long-term preservation at different temperature ( C)
1–2 year 3–7 year Near 10 year Near 20 year Near 30 yearr
Subkindom Division Class 5 12 70 5 12 70 5 12 70 5 12 70 5 12 70
Dikarya Ascomycota Dothideomycetes (27 98 99 100 83 86 97 64 41 96 38 4 95 23 0 93
genera, 77 species, 144
strains)
Eurotiomycetes (7 gen- 91 96 100 65 74 100 39 35 91 22 13 91 9 4 91
era, 12 species, 22
strains)
Insertae Sedis (6 genera, 89 100 100 67 56 100 22 11 89 11 11 67 11 0 56
9 species, 9 strains)
Leotiomycetes (5 gen- 81 89 93 48 63 85 30 19 67 4 0 63 4 0 63
era, 13 species, 27
strains)
Ascomycetes (1 genus, 100 100 100 100 100 100 100 100 100 0 0 100 0 0 100
1 species, 1 strains)
Saccharomycetes (1 100 100 100 100 100 100 100 0 100 0 0 100 0 0 100
genus, 1 species, 1
strain)
Sordariomycetes (32 94 100 100 75 68 99 55 34 93 39 1 92 23 0 91
strains)
Mucoromyceta Mortierellomycota Mortierellomycetes (1 78 86 100 41 53 96 17 16 94 4 4 74 0 0 74
genus, 18 species, 27
strains)
Mucoromycota Mucoromycetes (28 99 97 100 89 92 100 79 68 99 63 63 98 7 1 98
strains)
(continued)
Viability (%) after long-term preservation at different temperature ( C)
1–2 year 3–7 year Near 10 year Near 20 year Near 30 yearr
Subkindom Division Class 5 12 70 5 12 70 5 12 70 5 12 70 5 12 70
Umbelopsidomycetes (1 100 100 100 100 96 100 94 61 100 74 74 100 11 6 100
genus, 5 species, 18
strains)
Zoopagomyceta Entomophthoro- Entomophthoromycetes 67 67 67 22 33 67 0 22 67 0 0 67 0 0 67
mycota (2 genera, 3 species, 3
strains)
Kickxellomycota Kickxellomycetes (2 100 100 100 50 50 100 17 17 100 0 0 100 0 0 50
strains)
Zoopagomycota Zoopagomycetes (1 100 100 100 100 100 100 100 100 100 100 100 100 0 0 100
genus, 1 species, 1
strain)
100
90
80
70
Viability (%)
60
50 5°С
40
-12°С
30
20 -70°С
10
0
1-2 3-7 near 10 near 20 near 30
Time (year)
Fig. 1.1 The long-term preservation of zygomycetous fungi on silica gel at different temperatures
100
90
80
70
Viability (%)
60
5°С
50
-12°С
40
-70°С
30
20
10
0
1-2 3-7 near 10 near 20 near 30
Time (year)
Fig. 1.2 The long-term preservation of ascomycetous fungi on silica gel at different temperatures
The most acceptable temperature for the storage of mycelial fungi is the temper-
ature 70 C. In these conditions after 30 years of storage, 90% of strains were
viable (Fig. 1.1).
The advantages of storing mycelial fungi at different temperatures on silica gel
are obvious. On one hand, this method is so simple that the storage at 5 C and 12

C can be carried out for the most part in poorly equipped laboratories. On the other
hand, the presence of a low-temperature refrigerator ( 70 C) means it is possible to
support large numbers of cultures in a small area. The advantages of this method are
also a minimum of preparatory work, the rapid reconstituted part of the stored
material by transferring a few granules on appropriate culture medium, as well as the

possibility of using the same vial without defrosting for a long time.
The cooling equipment being used in VKM is ultralow temperature freezers
( 70–80 C, Sanyo, Japan) and household refrigerators (5 and 12 C).
1.6 Protocols
Protocols of cryopreservation, freeze-drying, and drying in sterile soil were

described earlier [15].
1.7 Protocol of Drying on Silica Gel

1.7.1 Preparation of Sterile Silica Gel and Ampoules
• Silica gel is pre-dried and sterilized by dry heat for 3 h at a temperature of 160 C,
conducting careful control of sterility.
• Plastic ampoules (Nunc) (3 for each culture) are labeled and sterilized by
autoclaving, at 121 C for 20 min.
• Sterile silica gel that has been washed with a concentration of cobalt chloride is
placed in the ampoules to indicate the humidity. The cobalt chloride is deep blue
when dry and turns pink when wet.
• A sterile cotton ball is placed on top of the indicator.
1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol
• Pour 5 mL of 10% glycerol into 12 mL glass tubes.

• Sterilized by autoclaving at 121 C for 20 min.
• Stored at +5 C for no longer than a month.
1.7.3 Preparation of Cultures
• Grow sporulating fungal cultures on slant agar under optimal growth conditions
and on suitable mediums (www.vkm.ru).
• Wash off spores from agar surface with 5 mL of cool sterile 10% glycerol.
• Titer of spores’ suspension should be not less than 106 spores/mL.
1.7.4 Silica Gel Inoculation
• Add 75–100 silica gel granules (40 grade, 9–16 mesh) in a sterile Petri dish.
• Add 1 mL spore suspension to sterile and dry silica gel.
• Shake the Petri dish with the granules.
• Put the Petri dish in desiccator and store in the refrigerator 12 h at 4–7 C.
1.7.5 Filling of Vials
• Add silica gel granules with fungal spores (20–25 pieces) to 3 plastic ampoules
with a sterile spoon.
• Place cryovials in the boxes and transfer them to the refrigerators (5 and 12 C)
and the ultralow temperature freezer ( 70 C).
1.7.6 Control of Viability
• Place ampoule in a special metal container, thermostatic inside by expanded

polystyrene, to prevent defrosting.
• Transfer one granule of silica gel from ampoule on fresh suitable agar medium
and incubate under optimal conditions.
• The remaining granules were resealed and stored as described. Thus, each
ampoule with fungal spores adsorbed on silica gel may be used repeatedly.
Result
The real storage time estimates obtained in VKM are given in Table 1 and Annex 2.
They are not final data: the cultures are still being stored, and we expect to get longer
storage times later on. Some cells of the table are empty; this is the case if the culture
is not stored by this method.
There is at present clear that more than 98% of fungal cultures preserved by
cryoconservation method remain viable after 20 years of storage. For lyophilization
and storage in sterile soil methods, these figures after 30 years of storage are 95 and
85%, respectively. For long-term storage of fungal cultures on silica gel, the
temperature 70 C should be chosen. At this temperature, over 90% of spore-
forming fungi retain their viability after 30 years of the experiment.
Conclusion
The conservation techniques used in VKM presents effective preservation of the
stock of filamentous fungi from different taxonomic groups. The possibility and
practical time estimates of secure long-term storage of fungal cultures belonging to
1600 species and 590 genera were shown. The represented information could be used
as a reference for researchers intending to maintain pure cultures of microorganisms
for a long time. The data produced are also accessible online on the VKM Web site.
Annexies
Annex 1: Fields Attributes in the Table «Database Preservation

Methods»
Code Counter
EntryDate Date/Time
Method Text
Col Text
Strain Numerical
dep Text
pat Text
Curator Text
Dubl-cart Text
Dubl fond Text
Dubl fond new Text
Ampules Numerical
Data Date/Time
Result Text
Data2 Date/Time
Result2 Text
Days Numerical
Year Numerical
Comments Text
Data3 Date/Time
Result3 Text
Data4 Date/Time
Result4 Text
Data5 Date/Time
Result5 Text
EditDate Date/Time
Protector Text
Programm Text
Location Text
Type Text
Annex 2: Maximal Preservation Times for VKM Fungal

Species
Cryopreservation Freeze-drying Soil
Number of Max storage Number of Max storage Number of Max storage
No. Name of species strains time (years) strains time (years) strains time (years)
1 Absidia caerulea Bainier 1889 5 19.70 5 45.95 5 12.42
2 Absidia cuneospora G.F. Orr et Plunkett 1959 1 25.41 1 27.38
3 Absidia cylindrospora Hagem 1908 2 31.17 2 37.64
4 Absidia glauca Hagem 1908 8 24.13 10 40.90 8 49.99
5 Absidia repens van Tieghem 1878 1 23.67 1 45.19
6 Absidia spinosa Lendner 1907 2 19.75 2 39.43 2 23.32
7 Achlya bisexualis Coker et Couch 1927 4 0.51
8 Achlya bonariensis Beroqui 1969 1 0.16
9 Achlya colorata Pringsheim 1882 2 6.32
10 Achlya intricata Beneke 1948 1 0.15
11 Achlya radiosa Maurizio 1899 1 28.12
12 Achlya sparrowii Reischer 1949 1 23.01
13 Acladium curvatum Bonorden 1851 1 35.41 1 0.73
14 Acremonium alternatum Link 1809 3 17.73 4 30.79 3 2.72
15 Acremonium arxii W. Gams 1971 2 19.46 2 27.32 2 3.20
16 Acremonium atrogriseum (Panasenko 1964) W. Gams 1971 3 17.47 3 32.65
17 Acremonium bacillisporum (Onions et G.L. Barron 1967) W. Gams 1971 2 22.13 3 22.08
18 Acremonium bactrocephalum W. Gams 1971 1 22.13 5 25.25 1 0.53
19 Acremonium biseptum W. Gams 1971 1 25.30
20 Acremonium breve (Sukapure et Thirumalachar 1966) W. Gams 1971 3 19.31 12 40.33 1 3.56
21 Acremonium cavaraeanum (Jasevoli 1924) W. Gams 1971 1 19.93 1 6.05
22 Acremonium cereale (P. Karsten 1887) W. Gams 1971 1 17.75 1 19.92 1 3.27
23 Acremonium charticola (J. Lindau 1907) W. Gams 1971 4 21.04 8 25.98 1 0.09
24 Acremonium chrysogenum (Schol-Schwarz 1965) W. Gams 1971 2 9.96
25 Acremonium crotocinigenum (Schol-Schwarz 1965) W. Gams 1971 4 32.50 3 2.72
26 Acremonium cymosum W. Gams 1971 2 6.54 2 28.44
27 Acremonium domschii W. Gams 1971 2 28.36 2 2.84
28 Acremonium egyptiacum (J.F.H. Beyma 1933) W. Gams 1971 1 16.93 1 29.72 1 0.09
29 Acremonium fuci Summerbell et al. 2004 3 2.15
30 Acremonium hyalinulum (Saccardo 1878) W. Gams 1971 2 31.29
31 Acremonium implicatum (J.C. Gilman et E.V. Abbott 1927) W. Gams 1975 2 19.57 5 27.42 3 0.68
32 Acremonium incrustatum W. Gams 1971 2 17.73 2 25.80 1 3.49
(continued)
33 Acremonium kiliense Gruetz 1925 4 22.13 4 26.11 2 0.09
34 Acremonium lichenicola W. Gams 1971 1 24.26
35 Acremonium murorum (Corda 1839) W. Gams 1971 11 5.74
36 Acremonium persicinum (Nicot 1958) W. Gams 1971 2 19.37 2 43.88 1 26.53
37 Acremonium polychromum (J.F.H. Beyma 1928) W. Gams 1971 5 29.82 5 35.47 3 0.09
38 Acremonium rutilum W. Gams 1971 1 8.02 1 19.10
39 Acremonium salmoneum W. Gams et Lodha 1975 4 2.70
40 Acremonium sclerotigenum (Moreau et R. Moreau 1941 ex Valenta 1948) W. 3 20.23 3 36.56 2 3.73
Gams 1971
41 Acremonium strictum W. Gams 1971 19 19.81 24 39.59 17 9.71
42 Acremonium tubakii W. Gams 1971 2 31.23
43 Acrophialophora fusispora (S.B. Saksena 1953) Samson 1970 1 17.49 1 30.36
44 Acrostalagmus albus Preuss 1851 1 19.59 1 32.45 1 3.51
45 Acrostalagmus luteoalbus (Link 1809) Zare et al. 2004 12 19.35 12 37.43 5 13.56
46 Acrothecium robustum J.C. Gilman et E.V. Abbott 1927 1 19.30 1 31.30 1 25.73
47 Actinomucor elegans (Eidam 1884) C.R. Benjamin et Hesseltine 1957 5 13.17 7 42.07 7 50.21
48 Agaricus arvensis Schaeffer 1774 1 20.04
49 Agaricus bisporus (J. Lange 1926) Imbach 1946 35 26.07
50 Agaricus squarrosus Oeder 1770 1 23.59
51 Albifimbria verrucaria (Albertini et Schweinitz 1805) L. Lombard et Crous 3 19.83 3 48.27 1 2.57
2016
52 Albonectria rigidiuscula (Berkeley et Broome 1875) Rossman et Samuels 1 19.85 1 34.34 1 7.45
1999
53 Alternaria alternariae (Cooke 1871) Woudenberg et Crous 2013 2 20.53 2 32.26
54 Alternaria alternata (Fries 1832) Keissler 1912 13 19.30 43 48.71
55 Alternaria atra (Preuss 1852) Woudenberg et Crous 2013 9 19.60 13 48.35 1 8.34
56 Alternaria botrytis (Preuss 1851) Woudenberg et Crous 2013 12 19.56 19 48.41 1 35.79
57 Alternaria brassicae (Berkeley 1836) Saccardo 1880 4 19.78 3 0.50
58 Alternaria brassicicola (Schweinitz 1832) Wiltshire 1947 6 19.28 7 43.57
59 Alternaria chartarum Preuss 1848 8 19.46 22 45.05
60 Alternaria cheiranthi (Libert 1827) P.C. Bolle 1924 1 19.79 1 19.67
61 Alternaria consortialis (Thuemen 1876) Groves et Hughes 1953 4 19.49 4 45.05
62 1 19.79
Alternaria cucumerina (Ellis et Everhart 1895) J.A. Elliott 1917 var.
cucumerina
63 Alternaria dauci (J.G. Kuehn 1855) J.W. Groves et Skolko 1944 5 19.79
64 Alternaria dianthicola Neergaard 1945 1 19.78
65 Alternaria geophila Daszewska 1912 1 12.56
66 Alternaria godetiae (Neergaard 1933) Neergaard 1945 1 12.56
67 Alternaria grandis E.G. Simmons 2000 1 1.74
68 Alternaria japonica Yoshii 1941 3 19.32 3 27.64
69 Alternaria leucanthemi Nelen 1962 1 19.78 1 24.95
70 Alternaria macrospora Zimmermann 1904 2 12.56 2 30.03
71 Alternaria multirostrata E.G. Simmons et C.R. Jackson 1968 1 17.80 1 11.28
72 Alternaria nobilis (Vize 1877) E.G. Simmons 2002 1 19.78
73 Alternaria oudemansii (E.G. Simmons 1967) Woudenberg et Crous 2013 2 19.32 1 42.78
74 Alternaria radicina Meier et al. 1922 2 17.60 2 38.20
75 Alternaria silybi Gannibal 2011 3 1.97
76 Alternaria simmonsii Gannibal 2011 2 1.97
77 Alternaria solani Sorauer 1896 6 19.78 4 30.40
78 Alternaria tenuissima (Kunze 1818) Wiltshire 1933 6 26.81
79 Alternariaster helianthi (Hansford 1943) E.G. Simmons 2007 2 7.12
80 Amanita citrina (Schaeffer 1762) Persoon 1797 1 17.92
81 Amauroascus aureus (Eidam 1887) Arx 1971 1 6.18 1 33.15
82 Amblyosporium botrytis Fresenius 1863 2 15.34 2 31.28 2 12.18
83 Amerosporium concinnum Petrak 1953 1 19.54 1 47.12
84 Ampelomyces artemisiae (Voglino 1905) Rudakov 1979 1 12.58 1 11.42
85 Ampelomyces heraclei (Dejeva 1967) Rudakov 1979 1 12.18 1 13.21
86 Ampelomyces humuli (Fautrey 1890) Rudakov 1979 1 16.55
87 Ampelomyces polygoni (Potebnia 1907) Rudakov 1979 2 12.28 2 34.50
88 Ampelomyces quercinus (Sydow 1915) Rudakov 1979 2 0.02
89 Ampelomyces quisqualis Cesati 1852 1 12.58 1 29.31
90 Ampelomyces ulicis (Adams 1907) Rudakov 1979 1 12.38 2 29.15
91 Ampelomyces uncinulae (Fautrey 1893) Rudakov 1979 1 12.58 1 40.64
92 Antrodia sinuosa (Fries 1821) P. Karsten 1881 1 8.16
93 Apenidiella antarctica Ivanushkina et al. 2019 1 7.34
94 Apenidiella strumelloidea (Milko et Dunaev 1986) W. Quaedvlieg et P.W. 1 29.09
Crous 2014
(continued)
95 Aphanoascus fulvescens (Cooke 1879) Apinis 1968 1 20.42 1 43.15 1 21.16
96 Aphanocladium album (Preuss 1848) W. Gams 1971 4 17.73 4 27.66
97 Aphanomyces helicoides Minden 1915 1 33.22
98 Apiospora montagnei Saccardo 1875 1 9.90
99 Aplanes treleaseanus (Humphrey 1893) Coker 1927 1 15.93
100 Aposphaeria caespitosa (Fuckel 1869) Jaczewski 1917 1 19.21 1 36.11
101 Arachniotus aurantiacus (Kamyschko 1967) Arx 1971 1 43.59 1 21.16
102 Arcticomyces warmingii (Rostrup 1888) Savile 1959 1 24.47 1 1.64
103 Armillaria cepistipes Velenovsky 1920 6 13.55
104 Armillaria lutea Gillet 1874 5 23.45
105 Armillaria mellea (Vahl 1790) P. Kummer 1871 5 33.22
106 Arthrinium arundinis (Corda 1838) Dyko et Sutton 1981 3 12.58 12 24.70
107 Arthrinium phaeospermum (Corda 1837) M.B. Ellis 1965 2 13.34
108 Arthrinium saccharicola F. Stevens 1917 1 16.91
109 Arthrinium sphaerospermum Fuckel 1874 1 19.21 3 47.40 1 3.85
110 Arthrobotrys arthrobotryoides (Berlese 1888) J. Lindau 1907 1 19.32 1 3.91
111 Arthrobotrys cladodes Drechsler 1937 2 15.74 2 23.73 2 7.77
112 Arthrobotrys conoides Drechsler 1937 4 19.32 4 9.44 3 13.48
113 Arthrobotrys longa Mekhtieva 1973 1 10.65 1 9.96 1 0.53
114 Arthrobotrys longispora Preuss 1853 1 25.39 1 3.91
115 Arthrobotrys oligospora Fresenius 1850 6 27.60 7 24.76 7 7.81
116 Arthrobotrys oviformis Soprunov 1958 1 2.40 1 0.19
117 Arthrobotrys robusta Duddington 1951 1 19.41 1 31.56 1 7.99
118 Arthrobotrys superba Corda 1839 7 26.36 7 26.32 7 3.91
119 Ascochyta cucumeris Fautrey et Roumeguere 1891 2 20.34 2 27.91
120 Ascochyta malvicola Saccardo 1878 1 19.22 1 11.74
121 Ascochyta pisi Libert 1830 3 19.45 1 44.50 1 8.67
122 Ascochyta viciae Libert 1837 1 20.31 1 26.76
123 Ascotricha chartarum Berkeley 1838 1 18.97 1 10.75
124 Aspergillus aculeatus Iizuka 1953 7 18.19 2 0.35
125 Aspergillus alliaceus Thom et Church 1926 6 41.09 5 30.10
126 Aspergillus amylovorus Panasenko 1964 ex Samson 1979 1 12.47 1 27.12 1 27.64
127 Aspergillus asperescens Stolk 1954 2 9.95 2 0.50
128 Aspergillus aureolatus Muntanola-Cvetkovic et Bata 1964 1 37.60 1 9.58
129 Aspergillus aureoterreus Samson et al. 2011 1 33.83 1 33.41
130 Aspergillus awamori Nakazawa 1915 13 45.82 13 37.03
131 Aspergillus awamori Nakazawa 1915 var. fumeus Nakazawa et al. 1936 1 17.96 1 30.17
132 Aspergillus brasiliensis Varga et al. 2007 2 36.25 2 22.13
133 Aspergillus brunneouniseriatus Suj. Singh et B.K. Bakshi 1961 1 21.90
134 Aspergillus caespitosus Raper et Thom 1944 2 30.61 2 19.26
135 Aspergillus calidoustus Varga et al. 2008 7 20.53
136 Aspergillus candidus Link 1809 11 39.94 6 46.75
137 Aspergillus carbonarius (Bainier 1880) Thom 1916 2 40.31 2 25.81
138 Aspergillus carneus (van Tieghem 1877) Blochwitz in Thom and Raper 1945 5 39.44 2 30.62
139 Aspergillus clavatus Desmazieres 1834 10 44.38 9 46.84
140 Aspergillus crustosus Raper et Fennell 1965 1 0.15
141 Aspergillus duricaulis Raper et Fennell 1965 1 21.55 1 21.92
142 Aspergillus echinulatus (Delacroix 1893) Thom et Church 1926 1 9.60 1 38.16
143 Aspergillus ficuum (Reichardt 1867) Thom et Currie 1916 2 15.58 1 20.92
144 Aspergillus fischeri Wehmer 1907 7 38.87 6 47.71
145 Aspergillus flavipes (Bainier et R. Sartory 1911) Thom et Church 1926 8 41.10 7 20.90
146 Aspergillus flavus Link 1809 16 41.63 12 46.87
147 Aspergillus flavus Link 1809 var. columnaris Raper et Fennell 1965 1 32.17 1 37.25
148 Aspergillus foetidus Thom et Raper 1945 2 30.88 2 27.21
149 Aspergillus fumigatus Fresenius 1863 15 44.89 10 46.83
150 Aspergillus giganteus Wehmer 1901 3 42.69 3 35.55
151 Aspergillus gorakhpurensis Kamal et Bhargava 1969 1 0.12
152 Aspergillus hennebergii Blochwitz 1935 1 6.46 1 0.14
153 Aspergillus heteromorphus Batista et H. Maia 1957 1 27.48 1 3.35
154 Aspergillus insuetus (Bainier 1908) Thom et Church 1929 1 15.90 1 37.09
155 Aspergillus janus Raper et Thom 1944 2 36.04 2 46.78
156 Aspergillus japonicus Saito 1906 10 34.48 8 32.73
157 Aspergillus kanagawaensis Nehira 1951 2 39.62 2 42.25
158 Aspergillus melleus Yukawa 1911 4 39.08 4 20.75
159 Aspergillus neoafricanus Samson et al. 2011 1 36.34 1 33.42
160 Aspergillus neoniveus Samson et al. 2011 1 0.37
161 Aspergillus nidulans (Eidam 1883) G. Winter 1884 13 48.25 9 47.04
(continued)
162 Aspergillus niger van Tieghem 1867 109 44.57 30 46.83
163 Aspergillus niveus Blochwitz 1929 6 37.67 4 37.78
164 Aspergillus nomius Kurtzman et al. 1987 2 0.07
165 Aspergillus nutans McLennan et Ducker 1954 2 46.76 2 37.38
166 Aspergillus ochraceus G. Wilhelm 1877 21 43.70 16 36.29
167 Aspergillus oryzae (Ahlburg 1878) E. Cohn 1884 25 43.68 19 42.05
168 Aspergillus oryzae (Ahlburg 1878) E. Cohn 1884 var. effusus (Tiraboschi 1 34.42 1 9.58
1908) Y. Ohara 1951
169 Aspergillus pallidus Kamyschko 1963 1 12.47 1 18.59 1 48.04
170 Aspergillus parasiticus Speare 1912 2 3.30
171 Aspergillus parvulus G. Smith 1961 1 46.73 1 30.34
172 Aspergillus penicilliformis Kamyschko 1963 1 38.97 1 28.21
173 Aspergillus penicillioides Spegazzini 1896 1 6.90 3 5.91
174 Aspergillus phoenicis (Corda 1840) Thom et Currie 1916 5 32.96 1 7.66
175 Aspergillus proliferans G. Smith 1943 1 10.75 1 11.13
176 Aspergillus pseudodeflectus Samson et Mouchacca 1975 1 36.19 1 38.11
177 Aspergillus puniceus Kwon-Chung et Fennell 1965 3 31.40 1 4.32
178 Aspergillus quadrilineatus Thom et Raper 1939 3 39.84 3 30.07
179 Aspergillus raperi Stolk et J.A. Meyer 1957 2 0.37
180 Aspergillus repens (Corda 1842) Saccardo 1882 11 47.00 11 46.92
181 Aspergillus restrictus G. Smith 1931 1 24.65 1 24.65
182 Aspergillus rugulosus Thom et Raper 1939 5 37.90 5 37.05
183 Aspergillus sclerotiorum G.A. Huber 1933 5 31.55 3 30.64
184 Aspergillus silvaticus Fennell et Raper 1955 1 36.62 1 33.75
185 Aspergillus sojae Sakaguchi et K. Yamada ex Murakami 1971 1 37.49 1 36.16
186 Aspergillus stellatus Curzi 1934 2 36.15 2 37.24
187 Aspergillus subsessilis Raper et Fennell 1965 1 12.44 2 46.65 1 26.30
188 Aspergillus sulphureus (Fresenius 1863) Wehmer 1901 3 40.12 1 42.08
189 Aspergillus sydowii (Bainier et R. Sartory 1913) Thom et Church 1926 30 38.84 7 46.83
190 Aspergillus tamarii Kita 1913 3 38.72 1 42.06
191 Aspergillus terreus Thom 1918 8 6.68 28 47.42 25 47.04
192 Aspergillus terricola Marchal et É.J. Marchal 1893 3 36.09 3 46.84
193 Aspergillus terricola Marchal et É.J. Marchal 1893 var. americanus Marchal 1 33.83 1 7.34
et É.J. Marchal 1921
194 Aspergillus tubingensis Mosseray 1934 1 12.64 1 0.19
195 Aspergillus umbrosus Bainier et R. Sartory 1912 1 13.10 1 0.23
196 Aspergillus unguis (Weill et L. Gaudin 1919) Dodge 1935 3 12.47 5 41.10 5 42.25
197 Aspergillus ustus (Bainier 1881) Thom et Church 1926 17 42.48 13 46.82
198 Aspergillus varians Wehmer 1897 1 18.57 1 25.81
199 Aspergillus versicolor (Vuillemin 1903) Tiraboschi 1908 46 42.57 12 46.87
200 Aspergillus viridinutans Ducker et Thrower 1954 2 28.31 1 47.20
201 Aspergillus wentii Wehmer 1896 12 40.01 6 27.64
202 Asterosporium orientale Melnik 1988 1 0.07
203 Athelia rolfsii (Curzi 1932) C.C. Tu et Kimbrough 1978 1 19.30
204 Aureobasidium melanogenum (Hermanides-Nijhof 1977) Zalar et al. 2014 9 46.31 2 12.60
205 Aureobasidium microstictum (Bubak 1907) W.B. Cooke 1962 1 19.85 3 35.55
206 Aureobasidium pullulans (de Bary 1866) G. Arnaud 1918 14 20.22 29 47.98 3 5.00
207 Backusella circina J.J. Ellis et Hesseltine 1969 2 23.57 2 44.37
208 Backusella indica (Baijal et B.S. Mehrotra 1965) G. Walther et de Hoog 1 20.66 1 25.49 1 20.12
2013
209 Backusella lamprospora (Lendner 1908) Benny et R.K. Benjamin 1975 2 25.41 4 39.34 3 8.78
210 Backusella oblongielliptica (H. Naganishi et al. ex Pidoplichko et Milko 1 20.62 1 34.63
1971) G. Walther et de Hoog 2013
211 Backusella recurva (E.E. Butler 1952) G. Walther et de Hoog 2013 1 25.31 1 19.65
212 Backusella tuberculispora (Schipper 1978) G. Walther et de Hoog 2013 1 25.31 1 36.53
213 Backusella variabilis (A.K. Sarbhoy 1965) G. Walther et de Hoog 2013 1 19.59 1 46.64 1 19.25
214 Basidiobolus magnus Drechsler 1964 1 20.06
215 Basidiobolus meristosporus Drechsler 1955 1 20.21
216 Beauveria bassiana (Balsamo-Crivelli 1835) Vuillemin 1912 12 29.66 14 46.45 12 22.79
217 Beauveria brongniartii (Saccardo 1892) Petch 1926 6 19.50 6 33.01 5 10.92
218 Beauveria caledonica Bissett et Widden 1988 1 3.00 1 0.70
219 Beauveria felina (De Candolle 1815) J.W. Carmichael 1980 1 13.51 1 0.60
220 Benjaminiella poitrasii (R.K. Benjamin 1960) Arx 1981 2 19.56 2 38.65 2 46.30
221 Berkeleyomyces basicola (Berkeley et Broome 1850) W.J. Nel et al. 2017 2 19.51 2 39.60
222 Bionectria ochroleuca (Schweinitz 1832) Schroers et Samuels 1997 1 26.61
223 Bipolaris australiensis (M.B. Ellis 1971) Tsuda et Ueyama 1981 6 19.30 14 49.80 2 8.53
224 Bipolaris bicolor (Mitra 1931) Shoemaker 1959 1 24.97
(continued)
225 Bipolaris cynodontis (Marignoni 1909) Shoemaker 1959 4 19.12 4 49.79 1 8.43
226 Bipolaris sorokiniana (Saccardo 1890) Shoemaker 1959 4 19.35 8 49.80
227 Bipolaris spicifera (Bainier 1908) Subramanian 1971 1 23.92
228 Bipolaris victoriae (F. Meehan et H.C. Murphy 1946) Shoemaker 1959 1 17.47 1 26.99
229 Biscogniauxia nummularia (Bulliard 1790) Kuntze 1891 1 19.32 1 7.17 1 0.10
230 Bispora antennata (Persoon 1801) E.W. Mason 1953 2 19.16 3 27.07
231 Bispora betulina (Corda 1838) S. Hughes 1958 2 17.45 2 34.55
232 Bispora effusa Peck 1891 1 20.22 1 15.09
233 Bjerkandera adusta (Willdenow 1787) P. Karsten 1879 7 8.31
234 Blakeslea trispora Thaxter 1914 14 26.15 16 48.52 7 11.74
235 Blumeriella jaapii (Rehm 1907) Arx 1961 1 8.61 1 9.62
236 Boeremia hedericola (Durieu et Montagne 1855) Aveskamp et al. 2010 1 4.61
237 Boeremia lycopersici (Cooke 1885) Aveskamp et al. 2010 1 14.02
238 Botryodiplodia malorum (Berkeley 1836) Petrak et Sydow 1926 1 20.00
239 Botryosphaeria rhodina (Berkeley et M.A. Curtis 1889) Arx 1970 1 19.98 1 26.19 1 0.12
240 Botryosporium longibrachiatum (Oudemans 1890) Maire 1903 2 21.41
241 Botryotinia narcissicola (P.H. Gregory 1941) N.F. Buchwald 1949 1 18.96 1 28.02 1 0.11
242 Botryotinia polyblastis (P.H. Gregory 1938) N.F. Buchwald 1949 1 27.67
243 Botryotrichum piluliferum Saccardo et Marchal 1885 6 19.23 7 48.07
244 Botryotrichum verrucosum (Pugh et al. 1964) X. Wei Wang et Houbraken 1 17.82
2018
245 Botryoxylon geniculatum (Corda 1839) Ciferri 1962 1 17.71 1 28.41
246 Botrytis aclada Fresenius 1850 2 19.30 2 37.80
247 Botrytis anthophila Bondartsev 1913 2 19.20 2 40.66
248 Botrytis bifurcata J.H. Miller et al. 1958 1 6.88
249 Botrytis cinerea Persoon 1794 14 19.23 24 37.74
250 Botrytis convallariae (Klebahn 1930) Ondrej 1972 ex Boerema et Hamers 3 15.32 3 15.42
1988
251 Botrytis convoluta Whetzel et Drayton 1932 2 19.23 2 47.64
252 Botrytis fabae Sardina 1929 1 19.12
253 Botrytis galanthina (Berkeley et Broome 1873) Saccardo 1886 1 19.23 1 27.67
254 Botrytis gladiolorum Timmermans 1941 2 19.30 2 38.79
255 Botrytis hyacinthi Westerdijk et J.F.H. Beyma 1928 1 19.20
256 Botrytis lutescens Saccardo et Roumeguere 1882 1 19.20
257 Botrytis squamosa J.C. Walker 1925 1 19.33
258 Botrytis tulipae (Libert 1830) Lind 1913 1 19.50 1 13.16
259 Bovista pusilla (Batsch 1789) Persoon 1801 1 16.61
260 Brachysporium nigrum (Link 1824) S. Hughes 1958 1 20.58 1 26.08
261 Burgoa anomala (Hotson 1912) Goidanich 1937 1 8.98
262 Byssochlamys nivea Westling 1909 2 19.44 2 36.44 2 44.03
263 Cadophora fastigiata Lagerberg et Melin 1928 6 19.41 7 28.68
264 Cadophora luteo-olivacea (J.F.H. Beyma 1940) T.C. Harrington et McNew 3 0.54
2003
265 Cadophora malorum (Kidd et Beaumont 1924) W. Gams 2000 7 19.76 9 45.39
266 Cadophora melinii Nannfeldt 1934 1 19.49 5 45.37
267 Calcarisporium arbuscula Preuss 1851 4 1.99 5 28.45
268 Calcarisporium griseum Spegazzini 1902 3 17.70 3 27.88
269 Celosporium sp. 1 6.56
270 Cephalotrichum gorgonifer (Bainier 1907) Sandoval-Denis et al. 2016 1 17.70 1 27.05
271 Cephalotrichum microsporum (Saccardo 1878) P.M. Kirk 1984 3 11.54
272 Cephalotrichum purpureofuscum (Schweinitz 1832) S. Hughes 1 19.39 2 31.05
273 Cephalotrichum stemonitis (Persoon 1801) Nees 1809 4 19.41 10 36.99 1 5.68
274 Ceratellopsis equiseticola (Boudier 1917) Corner 1950 1 23.34
275 Ceratocystis adiposa (E.J. Butler 1906) C. Moreau 1952 1 0.10
276 Ceratocystis paradoxa (Dade 1928) C. Moreau 1952 2 19.85 2 40.51 2 26.12
277 Ceratocystis pilifera (Fries 1822) C. Moreau 1952 2 18.86 2 39.58
278 Cercospora armoraciae Saccardo 1876 1 20.34 1 17.87 1 0.53
279 Cercospora beticola Saccardo 1876 2 25.47
280 Cercospora carotae (Passerini 1887) Kaznowski et Siemaszko 1929 1 20.34 1 23.40
281 Cercospora rosicola Passerini 1875 1 16.67 1 22.04
282 Cercospora violae Saccardo 1876 1 23.85
283 Ceriporiopsis gilvescens (Bresadola 1908) Domanski 1963 1 12.58
284 Cerrena unicolor (Bulliard 1788) Murrill 1903 1 12.58
285 Chaetocladium brefeldii van Tieghem et G. Le Monnier 1873 2 17.59 2 38.59 2 16.98
286 Chaetocladium jonesii (Berkeley et Broome 1854) Fresenius 1863 1 23.67 1 27.42
287 Chaetocytostroma sp. 1 20.53
288 Chaetomidium pilosum (C. Booth et Shipton 1966) Arx 1975 1 14.46 1 43.99 1 24.33
289 Chaetomium amesii Sergeeva 1965 1 19.28 1 31.01 1 24.33
(continued)
290 Chaetomium angustispirale Sergeeva 1956 1 19.28 1 43.64 1 23.33
291 Chaetomium aureum Chivers 1912 2 20.51 1 42.33 2 23.86
292 Chaetomium brasiliense Bat. et Pontual 1948 1 38.87
293 Chaetomium crispatum (Fuckel) Fuckel 1870 1 20.04
294 Chaetomium elatum Kunze 1817 3 20.51 3 43.64 3 24.33
295 Chaetomium fieberi Corda 1837 1 32.08 1 11.25
296 Chaetomium funicola Cooke 1873 1 19.28 1 26.15 1 0.72
297 Chaetomium globosum Kunze 1817 11 19.44 21 48.45 14 28.00
298 Chaetomium homopilatum Omvik 1953 1 19.33 2 35.37 2 0.32
299 Chaetomium indicum Corda 1840 1 19.28 2 42.28 1 23.47
300 Chaetomium megalocarpum Bainier 1910 2 19.28 2 43.64 2 23.47
301 Chaetomium nozdrenkoae Sergeeva 1961 1 18.87 1 35.25 1 0.11
302 Chaetomium perlucidum Sergeeva 1956 1 19.28 1 35.29 1 0.27
303 Chaetomium seminis-citrulli Sergeeva 1956 1 25.68 1 43.67 1 0.11
304 Chaetomium spirale Zopf 1881 1 19.28 1 37.32
305 Chaetomium subaffine Sergeeva 1961 1 19.28 1 35.29 1 23.47
306 Chaetomium subspirilliferum Sergeeva 1960 1 20.42 1 25.61 1 22.62
307 Chaetomium trilaterale Chivers 1912 1 11.80
308 Chaunopycnis alba W. Gams 1979 1 2.01
309 Chloridium caesium (Nees et T. Nees 1818) Réblová et Seifert 2016 1 27.64
310 Chloridium virescens (Persoon 1797) W. Gams et Holubova-Jechova 1976 1 19.67 1 30.31
var. caudigerum (Hoehnel 1903) W. Gams et Holubova-Jechova 1976
311 Chlorophyllum rhacodes (Vittadini 1835) Vellinga 2002 1 12.02
312 Choanephora conjuncta Couch 1925 1 3.78
313 Choanephora cucurbitarum (Berkeley et Ravenel 1875) Thaxter 1903 1 20.21 1 20.93
314 Choanephora infundibulifera (Currey 1873) Saccardo 1891 1 25.41 1 33.99
315 Chondrostereum purpureum (Persoon 1794) Pouzar 1959 1 20.10
316 Chordomyces antarcticus Bilanenko et al. 2015 6 8.00
317 Chromelosporium fulvum (Link 1824) McGinty et al. 1975 2 1.88 2 18.07
318 Chrysonilia sitophila (Montagne 1843) Arx 1981 1 43.95 1 22.14
319 Chrysosporium keratinophilum D. Frey 1959 ex J.W. Carmichael 1962 2 9.57 2 32.84 1 5.95
320 Chrysosporium lobatum Scharapov 1978 1 38.25 1 29.86
321 Chrysosporium lucknowense Garg 1966 6 5.62 6 22.03
322 Chrysosporium merdarium (Link 1818 ex Greville 1823) J.W. Carmichael 3 11.87 4 25.63 1 35.73
1962
323 Chrysosporium queenslandicum Apinis et R.G. Rees 1976 2 31.45 2 38.32 2 31.26
324 Chrysosporium tropicum J.W. Carmichael 1962 3 9.10 3 37.15 2 25.87
325 Chrysosporium undulatum P. Vidal et al. 1999 3 20.41 4 42.69 3 35.66
326 Circinella muscae (Sorokin 1870) Berlese et de Toni 1888 4 22.71 5 43.04 5 47.39
327 Circinella umbellata van Tieghem et G. Le Monnier 1873 1 19.68 1 40.08 1 37.94
328 Cistella sp. 2 1.87
329 Cladobotryum dendroides (Bulliard 1791) W. Gams et Hoozemans 1970 3 26.47 3 38.61 1 3.20
330 Cladobotryum multiseptatum de Hoog 1978 1 16.25
331 Cladobotryum varium Nees 1817 5 21.79 7 43.99 5 7.73
332 Cladobotryum verticillatum (Link 1809) S. Hughes 1958 2 43.99
333 Cladophialophora chaetospira (Grove 1886) Crous et Arzanlou 2007 1 33.14
334 Cladosporium aecidiicola Thuemen 1876 1 19.20 1 26.20
335 Cladosporium allicinum (Fries 1817: Fries 1832) Bensch et al. 2012 1 33.11
336 Cladosporium antarcticum K. Schubert et al. 2007 1 7.32
337 Cladosporium brevicompactum Pidoplichko et Deniak 1941 2 34.24
338 Cladosporium cladosporioides (Fresenius 1850) G.A. de Vries 1952 4 19.53 56 47.50 4 6.72
339 Cladosporium colocasiae Sawada 1916 1 19.35 1 19.16
340 Cladosporium cucumerinum Ellis et Arthur 1889 1 48.92
341 Cladosporium elegantulum Pidoplichko et Deniak 1938 2 26.47 1 7.58
342 Cladosporium gossypiicola Pidoplichko et Deniak 1941 2 34.83
343 Cladosporium halotolerans Zalar et al. 2007 2 28.46
344 Cladosporium herbarum (Persoon 1794) Link 1816 19 19.35 101 47.52 10 5.76
345 Cladosporium lycoperdinum Cooke 1883 1 41.76
346 Cladosporium macrocarpum Preuss 1848 4 19.01 5 47.72
347 Cladosporium pseudocladosporioides Bensch et al. 2010 1 23.56
348 Cladosporium sphaerospermum Penzig 1882 7 19.30 22 47.50 2 14.36
349 Cladosporium straminicola Pidoplichko et Deniak 1938 1 26.05
350 Cladosporium transchelii Pidoplichko et Deniak 1938 1 13.10
351 Clathrus archeri (Berkeley 1859) Dring 1980 1 1.65
352 Clavariadelphus pistillaris (Linnaeus 1753) Donk 1933 1 19.70
353 Claviceps paspali F. Stevens et J.G. Hall 1910 3 18.95
354 Claviceps purpurea (Fries 1823) Tulasne 1853 3 26.36 7 25.22
(continued)
355 Clitocybe odora (Bulliard 1784) P. Kummer 1871 1 0.01
356 Clonostachys byssicola Schroers 2001 2 3.67
357 Clonostachys rosea (Link 1816) Schroers et al. 1999 f. catenulata (J.C. 8 20.68 8 34.61 3 19.96
Gilman et E.V. Abbott 1927) Schroers 2001
358 Clonostachys rosea (Link 1816) Schroers et al. 1999 f. rosea 17 19.47 18 42.09 16 15.09
359 Clonostachys solani (Harting 1846) Schroers et W. Gams 2001 2 1.65
360 Clonostachys solani (Harting 1846) Schroers et W. Gams 2001 f. nigrovirens 2 32.42 1 4.92
(J.F.H.Beyma 1931) Schroers 2001
361 Coemansia aciculifera Linder 1943 1 21.05
362 Cokeromyces recurvatus Poitras 1950 3 24.14 3 42.75 3 22.74
363 Colletoconis aecidiophila (Spegazzini 1886) de Hoog et al. 1978 1 16.87 1 30.04
364 Colletotrichum coccodes (Wallroth 1833) S. Hughes 1958 4 20.10
365 Colletotrichum dematium (Persoon 1801) Grove 1918 1 6.08
366 Colletotrichum gloeosporioides (Penzig 1882) Penzig et Saccardo 1884 2 19.33 4 37.54
367 Colletotrichum musae (Berkeley et M.A. Curtis 1874) Arx 1957 1 19.32 1 24.90
368 Colpoma quercinum (Persoon 1796) Wallroth 1823 1 37.71
369 Conidiobolus coronatus (Costantin 1897) Batko 1964 4 15.93
370 Conidiobolus thromboides Drechsler 1953 2 6.78
371 Coniochaeta verticillata (van Emden 1973) Dania García et al. 2006 1 18.96 1 39.44 1 0.10
372 Coniophora puteana (Schumacher 1803) P. Karsten 1868 4 12.02
373 Coniothyrium concentricum (Desmazieres 1840) Saccardo 1878 1 19.01 1 12.63
374 Coniothyrium hellebori Cooke et Massee 1886 1 17.70 1 27.85
375 Coniothyrium rosarum Cooke et Harkness 1882 2 19.40 2 16.28
376 Coniothyrium wernsdorffiae Laubert 1905 1 19.01 1 20.34
377 Coprinellus disseminatus (Persoon 1801) J.E. Lange 1938 1 32.89
378 Coprinellus ephemerus (Bulliard 1786) Redhead et al. 2001 1 32.87
379 Coprinellus micaceus (Bulliard 1785) Vilgalys et al. 2001 3 32.83
380 Coprinellus radians (Desmazieres 1828) Vilgalys et al. 2001 1 32.83
381 Coprinopsis atramentaria (Bulliard 1783) Redhead et al. 2001 2 32.83
382 Coprinopsis gonophylla (Quélet 1884) Redhead et al. 2001 2 22.97
383 Coprinopsis kimurae (Hongo et Aoki 1966) Redhead et al. 2001 1 7.92
384 Coprinus comatus (O.F. Mueller 1780) Persoon 1797 2 32.83
385 Coprinus domesticus (Bolton 1788) Gray 1821 1 27.36
386 Coprinus sterquilinus (Fries 1821) Fries 1838 2 32.87
387 Coriolopsis trogii (Berkeley 1850) Domanski 1974 1 34.10
388 Coriolus sp. 1 19.42
389 Cortinarius bulbosus Gray 1821 1 22.97
390 Cortinarius caperatus (Persoon 1796) Fries 1838 1 23.53
391 Corynascella inaequalis (Pidoplichko et al. 1973) Arx 1975 1 18.95 1 42.84 1 27.97
392 Corynascus sepedonium (C.W. Emmons 1932) Arx 1973 1 20.42 1 39.91 1 21.16
393 Cosmospora arxii (W. Gams 1971) Gräfenhan et Schroers 2011 1 16.01 1 35.44 1 0.53
394 Cosmospora berkeleyana (P. Karsten 1891) Gräfenhan et al. 2011 8 24.26 1 21.27
395 Cosmospora lavitskiae (Zhdanova 1966) Gräfenhan et Seifert 2011 1 4.33 1 30.12 1 0.09
396 Crassicarpon hotsonii Koukol 2016 1 13.43
397 Cryphonectria parasitica (Murrill 1906) M.E. Barr 1978 5 19.35 5 41.47 1 0.19
398 Cryptococcus depauperatus (Petch 1932) Boekhout et al. 2015 1 23.96
399 Cunninghamella blakesleeana Lendner 1927 3 14.1 2 13.81
400 Cunninghamella echinulata (Thaxter 1891) Thaxter ex Blakeslee1905 13 25.18 15 42.96 12 49.77
401 Cunninghamella homothallica Kominami et Tubaki 1952 1 20.08
402 Cunninghamella japonica (Saito 1905) Pidoplichko et Milko 1971 6 25.18 7 37.02 6 35.84
403 Cunninghamella vesiculosa P.C. Misra 1966 1 21.95
404 Curvularia clavata B.L. Jain 1962 1 13.68
405 Curvularia comoriensis Bouriquet et Jauffret 1955 ex M.B. Ellis 1966 1 17.80 1 26.87
406 Curvularia fallax Boedijn 1933 1 13.60
407 Curvularia geniculata (Tracy et Earle 1896) Boedijn 1933 4 19.30 5 45.17
408 Curvularia inaequalis (Shear 1907) Boedijn 1933 5 19.76 6 28.20 1 4.33
409 Curvularia kusanoi (Y. Nisikado 1928) Manamgoda et al. 2014 1 18.74
410 Curvularia lunata (Wakker 1898) Boedijn 1933 6 19.35 9 44.30
411 Curvularia nodulosa (Saccardo 1886) Manamgoda et al. 2014 1 23.88
412 Curvularia protuberata Nelson et Hodges 1965 1 13.50
413 Cyathus olla (Batsch 1783) Persoon 1801 1 14.05
414 Cylindrium cordae Grove 1886 1 27.86 1 3.49
415 Cylindrocarpon album (Saccardo 1877) Wollenweber 1917 1 17.67 1 34.66 1 3.51
416 Cylindrocarpon chlamydospora Schischkina et Tzanava 1973 1 19.97 1 18.49
417 Cylindrocarpon congoense J.A. Meyer 1958 1 19.56 1 28.46 1 2.49
418 Cylindrocarpon destructans (Zinssmeister 1918) Scholten 1964 6 15.66 12 21.12 2 2.43
419 Cylindrocarpon didymum (Hartig 1846) Wollenweber 1926 2 19.56 3 21.12
420 Cylindrocarpon gracile Bugnicourt 1939 3 29.09 3 1.25 1 3.07
(continued)
421 Cylindrocarpon heteronema (Berkeley et Broome 1865) Wollenweber 1916 3 19.53 4 28.67 1 2.49
422 Cylindrocarpon lucidum C. Booth 1966 1 20.70
423 Cylindrocarpon obtusisporum (Cooke et Harkness 1884) Wollenweber 1926 1 25.95 1 18.94 1 2.45
424 Cylindrocarpon peronosporae (Fautrey et Lambotte 1896) Rudakov 1981 1 37.38 1 0.08
425 Cylindrocarpon tenue Bugnicourt 1939 1 3.81
426 Cylindrocephalum stellatum (Harz 1871) Saccardo 1886 1 12.09 1 38.72 1 3.20
427 Cylindrophora alba Bonorden 1851 1 19.78
428 Cylindrophora hoffmannii Daszewska 1912 1 17.73 1 28.06 1 5.65
429 Cytospora sp. 1 1.21
430 Dacrymyces stillatus Nees 1816 1 6.62
431 Dactylaria acerosa Matsushima 1975 1 0.77
432 Dactylaria dimorphospora Veenbaas-Rijks 1973 1 16.10 1 23.58
433 Dactylellina asthenopaga (Drechsler 1937) M. Scholler et al.1999 1 19.30 1 0.08
434 Daedalea quercina (Linnaeus 1753) Persoon 1801 2 34.05
435 Daedaleopsis confragosa (Bolton 1791) J. Schröter 1888 var. confragosa 1 10.36
436 Dematioscypha delicata (Berkeley et Broome 1859) Hosoya 2014 1 19.38 1 26.79
437 Dendrodochium toxicum Pidoplichko et Bilai 1947 1 19.33 1 28.29 1 4.79
438 Dendrostilbella macrospora W. Bally 1917 1 7.72 1 14.23 1 4.92
439 Dendrostilbella mycophila (Persoon 1822) Seifert 1985 1 37.05 1 2.89
440 Dendryphion nanum (Nees 1816) S. Hughes 1958 1 24.14 1 0.53
441 Dendryphion penicillatum (Corda 1838) Fries 1846 1 24.88 2 3.44
442 Dichobotrys sp. 1 19.28 1 18.88
443 Dichotomomyces cejpii (Milko 1964) D.B. Scott 1970 1 18.96 1 19.72 1 23.17
444 Dictyostelium discoideum (Bosc 1811) E. Fischer 1888 1 5.75 1 24.44
445 Dictyuchus monosporus Leitgeb 1870 2 12.57
446 Dicyma ampullifera Boulanger 1897 1 19.21 1 12.36
447 Dicyma olivacea (Emoto et Tubaki 1970) Arx 1982 1 19.40 1 48.10
448 Dicyma ovalispora (S. Hughes 1951) Arx 1982 1 6.69 1 12.32
449 Didymella glomerata (Corda 1840) Q. Chen et L. Cai 2015 11 19.47 8 46.45
450 Didymella musae (P. Joly 1961) Q. Chen et L. Cai 2015 4 19.59
451 Didymella pinodella (L.K. Jones 1927) Q. Chen et L. Cai 2015 1 20.54
452 Didymella pinodes (Berkeley et A. Bloxam 1861) Petrak 1924 1 19.22 1 22.41
453 Didymella pomorum (Thümen 1879) Q. Chen et L. Cai 2015 4 19.56 4 46.45
454 Didymopsis helvellae (Corda 1854) Saccardo et Marchall 1885 1 28.44 1 2.73
455 Dimargaris bacillispora R.K. Benjamin 1959 1 19.37 1 8.30
456 Dinemasporium strigosum (Persoon 1801) Saccardo 1881 1 17.80 1 20.15
457 Diplocladium majus Bonorden 1851 2 21.84 2 38.61 2 2.84
458 Diplocladium penicilloides Saccardo 1886 2 27.21 2 3.73
459 Diplodina acerina (Passerini 1875) B. Sutton 1980 1 6.56
460 Dipodascopsis tothii (Zsolt 1963) L.R. Batra et Millner 1978 1 42.60 1 0.10
461 Dipodascopsis uninucleata (Biggs 1937) L.R. Batra et Millner 1978 var. 2 4.03 2 42.60
uninucleata
462 Dipodascus aggregatus Francke-Grosmann 1952 2 18.88 2 42.63 2 0.10
463 Dipodascus armillariae W. Gams 1983 1 19.62 1 15.67
464 Discula brunneotingens E.I. Meyer 1953 1 20.32 1 29.45
465 Discula pinicola (Naumov 1926) Petrak 1927 var. mammosa Lagerberg et al. 1 19.33 1 39.74
1927
466 Dispira cornuta van Tieghem 1875 1 21.05
467 Dissoacremoniella silvatica Kirilenko 1970 1 28.88 1 22.90
468 Dothiora prunorum (Dennis et Buhagiar 1973) Crous 2016 1 16.93 1 27.42
469 Drechmeria coniospora (Drechsler 1941) W. Gams et H.-B. Jansson 1985 1 13.88 1 22.04
470 Drechslera avenacea (M.A. Curtis ex Cooke 1889) Shoemaker 1959 2 37.32
471 Drechslera campanulata (Leveille 1841) B. Sutton 1976 3 19.38 2 23.17
472 Drechslera poae (Baudys 1916) Shoemaker 1962 1 33.87
473 Duddingtonia flagrans (Duddington 1949) R.C. Cooke 1969 1 19.32 1 18.30 1 2.49
474 Echinobotryum rubrum Sorokin ex Jaczewski 1917 1 25.93 1 0.08
475 Eladia saccula (E. Dale 1926) G. Smith 1961 1 20.56 1 44.21
476 Emericellopsis alkalina Bilanenko et Georgieva 2013 3 7.77
477 Emericellopsis donezkii Beliakova 1974 7 20.48 7 46.22 6 0.09
478 Emericellopsis glabra (J.F.H. Beyma 1940) Backus et Orpurt 1961 1 19.33 3 46.54
479 Emericellopsis humicola (Cain 1956) Gilman 1956 1 19.33 1 48.87 1 23.47
480 Emericellopsis maritima Beliakova 1970 1 14.88 1 17.90
481 Emericellopsis minima Stolk 1955 10 20.42 10 46.48 9 22.75
482 Emericellopsis pallida Beliakova 1974 1 20.44 1 49.00 1 23.10
483 Emericellopsis robusta van Emden et W. Gams 1971 2 44.91
484 Emericellopsis terricola J.F.H. Beyma 1940 1 19.33 1 49.00 1 0.10
485 Engyodontium album (Limber 1940) de Hoog 1978 2 23.78 3 18.23
(continued)
486 Entomophthora dipterigena (Thaxter 1888) Saccardo et Traverso 1891 1 26.20
487 Entomophthora thaxteriana I.M. Hall et J. Bell 1963 5 23.20
488 Entyloma gaillardianum Vanky 1982 1 12.35 1 18.84
489 Epicoccum nigrum Link 1815 5 19.81 11 35.51
490 Epicoccum sorghinum (Saccardo 1878) Aveskamp et al. 2010 4 3.29
491 Epithyrium obscurum (Passerini 1885) Saccardo 1931 1 11.05
492 Eremascus fertilis Stoppel 1907 1 18.86 1 5.73
493 Eremothecium ashbyi Guilliermond 1935 5 17.32
494 Eremothecium gossypii (S.F. Ashby et W. Nowell 1926) Kurtzman 1995 2 16.33
495 Eupenicillium pinetorum Stolk 1968 1 0.10
496 Eurotium amstelodami L. Mangin 1909 16 43.08 9 46.64
497 Eurotium chevalieri L. Mangin 1909 8 42.86 5 46.87
498 Eurotium halophilicum C.M. Christensen et al. 1959 1 28.92 1 0.10
499 Eurotium herbariorum (F.H. Wiggers 1780) Link 1809 1 18.88 18 37.95 1 21.57
500 Eurotium rubrum Jos. König et al. 1901 7 43.26 6 37.41
501 Eurotium tonophilum Ohtsuki 1962 1 20.01 1 39.91 1 19.77
502 Eutypa sp. 1 0.04 1 2.69
503 Evlachovaea kintrischica B. Borisov et Tarasov 1999 1 15.54
504 Exobasidium bisporum Sawada ex Ezuka 1991 1 23.62 1 1.64
505 Exobasidium karstenii Saccardo et Trotter 1912 1 23.62
506 Exobasidium myrtilli Siegmund 1879 1 19.46 1 1.64
507 Exobasidium pachysporum Nannfeldt 1981 1 2.04
508 Exobasidium vaccinii (Fuckel 1861) Woronin 1867 2 23.62 2 1.81
509 Exophiala castellanii Iwatsu et al. 1984 2 20.40 2 23.19
510 Exophiala heteromorpha (Nannfeldt 1934) de Hoog et Haase 2003 1 19.54 1 21.63
511 Exophiala lecanii-corni (Benedek et Specht 1933) Haase et de Hoog 1999 1 18.56
512 Exophiala moniliae de Hoog 1977 1 18.56
513 Exophiala salmonis J.W. Carmichael 1966 1 17.78 1 10.32
514 Exophiala xenobiotica de Hoog et al. 2006 3 2.11
515 Exserohilum pedicellatum (A.W. Henry 1924) K.J. Leonard et Suggs 1974 1 19.48 1 40.52
516 Exserohilum rostratum (Drechsler 1923) K.J. Leonard et Suggs 1974 1 18.33
517 Farlowiella carmichaeliana (Berkeley 1836) Saccardo 1891 1 26.00 1 3.49
518 Farrowia seminuda (L.M. Ames 1949) D. Hawksworth 1975 1 38.09
519 Fennellomyces linderi (Hesseltine et Fennell 1955) Benny et R.K. Benjamin 1 19.64 1 15.02 1 29.69
1975
520 Fibroporia vaillantii (de Candolle 1815) Parmasto 1968 1 20.10
521 Flammulina velutipes (Curtis 1782) Singer 1951 6 34.05
522 Fomes fomentarius (Linnaeus 1753) J.J. Kickx 1867 4 34.14
523 Fomitopsis pinicola (Swartz 1810) P. Karsten 1881 8 20.04 1 0.99
524 Fomitopsis rosea (Albertini et Schweinitz 1805) P. Karsten 1881 1 20.10
525 Fonsecaea pedrosoi (Brumpt 1922) Negroni 1936 1 19.72 1 39.24
526 Fulvia fulva (Cooke 1883) Ciferri 1954 3 19.01 3 22.51
527 Fusarium agaricorum Sarrazin 1887 1 17.67 1 27.21 1 2.62
528 Fusarium aquaeductuum (Rabenhorst et Radlkofer 1861) Lagerheim et 3 0.19 2 33.19 2 2.52
Rabenhorst 1891
529 Fusarium aquaeductuum (Rabenhorst et Radlkofer 1861) Lagerheim et 1 23.04 1 5.64
Rabenhorst 1891 var. medium Wollenweber 1931
530 Fusarium arthrosporioides Sherbakoff 1915 1 13.96 1 21.18
531 Fusarium avenaceum (Fries 1832) Saccardo 1886 4 19.99 3 44.73 3 14.34
532 Fusarium avenaceum (Fries 1832) Saccardo 1886 var. herbarum (Corda 1 26.39 1 2.90
1839) Saccardo 1886
533 Fusarium cerealis (Cooke 1878) Saccardo 1886 1 2.21 1 26.52
534 Fusarium chlamydosporum Wollenweber et Reinking 1925 2 29.57
535 Fusarium concolor Reinking 1935 1 1.88 2 16.36
536 Fusarium culmorum (W.G. Smith 1884) Saccardo 1895 3 17.67 3 32.72 3 11.43
537 Fusarium decemcellulare Brick 1908 2 19.50 2 44.40 2 11.93
538 Fusarium epistroma (Hoehnel 1909) C. Booth 1971 2 32.43 2 3.04
539 Fusarium equiseti (Corda 1838) Saccardo 1886 5 19.28 7 44.85 3 26.61
540 Fusarium expansum Schlechtendal 1824 1 29.47 1 2.68
541 Fusarium fujikuroi Nirenberg 1976 1 7.65 1 39.07 1 24.70
542 Fusarium graminearum Schwabe 1839 4 15.66 4 35.19 3 3.98
543 Fusarium graminearum Schwabe 1839 f. oxalis 1 38.91 1 7.09
544 Fusarium heterosporum Nees et T. Nees 1818 3 31.04 1 7.09
545 Fusarium heterosporum Nees et T. Nees 1818 var. pucciniophilum Saccardo 1 17.73 1 23.19 1 2.62
et Sydow 1899
546 Fusarium incarnatum (Roberge 1849) Saccardo 1886 4 16.56 4 28.98 4 2.97
547 Fusarium javanicum Koorders 1907 2 13.36 2 35.60 2 7.90
548 Fusarium lateritium Nees 1816 7 20.47 8 39.14 4 21.85
(continued)
549 Fusarium merismoides Corda 1838 3 17.90 3 33.20 1 3.64
550 Fusarium oxysporum Schlechtendal 1824 14 31.06 28 38.36 7 24.51
551 Fusarium oxysporum Schlechtendal 1824 f. sp. batatas (G.F. Atkinson 1892) 1 18.10 1 25.21
W.C. Snyder et H.N. Hansen 1940
552 Fusarium oxysporum Schlechtendal 1824 f. sp. conglutinans W.C. Snyder et 2 23.35 1 25.52
H.N. Hansen 1940
553 Fusarium oxysporum Schlechtendal 1824 f. sp. lycopersici W.C. Snyder et 2 29.35 2 23.85
H.N. Hansen 1940
554 Fusarium oxysporum Schlechtendal 1824 f. sp. vasinfectum W.C. Snyder et 1 25.04 1 26.69
H.N. Hansen 1940
555 Fusarium poae (Peck 1903) Wollenweber 1913 3 17.81 4 45.15 3 12.68
556 Fusarium redolens Wollenweber 1913 3 21.67 3 21.54
557 Fusarium sambucinum Fuckel 1863 11 7.69 9 37.19 7 13.96
558 Fusarium sambucinum Fuckel 1863 var. ossicola (Berkeley et M.A.Curtis 1 11.59 1 0.73
1875) Bilai 1955
559 Fusarium sarcochroum (Desmazieres 1850) Saccardo 1879 1 4.99 1 21.31 1 2.90
560 Fusarium solani (Martius 1842) Saccardo 1881 12 16.16 23 34.24 10 16.90
561 Fusarium sporotrichioides Sherbakoff 1915 6 17.73 6 47.20 6 18.61
562 Fusarium tricinctum (Corda 1838) Saccardo 1886 7 17.47 7 37.16 4 4.75
563 Fusarium ventricosum Appel et Wollenweber 1913 3 29.13 2 12.68
564 Fusarium verticillioides (Saccardo 1881) Nirenberg 1976 25 30.64 26 42.97 23 28.25
565 Fusarium viride (Lechmere 1912) Wollenweber 1917 1 17.67 1 23.96 1 2.62
566 Fusarium wolgense Rodigin 1942 1 37.26 1 3.04
567 Fusicladium peltigericola Crous et Diederich 2010 1 2.12
568 Fusicladium pomi (Fries 1825) Lind 1913 1 19.82
569 Fusicoccum castaneum Saccardo 1882 1 11.21
570 Fusicolla epistroma (Höhn. 1909) Gräfenhan and Seifert 2011 1 3.70
571 Gabarnaudia betae (Delacroix 1897) Samson et W. Gams 1974 2 11.05 3 33.00
572 Gaeumannomyces caricis J. Walker 1980 1 5.62
573 Gaeumannomyces graminis (Saccardo 1875) Arx et D.L. Olivier 1952 var. 1 25.68
graminis
574 Galactomyces geotrichum (E.E. Butler et L.J. Petersen 1972) Redhead et 3 45.89 3 3.45
Malloch 1977
575 Galactomyces reessii (van der Walt 1959) Redhead et Malloch 1977 1 19.27 1 46.16 1 3.45
576 Ganoderma lipsiense (Batsch 1786) G.F. Atkinson 1908 3 17.39
577 Ganoderma lucidum (Curtis 1781) P. Karsten 1881 1 9.55
578 Geastrum fimbriatum Fries 1829 1 16.63
579 Geomyces asperulatus Sigler et J.W. Carmichael 1976 1 8.54
580 Geomyces pannorum (Link 1824) Sigler et J.W. Carmichael 1976 38 20.25 166 39.85 8 3.20
581 Geosmithia lavendula (Raper et Fennell 1948) Pitt 1980 1 44.17 1 5.31
582 Geosmithia namyslowskii (K.M. Zalessky 1927) Pitt 1980 1 43.86 1 22.92
583 Geotrichum amycelicum Redaelli et Ciferri 1935 1 18.86 1 11.15
584 Geotrichum bipunctatum Rolland et Fautrey 1894 1 38.59 1 2.90
585 Geotrichum candidum Link 1809 24 31.24 42 45.14 15 2.90
586 Geotrichum fragrans (Berkhout 1923) Morenz 1960 ex Morenz 1964 4 16.13 4 31.67 1 10.53
587 Geotrichum klebahnii (Stautz 1931) Morenz 1964 3 19.79 3 29.62
588 Gibberella fujikuroi (Sawada 1917) Wollenweber 1931 3 19.32 3 26.87 3 30.36
589 Gibberella zeae (Schweinitz 1821) Petch 1936 2 19.77 3 16.40 1 6.20
590 Gibellula pulchra Cavara 1894 1 6.13
591 Gibellulopsis nigrescens (Pethybridge 1919) Zare et al. 2007 6 41.27 6 13.62
592 Gilbertella persicaria (E.D. Eddy 1925) Hesseltine 1960 1 11.41 1 27.13 1 37.52
593 Gilmaniella humicola G.L. Barron 1964 2 13.68
594 Gliocephalotrichum bulbilium J.J. Ellis et Hesseltine 1962 1 15.86
595 Gliocladiopsis tenuis (Bugnicourt 1939) Crous et M.J. Wingfeld 1993 1 14.46
596 Gliocladium album (Preuss 1851) Petch 1926 2 26.90 2 2.49
597 Gliocladium ammoniphilum Pidoplichko et Bilai 1953 1 19.26 1 28.55 1 9.57
598 Gliocladium aurifilum (W. Gerard 1874) Seifert et al. 1985 1 0.54 1 14.41
599 Gliocladium cholodnyi Pidoplichko 1931 2 16.15 2 26.27 2 9.13
600 Gliocladium comtus Rudakov 1981 1 7.81 1 30.99 1 3.73
601 Gliocladium viride Matruchot 1893 4 32.23 1 3.32
602 Gliomastix cerealis (P. Karsten 1887) C.H. Dickinson 1968 2 19.22 2 36.53 1 10.70
603 Gliomastix inflata C.H. Dickinson 1968 2 23.17 2 3.22
604 Gliomastix luzulae (Fuckel 1870) E.W. Mason 1953 ex S. Hughes 1958 2 6.22 3 29.44 3 2.74
605 Gliomastix murorum (Corda 1838) S. Hughes 1958 var. felina (Marchal 5 30.42 6 44.79 4 9.25
1895) S. Hughes 1958
606 Gliomastix murorum (Corda 1838) S. Hughes 1958 var. murorum 10 30.71 9 33.64 6 14.07
607 Gloeophyllum odoratum (Wulfen 1788) Imazeki 1943 1 23.78
608 Gloeophyllum sepiarium (Wulfen 1786) P. Karsten 1882 5 34.14
609 Gongronella butleri (Lendner 1926) Peyronel et Dal Vesko 1955 6 24.55 6 32.12 3 5.42
(continued)
610 Gongronella lacrispora Hesseltine et J.J. Ellis 1961 1 15.38 1 27.66
611 Gonytrichum macrocladum (Saccardo 1880) S. Hughes 1951 3 17.68 4 30.36
612 Graphium penicillioides Corda 1837 1 18.14
613 Graphium putredinis (Corda 1839) S. Hughes 1958 1 23.88
614 Grifola frondosa (Dickson 1785) Gray 1821 2 12.58
615 Guepiniopsis buccina (Persoon 1801) L.L. Kennedy 1959 1 25.88
616 Gymnoascus reessii Baranetzky 1872 1 36.56 1 3.35
617 Gymnopilus sapineus (Fries 1821) Murrill 1912 1 22.97
618 Gymnostellatospora japonica Udagawa 1993 1 2.30
619 Hansfordia pulvinata (Berkeley et M.A. Curtis 1875) S. Hughes 1958 2 19.44 2 26.42
620 Hansfordia triumfettae (Hahsford 1943) S. Hughes 1952 1 27.00
621 Haplotrichum capitatum (Link 1809) Link 1824 2 30.60 2 30.56 2 25.26
622 Hapsidospora milkoi Beliakova 1975 1 2.27 1 34.82 1 23.86
623 Harposporium lilliputanum Dixon 1952 1 19.30 1 21.69
624 Harposporium sinense C.Y. Wang et K.Q. Zhang 2007 1 20.85
625 Harzia acremonioides (Harz 1871) Costantin 1888 4 19.49 4 45.20
626 Hebeloma versipelle (Fries 1838) Gillet 1876 1 22.96 1 6.77
627 Helicodendron tubulosum (Riess 1853) Linder 1929 1 19.31 1 11.44
628 Helicostylum elegans Corda 1842 1 17.59 1 44.19 1 15.39
629 Helicostylum pulchrum (Preuss 1851) Pidoplichko et Milko 1971 2 25.41 2 27.11 2 38.98
630 Helminthosporium solani Durieu et Montagne 1849 1 19.61 1 9.56
631 Hemicarpenteles ornatum (Subramanian 1972) Arx 1974 1 39.58 1 5.09
632 Hericium coralloides (Scopoli 1772) Persoon 1794 4 32.55
633 Hericium erinaceus (Bulliard 1781) Persoon 1797 4 25.07
634 Hesseltinella vesiculosa H.P. Upadhyay 1970 1 21.26 1 40.75
635 Heterobasidion annosum (Fries 1821) Brefeld 1888 1 20.10
636 Hirsutella thompsonii F.E. Fischer 1950 1 15.26
637 Holwaya mucida (Schulzer 1860) Korf et Abawi 1971 var. mucida 1 2.30
638 Hormiactis alba Preuss 1851 1 23.26 1 2.73
639 Hormoconis resinae (Lindau 1906) Arx et G.A. de Vries 1973 12 19.49 12 47.50 2 17.28
640 Hormonema macrosporum L. Voronin 1986 1 19.81 1 26.36
641 Humicola fuscoatra Traaen 1914 3 28.79 4 32.96 1 33.60
642 Humicola grisea Traaen 1914 var. thermoidea Cooney et Emerson 1964 1 11.62
643 Humicola insolens Cooney et R. Emerson 1964 1 16.82
644 Hydnoporia tabacina (Sowerby 1797) V. Spirin et al. 2019 1 19.56
645 Hyphopichia burtonii (Boidin et al. 1964) Arx et Van der Walt 1976 1 38.10
646 Hyphozyma sanguinea (C. Ramirez 1952) de Hoog et M.T. Smith 1981 1 28.74 1 20.45
647 Hyphozyma variabilis de Hoog et M.T. Smith 1981 2 19.30
648 Hyphozyma variabilis de Hoog et M.T. Smith 1981 var. odora de Hoog et M. 1 21.64
T. Smith 1981
649 Hyphozyma variabilis de Hoog et M.T. Smith 1981 var. variabilis 1 20.45
650 Hypomyces ochraceus (Persoon 1801) Tulasne et C. Tulasne 1865 3 22.53
651 Inocutis dryophila (Berkeley 1904) Fiasson et Niemelä 1984 1 20.73
652 Inonotus obliquus (Acharius ex Persoon 1801) Pilat 1942 2 34.08
653 Inonotus rheades (Persoon 1825) Bondartsev et Singer 1941 2 15.29
654 Irpex lacteus (Fries 1818) Fries 1828 1 12.40
655 Isaria farinosa (Holmskjold 1781) Fries 1832 7 20.34 8 35.81 3 18.38
656 Isaria fumosorosea Wize 1904 6 21.44 6 43.70 1 2.32
657 Isaria javanica (Friedrichs et Bally 1923) Samson et Hywel-Jones 2005 1 6.21
658 Isaria tenuipes Peck 1879 1 6.84
659 Juxtiphoma eupyrena (Saccardo 1879) Valenzuela-Lopez et al. 2017 3 17.62
660 Kickxella alabastrina Coemans 1862 1 6.69 1 6.46
661 Kuehneromyces lignicola (Peck 1872) Redhead 1984 1 23.50
662 Kuehneromyces mutabilis (Schaeffer 1774) Singer et A.H. Smith 1946 6 24.26
663 Laccaria bicolor (Maire 1937) P.D. Orton 1960 1 14.95
664 Laccaria laccata (Scopoli 1772) Cooke 1884 2 22.96
665 Lactarius helvus (Fries 1821) Fries 1838 1 24.01
666 Laetiporus sulphureus (Bulliard 1789) Murrill 1920 6 19.99 1 8.56
667 Lasiodiplodia theobromae Patouillard 1892) Griffon et Maublanc 1909 1 7.36 1 17.95
668 Lecanicillium dimorphum (J.D. Chen 1985) Zare et W. Gams 2001 2 38.36
669 Lecanicillium fungicola (Preuss 1851) Zare et W. Gams 2008 3 20.40 3 39.64
670 Lecanicillium fusisporum (W. Gams 1971) Zare et W. Gams 2001 1 25.03
671 Lecanicillium lecanii (Zimmermann 1898) Zare et W. Gams 2001 4 19.84 4 39.18 1 4.47
672 Lecanicillium longisporum (Petch 1925) Zare et W. Gams 2001 1 19.26 1 37.00 1 2.54
673 Lecanicillium muscarium (Petch 1931) Zare et W. Gams 2001 14 22.33 15 36.34 3 4.97
674 Lecanicillium psalliotae (Treschew 1941) Zare et W. Gams 2001 6 20.44 7 36.72
675 Leccinum scabrum (Bulliard 1783) Gray 1821 1 12.08
(continued)
676 Lecythophora decumbens (J.F.H. Beyma 1942) E. Weber et al. 2002 1 20.40 1 38.10
677 Lecythophora fasciculata (J.F.H. Beyma 1939) E. Weber et al. 2002 1 20.40 1 42.03
678 Lecythophora hoffmannii (J.F.H. Beyma 1939) W. Gams et McGinnis 1983 3 20.52 5 42.40
679 Lecythophora mutabilis (J.F.H. Beyma 1944) W. Gams et McGinnis 1983 1 20.40 4 45.70
680 Lentinula edodes (Berkeley 1878) Pegler 1976 5 26.21
681 Lentinus sulcatus Berkeley 1845 1 12.13
682 Lentinus tigrinus (Bulliard 1782) Fries 1825 3 20.98
683 Lenzites betulina (Linnaeus 1753) Fries 1838 3 13.47
684 Lepista luscina (Fries 1818) Singer 1951 1 0.21
685 Lepista nuda (Bulliard 1790) Cooke 1871 1 1.82
686 Leptobacillium leptobactrum (W. Gams 1971) Zare et W. Gams 2016 2 3.35 2 21.46
687 Leptographium lundbergii Lagerberg et Melin 1927 1 11.42
688 Leptosphaeria coniothyrium (Fuckel 1870) Saccardo 1875 1 18.96 1 6.03
689 Leucoagaricus leucothites (Vittadini 1835) Wasser 1977 1 3.04
690 Leucoagaricus nympharum (Kalchbrenner 1873) Bon 1977 1 12.07
691 Leuconeurospora pulcherrima (G. Winter 1876) Malloch et Cain 1970 1 0.54
692 Lichtheimia blakesleeana (Lendner 1924) Kerst. Hoffmann et al. 2009 4 28.53 4 44.36 4 48.92
693 Lichtheimia corymbifera (Cohn 1884) Vuillemin 1903 12 23.06 18 40.93 18 50.21
694 Lichtheimia hyalospora (Saito 1906) Kerst. Hoffmann et al. 2009 1 19.38 1 19.04 1 29.82
695 Linderina pennispora Raper et Fennell 1952 1 11.78 1 46.29
696 Lobosporangium transversale (Malloch) M. Blackwell et Benny 2004 1 7.17
697 Lycoperdon perlatum Persoon 1796 1 21.10
698 Lycoperdon pyriforme Schaeffer 1774 2 20.04
699 Macrolepiota mastoidea (Fries 1821) Singer 1951 1 12.20
700 Macrolepiota procera (Scopoli 1772) Singer 1948 1 12.32
701 Macrophoma mantegazziana (Penzig 1882) Berlese et Voglino 1886 1 0.97 1 26.65
702 Magnusiomyces magnusii (F. Ludwig 1886) Redhead et Malloch 1977 1 31.43 1 0.11
703 Malbranchea flavorosea Sigler et J.W. Carmichael 1976 1 7.19
704 Mammaria echinobotryoides Cesati 1854 1 1.99
705 Marasmius oreades (Bolton 1792) Fries 1836 1 34.16
706 Mariannaea elegans (Corda 1838) Samson 1974 6 31.50 6 45.32 4 9.56
707 Melanconium apiocarpum Link 1825 2 18.22 1 24.70
708 Melanconium bicolor Nees 1817 1 24.70
709 Melanocarpus albomyces (Cooney et R. Emerson 1964) Arx 1975 2 18.61 2 34.85
710 Melanospora betae Panasenko 1938 1 4.07 1 44.99 1 22.75
711 Melanospora damnosa (Saccardo 1895) Lindau 1897 2 29.97 2 22.29
712 Melanospora phaseoli Roll-Hansen 1948 1 0.12 1 45.85
713 Memnoniella echinata (Rivolta 1884) Galloway 1933 2 20.54 3 32.39
714 Menispora ciliata Corda 1837 1 19.25 1 45.78
715 Menispora tortuosa Corda 1839 1 9.82
716 Merimbla ingelheimense (J.F.H. Beyma 1942) Pitt 1980 2 36.14 1 22.89
717 Metarhizium anisopliae (Metschnikoff 1879) Sorokin 1883 6 19.56 6 32.32 4 15.82
718 Microascus cirrosus Curzi 1930 1 19.32
719 Microascus trigonosporus C.W. Emmons et B.O. Dodge var. terreus 1 20.42 1 36.69 1 21.16
Kamyschko 1966
720 Microbotryum silenes-inflatae (de Candolle 1815 ex Liro 1924) G. Deml et 2 26.41 2 19.05
Oberwinkler 1982
721 Microbotryum vinosum (Tulasne et C. Tulasne 1847) Denchev 1994 1 23.70 1 19.05
722 Microbotryum violaceum (Persoon 1797) G. Deml et Oberwinkler 1982 2 21.38 2 19.05
723 Microdiplodia pruni Diedicke 1914 1 19.90 1 9.94
724 Microdochium nivale (Fries 1825) Samuels et I.C. Hallett 1983 1 6.04 1 31.05 1 23.41
725 Microsphaeropsis olivacea (Bonorden 1869) Höhnell 1917 1 19.40 1 12.05
726 Mirandina corticola G. Arnaud 1952 ex Matsushima 1975 1 19.27 1 31.46
727 Monascus floridanus P.F. Cannon et E.L. Barnard 1987 1 6.75
728 Monilia brunnea J.C. Gilman et E.V. Abbott 1927 1 15.22 1 27.24
729 Monilia diversispora J.F.H. Beyma 1933 1 15.22 1 35.69
730 Monilia medoacensis (Saccardo 1913) J.F.H. Beyma 1933 1 28.21
731 Monilia megalospora (Berkeley et M.A. Curtis 1869) Saccardo 1886 1 26.72 1 22.73
732 Moniliella suaveolens (Lindner 1895 ex Lindner 1906) Arx 1972 var. nigra 4 15.42 4 23.99
(Burri et Staub 1909) de Hoog 1979
733 Moniliella suaveolens (Lindner 1895 ex Lindner 1906) Arx 1972 var. 1 18.97 1 35.38
suaveolens
734 Monilinia fructigena (Aderhold et Ruhland 1905) Honey 1936 2 20.03
735 Monochaetia concentrica (Berkeley et Broome 1874) Saccardo et D. 1 9.21
Saccardo 1906
736 Monochaetia dimorphospora T. Yokoyama 1975 1 24.55
737 Monochaetia karstenii (Corda 1839) Nag Raj 1985 3 13.39
738 Monocillium dimorphosporum W. Gams 1971 2 27.09
(continued)
739 Monocillium indicum S.B. Saksena 1955 1 16.32 2 15.10
740 Monocillium nordinii (Bourchier 1961) W. Gams 1971 1 19.84 2 22.59
741 Monocillium tenue W. Gams 1971 2 24.20 1 0.53
742 Monodictys paradoxa (Corda 1938) S. Hughes 1958 1 17.68 1 11.72
743 Monosporium meliolicola Spegazzini 1910 1 23.10
744 Mortierella alliacea Linnemann 1953 1 13.86
745 Mortierella alpina Peyronel 1913 5 21.33 3 43.35 2 27.46
746 Mortierella ambigua B.S. Mehrotra 1963 1 11.73 1 35.99
747 Mortierella angusta Linnemann 1969 1 23.44
748 Mortierella beljakovae Milko 1973 1 3.79
749 Mortierella bisporalis (Thaxter 1914) Bjoerling 1936 2 13.09 2 2.74
750 Mortierella capitata Marchal 1891 1 21.26 1 35.16 1 13.30
751 Mortierella dichotoma Linnemann 1936 ex W. Gams 1977 1 22.23 1 25.53 1 1.08
752 Mortierella elasson Sideris et G.E. Paxton 1929 2 10.95 2 5.88
753 Mortierella elongata Linnemann 1941 2 28.89 5 4.40
754 Mortierella exigua Linnemann 1941 3 34.07 3 36.68
755 Mortierella gamsii Milko 1974 7 18.90
756 Mortierella gemmifera M. Ellis 1940 3 23.25 5 25.48 2 21.69
757 Mortierella globalpina W. Gams et Veenbaas-Rijks 1976 1 13.86
758 Mortierella globulifera O. Rostrup 1916 3 33.28 1 2.76 2 41.39
759 Mortierella horticola Linnemann 1941 2 32.84
760 Mortierella humilis Linnemann 1936 ex W. Gams 1977 5 14.00
761 Mortierella hyalina (Harz 1871) W. Gams 1970 var. hyalina 3 23.57 2 31.26 3 1.08
762 Mortierella jenkinii (A.L. Smith 1898) Naumov 1935 3 13.28 2 36.62
763 Mortierella lignicola (G.W. Martin 1937) W. Gams et R. Moreau 1959 1 13.97 1 45.23 1 23.94
764 Mortierella minutissima van Tieghem 1878 4 34.13 3 14.31 1 1.02
765 Mortierella mutabilis Linnemann 1941 2 22.07 2 14.53
766 Mortierella nigrescens van Tieghem 1878 1 18.26
767 Mortierella oligospora Bjoerling 1936 1 27.05 1 2.52 1 1.08
768 Mortierella parvispora Linnemann 1941 6 33.99 5 27.66 4 22.30
769 Mortierella polycephala Coemans 1863 1 24.88
770 Mortierella pulchella Linnemann 1941 1 23.24 1 25.48
771 Mortierella pusilla Oudemans 1902 1 33.99 1 25.11
772 Mortierella reticulata van Tieghem et G. Le Monnier 1873 1 6.98 1 45.16 1 45.75
773 Mortierella sarnyensis Milko 1973 1 13.86
774 Mortierella sclerotiella Milko 1967 1 22.97
775 Mortierella strangulata van Tieghem 1875 1 3.79 1 25.26
776 Mortierella stylospora Dixon-Stewart 1932 1 19.96 1 27.89 1 34.43
777 Mortierella turficola Y. Ling 1930 1 11.03
778 Mortierella verticillata Linnemann 1941 8 23.25 8 39.71 4 24.60
779 Mortierella zonata Linnemann 1936 ex W. Gams 1977 1 13.96 1 5.09
780 Mortierella zychae Linnemann 1941 5 26.76 5 31.68 3 20.46
781 Mucobasispora tarikii Moustafa et Abdul-Wahid 1990 1 23.92
782 Mucor abundans Povah 1917 1 24.09 1 29.37 1 5.99
783 Mucor aligarensis B.S. Mehrotra et B.R. Mehrotra 1969 1 23.28
784 Mucor amphibiorum Shipper 1978 1 15.38 1 27.73 1 0.58
785 Mucor bacilliformis Hesseltine 1954 1 20.55 1 28.79
786 Mucor bainieri B.S. Mehrotra et Baijal 1963 1 20.70 1 32.41
787 Mucor circinelloides van Tieghem 1875 var. circinelloides 15 25.31 17 44.90 14 45.75
788 Mucor circinelloides van Tieghem 1875 var. janssenii (Lendner 1907) 6 22.62 7 44.93 6 49.10
Schipper 1976
789 Mucor circinelloides van Tieghem 1875 var. lusitanicus (Bruderlein 1916) 6 25.18 8 44.18 7 28.44
Schipper 1976
790 Mucor durus G. Walther et de Hoog 2013 1 19.68 1 42.97 1 37.90
791 Mucor exponens (Burgeff 1924) G. Walther et de Hoog 2013 4 25.41 4 30.87
792 Mucor flavus Bainier 1903 19 28.49 19 47.04 16 26.71
793 Mucor fragilis Bainier 1884 1 24.09 1 37.17 1 30.73
794 Mucor fuscus Bainier 1903 3 20.68 3 45.03 3 32.05
795 Mucor genevensis Lendner 1908 4 25.31 4 37.18
796 Mucor griseocyanus Hagem 1908 3 25.41 3 38.26 3 24.30
797 Mucor guilliermondii Nadson et Philippow 1925 1 24.48 1 37.53 1 30.03
798 Mucor heterogamus Vuillemin 1903 1 27.33 1 1.66
799 Mucor hiemalis Wehmer 1903 var. corticolus (Hagem 1910) Schipper 1973 3 20.71 3 43.93 2 9.59
800 Mucor hiemalis Wehmer 1903 var. hiemalis 18 33.53 19 46.92 14 45.95
801 Mucor hiemalis Wehmer 1903 var. silvaticus (Hagem 1908) Schipper 1973 3 20.56 3 38.71 3 2.61
802 Mucor inaequisporus Dade 1937 1 19.64 1 39.72 1 0.10
803 Mucor indicus Lendner 1930 3 33.53 3 40.30 3 47.89
(continued)
804 Mucor laxorrhizus Y. Ling 1930 5 19.59 5 31.36 5 20.52
805 Mucor luteus Linnemann 1936 2 19.66 2 41.88 2 16.22
806 Mucor megaolocarpus G. Walther et de Hoog 2013 1 19.75 1 9.93
807 Mucor microsporus Namyslowski 1910 1 19.57 1 28.08
808 Mucor moelleri (Vuillemin 1903) Lendner 1908 5 23.57 5 47.23 5 14.60
809 Mucor mousanensis Baijal et B.S. Mehrotra 1966 1 19.70 1 45.34 1 24.54
810 Mucor mucedo Linnaeus 1753 9 25.41 9 46.64 7 29.90
811 Mucor odoratus Treschew 1940 2 25.31 2 16.18
812 Mucor piriformis A. Fischer 1892 5 25.41 5 35.21
813 Mucor plasmaticus van Tieghem 1875 1 19.68 1 44.21 1 5.23
814 Mucor plumbeus Bonorden 1864 11 24.15 18 40.64 16 49.99
815 Mucor psychrophilus Milko 1971 1 25.41 1 14.88
816 Mucor racemosus Fresenius 1850 var. chibinensis (Neophytova 1955) 2 11.86 4 40.59 3 34.29
Schipper 1976
817 Mucor racemosus Fresenius 1850 var. racemosus 23 24.25 34 43.94 29 50.21
818 Mucor racemosus Fresenius 1850 var. sphaerosporus (Hagem 1908) 3 25.41 3 44.91 3 47.36
Schipper 1970
819 Mucor ramosissimus Samoutsevitch 1927 1 19.64 1 17.67
820 Mucor saturninus Hagem 1910 1 20.72 1 35.92 1 8.11
821 Mucor sinensis Milko et Beliakova 1971 2 21.24 2 36.82 2 33.55
822 Mucor strictus Hagem 1908 2 24.09 2 32.51 2 8.38
823 Mucor ucrainicus Milko 1971 1 23.18 1 25.68
824 Mucor zonatus Milko 1967 2 19.75 2 27.40 1 5.69
825 Mucor zychae Baijal et B.S. Mehrotra 1965 var. zychae 2 25.31 2 44.13
826 Mutinus caninus (Hudson 1778) Fries 1849 1 13.37
827 Myceliophthora fergusii (Klopotek 1974) Oorschot 1977 2 7.38
828 Myceliophthora lutea Costantin 1892 1 34.75 1 3.73
829 Myceliophthora thermophila (Apinis 1962) van Oorschot 1977 3 20.51 3 20.37 2 19.25
830 Mycena epipterygia (Scopoli 1772) Gray 1821 1 12.20
831 Mycena pura (Persoon 1794) P. Kummer 1871 1 34.16
832 Mycogone cervina Ditmar 1817 1 19.86
833 Mycogone nigra (Morgan 1895) C.N. Jensen 1912 4 19.54 4 47.62
834 Mycogone rosea Link 1809 4 19.38 4 38.41
835 Mycosticta cytosporicola Frolov 1968 2 17.68 2 37.24
836 Mycotypha africana R.O. Novak et Backus 1963 1 21.41
837 Mycotypha indica P.M. Kirk et Benny 1985 1 22.42
838 Myrothecium sp. 2 19.19 2 43.47
839 Myxotrichum setosum (Eidam 1882) G.F. Orr et Plunkett 1963 2 38.43
840 Myxotrichum stipitatum (Eidam 1882) G.F. Orr et Kuehn 1963 1 4.07 1 45.26
841 Nadsoniella nigra Issatschenko 1914 var. hesuelica Lyakh et Ruban 1970 1 18.86 1 12.62
842 Nakataea sigmoidea (Cavara 1889) Hara 1939 1 19.31
843 Nectria cosmariospora Cesati et de Notaris 1863 2 18.95 2 35.78 2 3.45
844 Nectria inventa Pethybridge 1919 1 27.90 1 3.49
845 Nematogonum mycophilum (Saccardo 1886) Rogerson et W. Gams 1981 1 21.73 1 20.12
846 Neoantrodia serialis (Fries 1821) Audet 2017 1 17.47
847 Neocamarosporium betae (Berlese 1888) Ariyawansa et K.D. Hyde 2015 2 19.33 2 34.74
848 Neocosmospora vasinfecta E.F. Smith 1899 var. africana (von Arx 1955) 2 20.10 2 48.22
Cannon et D. Hawksworth 1984
849 Neonectria galligena (Bresadola 1901) Rossman et Samuels 1999 1 18.96
850 Neoscytalidium dimidiatum (Penzig 1887) Crous et Slippers 2006 1 19.40
851 Neottiospora caricina (Desmazieres 1836) Hoehnel 1924 1 31.07
852 Neovossia setariae (Ling 1945) Yu et Lou 1962 1 19.41
853 Neurospora crassa Shear et B.O. Dodge 1927 71 18.95 77 44.92 9 23.18
854 Neurospora sitophila Shear et B.O. Dodge 1927 4 19.44 4 42.28 4 22.14
855 Neurospora toroi F.L. Tai 1935 1 18.97 1 41.71 1 21.16
856 Newbya pascuicola M.C. Vick et M.W. Dick 2002 1 20.71
857 Niesslia exilis (Albertini et Schweinitz 1805) G. Winter 1885 1 18.96 1 7.35 1 4.37
858 Nigrospora gorlenkoana Novobranova 1972 2 19.47 2 39.55
859 Nigrospora gossypii Jaczewski 1929 1 17.47 1 33.25
860 Nigrospora oryzae (Berkeley et Broome 1873) Petch 1924 4 42.86 1 35.79
861 Nodulisporium verrucosum (J.F.H. Beyma 1929) G. Smith 1954 1 19.72
862 Nomuraea rileyi (Farlow 1883) Samson 1974 1 9.85 1 12.70
863 Ochrocladosporium elatum (Harz 1871) Crous et U. Braun 2007 1 12.58 1 27.80 1 7.91
864 Ochroconis constricta (E.V. Abbott 1927) de Hoog et Arx 1973 1 18.32
865 Oedocephalum sp. 1 15.56 1 28.99 1 15.98
866 Oidiodendron cereale (Thuemen 1880) G.L. Barron 1962 5 0.18 4 28.60
867 Oidiodendron echinulatum G.L. Barron 1962 2 19.58 2 31.49
868 Oidiodendron griseum Robak 1934 2 13.34
(continued)
869 Oidiodendron periconioides Morrall 1968 1 1.79
870 Oidiodendron sulphureoochraceum 1 1.95
871 Oidiodendron truncatum G.L. Barron 1962 3 8.70
872 Olpitrichum sp. 1 20.47 1 19.95 1 2.49
873 Oospora nicotianae Pezzolato 1899 1 19.77 1 29.63 1 2.89
874 Oospora oryzae Ferraris 1902 1 19.95 1 1.05 1 2.90
875 Oospora sajanica Ogarkov 1979 1 16.12 1 25.73
876 Oospora sulphurea (Preuss 1852) Saccardo et Voglino 1886 1 28.81 1 19.34
877 Oospora sulphurella (Saccardo et Roumeguere 1881) Saccardo 1886 1 19.56 1 26.34
878 Oospora tenuis (P. Maze 1910) Berkhout 1923 1 31.12 1 30.92 1 0.09
879 Oospora variabilis (Lindner 1898) J. Lindau 1907 1 19.86 1 14.69
880 Ophiostoma piceae (Münch 1907) Sydow et P. Sydow 1919 2 22.04
881 Ovadendron sulphureo-ochraceum (J.F.H. Beyma 1933) Sigler et J.W. 1 19.79 1 43.59
Carmichael 1976
882 Paecilomyces borysthenicus B.A. Borisov et Tarasov 1997 2 5.07 2 12.90
883 Paecilomyces carneus (Duche et R. Heim 1931 ) A.H.S. Brown et G. Smith 2 4.41 2 8.12 2 0.21
1957
884 Paecilomyces fulvus Stolk et Samson 1971 1 5.53
885 Paecilomyces inflatus (Burnside 1927) J.W. Carmichael 1962 3 8.19
886 Paecilomyces marquandii (Massee 1898) S. Hughes 1951 4 19.56 5 44.40 3 21.91
887 Paecilomyces penicillatus (Höhnel 1904) Samson 1974 1 5.44
888 Paecilomyces suffultus (Petch 1944) Samson 1974 1 6.10
889 Paecilomyces variotii Bainier 1907 20 19.41 30 45.44 16 47.90
890 Paecilomyces zollerniae Stolk et Samson 1971 1 12.37 1 12.56
891 Panus conchatus (Bulliard 1787) Fries 1838 1 23.84
892 Papulaspora biformospora Kirilenko 1971 1 19.64 1 20.69
893 Paraconiothyrium fuckelii (Saccardo 1878) Verkley et Gruyter 2012 1 9.65
894 Paraconiothyrium sporulosum (W. Gams et Domsch 1969) Verkley 2004 2 19.40 2 16.28
895 Paradendryphiella salina (G.K. Sutherland 1916) Woudenberg et Crous 4 10.24
2013
896 Paramyrothecium roridum (Tode 1790) L. Lombard et Crous 2016 3 19.49 4 45.17
897 Paraphoma fimeti (Brunaud 1889) Gruyter et al. 2010 6 2.11
898 Parasitella parasitica (Bainier 1884) Sydow 1903 2 33.43 2 46.09
899 Penicillium adametzii K.M. Zalessky 1927 4 40.39 4 45.81
900 Penicillium albicans Bainier 1907 2 22.03 2 37.14
901 Penicillium albidum Sopp 1912 1 4.99 1 0.95
902 Penicillium alicantinum C. Ramirez et A.T. Martinez 1980 1 38.03 1 12.20
903 Penicillium anatolicum Stolk 1968 1 33.19 1 15.32
904 Penicillium aragonense C. Ramirez et A.T. Martinez 1981 1 38.03 1 12.20
905 Penicillium arenicola Chalabuda 1950 1 22.92 1 26.18
906 Penicillium atramentosum Thom 1910 1 21.02 1 2.56
907 Penicillium aurantioflammiferum C. Ramirez et al. 1980 1 27.31 1 12.20
908 Penicillium aurantiogriseum Dierckx 1901 9 20.52 62 44.47 36 42.96
909 Penicillium bilaiae Chalabuda 1950 1 43.15 1 18.52
910 Penicillium brevicompactum Dierckx 1901 6 20.50 23 45.39 15 18.10
911 Penicillium brunneum Udagawa 1959 1 37.98 1 12.07
912 Penicillium camemberti Thom 1906 11 42.00 10 24.37
913 Penicillium canescens Sopp 1912 31 20.50 60 47.85 44 49.17
914 Penicillium capsulatum Raper et Fennell 1948 2 19.51 3 44.92 3 28.71
915 Penicillium castellonense C. Ramirez et A.T. Martinez 1981 1 38.03 1 12.20
916 Penicillium chermesinum Biourge 1923 4 42.92 4 45.97
917 Penicillium chrysogenum Thom 1910 17 18.95 96 45.01 53 55.63
918 Penicillium cinerascens Biourge 1923 1 41.75 1 28.22
919 Penicillium citreonigrum Dierckx 1901 3 18.95 13 43.65 10 46.51
920 Penicillium citrinum Thom 1910 9 18.31 27 45.08 17 33.21
921 Penicillium commune Thom 1910 10 2.52 23 44.45 22 38.12
922 Penicillium coprophilum (Berkeley et M.A. Curtis 1868) Seifert et Samson 2 0.98
1986
923 Penicillium cordubense C. Ramirez et A.T. Martinez 1981 1 38.01 1 12.20
924 Penicillium corylophilum Dierckx 1901 3 28.97 2 15.77
925 Penicillium crustosum Thom 1930 31 45.20 10 47.85
926 Penicillium cyaneum (Bainier et R. Sartory 1913) Biourge 1923 ex Thom 1 44.58 1 9.56
1930
927 Penicillium daleae K.M. Zalessky 1927 1 2.27 2 29.98 1 22.64
928 Penicillium decumbens Thom 1910 4 18.08 17 43.07 8 37.99
929 Penicillium dierckxii Biourge 1923 2 18.31 7 44.77 7 28.88
930 Penicillium digitatum (Persoon 1801) Saccardo 1881 3 44.23 3 21.28
931 Penicillium diversum Raper et Fennell 1948 3 40.50 1 38.07
(continued)
932 Penicillium dodgei Pitt 1980 1 27.75 1 12.58
933 Penicillium duclauxii Delacroix 1892 7 44.65 7 32.90
934 Penicillium expansum Link 1809 27 42.88 7 41.58
935 Penicillium fagi A.T. Martinez et C. Ramirez 1978 1 38.05 1 12.07
936 Penicillium fellutanum Biourge 1923 3 1.96 1 1.15
937 Penicillium funiculosum Thom 1910 5 20.50 14 44.53 9 38.16
938 Penicillium glabrum (Wehmer 1893) Westling 1911 23 40.77 9 31.15
939 Penicillium gladioli Machacek 1928 2 20.46 2 36.75 2 7.91
940 Penicillium glaucum Link 1805 1 20.52 1 44.50 1 48.00
941 Penicillium grancanariae C. Ramirez et al. 1978 1 38.05 1 5.70
942 Penicillium granulatum Bainier 1905 17 45.20 5 21.61
943 Penicillium griseofulvum Dierckx 1901 11 43.90 8 43.32
944 Penicillium herqueri Bainier et R. Sartory 1912 4 45.41 4 28.33
945 Penicillium hirayamae Udagawa 1959 1 20.37 1 20.18
946 Penicillium hirsutum Dierckx 1901 var. hirsutum 1 2.77 1 5.41
947 Penicillium hispanicum C. Ramirez et al. 1978 1 38.05 1 12.07
948 Penicillium humuli J.F.H. Beyma 1937 1 44.47 1 47.94
949 Penicillium ilerdanum C. Ramirez et al. 1980 1 26.66 1 12.20
950 Penicillium indonesiae Pitt 1980 2 38.31 2 37.22
951 Penicillium inflatum Stolk et Malla 1971 2 2.95 1 0.15
952 Penicillium insectivorum (Sopp 1912) Biourge 1923 1 44.21 1 38.19
953 Penicillium islandicum Sopp 1912 1 2.23 3 33.12 3 23.12
954 Penicillium italicum Wehmer 1894 3 39.11 3 22.63
955 Penicillium janczewskii K.M. Zalessky 1927 13 44.47 8 47.96
956 Penicillium jensenii K.M. Zalessky 1927 9 47.35 8 43.44
957 Penicillium lagena (Delitsch 1943) Stolk et Samson 1983 3 19.46 4 39.10 4 25.92
958 Penicillium lanosum Westling 1911 3 44.45 3 38.02
959 Penicillium lapidosum Raper et Fennell 1948 5 44.45 4 42.21
960 Penicillium lehmanii Pitt 1980 2 40.41 2 47.99
961 Penicillium lineatum Pitt 1980 1 10.51 1 11.55
962 Penicillium lividum Westling 1911 15 38.20 1 15.33
963 Penicillium malacaense C. Ramirez et A.T. Martinez 1980 1 38.01 1 12.20
964 Penicillium martensii Biourge 1923 var. moldavicum Solovei 1975 1 2.27 1 20.16 1 10.71
965 Penicillium megasporum Orpurt et Fennell 1955 3 20.18 2 20.34
966 Penicillium melanoconidium Dierckx 1901 2 12.89 2 0.10
967 Penicillium melinii Thom 1930 1 3.99 6 41.94 4 37.14
968 Penicillium miczynskii K.M. Zalessky 1927 1 4.02 7 43.38 6 47.84
969 Penicillium minioluteum Dierckx 1901 37 38.03 2 12.20
970 Penicillium mirabile Beliakova et Milko 1972 1 36.90 1 9.56
971 Penicillium multicolor Grigorieva-Manoilova et Poradielova 1915 1 42.24 1 20.77
972 Penicillium multicolor Novobranova 1972 1 12.94 1 15.09
973 Penicillium murcianum C. Ramirez et A.T. Martinez 1981 1 31.98 1 32.01
974 Penicillium nalgiovense Laxa 1932 7 9.24 7 0.08
975 Penicillium novae-zeelandiae J.F.H. Beyma 1940 5 44.78 5 9.70
976 Penicillium ochrochloron Biourge 1923 7 44.30 5 26.52
977 Penicillium olivicolor Pitt 1980 1 0.32
978 Penicillium olsonii Bainier et R. Sartory 1912 2 0.95
979 Penicillium onobense C. Ramirez et A.T. Martinez 1981 1 38.05 1 12.20
980 Penicillium ovetense C. Ramirez et A.T. Martinez 1981 1 38.03 1 12.20
981 Penicillium oxalicum Currie et Thom 1915 6 38.05 6 38.11
982 Penicillium palitans Westling 1911 9 9.63 4 0.25
983 Penicillium palmense C. Ramirez et al. 1978 1 38.05 1 12.20
984 Penicillium paxilli Bainier 1907 3 19.51 8 44.47 7 18.52
985 Penicillium phoeniceum J.F.H. Beyma 1933 4 44.92 4 15.02
986 Penicillium piceum Raper et Fennell 1948 1 20.55 3 41.96 3 47.78
987 Penicillium pinophilum Thom 1910 3 37.80 2 32.70
988 Penicillium polonicum K.M. Zalessky 1927 6 6.49 6 0.08
989 Penicillium purpurogenum Stoll 1904 19 46.91 9 50.92
990 Penicillium quercetorum Baghdadi 1968 1 4.06 1 44.72 1 38.26
991 Penicillium raistrickii G. Smith 1933 5 36.68 3 20.13
992 Penicillium resticulosum Birkinshaw et al. 1942 1 26.72 1 44.28
993 Penicillium restrictum J.C. Gilman et E.V. Abbott 1927 2 18.31 14 44.23 8 38.51
994 Penicillium roqueforti Thom 1906 7 3.99 16 45.00 15 43.04
995 Penicillium roseopurpureum Dierckx 1901 8 44.69 5 28.62
996 Penicillium rubrum Stoll 1904 5 20.50 13 45.08 13 43.02
997 Penicillium rugulosum Thom 1910 3 28.56 23 44.08 17 43.08
998 Penicillium sclerotiorum J.F.H. Beyma 1937 7 42.22 7 34.33
(continued)
999 Penicillium severskii Schechovtsov 1981 1 11.10 1 5.72
1000 Penicillium simplicissimum (Oudemans 1903) Thom 1930 3 18.31 26 45.25 19 47.55
1001 Penicillium solitum Westling 1911 13 18.00 15 44.01 15 38.32
1002 Penicillium spinulosum Thom 1910 6 4.02 25 43.47 17 46.00
1003 Penicillium terraconense C. Ramirez et A.T. Martinez 1980 1 38.04 1 2.55
1004 Penicillium thomii Maire 1917 4 2.27 9 42.32 9 36.25
1005 Penicillium thymicola Frisvad et Samson 2004 1 6.41 1 0.31
1006 Penicillium turbatum Westling 1911 1 28.44 1 43.16
1007 Penicillium turolense C. Ramirez et A.T. Martinez 1981 1 38.04 1 12.20
1008 Penicillium umbonatum Sopp 1912 1 32.95
1009 Penicillium valentinum C. Ramirez et A.T. Martinez 1980 1 38.03 1 12.20
1010 Penicillium vanbeymae Pitt 1980 1 42.32 1 7.00
1011 Penicillium variabile Sopp 1912 37 42.19 17 42.68
1012 Penicillium vasconiae C. Ramirez et A.T. Martinez 1980 1 37.93 1 12.20
1013 Penicillium velutinum J.F.H. Beyma 1935 11 44.65 10 38.52
1014 Penicillium verrucosum Dierckx 1901 6 0.58 18 42.32 9 32.75
1015 Penicillium verruculosum Peyronel 1913 14 18.08 23 42.33 13 28.85
1016 Penicillium vinaceum J.C. Gilman et E.V. Abbott 1927 4 44.59 4 43.16
1017 Penicillium viridicatum Westling 1911 5 6.06 1 0.09
1018 Penicillium vulpinum (Cooke et Massee 1888) Seifert et Samson 1985 10 44.53 10 42.56
1019 Penicillium waksmanii K.M. Zalessky 1927 1 2.27 10 44.57 4 47.96
1020 Penicillium westlingii K.M. Zalessky 1927 1 29.52 1 21.65
1021 Penicillium zacinthae C. Ramírez et A.T. Martínez 1981 1 26.64 1 12.20
1022 Penidiella sp. 5 2.10
1023 Perenniporia medulla-panis (Jacquin 1778) Donk 1967 1 18.55
1024 Periconia igniaria E.W. Mason et M.B. Ellis 1953 1 9.90
1025 Periconia macrospinosa Lefebvre et Aar.G. Johnson 1949 2 19.49 2 28.22
1026 Periconiella cocoes M.B. Ellis 1967 1 18.12
1027 Pestalotia pezizoides de Notaris 1841 2 19.86 2 39.30
1028 Pestalotiopsis guepinii (Desmazieres 1840) Steyaert 1949 6 10.76
1029 Pestalotiopsis sydowiana (Bresadola 1895) B. Sutton 1961 1 19.29
1030 Petriella sordida (Zukal 1890) G.L. Barron et J.C. Gilman 1961 1 10.23
1031 Phacidium lacerum Fries 1818 1 0.33
1032 Phaeococcomyces nigricans (Rich et Stern 1958) de Hoog 1979 1 23.58 1 0.53
1033 Phaeoisaria triseptata Holubova-Jechova 1988 1 18.69
1034 Phaeosphaeria sp. 1 1.56
1035 Phallus hadriani Ventenat 1798 1 29.07
1036 Phallus impudicus Linnaeus 1753 var. togatus (Kalchbrenner 1883) 3 21.77
Costantin et L.M. Dufour 1895
1037 Phanerochaete sanguinea (Fries 1828) Pouzar 1973 1 3.25
1038 Phellinus igniarius (Linnaeus 1753) Quelet 1886 6 21.17
1039 Phellinus lundellii Niemelae 1972 3 20.02
1040 Phellinus populicola Niemelae 1975 3 27.22
1041 Phialophora atrovirens (J.F.H. Beyma 1935) Schol-Schwarz 1970 1 20.40 1 28.65
1042 Phialophora bubakii (Laxa 1930) Schol-Schwarz 1970 2 20.42 10 36.70
1043 Phialophora cyclaminis J.F.H. Beyma 1942 2 6.36
1044 Phialophora lagerbergii (Melin et Nannfeldt 1934) Conant 1937 1 19.38 1 13.60
1045 Phialophora melinii (Nannfeldt 1934) Conant 1937 13 5.86
1046 Phialophora verrucosa Medlar 1915 1 19.76 1 19.52
1047 Phlebia ochraceofulva (Bourdot et Galzin 1911) Donk 1957 1 16.82
1048 Phlebia rufa (Persoon 1801) M.P. Christiansen 1960 1 14.95
1049 Phlebia tremellosa (Schrader 1794) Nakasone et Burdsall 1984 2 19.68
1050 Phlebiopsis gigantea (Fries 1815) Juelich 1978 3 20.04
1051 Pholiota adiposa (Batsch 1786) P. Kummer 1871 1 20.10
1052 Pholiota aurivella (Batsch 1786) P. Kummer 1871 1 19.72
1053 Pholiota lenta (Persoon 1801) Singer 1951 2 14.04
1054 Pholiota microspora (Berkeley 1850) Saccardo 1887 1 8.08
1055 Phoma herbarum Westendorp 1852 5 4.99
1056 Phoma leveillei Boerema et G.J. Bollen 1975 4 13.49
1057 Phoma lingam (Tode 1791) Desmazieres 1849 4 19.59
1058 Phomatospora sp. 1 18.86 1 26.61
1059 Phomopsis castanea (Saccardo 1879) Petrak 1921 1 11.21
1060 Phomopsis castaneae Moriondo 1963 1 11.10
1061 Phomopsis helianthi Muntanola-Cvetcovic et al. 1981 1 8.26 1 5.68
1062 Phycomyces blakesleeanus Burgeff 1925 8 24.13 8 31.79 2 1.51
1063 Phycomyces nitens (C. Agardh 1823) Kunze 1823 2 23.06 2 43.00 1 0.34
1064 Phyllosticta castaneae Ellis et Everhart 1894 1 11.05
(continued)
1065 Phyllosticta pucciniospila C. Massalongo 1900 1 11.45 1 23.98
1066 Phytophthora cactorum (Lebert et Cohn 1870) J. Schroeter 1886 1 17.05
1067 Phytophthora capsici Leonian 1922 2 28.12
1068 Phytophthora cinnamomi Rands 1922 4 18.78
1069 Phytophthora cryptogea Pethybridge et Lafferty 1919 1 18.03
1070 Phytophthora drechsleri Tucker 1931 3 26.22
1071 Phytophthora megasperma Drechsler 1931 1 0.19
1072 Pidoplitchkoviella terricola Kirilenko 1975 1 20.22 1 42.69
1073 Piedraia hortae Fonseca et Leao 1928 2 19.35 1 34.74 1 23.85
1074 Piedraia hortae Fonseca et Leao 1928 var. paraguayensis Fonseca et Leao 1 32.46 1 0.11
1928
1075 Piedraia sarmentoi M.J. Pereira 1930 1 19.35 1 25.02 1 21.46
1076 Pilaira anomala (Cesati 1851) J. Schroeter 1886 1 26.00 1 1.11
1077 Pilaira caucasica Milko 1970 1 24.12 1 16.20
1078 Pilaira moreaui Y. Ling 1926 1 23.57 1 27.53
1079 Pilobolus crystallinus (F.H. Wiggers 1780) Tode 1784 1 15.64
1080 Pilobolus longipes van Tieghem 1878 1 3.68 1 15.64
1081 Pilobolus umbonatus Buller 1934 1 15.65
1082 Piptoporus betulinus (Bulliard 1788) P. Karsten 1881 3 26.12
1083 Pirella circinans Bainier 1882 2 24.09 1 46.10 1 14.40
1084 Pirella circinans Bainier 1882 var. volgogradensis (Milko 1974) Benny et 1 37.71
Schipper 1988
1085 Pirella naumovii (Milko 1970) Benny et Schipper 1992 1 19.32 1 15.35 1 46.87
1086 Pithoascus schumacheri (E.C. Hansen 1877) Arx 1973 1 0.32
1087 Plectosphaerella cucumerina (Lindfors 1919) W. Gams 1968 1 20.79 1 39.44
1088 Plenodomus tracheiphila (Petri 1929) Gruyter et al. 2013 1 12.56
1089 Pleotrichocladium opacum (Corda 1837) Hernández-Restrepo et al. 2017 3 19.43 4 32.79
1090 Pleurocytospora sp. 1 7.59
1091 Pleurodesmospora coccorum (Petch 1924) Samson et al. 1980 1 28.25
1092 Pleurophoma cava (Schulzer 1871) Boerema 1996 3 19.81 3 44.72
1093 Pleurotus cornucopiae (Paulet 1793) Rolland 1910 1 20.02
1094 Pleurotus eryngii (De Candolle 1815) Quelet 1872 1 18.92
1095 Pleurotus ostreatus (Jacquin 1774) P. Kummer 1871 70 34.08
1096 Pleurotus pulmonarius (Fries 1821) Quelet 1872 4 8.16
1097 Pochonia bulbillosa (W. Gams et Malla 1988) Zare et W. Gams 2001 5 19.99 7 28.36 2 2.72
1098 Pochonia chlamydosporia (Goddard 1913) Zare et W. Gams 2001 7 19.47 7 31.72
1099 Poitrasia circinans (H. Naganishi et N. Kawakami 1955) P.M. Kirk 1984 1 20.08 1 44.36
1100 Polycephalomyces tomentosus (Schrad. 1799) Seifert 1985 1 19.54 1 40.41 1 1.55
1101 Polyporus ciliatus Fries 1815 1 10.36
1102 Polyporus tomentosus Fries 1821 1 9.07
1103 Polyscytalum pustulans (M.N. Owen et Wakefield 1919) M.B. Ellis 1976 1 15.57 1 38.53
1104 Porodaedalea pini (Brotero 1804) Murrill 1905 1 22.97
1105 Preussia fleischhakii (Auerswald 1866) Cain 1961 1 43.78 1 23.47
1106 Protomyces macrosporus Unger 1834 1 19.01
1107 Pseudallescheria boydii (Shear 1922) McGinnis et al. 1982 3 20.28 3 44.50 2 22.75
1108 Pseudallescheria ellipsoidea (Arx et Fassatiova 1973) McGinnis et al. 1982 1 42.46 1 23.42
1109 Pseudeurotium bakeri C. Booth 1961 1 20.48 1 43.04
1110 Pseudeurotium desertorum Mouchacca 1971 1 18.88 1 41.28
1111 Pseudeurotium hygrophilum (Sogonov et al. 2005) Minnis et D.L. Lindner 9 1.67
2013
1112 Pseudeurotium ovale Stolk 1955 var. milkoi Beliakova 1969 2 44.73
1113 Pseudeurotium ovale Stolk 1955 var. ovale 3 34.44 1 45.50 1 0.11
1114 Pseudeurotium zonatum J.F.H. Beyma 1937 9 20.42 20 44.28 10 24.97
1115 Pseudogymnoascus caucasicus Cejp et Milko 1966 1 29.35 1 39.38 1 25.13
1116 Pseudogymnoascus roseus Raillo 1929 2 22.61 5 47.77
1117 Puccinia adoxae R. Hedwig 1805 1 19.60 1 19.05
1118 Puccinia bupleuri (Opiz 1852) F. Rudolphi 1829 1 19.60 1 1.84
1119 Puccinia punctiformis (F. Strauss 1811) Roehling 1813 1 23.64 1 19.05
1120 Purpureocillium lilacinum (Thom 1910) Luangsa-ard et al. 2011 16 22.80 28 44.55 17 19.37
1121 Pycnidiella resinae (Ehrenberg 1818) Hoehnel 1915 1 25.43
1122 Pycnoporus cinnabarinus (Jacquin 1776) P. Karsten 1881 2 23.74
1123 Pyrenochaeta sp. 1 6.54
1124 Pyrenophora biseptata (Saccardo et Roumeguere 1881) Crous 2013 1 19.30 1 33.34 1 4.33
1125 Pyrenophora triseptata (Drechsler 1923) Rossman et K.D. Hyde 2015 1 4.24
1126 Pyricularia grisea Saccardo 1880 3 19.85 1 13.76
1127 Pyronema omphalodes (Bulliard 1791) Fuckel 1870 1 18.96 1 17.28
1128 Pythium heterothallicum W.A. Campbell et F.F. Hendrix 1968 2 33.45
1129 Pythium intermedium de Bary 1881 1 6.50
(continued)
1130 Pythium irregulare Buisman 1927 2 33.47
1131 Pythium mamillatum Meurs 1928 1 17.05
1132 Pythium oedichilum Drechsler 1930 1 33.28
1133 Pythium paroecandrum Drechsler 1930 1 33.58
1134 Pythium spinosum Sawada 1926 1 0.15
1135 Pythium sylvaticum W.A. Campbell et F.F. Hendrix 1967 2 20.59
1136 Quambalaria cyanescens (de Hoog et G.A. de Vries 1973) Z.W. de Beer et 3 6.48
al. 2006
1137 Radiomyces embreei R.K. Benjamin 1960 2 44.93 2 46.27
1138 Radiomyces spectabilis Embree 1959 1 25.31 1 39.17 1 24.89
1139 Remotididymella destructiva (Plowright 1881) Valenzuela-Lopez et al. 2017 3 16.86
1140 Rhinocladiella atrovirens Nannfeldt 1934 3 17.70 5 13.39
1141 Rhinotrichum aureum Cooke et Massee 1889 1 28.38
1142 Rhinotrichum lanosum Cooke 1871 1 19.47 1 21.52
1143 Rhizoctonia solani J.G. Kuehn 1858 22 20.04 1 5.53 4 21.24
1144 Rhizoctonia tuliparum (Klebahn 1905) Whetzel et J.M. Arthur 1924 1 14.94
1145 Rhizomucor miehei (Cooney et R. Emerson 1964) Schipper 1978 1 19.68 1 26.51 1 46.10
1146 Rhizomucor pusillus (Lindt 1886) Schipper 1978 4 19.70 4 43.33 4 43.99
1147 Rhizomucor tauricus (Milko et Schkurenko 1970) Schipper 1978 2 19.64 2 45.07 2 24.69
1148 Rhizopus arrhizus A. Fischer 1892 23 20.72 26 45.19 25 50.01
1149 Rhizopus microsporus van Tieghem 1875 var. chinensis (Saito 1904) 5 20.77 5 45.28 5 48.43
Schipper et Stalpers 1984
1150 Rhizopus microsporus van Tieghem 1875 var. microsporus 8 20.75 11 39.38 10 49.99
1151 Rhizopus microsporus van Tieghem 1875 var. oligosporus (Saito 1905) 2 12.43 2 44.83 2 49.50
1152 Rhizopus microsporus van Tieghem 1875 var. rhizopodiformis (Cohn 1884) 4 15.00 3 12.48
1153 Rhizopus stolonifer (Ehrenberg 1818) Vuillemin 1902 var. stolonifer 20 24.15 24 45.19 19 50.21
1154 Rhodocollybia butyracea (Bulliard 1792) Lennox 1979 1 2.82
1155 Robillarda sessilis (Saccardo 1878) Saccardo 1880 1 18.99
1156 Rosellinia mammiformis (Persoon 1801) Cesati et de Notaris 1863 1 19.28 1 31.68
1157 Russula aurora (Krombholz 1836) Bresadola 1892 1 19.40
1158 Russula decolorans (Fries 1821) Fries 1838 1 34.23
1159 Russula grisea (Batsch 1786) Fries 1838 1 34.03
1160 Saksenaea vasiformis S.B. Saksena 1953 1 27.79
1161 Saprochaete gigas (Smit et L. Meyer 1928) de Hoog et M.T. Smith 2004 1 19.87 1 43.67 1 0.12
1162 Saprolegnia asterophora de Bary 1860 1 15.20
1163 Saprolegnia blelhamensis (M.W. Dick 1969) Milko 1979 3 13.34
1164 Saprolegnia ferax (Gruithuisen 1821) Nees 1843 2 13.81
1165 Saprolegnia litoralis Coker 1923 1 13.10
1166 Saprolegnia mixta de Bary 1883 1 0.17
1167 Saprolegnia terrestris Cookson 1937 ex R.L. Seymour 1970 1 0.17
1168 Saprolegnia unispora (Coker et Couch 1923) R.L. Seymour 1970 2 0.17
1169 Sarocladium strictum (W. Gams 1971) Summerbell 2011 4 19.91 4 26.96 2 23.67
1170 Schizophyllum commune Fries 1815 4 34.08
1171 Sclerotinia borealis Bubák et Vleugel 1917 19 3.85
1172 Sclerotinia nivalis I. Saito 1997 25 3.85
1173 Sclerotinia ricini G.H. Godfrey 1919 1 18.97 1 27.35 1 0.11
1174 Sclerotinia sclerotiorum (Libert 1837) de Bary 1884 2 31.23
1175 Scopulariopsis acremonium (Saccardo 1882) Bainier 1907 (Thom 1910) 1 19.33 1 23.60 1 4.86
Thom 1930
1176 Scopulariopsis asperula (Saccardo 1882) S. Hughes 1958 1 19.26 1 39.91 1 18.38
1177 Scopulariopsis brevicaulis (Saccardo 1882) Bainier 1907 15 20.49 17 48.08 12 46.09
1178 Scopulariopsis brumptii Salvanet-Duval 1935 3 15.25 4 21.54 1 3.33
1179 Scopulariopsis coprophila (Cooke et Massee 1887) W. Gams 1971 1 31.33
1180 Scopulariopsis croci J.F.H. Beyma 1944 1 35.42 1 23.92
1181 Scopulariopsis flava (Sopp 1912) F.J. Morton et G. Smith 1963 (Thom 1910) 1 19.30 1 23.56 1 20.68
Thom 1930
1182 Scopulariopsis halophilica Tubaki 1973 1 28.84
1183 Scopulariopsis koningii (Oudemans 1902) Vuuillemin 1911 1 30.89
1184 Scytalidium terminale G.V. Rao et de Hoog 1975 1 19.50 1 29.45
1185 Seimatosporium pestalozzioides (Saccardo 1884) B. Sutton 1975 1 9.19
1186 Sepedonium macrosporum Saccardo et Cavara 1900 1 39.83 1 25.33
1187 Septoria lycopersici Spegazzini 1881 1 17.25
1188 Septoria rosarum Westendorp 1851 19.42
1189 Serpula lacrymans (Wulfen 1781) J. Schroeter 1885 2 20.11
1190 Simplicillium lamellicola (F.E.W. Smith 1924) Zare et W. Gams 2001 4 19.57 4 24.26
1191 Simplicillium obclavatum (W. Gams 1984) Zare et W. Gams 2001 1 3.83
(continued)
1192 Sistotrema brinkmannii (Bresadola 1903) J. Eriksson 1948 1 0.16
1193 Sordaria fimicola (Roberge ex Desmazières 1849) Cesati et de Notaris 1863 2 19.37 2 43.76 2 23.47
1194 Sorosporium saponariae F. Rudolphi 1830 1 17.70
1195 Spadicesporium acrosporum V.N. Borisova et Dvoinos 1982 1 19.43 1 43.13
1196 Spadicesporium acrosporum-majus V.N. Borisova et Dvoinos 1982 1 19.43 1 11.45
1197 Spadicesporium bifurcatum V.N. Borisova et Dvoinos 1982 1 19.43 1 19.24
1198 Spadicesporium bifurcatum-majus V.N. Borisova et Dvoinos 1982 1 19.43 1 13.52
1199 Spadicesporium copiosum V.N. Borisova et Dvoinos 1982 1 19.43 1 32.34
1200 Spadicesporium persistens V.N. Borisova et Dvoinos 1982 1 19.43 1 40.85
1201 Spadicesporium ramosum V.N. Borisova et Dvoinos 1982 1 19.78 1 43.13
1202 Sparassis crispa (Wulfen 1781) Fries 1821 1 7.49
1203 Sphaceloma sp. 1 23.55
1204 Sphaeropsis sapinea (Fries 1823) Dyko et B. Sutton 1980 2 19.90
1205 Sphaerostilbella aureonitens (Tulasne et C. Tulasne 1865) Seifert et al. 1985 1 22.34
1206 Sphaerostilbella penicillioides (Corda 1840) Rossman et al. 2015 6 19.19 6 34.71 2 3.20
1207 Sporocadus lichenicola Corda 1839 1 10.24
1208 Sporodiniopsis dichotoma van Hoehnel 1903 1 15.90 1 15.95 1 10.80
1209 Sporormiella australis (Spegazzini 1887) S.I. Ahmed et Cain 1972 1 2.41 1 5.07
1210 Sporormiella intermedia (Auerswald 1868) S.I. Ahmed et Cain ex Kobayasi 1 38.09
1969
1211 Sporothrix fungorum de Hoog et G.A. de Vries 1973 1 1.94
1212 Sporotrichum aeruginosum Schweinitz 1886 var. microsporum Karsten 1 32.35
1905
1213 Sporotrichum bombycinum (Corda 1839) Rabenhorst 1844 3 19.31 3 29.59 2 35.25
1214 Sporotrichum gorlenkoanum Kuritzina et Sizova 1967 1 23.64 1 20.27
1215 Sporotrichum laxum Nees 1816 1 19.31 1 26.27
1216 Sporotrichum mycophilum Link 1818 1 27.68
1217 Sporotrichum pruinosum J.C. Gilman et E.V. Abbott 1927 7 19.82 13 42.71 5 35.05
1218 Sporotrichum roseolum Oudemans et Beijerinck 1903 1 15.20 1 26.16 1 2.28
1219 Stachybotrys chartarum (Ehrenberg 1818) S. Hughes 1958 10 19.32 14 48.10 2 9.56
1220 Stachybotrys cylindrospora C.N. Jensen 1912 1 29.41
1221 Stachylidium variabile Schulzer et Saccardo 1884 1 1.59
1222 Stagonospora paludosa (Saccardo et Spegazzini 1879) Saccardo 1884 1 19.90
1223 Stagonosporopsis hortensis (Saccardo et Malbranche 1882) Petrak 1921 1 20.34 1 27.54
1224 Stagonosporopsis trachelii (Allescher 1895) Aveskamp et al. 2010 1 20.34
1225 Stemphyliomma sp. 1 26.05 1 35.79
1226 Stemphylium botryosum Wallroth 1833 1 17.06 1 23.43
1227 Stemphylium sarciniforme (Cavara 1890) Wiltshire 1938 3 17.80 3 25.45
1228 Stenocarpella maydis (Berkeley 1847) B. Sutton 1980 1 21.31 1 11.57
1229 Stephanoma sp. 1 16.24 1 16.03
1230 Stereum hirsutum (Willdenow 1787) Persoon 1800 3 20.04
1231 Stereum sanguinolentum (Albertini et Schweinitz 1805) Fries 1838 1 23.21
1232 Stilbella bulbicola Hennings 1905 1 19.46 1 44.40
1233 Stilbotulasnella conidiophora Bandoni et Oberwinkler 1982 1 23.91
1234 Striaticonidium brachysporum (Nicot 1961) L. Lombard et Crous 2016 1 12.92 1 18.96
1235 Striaticonidium cinctum (Corda 1842) L. Lombard et Crous 2016 1 19.30 1 28.18
1236 Strobilomyces strobilaceus (Scopoli 1770) Berkeley 1851 1 26.53
1237 Stropharia rugosoannulata Farlow ex Murrill 1922 1 12.21
1238 Syncephalastrum racemosum Cohn ex J. Schroeter 1886 12 19.75 12 42.96 12 50.78
1239 Syncephalis cornu van Tieghem et G. Le Monnier 1873 1 46.84 1 45.50
1240 Syncephalis nodosa van Tieghem 1875 1 24.13 1 23.87 1 32.34
1241 Taeniolella aquatilis (Woronichin 1925) Milko 1985 1 19.33 1 31.75
1242 Talaromyces emersonii Stolk 1965 1 30.14 1 37.44
1243 Talaromyces flavus (Kloecker 1902) Stolk et Samson 1972 2 18.08 4 43.58 4 46.02
1244 Talaromyces luteus (Zukal 1889) C.R. Benjamin 1955 9 43.25 9 36.49
1245 Talaromyces stipitatus (Thom 1935) C.R. Benjamin 1955 2 33.85 2 33.96
1246 Talaromyces thermophilus Stolk 1965 1 31.21 1 1.25
1247 Talaromyces ucrainicus Udagawa 1966 2 18.08 3 44.17 3 36.26
1248 Talaromyces wortmannii (Kloecker 1903) C.R. Benjamin 1955 3 22.58 3 12.07
1249 Taphrina bergeniae Döbbeler 1979 1 19.01 1 10.22
1250 Taphrina betulina Rostrup 1883 1 13.27
1251 Taphrina carnea Johanson 1886 1 8.12
1252 Taphrina coerulescens Lindquist et Wright 1 13.25
1253 Taphrina flavorubra W.W. Ray 1939 1 13.27
1254 Taphrina pruni (Fuckel 1861) Tulasne 1866 1 19.01
1255 Taphrina purpurascens B.L. Robinson 1887 1 13.27 1 8.72
1256 Taphrina robinsoniana Giesenhagen 1892 1 13.25
1257 Taphrina sadebeckii Johanson 1885 1 13.25
(continued)
1258 Taphrina tosquinetii (Westendorp 1861) Tulasne 1866 1 18.91 1 9.59
1259 Tapinella panuoides (Batsch 1783) E.-J. Gilbert 1931 2 7.02
1260 Teberdinia hygrophila Sogonov et al. 2005 1 4.67
1261 Tetraploa aristata Berkeley et Broome 1850 1 30.33
1262 Thamnidium elegans Link 1809 3 17.72 3 40.96 3 33.55
1263 Thamnostylum piriforme (Bainier 1880) Arx et H.P. Upadhyay 1970 4 24.14 4 37.27 4 48.92
1264 Thelebolus microsporus (Berkeley et Broome 1865) Kimbrough 1967 3 6.54
1265 Thelebolus polysporus (P. Karsten 1871) Otani et Kanzawa 1970 1 20.60 2 33.25
1266 Thermomyces ibadanensis Apinis et Eggins 1966 2 28.76
1267 Thielavia appendiculata Srivastava et al. 1966 1 46.06
1268 Thielavia hyrcaniae Nicot 1961 1 4.06 1 42.75
1269 Thielavia inaequalis Pidoplichko et al. 1973 2 4.07 2 45.58 2 0.10
1270 Thielavia ovispora Pidoplichko et al. 1973 3 19.33 3 43.57 3 24.15
1271 Thielavia terrestris (Apinis 1963) Malloch et Cain 1972 1 20.52 1 12.62
1272 Thielavia terricola (J.C. Gilman et E.V. Abbott 1927) Emmons 1930 5 19.24 5 43.99 5 24.81
1273 Thyrostroma carpophilum (Leveille 1843) B. Sutton 1997 1 19.92
1274 Thysanophora canadensis Stolk et Hennebert 1968 1 17.70 1 7.31
1275 Thysanophora penicillioides (Roumeguere 1890) W.B. Kendrick 1961 6 19.21 6 32.25
1276 Tilachlidium pinnatum Preuss 1851 1 29.01
1277 Tilletia laevis J.G. Kühn 1873 1 23.91 1 19.05
1278 Tilletiopsis albescens Gokhale 1972 1 16.41 1 20.08
1279 Tilletiopsis washingtonensis Nyland 1950 3 28.64 3 29.65
1280 Tolypocladium cylindrosporum W. Gams 1971 5 20.73 5 36.11 1 5.13
1281 Tolypocladium geodes W. Gams 1971 6 13.72 6 13.67
1282 Tolypocladium inflatum W. Gams 1971 9 19.28 13 35.21 2 0.09
1283 Tolypocladium microsporum (Jaap 1916) Bissett 1983 1 3.92
1284 Torula ligniperda (Willkomm 1866) Saccardo 1906 1 12.49 1 30.12
1285 Trametes gibbosa (Persoon 1795) Fries 1838 1 23.84
1286 Trametes hirsuta (Wulfen 1788) Lloyd 1924 5 26.58
1287 Trametes ochracea (Persoon 1794) Gilbertson et Ryvarden 1987 1 8.25
1288 Trametes pubescens (Schumacher 1803) Pilat 1939 3 34.10
1289 Trametes versicolor (Linnaeus 1753) Lloyd 1920 10 34.10
1290 Trametes zonatella Ryvarden 1978 1 0.41
1291 Tricellula aquatica J. Webster 1959 1 16.22 1 29.30
1292 Tricellula aurantiaca (Haskins 1958) Arx 1970 1 31.01 1 34.22
1293 Trichaptum abietinum (Dickson 1793) Ryvarden 1972 2 25.23
1294 Trichocladium asperum Harz 1871 2 19.56 2 47.73
1295 Trichocladium griseum (Traaen 1914) X. Wei Wang et Houbraken 2018 2 19.30 7 48.38
1296 Trichocladium nigrospermum (Schweinitz 1832) X. Wei Wang et 3 17.78 3 32.27 1 35.79
Houbraken 2018
1297 Trichoderma asperellum Samuels et al. 1999 2 7.71 1 8.48
1298 Trichoderma atroviride P. Karsten 1892 5 20.24 9 46.30 4 30.59
1299 Trichoderma aureoviride Rifai 1969 4 19.82 4 41.99 4 29.94
1300 Trichoderma citrinoviride Bissett 1984 3 1.92
1301 Trichoderma deliquescens (Sopp 1912) Jaklitsch 2011 1 21.12 1 12.36
1302 Trichoderma flavofuscum (J.H. Miller et al. 1957) Bissett 1991 1 25.05 1 0.09
1303 Trichoderma ghanense Yoshim. Doi et al. 1987 1 25.05 1 0.09
1304 Trichoderma hamatum (Bonorden 1851) Bainier 1906 1 20.36 1 25.62 1 10.01
1305 Trichoderma harzianum Rifai 1969 24 19.96 32 45.53 12 12.42
1306 Trichoderma koningii Oudemans 1902 4 19.82 4 42.75 4 35.83
1307 Trichoderma lignorum (Tode 1790) Harz 1872 1 2.99
1308 Trichoderma longibrachiatum Rifai 1969 16 21.37 22 46.32 9 37.65
1309 Trichoderma polysporum (Link 1816) Rifai 1969 4 19.99 4 46.07 3 3.56
1310 Trichoderma pseudokoningii Rifai 1969 8 19.81 9 45.02 8 38.35
1311 Trichoderma reesei E.G. Simmons 1968 6 19.82 6 36.31 5 35.98
1312 Trichoderma saturnisporum Hammill 1970 2 0.54 2 25.05 1 4.92
1313 Trichoderma virens (J.H. Miller et al. 1957) Arx 1987 2 18.70 3 44.87 2 18.38
1314 Trichoderma viride Persoon 1794 13 20.47 12 40.53 11 29.81
1315 Trichoderma viride Persoon 1794 var. kizhanicum Krapivina 1975 1 32.63 1 8.12
1316 Trichoderma viridescens (A.S. Horne et H.S. Williamson 1923) Jaklitsch et 5 26.99 4 26.87
Samuels 2006
1317 Trichosporiella cerebriformis (G.A. de Vries et Kleine-Natrop) W. Gams 2 19.59 3 19.22
1971
1318 Trichosporon dulcitum (Berkhout 1923) Weijman 1979 1 27.05
1319 Trichosporum herbarum Jaap 1916 1 0.54 1 27.82 1 3.73
1320 Trichothecium roseum (Persoon 1794) Link 1809 12 20.43 13 47.13 9 10.41
1321 Tritirachium oryzae (Vincens 1910) de Hoog 1972 5 17.67 5 45.93 4 3.38
(continued)
1322 Tropicoporus linteus (Berkeley et M.A. Curtis 1858) L.W. Zhou et Y.C. Dai 1 22.97
2015
1323 Truncatella angustata (Persoon 1801) S. Hughes 1958 3 19.47 4 46.76
1324 Tympanosporium parasiticum W. Gams 1974 1 18.61 1 35.89 1 0.53
1325 Ugola praticola (Pidoplichko 1950) Stalpers 1984 1 30.96 1 8.47
1326 Umbelopsis isabellina (Oudemans 1902) W. Gams 2003 7 23.57 8 42.26 7 47.18
1327 Umbelopsis longicollis (Dixon-Stewart 1932) Y.N. Wang et al. 2015 4 19.43 4 38.84 4 50.23
1328 Umbelopsis nana (Linnemann 1941) Arx 1984 3 24.12 2 35.21 2 27.28
1329 Umbelopsis ramanniana (Moeller 1903) W. Gams 2003 11 19.43 11 40.99 11 50.21
1330 Umbelopsis vinacea (Dixon-Stewart 1932) Arx 1984 3 24.73 3 39.93 3 34.51
1331 Ustilago avenae (Persoon 1801) Rostrup 1890 1 18.51 1 1.64
1332 Ustilago cordae Liro 1924 1 19.56 1 1.84
1333 Ustilago cynodontis (Passerini 1870) Hennings 1893 1 21.45 1 18.84
1334 Ustilago filiformis (Schrank 1793) Rostrup 1890 1 9.34 1 18.84
1335 Ustilago hordei (Persoon 1801) Lagerheim 1889 1 12.32 1 18.84
1336 Ustilago maydis (de Candolle 1815) Corda 1842 2 13.39
1337 Valsa sordida Nitschke 1870 1 17.51
1338 Venturia tremulae Aderhold 1897 1 2.11
1339 Verticillium albo-atrum Reinke et Berthold 1879 3 19.33 3 29.00 3 5.02
1340 Verticillium bulbillosum W. Gams et Malla 1971 4 29.29
1341 Verticillium candidulum W. Gams et Malla 1971 1 29.62
1342 Verticillium cellulose W. Gams et Malla 1971 1 9.19
1343 Verticillium dahliae Klebahn 1913 4 19.37 4 43.73 4 13.62
1344 Verticillium epiphytum Hansford 1943 1 38.28
1345 Verticillium fumosum Seman 1968 1 19.27 1 31.86 1 4.74
1346 Verticillium lecanii (Zimmermann 1898) Viegas 1939 6 19.37 13 39.89 4 3.33
1347 Verticillium longisporum (C. Stark 1961) Karapapa et al. 1997 1 2.97
1348 Verticillium psalliotae Treschew 1941 1 24.57
1349 Verticillium tenerum Nees 1816 2 24.68
1350 Verticillium tricorpus I. Isaac 1953 2 19.33 2 37.17 2 7.30
1351 Verticillium villosum Rudakov 1981 1 41.45
1352 Verticillium zaregamsianum Inderbitzin et al. 2011 7 20.76 7 35.67 5 6.53
1353 Viennotidia humicola (Samson et W. Gams 1974) P.F. Cannon et D. 1 27.31 1 27.65
Hawksworth 1982
1354 Volutella ciliata (Albertini et Schweinitz 1805) Fries 1832 2 19.84 2 21.53
1355 Volutella roseola Cooke 1872 1 21.38 1 15.43
1356 Wallemia sebi (Fries 1832) Arx 1970 3 19.60 2 15.96
1357 Wallrothiella subiculosa Hoehnel 1912 1 21.53
1358 Wardomyces anomalus Brooks et Hansford 1923 1 13.50
1359 Westerdykella dispersa (Clum 1955) Cejp et Milko 1964 1 19.35 1 34.61 1 25.11
1360 Westerdykella multispora (Saito et Minoura ex Cain 1961) Cejp et Milko 1 3.99 1 43.57 1 23.47
1964
1361 Xeromyces bisporus L.R. Fraser 1953 1 18.86 1 15.48 1 0.21
1362 Xylobolus frustulatus (Persoon 1801) Boidin 1958 1 20.01
1363 Zasmidium biverticillatum (Arzanlou et P.W. Crous 2007) S.I.R. Videira et 1 25.54
P.W. Crous 2017
1364 Zygosporium echinosporum Bunting et E.W. Mason 1941 1 19.56 1 36.47 1 1.00
1365 Zymoseptoria passerinii (Saccardo 1884) Quaedvlieg et Crous 2011 1 1.74
1366 Zymoseptoria pseudotritici B. McDonald et al. 2012 1 6.45
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s00253-015-6464-x
Chapter 2
Sabouraud Agar and Other Fungal
Growth Media
Tankeshwar Acharya and Janelle Hare
Contents
2.1 Sabouraud Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.1.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.1.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.1.3 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2.1.4 Method of Sabouraud Agar Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
2.1.5 Methods of Inoculation and Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.1.6 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.2 Potato Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2.2.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2.2.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.2.3 Methods of Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.2.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.3 Bird Seed Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.3.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.3.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.3.3 Methods of Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.3.4 Method of Inoculation and Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.3.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.4 Dermatophyte Test Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.4.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.4.2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.4.3 Method of Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
T. Acharya
Department of Microbiology and Immunology, Patan Academy of Health Sciences, Lalitpur,
Nepal
e-mail: tankeshwaracharya@pahs.edu.np
J. Hare (*)
Department of Biology & Chemistry, Morehead State University, Morehead, KY, USA
e-mail: jm.hare@moreheadstate.edu

70 T. Acharya and J. Hare
2.4.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.5 Safety Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.6 Additional Fungal Growth Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.1 Sabouraud Dextrose Agar
2.1.1 History
Sabouraud agar medium was developed by the French dermatologist Raymond J. A.

Sabouraud (pronounced sah-bū-rō0 ) in the late 1800s to support the growth of fungi,
particularly dermatophytes [1, 2]. Sabouraud’s medical investigations focused on
bacteria and fungi that cause skin lesions, and he developed many agars and
techniques to culture pathogens such as dermatophytes and Malassezia species.
The long incubation period (multiple weeks) of dermatophytes and the need to
avoid bacterial contamination while culturing them, was one driving force behind
the development of this medium. Additionally, Sabouraud sought to provide a
medium that would yield reliable results for fungal identification across laboratories.
He recommended that all mycologists detail their exact media formulations and
sources of ingredients as well as the temperatures and times of specimen incubation,
in order to standardize observations and reduce media-derived sources of differences
in appearance [3].
Ironically, given Sabouraud’s original desire to standardize the construction of
fungal media, there are currently many sources of confusion and variation in both the
names and ingredients associated with Sabouraud agar, also called Sabouraud’s agar
(abbreviated either SDA or SAB). Due to the old-fashioned use of the term “dex-
trose” to refer to D-glucose, the medium has been referred to as Sabouraud dextrose
agar as well as Sabouraud glucose agar, the name most appropriate and consistent
with standard chemical nomenclature [3]. Finally, a more recent modification of
Sabouraud agar by Emmons is either called Sabouraud agar (Modified), or
Sabouraud agar, Emmons [4]. Many of the historical details behind these names
and ingredient variations are described in Odds’ excellent review article [3].
2.1.2 Theory
Sabouraud agar is a selective medium that is formulated to allow the growth of fungi
and inhibit the growth of bacteria. The available means of inhibiting bacterial growth
in Sabouraud’s pre-antibiotic era was via an acidic medium (pH 5.6). Currently, the
addition of antibiotics or antimicrobials to the acidic medium is used to inhibit
2 Sabouraud Agar and Other Fungal Growth Media 71
bacterial growth (and sometimes saprophytic fungi, depending on the particular

antimicrobial used).
Sabouraud agar medium is complex and undefined but contains few ingredients.
Peptones, as soluble protein digests, are sources of nitrogenous growth factors that
can vary significantly according to the particular protein source. The most variation
is present in the source and method of these protein digests. Both Difco and BBL
brand Sabouraud agars use pancreatic digests of casein as their peptone source, but
they and other vendors also use a combination of pancreatic digest of casein and
peptic digest of animal tissues. Sabouraud’s original formulation contained a pep-
tone termed “Granulée de Chassaing,” which is no longer available. Mold morphol-
ogy can vary slightly based on the peptones used, but pigmentation and sporulation
can be consistent if one uses a consistent method of medium preparation, with the
ingredients from the same source each time. Researchers should also explicitly
describe the commercial or laboratory-prepared components used in their medium.
Sabouraud originally used sugar maltose as an energy source, and although this
medium is still commercially available, glucose (formerly referred to as dextrose) is
currently used most frequently. Glucose is present at a high level of 4% in
Sabouraud’s formulation to assist in vigorous fermentation and acid production by
any bacteria present, inhibiting later bacterial growth [5].
In 1977, Emmons formulated an alternative version of Sabouraud’s agar, which
contains half the amount of glucose (2%) and a neutral pH of 6.8–7.0. The neutral
pH seems to enhance the growth of some pathogenic fungi, such as dermatophytes.
Agar concentrations ranging from 1.5–2.0% are found in commercial preparations of
Sabouraud agar in both the original formula and Emmons modification and serve to
solidify the medium in tube and plate medium.
2.1.3 Materials
Sabouraud agar can be either made from individual ingredients (see Tables 2.1 and
2.2), purchased either as a dehydrated powder that must be dissolved in water,
autoclaved, and dispensed, or as a prepared medium that can be purchased in a
tube, plate, or broth form from a variety of commercial sources. Various antimicro-
bials can be added to either the original recipe (Sabouraud agar) or Sabouraud agar,
Modified/Emmons (see Table 2.2).
Table 2.1 Ingredients for Sabouraud agar and Sabouraud agar (Emmons)
Ingredient Sabouraud agar (per liter) Sabouraud agar (Emmons) (per liter)
Pancreatic digest of casein 10 g 10 g
Glucose 40 g 20 g
Agar 15–20 g 15–20 g
Table 2.2 Antimicrobial and other additives to Sabouraud agar

Amount (per
Ingredient liter) Notes on preparation for use
Chloramphenicol* 50 mg Dissolve in 10 mL 95% ethanol
Cycloheximide* 0.5 g Dissolve in 2 mL acetone
Gentamicin 50 mg Dissolve in 5 mL water; add before autoclaving
sulfate
Lecithin 0.7 g Add directly with other powdered medium ingredients
Tween 80 5g before autoclaving
Olive oil N/A Spread 0.1 mL sterile olive oil on surface of each agar plate
*Add these to molten, autoclaved media once it has been tempered in water bath to 45–50 C
1. Deionized, distilled water.

2. Autoclave.
3. Graduated cylinder, 1000 mL.
4. Erlenmeyer flask (2 L if making 1 L of medium).
5. Analytical balance (if using antimicrobial agents).
6. Balance for weighing media ingredients.
7. Stir bar.
8. Stirring hotplate.
9. Slant tube rack for holding media tubes after autoclaving to solidify with a
slanted surface.
10. Pancreatic digest of casein.
11. Glucose.
12. Chloramphenicol.
13. Gentamicin sulfate.
14. Cycloheximide.
15. Tween 80 (polysorbate 80).
16. Lecithin.
17. Olive oil, sterilized by autoclaving.
18. Sterile glass test tubes with caps.
19. Sterile Petri dishes, 100 mm diameter.
2.1.4 Method of Sabouraud Agar Preparation
2.1.4.1 Standard Preparation
1. Combine all ingredients, except any antimicrobials to be used, in ~900 mL of

deionized water in a graduated cylinder while stirring with a magnetic stir bar.
2. Adjust to pH 5.6 with hydrochloric acid and adjust final volume to 1 L.
3. Transfer contents to a 2 L flask and boil on a heating/stirring plate while stirring,
for 1 min.
4. Cover the opening of the flask loosely with aluminum foil and autoclave for
15 minutes at 121 C under pressure of 15 lb. in2.
5. Cool to ~45–50 C (roughly until one can support the flask underneath with an
ungloved hand). If the antimicrobials chloramphenicol or cycloheximide are to be
added, aseptically add them at this point and swirl medium gently (see
Sect. 2.1.4.3).
6. Pour into Petri dishes or tubes and leave them at room temperature (15–30 C)
overnight to solidify and dry. When pouring plates, fill each Petri dish with at
least 25 mL of medium to allow for medium dehydration during the longer
incubation period required for fungi. If preparing tubes, slant the rack of covered
tubes immediately after pouring in a slant tube rack, either at a 5 or 20 slant.
7. Store all media at 4 C, regardless of whether they contain antimicrobials.
2.1.4.2 Method of Sabouraud Agar, Emmons Modification Preparation
1. Combine all ingredients, except any antimicrobials to be used, in ~900 mL of

deionized water in a graduated cylinder while stirring with a magnetic stir bar.
2. Adjust to pH 6.8–7.0 with hydrochloric acid and adjust final volume to 1 L.
3. Transfer contents to a 2 L flask and boil on a heating/stirring plate while stirring,
for one minute.
4. Cover the opening of the flask loosely with aluminum foil and autoclave for
15 minutes at 121 C under pressure of 15 lb. in2.
5. Cool to ~45–50 C (roughly until one can support the flask underneath with an
ungloved hand). If the antimicrobials chloramphenicol or cycloheximide are to be
added, aseptically add them at this point and swirl medium gently (see
Sect. 2.1.4.3).
6. Pour into Petri dishes or tubes and leave at room temperature overnight to solidify
and dry. Fill each Petri dish with at least 25 mL of medium to allow for medium
dehydration during the longer incubation period required for fungi. If preparing
tubes, slant the rack of covered tubes immediately after pouring in a slant tube
rack, either at a 5 or 20 slant.
7. Store all media at 4 C, regardless of whether they contain antimicrobials.
2.1.4.3 Variations on Standard Sabouraud Agar
Either Sabouraud agar or its Emmons version can be made more selective by adding
antimicrobials (see Table 2.2). Antimicrobials commonly used are the
aminoglycoside gentamicin, which inhibits gram-negative bacteria, chlorampheni-
col, which inhibits a wide range of gram-positive and gram-negative bacteria, and
cycloheximide, which inhibits primarily saprophytic fungi, but not dermatophytes or
yeasts [6, 7]. Chloramphenicol and gentamicin are used at 50 mg L1 and cyclo-
heximide at 0.5 g L1 (See Table 2.2) [8]. Chloramphenicol and cycloheximide
should only be added after the media has been autoclaved and then cooled to
~45–50 C (See step 5 in Sect. 2.1.4.1). Gentamicin may be added to the medium
ingredients before autoclaving.
Lecithin and Tween 80 are added to Sabouraud agar (Table 2.2) that is used in
monitoring environmental surfaces that may have been treated with antiseptics and
quaternary ammonium compounds, as these additives neutralize the cleaning com-
pounds [9]. Sterile olive oil can be spread on the surface of Sabouraud agar plates to
grow lipophilic Malassezia species [10].
2.1.5 Methods of Inoculation and Incubation
Sabouraud agar plates can be inoculated by streaking for isolation, as with standard
bacteriological media, by exposing the medium to ambient air, or by tamping clinical
sample material (e.g., hair, skin scrapings) onto the surface of the agar medium.
When growing cultures in tubes, the caps should be screwed on loosely to admit air,
as dermatophytes and most molds are obligate aerobes. Isolation of fungi is
performed on plates, while slants are primarily used for maintaining pure, or stock,
cultures once isolated. If using selective Sabouraud media, a control plate/tube
without antimicrobials should also be inoculated for comparison. Typically, molds
are incubated at room temperature or slightly warmer (25–30 C), yeasts are
incubated at 28–30 C or both 30 C and 37 C if suspected to be dimorphic fungi.
Incubation times will vary, from approximately 2 days for the growth of yeast
colonies such as Malasezzia to 2–4 weeks for growth of dermatophytes or dimorphic
fungi such as Histoplasma capsulatum. Indeed, the incubation time required to
acquire fungal growth is one diagnostic indicator used to identify or confirm fungal
species. Dermatophytes, in particular, show characteristic incubation times ranging
from 5–7 days (some Epidermophyton or Microsporum species) to 3–4 weeks for
some Trichophyton species [11]. Cultures should be examined twice weekly and be
held for 4–6 weeks before being reported as negative if infection by systemic agents
such as Histoplasma, Blastomyces, or Coccidioides species is suspected.
2.1.6 Results
Depending on the antimicrobials used, different types of microorganisms and groups

of fungi may grow on Sabouraud agar (See Table 2.3). Typically, saprophytic fungi
are inhibited by cycloheximide and/or chloramphenicol, but yeasts and dermato-
phytes grow well in their presence. Conversely, even Sabouraud agar is unable to
support the growth of a few dermatophytes in the absence of additives. For example,
some Trichophyton species require additional growth factors, such as thiamine and
inositol (T. verrucosum) or nicotinic acid (T. equinum), and may not grow well, if at
all, on Sabouraud agar [12]. T. mentagrophytes and T. rubrum, however, grow well
on Sabouraud agar. Similarly, the growth of Malassezia species is significantly
impaired without the addition of olive oil overlaid on the surface of a Sabouraud
agar plate [10].
Table 2.3 Expected growth of various microbes on Sabouraud agar containing antimicrobials
Microbe Growth on SAB + CAMa Growth on SAB + CHXb
Candida albicans Yes Yes
Cryptococcus neoformans Yes No
Aspergillus Niger Yes No
Trichophyton mentagrophytes Yes Yes
Microsporum audouinii Yes Yes
Blastomyces dermatitidis Yes (mold phase at 25 C) Yes (mold phase at 25 C)
No (yeast phase at 37 C) No (yeast phase at 37 C)
Histoplasma capsulatum Yes (mold phase at 25 C) Yes (mold phase at 25 C)
No (yeast phase at 37 C) No (yeast phase at 37 C)
Rhizopus spp. Yes No
Sporothrix schenckii Yes Yes
Penicillium roquefortii Yes No
Escherichia coli No No
a
SAB + CAM ¼ Sabouraud agar plus chloramphenicol
b
SAB + CHX ¼ Sabouraud agar plus cycloheximide
Mold morphology should be observed on both the top (obverse) and bottom
(reverse) surfaces, as differences can be seen on each surface.
Variation from lot to lot as well as between commercial vendors of Sabouraud
agar can significantly impact the qualitative and quantitative growth of fungi. One
study comparing five different commercial preparations of Sabouraud glucose agar
observed significant differences in the quantitation of yeasts as well as the color of
Aspergillus colonies; however, the dermatophytes yielded reliably similar appear-
ances on the five media sources tested [13].
2.2 Potato Dextrose Agar
2.2.1 History
The use of potato extract as a growth source in fungal media was published by
New Zealand mycologist Ross Beever in 1970 [14], as part of a detailed study into
the most efficacious component of potatoes as a growth medium. After analysis of
the carbon, nitrogen, mineral salts, and other growth factor components of the potato
extract medium, it was concluded that no one component was responsible for the
stimulation of the growth of fungi.
2.2.2 Theory
Potato Dextrose Agar (PDA) contains dextrose as a carbohydrate source which

serves as a growth stimulant and potato infusion that provides a nutrient base for
luxuriant growth of most fungi. Agar is added as the solidifying agent. Potato
Dextrose Agar (PDA) is a general-purpose medium for the identification, cultivation,
and enumeration of fungi in foods and dairy products. Potato dextrose broth is a
general-purpose broth medium for yeasts and molds.
Certain additives like tartaric acid, chloramphenicol, and chlortetracycline can be
added as selective agents (Table 2.4). As recommended by the Food and Drug
Administration, the American Public Health Association, and the Association of
Analytical Chemists [15–18], PDA with tartaric acid is used for the plate count
microbial examination of food and dairy products. The addition of chlortetracycline
is recommended for the microbial enumeration of yeast and mold from cosmetics.
PDA with chloramphenicol is recommended for the selective cultivation of fungi
from mixed samples. PDA is also recommended by the U.S. Pharmacopeia for the
preparation and maintenance of test strains used for microbial limit tests.
Potato infusion and dextrose promote luxuriant fungal growth, so PDA is also
used for primary isolation of yeasts and molds from clinical specimens. Since it
stimulates sporulation and pigmentation, it is also used for the differentiation of
typical varieties of dermatophytes (species belonging to the genera Epidermophyton,
Microsporum or Trichophyton) based on their pigment production and for the
maintenance of their stock cultures [19].
2.2.3 Methods of Preparation
See Sect. 2.1.3 for general comments regarding needed materials.

Potato Dextrose Agar can be either made from individual ingredients, purchased
as a dehydrated powder that must be dissolved in water, autoclaved, and dispensed,
or as a prepared medium in a tube, plate, or broth format from a variety of
commercial sources.
Table 2.4 Antimicrobial and other additives to Potato Dextrose Agar

Amount (per
Ingredient liter) Purpose in medium
Tartaric acid 1.4 g Lowers pH to 3.5; antibacterial for testing food products
Chlortetracyclinea 40 mg Antibacterial; for cosmetics testing
Sodium chloride 75 g Inhibition of fast- growing molds
Chloramphenicola 25 mg Antibacterial
Copper sulfate 1 mg Supports pigmentation
a
See Table 2.2 for preparation information on adding antimicrobials to media
Table 2.5 Ingredients for Potato Dextrose Agar (commercial and manual)
Potato Dextrose Agar (commercial) Potato Dextrose Agar (manual)
Ingredient (g/L) (g/L)
Potato extract* 4g –
Potato infusion – 200 g
Dextrose 20 g 20 g
Agar 15 g 15 g
Final pH at 5.6 0.2 5.6 0.2
25 C
*4 g of potato extract is equivalent to infusion from 200 g of potatoes
A survey of 10 companies’ media found that five media sources contained

insufficient copper, which reduced the pigmentation of the resulting colonies.
Therefore, supplementation of the other ingredients of PDA with 1 μg mL1 copper
sulfate, or a trace minerals mixture, is recommended [20].
1. Suspend 39 g of dehydrated media (if commercially supplied) in 1 L of deionized
water and mix thoroughly in a 2 L flask.
Alternatively, to make medium from individual ingredients (See Table 2.5):
– Boil 200 g of sliced potatoes in 500 mL distilled water until thoroughly cooked
(about 1 h).
– Filter infusion through cheesecloth/gauze, saving the filtrate, which is potato
infusion.
– In a 2 L flask, combine the potato infusion, dextrose (20 g), and agar (15 g),
and bring the volume to 1 L.
2. Boil on a heating/stirring plate while stirring, for 1 min to dissolve the powder
completely.
3. Cover the opening of the flask loosely with aluminum foil and autoclave 15 min
at 121 C under pressure of 15 lb. in2.
4. Cool to around 45–50 C (roughly until one can support the flask underneath with
an ungloved hand).
5. When modifications are needed in the standard media:
(i) To decrease the pH of the agar medium to pH 3.5, add the specified amount
of sterile tartaric acid. The amount of acid required for 1 L of sterile, cooled
medium is approximately 10 mL of a 10% solution.
Note: Do not reheat the medium after adjusting pH as it may hydrolyze the
agar which can render the agar unable to solidify.
(ii) If the antimicrobials, such as chloramphenicol or chlortetracycline or other
additives like sodium chloride are to be added, aseptically add them at this
point and swirl medium gently.
6. Pour into Petri dishes or tubes and leave at room temperature overnight to solidify
and dry. When pouring plates, fill each Petri dish with at least 25 mL of medium
to allow for medium dehydration during the longer incubation period required for
fungi. If preparing tubes, slant the rack of covered tubes immediately after
pouring in a slant tube rack, either at 5 or 20 slant.
7. Store prepared media away from direct light at 4 C to 8 C with the medium side
uppermost to prevent excessive accumulation of moisture on the agar surface.
Under these conditions, this medium has a shelf life of 12 weeks.
2.2.4.1 Quality Control
After checking for correct pH, color, depth, and sterility, the following organisms are
used to determine the growth performance of the completed medium: Candida
albicans ATCC 14053 and Aspergillus niger ATCC 16404 will each produce
growth on this medium.
2.2.4.2 Inoculation and Incubation
1. For yeast and mold counts in foods, a standard pour plate technique should be
used, and the pH of the medium should be adjusted to approximately 3.5 with
sterile tartaric aid.
2. For the cultivation and maintenance of pure cultures, tubed slants are used. They
should be inoculated and incubated the same as a plated medium, below.
3. For other specimen processing, streak the specimen onto the medium with a
sterile inoculating loop to obtain isolated colonies.
4. Plates can be incubated at various temperatures, depending on the application
(molds typically use a lower temperature such as room temperature 20–25 C
while yeasts may require 25–30 C) in an inverted position (agar side up) with
increased humidity.
5. Cultures should be examined daily for fungal growth, with extended time periods
up to at least one week for lower temperature incubations and several days for
higher temperature incubations.
2.2.5 Results
After sufficient incubation culture plates should show isolated colonies in streaked
areas and confluent growth in areas of heavy inoculation. Yeasts will grow as creamy
to white colonies. Molds will grow as filamentous colonies of various colors. Further
microscopic examination and biochemical testing are required to identify the genus
and species of the isolate.
The number of yeast or mold present in the particular test sample is determined by
counting the colonies in pour plates and multiplying with the applicable dilution
factor.
2.3 Bird Seed Agar
2.3.1 History
In 1962, Staib et al. described that incorporation of an extract of Guizotia abyssinica

seed in a fungal medium resulted in the formation of brown colonies of Cryptococ-
cus neoformans [21]. In 1966, Shields and Ajello modified Staib’s G. abyssinica
seed agar formulation by making the medium selective with the addition of the
antimicrobial agent chloramphenicol [22]. In the literature, this medium is variously
referred to as Staib medium, Bird seed agar, Guizotia abyssinica creatinine agar,
niger seed creatinine agar, or thistle seed medium [23].
2.3.2 Theory
Bird Seed Agar is a selective and differential medium for the isolation of Crypto-
coccus neoformans from clinical specimens and differentiation of it from other
microbes. The use of Bird Seed Agar as the primary culture medium for sputum
and urine specimens from AIDS patients increases sensitivity for C. neoformans
[24]. Cryptococcus neoformans produces dark colonies on this agar, unlike Candida
albicans, which produces white colonies. C. neoformans is the only yeast known to
produce this pigmentation [25].
The extract of Guizotia abyssinica seeds contains caffeic acid
(3, 4-dihydroxycinnamic acid, an o-diphenol). Phenoloxidase enzyme produced by
C. neoformans uses caffeic acid as a substrate and produces melanin. Melanin is
absorbed by the yeast cell wall, yielding tan to reddish-brown pigmentation. It is also
known as the phenoloxidase test.
Glucose is the energy source in the medium. Cryptococcus species use creatinine
as their sole source of nitrogen [22]. Creatinine also enhances the melanization of
some strains of C. neoformans. Agar is the solidifying agent. Chloramphenicol
improves the recovery of Cryptococus species from specimens containing mixed
flora by selecting against bacterial growth.
2.3.3 Methods of Preparation

Bird Seed Agar can be prepared from individual ingredients (Table 2.6) by
purchasing the seeds of Guizotia abyssinica (niger seed), grinding them, and adding
filtrate with necessary ingredients, or by using a commercially available powder
media that includes bird seed filtrate. Prepared plates or tubes are also commercially
available.
The exact composition of the media supplied by different commercial suppliers
varies; each mentions that they modified the original formula to suit performance
parameters. Some of the modifications include a substitution of chloramphenicol for
penicillin and streptomycin sulfate, preparing modified G. abyssinicia seed-based
agar media by depleting or removing its constituents such as diphenyl, glucose,
creatinine, etc. It was found that decreasing the sugar concentration resulted in the
rapid development of brown pigmentation by C. neoformans [26].
2.3.3.1 Preparation of Bird Seed Agar
1. Suspend required quantity powder media (as per manufacturer’s instruction), OR

items a-c and f-g in Table 2.6, in a total of 1 l of distilled water in a 2 L flask.
2. Heat to boiling on a heating/stirring plate to dissolve the medium completely.
3. Cover opening of flask loosely with aluminum foil and autoclave 15 min at
121 C under pressure of 15 lb. in2.
4. Cool to 45 C before adding additives if needed (e.g., diphenyl can be added to
inhibit saprotrophic fungi).
5. Mix well and pour into sterile Petri plates or tubes (see Sect. 2.1.4.1, step 6).
6. Store prepared medium at 2–8 C with the medium side uppermost to prevent
excessive accumulation of moisture on the agar surface.
Table 2.6 Ingredients for Bird Seed Agar

Shield’s and Ajello’s formulation Commercial preparation
Ingredients (22) (7)
a) Guizotia abyssinica seeds 200 mL filtrate 50 g
b) Glucose/dextrose 10 g 1g
c) Creatinine 780 mg 1g
d) Chloramphenicol 50 mg 50 g
e) Diphenyl 100 mg –
f) Monopotassium – 1g
phosphate
g) Agar 20 g 15 g
Final volume 1L 1 liter
Final pH at 25 C 6.7 0.2 6.7 0.2
2.3.4 Method of Inoculation and Incubation
2.3.4.1 Quality Control
After checking for correct pH, color, depth, and sterility, the following organisms are
used to determine the growth performance of the completed medium: Cryptococcus
neoformans ATCC 32045 will produce brown to black pigmented colony growth on
this medium. Escherichia coli ATCC 25922 serves as a negative control, being
partial to completely inhibited in its growth on Bird Seed Agar.
2.3.4.2 Inoculation and Incubation
1. Streak the specimen onto the medium with a sterile inoculating loop to obtain
isolated colonies.
2. Incubate the plates at 25–30 C in an inverted position (agar side up) with
increased humidity.
3. Cultures should be examined at least daily for fungal growth and should be held
for up to 4 weeks before being reported as negative.
After inoculation of the clinical specimen, and periodically examined for brown-
colored, mucoid colonies, which later turn black or brown in the case of the genus
Cryptococcus.
2.3.5 Results
Plates inoculated with suspected samples are observed after incubation at 25–30 C
for 2 weeks. The presence of golden brown to black pigmented smooth or mucoid
colonies is indicative of Cryptococcus neoformans. Other species like Cryptococcus
laurentii, Saccharomyces cerevisiae, etc., produce non-pigmented colonies, and
Candida species appear as white colonies. However, pigmented colonies arising
on Bird Seed Agar should be also grown on a non-differential medium such as
Sabouraud dextrose agar to rule out a naturally pigmented strain or species [19].
2.4 Dermatophyte Test Medium
2.4.1 History
Dermatophyte Test Medium (DTM) was formulated by Taplin et al. in 1969 to

rapidly diagnose dermatophytic (ringworm) infections in Vietnam War soldiers,
under conditions where experienced laboratory personnel and incubators were

typically unavailable [27].
2.4.2 Theory
Dermatophyte Test Medium, or DTM, is essentially a selective and differential

version of Sabouraud dextrose agar designed to indicate the presence of
Epidermophyton, Microsporum, and Trichophyton spp. that cause dermatophytic
infections. Amino acids, nitrogen- and carbon-containing compounds are provided
by the soy peptone, and dextrose is the energy source for fungal growth. The acidic
pH 5.6 favors fungal growth.
The original formulation included chlortetracycline HCl and gentamicin as
antibacterial agents [27]. Current formulations include the antibiotics chloramphen-
icol (due to the relative unavailability of chlortetracycline) and sometimes gentami-
cin, which select against a wide range of bacteria. Cycloheximide selects against
saprophytic fungi. The medium is made differential through the addition of the
indicator dye phenol red. At acidic pH values below 6.8, it is yellow, and at alkaline
pH values of 8.2 and above, is bright pink to red. Dermatophytes produce alkaline
metabolites that turn the media bright pink or red color.
2.4.3 Method of Preparation

DTM can be either made from individual ingredients (see Table 2.7), purchased
as a dehydrated powder that must be dissolved in water, autoclaved, and dispensed
(after addition of antimicrobials), or as a prepared medium that can be purchased in
tube or plate form.
Table 2.7 Ingredients for Ingredient Amount (per liter)

Dermatophyte Test Medium
Soy peptone 10.0 g
Dextrose 10.0 g
Cycloheximidea 0.5 g
Chloramphenicola 0.05 g
Gentamicin sulfatea 0.1 g
Phenol red 0.2 g
Agar 20.0 g
a
See Table 2.2 for preparation information on adding antimicro-
bials to media
Follow steps 1–5 listed in Sect. 2.1.4.1 Standard Preparation. After cooling the
sterilized media, add the appropriate antimicrobials for DTM (Table 2.7) and pour
them into plates or tubes.
There is a modification of DTM, called dermatophyte isolation medium DIM
[28], which uses the pH indicator dye bromocresol purple in place of phenol red, as
well as penicillin and streptomycin as its antibiotics. It also uses cycloheximide, at
4 g L1, which is eight times the amount found in DTM. However, at its
recommended incubation temperature of 37 C (but not a lower temperature of
30 C), high false-negative rates for common Trichophyton species were noted,
perhaps due to the high levels of cycloheximide [29]. Furthermore, the dimorphic
fungus Coccidioides immitis grew as a white mold at 37 C that resembled the
hallmark dermatophyte appearance, thereby constituting a false-positive issue with
this medium [29]. Consequently, this medium formulation, as published, is not the
most useful for presumptive identification of dermatophyte infections.
Suitable samples for DTM inoculation include hair, nails, and skin scrapings. Clean
the skin or body surface with alcohol before using a new toothbrush or other small
brush to obtain a surface sample. Bringing the medium to room temperature helps
facilitate more rapid fungal growth. Follow the instructions listed in Sect. 2.1.5. Lay
the sample firmly on top of, but not into, the medium.
Loosely capped, inoculated tubes or plates should be incubated at room temper-
ature or around 25 C, and should be evaluated for red medium color change daily,
up to 10–14 days.
2.4.5 Results
Two observations constitute a true positive reaction on DTM: white or buff-colored

mold growth (containing aerial hyphae), and red-colored medium. Both the fungal
growth and the medium color change to pink/red should appear at the same time,
which may be as short as three to 5 days, depending on the inoculum. A red-colored
medium arising after approximately 10–14 days likely represents contaminating
fungal growth [27]. Various false-negative appearances are possible, such as sapro-
phytes that grow as dark-colored molds although they turn the medium red/pink.
Alternately, bacterial or yeast will grow as white- or light-colored creamy colonies,
and thus easily distinguished from the mold-like appearance of dermatophytes.
DTM is primarily a screening medium and is not suitable for identification
beyond presumptively belonging to the three species of dermatophytes.
2.5 Safety Notes
Fungi often produce spores that are easily dispersed into the laboratory upon the
opening of plates.
Plates should be incubated with the lid on the top (as opposed to the typical
practice of inverting microbiological plates for incubation) to avoid spreading spores
when the plates are opened. After growth, plates should be wrapped in Parafilm to
maintain them securely closed for storage and transport. Plate or tube cultures should
be opened only within a class II biological safety cabinet to avoid contamination of
laboratory spaces with fungal spores, possible infection of individuals by pathogenic
fungi, or induction of allergic responses. See Chap. 1 for detailed procedures and
guidelines.
Because the growth of large numbers of fungi can pose a potential infection
hazard, measures must also be taken to prevent infection of laboratory researchers.
Note that some fungi are biosafety level one (BSL-1) while most are BSL-2
[30]. The American Society for Microbiology strongly recommends that environ-
mental enrichment experiments should only be performed in BSL-2 laboratories
[31]. The following precautions apply to the use of any fungal medium:
1. Soil, water, and other materials directly obtained from the environment that
typically contain infectious organisms should be handled according to the bio-
safety level of that infectious agent.
2. Cultures of enriched microorganisms derived from environmental samples should
be handled using BSL-2 precautions.
3. Mixed, enriched, or pure cultures of microorganisms from environmental samples
with a significant probability of containing infectious agents should be manipu-
lated in a Class II biosafety cabinet if available.
4. Researchers should be aware of working in regions with endemic fungi capable of
causing systemic infections and should avoid environmental isolations.
Some safe (BSL-1) fungi for student experimentation and handling include the
molds Penicillium camemberti and P. roqueforti (used in making cheeses), Rhizopus
stolonifor (used in making tempeh), Aspergillus species (except A. fumigatus and
A. flavus), the yeasts Saccharomyces cerevisiae, Rhodotorula rubrum, and Neuros-
pora crassa.
2.6 Additional Fungal Growth Media
Table 2.8 Other commonly used fungal isolation and growth media
Culture
Media Essential ingredients Intended use
Inhibitory Tryptone, beef extract, yeast extract, Recovery of fungi from specimens that
Mold agar starch, dextrin, chloramphenicol, contain bacterial microbiota
(IMA) gentamicin, and saline buffer
(continued)

Culture
Media Essential ingredients Intended use
Mycosel/ Papaic digest of soybean meal, dex- Highly selective medium; recommended
Mycobiotic trose, cycloheximide, chlorampheni- for isolation of pathogenic fungi from
agar col, agar materials containing a large amount of
fungal and bacterial flora
Potato flake Potato flakes, dextrose, agar Primary recovery of saprophytic and
agar dimorphic fungi, particular fastidious
and slow-growing strains
Cornmeal Cornmeal, tween 80, agar Stimulation of chlamydospore forma-
agar/cornmeal tion in yeasts
tween agar
Rice starch Cream of rice, tween 80, agar Production of chlamydospore in Can-
agar dida albicans
Brain-heart Brain heart infusion, glucose, Growth of fastidious pathogenic fungi
infusion agar L-cysteine hydrochloride, agar such as Histoplasma capsulatum and
Blastomyces dermatitidis
Czapek-dox NaNO3, K2HO4, KCl, MgSO4, Identification of aspergillus and Peni-
agar FeSO4, glucose, agar cillium species
CHROMagar Peptone, glucose, chloramphenicol, Selective and differential chromogenic
Candida ‘chromogenic ix’, agar medium for the isolation and identifica-
medium tion of various Candida species
References
1. Raymond Sabouraud (1896) La question des teignes. In: Annales de Dermatologie 3rd series
VII. p. 87–135. (3; vol. 7)
2. Raymond Sabouraud (1896) Recherche des milieux de culture propres a la différenciation des
espèces trichophytiques a grosse spore. In: Les trichophyties Humaines. Masson et Cie, Paris,
pp 49–55
3. Odds FC (1991) Sabouraud(‘s) agar. J Med Vet Mycol 29:355–359
4. Emmons CW, Binford CH, Utz JP, Kwon-Chung KJ (1977) Culture media. In: medical
mycology, 3rd edn. Lea & Febiger, Philadelphia
5. Jarrett L, Sonnenwirth AC (1980) Gradwohl’s and parasitic infections, 7th edn. American
Public Health Association, Washington, DC
6. McDonough ES, Ajello L, Georg LK, Brinkman S (1960 Jan 1) In vitro effects of antibiotics on
yeast phase of Blastomyces dermatitidis and other fungi. J Lab Clin Med 55(1):116–119
7. Lorian V (2005) Antibiotics in laboratory medicine. Lippincott, Williams & Wilkins, Baltimore
8. Hungerford LL, Campbell CL, Smith AR (1998) Veterinary mycology lab manual. Iowa State
University Press, Ames
9. Curry AS, Graf JG, McEwen GN Jr (1993) CFTA microbiology guidelines. The Costmetic,
Toiletry and Fragrane Association, Washington, DC
10. Kwon-Chung KJ, Bennett JE (1992) Infections caused by Malassezia species. In: Medical
mycology. Lea and Febiger, Philadelphia, pp 70–182
11. Robert R, Pihet M (2008 Dec) Conventional methods for the diagnosis of dermatophytosis.
Mycopathologia 166(5–6):295–306
12. Georg LK, Camp LB (1957 Aug) Routine nutritional tests for the identification of dermato-
phytes. J Bacteriol 74(2):113–121
13. Brun S, Bouchara JP, Bocquel A, Basile AM, Contet-Audonneau N, Chabasse D (2001 Oct)
Evaluation of five commercial Sabouraud gentamicin-chloramphenicol agar media. Eur J Clin
Microbiol Infect Dis 20(10):718–723
14. Beever RE, Bollard EG (1970) The nature of the stimulation of fungal growth by potato extract.
J Gen Microbiol 60:273–279
15. US Food and Drug Administration (2019) Bacteriological Analytical Manual [Internet].
AOAC; [cited 2019 Aug 14]. Available from: https://www.fda.gov/food/laboratory-methods-
food/bacteriological-analytical-manual-bam
16. Association of Official Analytical Chemists (2012) Official methods of analysis, Report No.:
19. AOAC, Washington, DC
17. American Public Health Assocation2 Standard methods for the examination of dairy products,
22nd edn. APHA, 2012, Washington, DC
18. APHA (2001) Technical committee on microbiological methods for foods. Compendium of
methods for the microbiological examination of foods, 4th edn. Washington, DC, APHA
19. MacFaddin JF (1985) Media for isolation-cultivation-identification-maintenance of medical
bacteria [Internet]. Williams and Wilkins, Baltimore (Md.). Available from: http://lib.ugent.
be/catalog/rug01:000079718
20. Griffith GW, Easton G, Detheridge A, Roderick K, Edwards A, Worgan H et al (2007 Dec 1)
Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various
fungi. FEMS Microbiol Lett 276:165–171
21. Staib F (1962) Cryptococcus neoformans and Guizotia abyssinica (syn. G. oleifera D.C.).
(Colour reaction for Cr. Neoformans.). Zeitschrift fur Hygiene und Infektionskrankheiten
148(5):466–475
22. Shields AB, Ajello L (1966 Jan 14) Medium for selective isolation of Cryptococcus
neoformans. Science 151(3707):208–209
23. Staib F, Seibold M, Antweiler E, Fröhlich B (1989 Sep 1) Staib agar supplemented with a triple
antibiotic combination for the detection of Cryptococcus neoformans in clinical specimens:
Staib-agar mit einer Dreifach-Antibiotika-Kombination für den Nachweis von Cryptococcus
neoformans in klinischem Untersuchungsmaterial. Mycoses 32(9):448–454
24. Denning DW, Stevens DA, Hamilton JR (1990 Nov) Comparison of Guizotia abyssinica seed
extract (birdseed) agar with conventional media for selective identification of Cryptococcus
neoformans in patients with acquired immunodeficiency syndrome. J Clin Microbiol 28(11):
2565–2567
25. Warren NG, Hazen KC (1999) Candida, Cryptococcus and other yeasts of medical
importance. In: Manual of clinical microbiology, 7th edn. American Society for Microbiology,
Washington, DC, pp 1184–1199
26. Paliwal DK, Randhawa HS (1978 Apr) Evaluation of a simplified Guizotia abyssinica seed
medium for differentiation of Cryptococcus neoformans. J Clin Microbiol 7(4):346–348
27. Taplin D, Zaias N, Rebell G, Blank H (1969 Feb) Isolation and recognition of dermatophytes on
a new medium (DTM). Arch Dermatol 99(2):203–209
28. Salkin IF, Padhye AA, Kemna ME (1997 Oct) A new medium for the presumptive identification
of dermatophytes. J Clin Microbiol 35(10):2660–2662
29. Gromadzki S, Ramani R, Chaturvedi V (2003 Jan 1) Evaluation of new medium for identifi-
cation of dermatophytes and primary dimorphic pathogens. J Clin Microbiol 41(1):467
30. Centers for Disease Control and Prevention (2009) Section VIII-B: Fungal agents. In: Biosafety
in Microbiological and Biomedical Laboratories (BMBL) [Internet], 5th edn, pp 170–181.
Available from: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
31. Emmert EAB (2013) ASM task committee on laboratory biosafety. Biosafety guidelines for
handling microorganisms in the teaching laboratory: Development and rationale. J Microbiol
Biol Educ 14(1):78–83
Chapter 3
Fluorescence In Situ Hybridization
of Uncultured Zoosporic Fungi
Télesphore Sime-Ngando, Marlène Jobard, and Serena Rasconi
Contents
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.3.1 Classical FISH Probing (See Note 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.3.2 CARD-FISH Probing (See Note 7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.1 Introduction
Molecular surveys of microbial eukaryotes have revealed overlooked uncultured

environmental fungi with novel putative functions [1–3], among which zoosporic
forms (i.e. chytrids) are the most important in terms of diversity, abundance, and
functional roles, primarily as infective parasites of phytoplankton [4, 5] and as
valuable food sources for zooplankton via massive zoospore production, particularly
in freshwater lakes [6–8]. However, due to their small size (2–5 μm), their lack of
distinctive morphological features, and their phylogenetic position, traditional
microscopic methods are not sensitive enough to detect fungal zoospores among a
mixed assemblage of microorganisms. Chytrids occupy the most basal branch of the
kingdom Fungi, a finding consistent with choanoflagellate-like ancestors [9]. The
above reasons may help explain why both infective (i.e. sporangia) and
T. Sime-Ngando (*) · M. Jobard · S. Rasconi

LMGE, Laboratoire Microorganismes: Génome et Environnement, UMR CNRS 6023,
Université Clermont Auvergne, Aubière Cedex, France
e-mail: telesphore.sime-ngando@uca.fr

88 T. Sime-Ngando et al.
disseminating (i.e. zoospores) life stages of chytrids have been misidentified in

previous studies, respectively as phagotrophic sessile flagellates
(e.g. choanoflagellates, bicosoecids) and as ‘small undetermined’ cells. These cells
often dominate the abundance of free-living heterotrophic nanoflagellates (HNFs)
and are considered the main bacterivores in aquatic microbial food webs
[2, 10]. Their contribution ranges from 10% to 90% of the total abundance of
HNFs in pelagic systems (see review in [11]). Preliminary data have provided that
up to 60% of these unidentified HNFs can correspond to fungal zoospores [12],
establishing the HNF compartment as a black box in the context of microbial food
web dynamics [4]. A simulation analysis based on Lake Biwa (Japan) inverse model
indicated that the presence of zoosporic fungi leads to (i) an enhancement of the
trophic efficiency index, (ii) a decrease of the ratio detritivory/herbivory, (iii) a
decrease of the percentage of carbon flowing in cyclic pathways, and (iv) an increase
in the relative ascendency (indicates trophic pathways more specialized and less
redundant) of the system [13]. Unfortunately, because a specific methodology for
their detection is not available, quantitative data on zoosporic fungi are missing.
Sporangia and the associated rhizoidal system are characterized by a chitinaceous
wall (a common fungal structure element for many species) which can be targeted by
specific fluorochromes such as calcofluor white [14]. In contrast, because the
chitinaceous wall springs out after zoospore encystment, chytrid zoospores
completely lack cell wall and chitin, precluding any simple use of fluorochromes
for their quantitative assessment in natural environments [12]. Molecular
approaches, primarily fluorescence in situ hybridization (FISH), offer an alternative
for quantitatively probing both chytrid sporangia and zoospores in nature. FISH
method is based on the detection of targeted nucleic acid sequences by the use of
oligonucleotide probes labelled by a fluorochrome, usually Cy3 [15, 16]. One of the
major limitations of FISH-based methods for natural samples is the autofluorescence
interference from autotrophic organisms. During the past few years, numerous
efforts have been made to improve the sensitivity of monolabelled probes for
FISH assay, including the use of brightener fluorochromes [17], or of signal ampli-
fication with reporter enzymes [18]. Of particular interest is the hybridization
method using horseradish peroxidase (HRP)-labelled probes activated by fluorescent
tyramide (also known as catalyzed reporter deposition, CARD-FISH), which is very
efficient in overcoming the interference from natural fluorescence [19]. The method
is based on the fact that each HRP-labelled probe catalyzes the deposition of many
labelled tyramides, so that numerous fluorescent molecules are introduced at the
hybridization site, resulting in net fluorescence signal amplification, compared to the
classical Cy3-monolabelled FISH probes [20].
The main objective of this chapter is to provide, in a simplified step-by-step
format, classical FISH and CARD-FISH protocols for the identification and quan-
titative assessment of uncultured zoosporic fungi and other zoosporic microbial
eukaryotes in natural aqueous environments (cf. 12), together with practical advices
on how to apply the methods.
3 Fluorescence In Situ Hybridization of Uncultured Zoosporic Fungi 89
3.2 Materials
1. Gloves (should be worn when manipulating most of the materials below).

2. 0.6 μm pore size polycarbonate white filters (e.g. catalog no. DTTP02500,
Millipore, Billerica, MA, USA)
3. Appropriate Cy3-labelled oligonucleotidic probe and its reverse complement
stored at 20 C (see Note 1).
4. Sodium dodecyl sulphate (SDS).
5. Formaldehyde 37%.
6. FISH hybridization buffer (0.9 M NaCl, 20 mM Tris–HCl (pH 7.2), 0.01% SDS)
containing 30% formamide and 2.5 ng μl1 of Cy3-labelled probe (see Note 2).
7. Washing buffer - 20 mM Tris–HCl (pH 7.2, 5 mM EDTA, 0.01% SDS, 112 mM
NaCl [21].
8. Appropriate filtration columns equipped with a peristaltic pump.
9. DAPI – 4.6-diamidino-2-phenylindole.
10. Glass slides and coverslips.
11. Non-fluorescent immersion oil.
12. Epifluorescence microscope equipped with appropriate filter sets (blue and UV)
and Neofluar objective lens (optional).
13. CARD-FISH hybridization buffer: 30% deionized formamide, 0.9 M NaCl,
20 mM Tris-HCl (pH 7.5), 0.01% SDS, and 10% blocking reagent
(e.g. Roche Diagnostics/Boehringer).
14. Appropriate oligonucleotide probe labelled with HRP (in our case, commer-
cially synthesized by Biomers, Germany).
15. TNT buffer: 0.1 M Tris-Hcl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20.
16. TSA mixture: (1:1) of 40% dextran sulphate (Sigma-Aldrich, St. Louis, MO,
USA) and 1X amplification diluent (PerkinElmer LAS, Waltham, MA, USA).
17. Fluorescein isothiocyanate coupled with tyramide (1, Perkin-Elmer LAS).
3.3 Methods
3.3.1 Classical FISH Probing (See Note 3)
Fixe experimental samples with 2% formaldehyde, vol:vol final concentration. The

fixation step is facultative and can be avoided when observations are made without
delay.
1. Filter-collect appropriate volumes (x 3 replicates) of cultures, enriched cultures,
or natural samples containing zoosporic organisms onto 0.6 μm pore size poly-
carbonate white filters (see Note 4) by using gentle vacuum (< 20 kPa).
2. In the dark, poor the filters with targeting fungal zoospores and sporangia and
perform hybridization in the standard FISH hybridization buffer (containing 30%
formamide and 2.5 ng μl1 of the Cy3-labelled oligonucleotide probe) for 3 h at

46 C (see Note 5).
3. Use the reverse complement probe in a negative control to check for the
autofluorescence interference from fungi and other natural plankton present in
natural samples.
4. After hybridization, thoroughly rinse the filters in the washing buffer for 30 min at
48 C.
5. Counterstained the filters in the dark at room temperature for 5 min with DAPI
0.5 μg ml1, and repeat the washing step.
6. Mount the filters between glass slides and coverslips using appropriate
non-fluorescent immersion oil (see Note 6). At this stage, mounted filters can
be conserved at 20 C until microscopic observation.
7. In a dark room, examine the filters under an epifluorescence microscope equipped
with appropriate set of filters and objective lens. Shift between blue and UV light
to distinguish between Cy3 stain and DAPI, use different convenient magnifica-
tions for sporangia and zoospores, and apply a standard procedure for micro-
scopic counting.
3.3.2 CARD-FISH Probing (See Note 7)
Perform steps 1 and 2 in classical FISH probing.

1. In the dark, poor the filters with targeting fungal zoospores and sporangia and
perform hybridization in the CARD-FISH hybridization buffer (containing 30%
formamide and 2.5 ng μl1 of HRP labelled oligonucleotide probe) for 3 h at
35 C (see Note 5).
2. After hybridization, thoroughly rinse the filters in the washing buffer for 2 x
20 min at 37 C.
3. Equilibrate samples to increase enzyme activity in TNT buffer at room temper-
ature for 15 min.
4. Perform signal amplification by 30 min incubation in TSA mixture, to which
fluorescein isothiocyanate coupled with tyramide was added (1:50 vol/vol).
5. Transfer filters in two successive 5-ml TNT buffer baths at 55 C for 20 min, in
order to stop the enzymatic reaction and remove the dextran sulphate.
6. Follow steps 6 and 8 in classical FISH probing.
3.4 Notes
1. We propose to use a probe named Chyt1061 (sequence 50 > 30 : CATAAGGTG

CCGAACAAGTG ), because of the sequence position (1061 base pairs) on
Saccharomyces cerevisiae small-subunit rDNA molecule (GenBank accession
no. J01353). According to Behrens and collaborators [22], this position provides
good accessibility for FISH probing. There were two mismatches in the middle of
the probe with sequences of Chytridiales species (cf. 12), which did not result in
loss of positive signal. Chyt1061 was designed in silico for targeting fungal
species in the order Chytridiales, i.e. the largest order of the division
Chytridiomycota (chytrids) mainly represented by phytoplanktonic parasites in
aquatic environments [12]. The design was based on the alignment of rDNA
sequences of Chytridiales obtained from GenBank, together with 106 sequences
derived from18S rDNA PCR surveys of freshwater picoeukaryotes conducted in
French Lakes Pavin, Godivelle, and Aydat [12]. Distinct rDNA sequence unique
to target organisms was localized and imported in Primer3 software (http://
fokker.wi.mit.edu/primer3/input.htm) in order to design a probe with a size
between 18 and 27 bases, probe melting temperature (Tm) between 57 C and
63 C, and GC percentage at about 50%. The probe was analyzed for potential
complementarities, and no dimers or hairpins were found using Netprimer soft-
ware (http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html).
The probe was commercially synthesized by MWG-biotech Company (Germany)
and labelled with the fluorochrome Cy3 for classical FISH or application to
environmental samples using the CARD-FISH approach [12].
2. In the case you design your one probe because of the increasing availability of
sequences in the database, hybridization stringency should be tested and validated
using appropriate positive and negative cultures, before application to natural
samples. In the absence of laboratory cultures, our probe Chyt1061 was evaluated
from an adaptation of an alternative approach called clone-FISH, known from
prokaryotes [23]. This approach is based on the genetic modification of a clone of
Escherichia coli by inserting a plasmid vector containing the target rDNA
sequence. In our adaptation of the approach, cells of E. coli clone BL21 star
were genetically transformed by a inserting plasmid vector containing rDNA
sequence from several different target fungal cells. Specific plasmid inserts came
from freshwater lake surveys of picoeukaryote 18S rDNA and fungal 18S-ITS
rDNA as well (cf. 12).
3. The specificity of the designed probe should be checked both in silico by using a
basic local alignment search tool (e.g. BLAST) [24] and in vivo by screening
clone libraries with classical FISH (the clone-FISH approach can be used here). In
our case, clones containing rRNA gene inserts from different eukaryotes closely
related to microorganisms of interest and negative controls as well were FISH-
targeted following the protocol described in this chapter (i.e. with 30% formam-
ide in the hybridization buffer). In addition, the in vivo transcription of the 18S
rRNA gene insert was induced with IPTG (1 mM) for 1 h. The designed probe or
its reverse complement probe was used, depending on the orientation of insertion
into the vector, i.e. 30 ! 50 or 50 ! 30 way downstream the T7 promoter (cf. 12).
4. In vivo tests using the clone-FISH approach yield the best fluorescence signal
when hybridization was performed at 30% formamide concentration in the
hybridization buffer. Assuming an increase of the effective hybridization tem-
perature of 0.5 C per 1% of added formamide, the melting temperature (Tm) of
the probe Chyt1061 was experimentally calculated at 61 C [12].
5. This protocol is suitable for cultures and enriched cultures (i.e. concentrates of
targeted zoosporic organisms during host blooms) (cf. 12). However, the FISH
resolution for fungal images and species identification based on sporangium
features is poor, compared to the calcofluor approach which is more appropriate
for the identification of zoosporic organisms [14]. In addition, the
Cy3-monolabelled FISH probing of natural samples clearly showed that the
fluorescence of targeted chytrid zoospores may be quite similar to the
autofluorescence from natural picoautotrophs. That is why CARD-FISH is
more appropriate for environmental samples.
6. For natural waters, the appropriate volume depends on the trophic status of the
natural waters, whether the sampling period corresponds to a bloom period, and
on the nature of the phytoplankton species. Zoosporic parasites are much more
abundant when large-size phytoplankton hosts such as diatoms or filamentous
cyanobacteria develop [5, 14]. In oligotrophic waters, concentration of natural
samples could be required before harvesting targeted organisms onto polycar-
bonate filters [15].
7. One filter corresponding to one sampling time point can be cut in pieces before
hybridization when several probes are used.
8. The mounting medium should not be fluorescent and will minimize the fading of
fluorochromes. An example of mountant is a solution composed of 50% glycerol,
50% phosphate-buffered saline (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5), and
0.1% p-phenylenediamine (made fresh daily from a frozen 10% aqueous stock
solution; Sigma-Aldrich, St. Louis, MO, USA).
9. The CARD-FISH protocol is suitable for natural samples, primarily in oligotro-
phic waters where its application improves the detection and the recognition of
chytrid, because of the enhanced signal conferred by HRP-labelled probes,
compared to monolabelled oligonucleotides. In addition, the choice of fluorescein
as a stain (emission in the green spectrum at 520 nm) significantly reduces the
interference from natural fluorescence of autotrophic organisms, thereby
preventing the use of the deductive approach based on double counting of the
same sample, i.e. with and without hybridization [25]. However, similar to the
simple FISH approach, the CARD-FISH resolution for fungal images and species
identification based on sporangium features is poor, compared to the calcofluor
approach which is more appropriate for the identification of zoosporic
organisms [14].
Acknowledgements MJ and SR were supported by PhD Fellowships from the Grand Duché du
Luxembourg (Ministry of Culture, High School, and Research) and from the French Ministère de la
Recherche et de la Technologie (MRT), respectively. This study receives grant-aided support from
the French ANR Programme Blanc # ANR 07 BLAN 0370 titled DREP: Diversity and Roles of
Eumycetes in the Pelagos.
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Chapter 4
Technique for Identifying and Counting
Infective Chytrid Sporangia Using
the Chitinaceous Fluorochrome Calcofluor
White
Télesphore Sime-Ngando, Serena Rasconi, and Mélanie Gerphagnon
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
4.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.3.1 Concentrations of Cells (See Note 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.3.2 Preparation of Calcofluor Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.3.3 Staining and Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.1 Introduction
Fungal infections are recurrent in aquatic ecosystems [1, 2]. The most described
aquatic fungi in freshwater ecosystems belong to Chytridiomycota (or chytrids).
Chytrids infect a wide variety of hosts, including fish, eggs, zooplankton, and other
aquatic fungi but especially phytoplankton. Typical phytoplankton hosts include
prokaryotes and eukaryotes, primarily large-size diatoms and filamentous species
[3]. Associated chytrids are external eucarpic parasites which produce a specialized
rhizoidal system within host cells, i.e. the diet conveying system that leads to the
formation of the chitinous fruit bodies: the sporangium. This parasitic stage produces
T. Sime-Ngando (*) · S. Rasconi · M. Gerphagnon

LMGE, Laboratoire Microorganismes: Génome et Environnement, UMR CNRS 6023,

numerous uniflagellate spores: the zoospores, which constitute the dissemination

phase of the life cycle [4].
Various approaches have been used to study fungal parasites, but routine tech-
niques for reliably identifying and counting these organisms are missing in the
context of aquatic microbial ecology [5, 6]. So far, observations of parasitic fungi
were obtained by using phase contrast light microscopy with live or Lugol’s iodine
preserved samples [7–9]. Such conventional microscopy allows observation of
fungal sporangia or similar forms (especially in laboratory cultures) but is a poor
approach for characterizing chytrid parasites in natural samples, at the complex
community level. For example, a simple light microscopy observation of fungal
rhizoidal systems, i.e. a pertinent criterium for identifying chytrids [4, 10, 11], is
very difficult. This situation may help explain the confusion of chytrids with
protistan flagellates such as choanoflagellates or other bacterivorous flagellates in
the group of Bicosoeca, which are attached to phytoplankton but do not harm their
host [5–7].
Earlier studies on chytrids were restricted to morphological descriptions and
focused on few species [12–15]. Electron microscopy was used to describe different
life stages and the ultrastructural cytology of fungal zoospores and spore differen-
tiation [16–18], providing the basis for chytrid taxonomy [19, 20]. Studies on pelagic
chytrids started in the British lakes [21], and different authors have provided
descriptions of morphological characters [22–26]. However, few attempts have
been made to include the related parasitism pathway in the aquatic food web
dynamics and to understand environmental factors that trigger epidemics as well
[27]. Some authors have also investigated the effects of parasitism on the growth of
algal host species and on the genetic structure of infected populations [28]. Parasites
are thus considered relevant not only for the evolution of their hosts but also for the
population dynamics such as successions of phytoplankton communities and for
structuring microbial communities in general [9, 29]. Moreover, chytrids can repre-
sent interesting key intermediates in the food chain [30, 31]. The nutrients from
infected large-size algae which could not be directly fed by zooplankton can be
transferred from sporangium to grazers via fungal zoospore production. Fungal
zoospores have suitable dimensions and represent a valuable food source for zoo-
plankton [32]. The activity of zoosporic fungi and the related biogeochemical
processes can thus be crucial in matter and energy transfer in aquatic systems
[29]. Methodological limitations for the study of the ecological dynamics of chytrid
populations can be overcome with epifluorescence microscopy coupled to a specific
fluorochrome targeting molecular tracers (i.e. some types of polysaccharides) of the
fungal chitinous structures, including sporangium and the rhizoidal system.
The protein stain fluorescein isothiocyanate (FITC) and, in particular, the chitin
stain calcofluor white (CFW) were suggested as good markers that offer useful tools
for the investigation of fungal dynamics in aquatic samples [33]. CFW binds to β1–3
and β1–4 polysaccharides such as those found in cellulose or in chitin which
commonly occur in the fungal cell wall [1, 2]. It fluoresces when exposed to UV
light and is currently used in clinical mycology for direct microscopic examination
of skin scrapings, hairs, nails, and other clinical specimens for fungal elements
4 Technique for Identifying and Counting Infective Chytrid Sporangia Using. . . 97
Fig. 4.1 Calcofluor white

(CFW) staining of a chytrid
infected filamentous
cyanobacteria observed
under an epifluorescence
microscope using the
proposed protocol. CFW
penetrates well into infected
host cells and is more
efficient for the observation
of both the fungal
chitinaceous sporangium
and the complete rhizoidal
system within the host
[34, 35]. In contrast to FITC, CFW penetrates into infected host cells and is more
efficient for the observation of the complete rhizoidal system of parasites (Fig. 4.1),
i.e. a pertinent criterion for chytrid identification [4, 10, 11].
format, a routine protocol based on size fractionation of pelagic samples and the use
of the fluorochrome calcofluor white for diagnosing, identifying, and counting
chitinous fungal parasites (i.e. sporangia of chytrids) within phytoplanktonic com-
munities [3], together with practical advice on how to apply the method.
4.2 Materials
1. 25 μm nylon filter
2. 0.2 μm filters
3. High-performance concentration/diafiltration system. As an example, we use the
system Amicon model DC 10LA (Epernon, France) equipped with a reusable
hollow fibre cartridge (0.2 μm cutoff, surface area of 0.45 m2).
4. 36% Formaldehyde.
5. Calcofluor white (C40H44N12O10S2 fluorescent brightener 28; Sigma catalog
no. F3543).
6. 10 N NaOH
7. Balance.
8. Distilled water.
9. 15 ml and 0.2 ml tubes
10. Glass slides and coverslips.
11. Epifluorescence microscope equipped with appropriate UV filter sets and
Neofluar objective lens (optional).
4.3 Methods
4.3.1 Concentrations of Cells (See Note 1)
1. Pass the sample (ca. 20 L) through the 25 μm pore size nylon filter (see Note 2).
2. Collect large phytoplankton cells in the >25 μm size fraction by washing the filter
with 40 mL of 0.2 μm filtered natural sample.
3. Fix the concentrate sample with formaldehyde (2% final conc.), before staining
and analysis.
4. Concentrate nanoplanktonic cells in the <25 μm size fraction (i.e. the 20 L filtrate)
ca 20x by ultrafiltration to a volume of approximately 1 L, entry pressure 0.9 bar.
5. Fix about 180 mL of the ultrafiltrate retentate with formaldehyde (2% final conc.),
before staining and analysis.
4.3.2 Preparation of Calcofluor Stock Solution
1. Weigh 35 mg of calcofluor white into a 15 mL tube.

2. Add 7 mL of sterile distilled water and 2–3 drops of 10 N NaOH (to increase pH
to 10–11). Calcofluor does not dissolve well in neutral solutions.
3. Dissolve the calcofluor.
4. Adjust the volume to 10 mL by adding sterile distilled water.
5. Distribute the stock solution in 0.2 ml tubes and store in a lightproof tube at
20 C.
4.3.3 Staining and Visualization
1. In the dark, stain aliquots (about 200 μl) of concentrated and fixed materials by
adding 1–2.5% (vol/vol) of CFW stock solution directly in solution for 10 min.
2. Mount drop (5–10 μl) of the stained samples between glass slides and coverslips
for observations and counting.
3. In a dark room, examine the slides under an epifluorescence microscope equipped
with an appropriate set of filters and objective lenses. Shift between white and UV
light to visualize and determine parasites and phytoplankton cells, and check the
viability of the host cell, e.g. presence of chloroplast.
4. Applied a standard procedure for microscopic counting (see Notes 3 and 4).
4.4 Notes
1. Different approaches were tested to concentrate samples: the total community

approach and the size-fractionated community approach. For the former
approach, 180 ml of experimental samples were fixed with formaldehyde (2%
final conc.) and aliquots concentrated in three different ways: (i) by simple gravity
following Utermöhl’s [36] method before staining the chyrids, (ii) by vacuum
pressure on two different filters before staining directly onto filters, and (iii) by
vacuum pressure on the same two types of filters but after staining in solution.
For the Utermöhl method, 100 ml of fixed samples were settled for at least 24 h.
For each of the two filter-vacuum pressure methods, 10 ml 2 of fixed samples
were filtered onto polycarbonate white filters (pore size 0.6 μm, catalog
no. DTTP02500, Millipore) and nuclepore polycarbonate black filters (pore size
0.8 μm, catalog no. 110659, Whatman), by using gentle vacuum (< 0.2 bar or
20 kPa). For the total community approach using the classical Utermöhl [36]
method, visualization of fungal parasites was very difficult and most of the time
practically impossible for all the stain concentrations tested. The main reason was
that staining directly in the Utermöhl chamber resulted in very poor-quality
specimens of parasites observed in any given sample. Other disadvantages of
the procedure include the relatively long sedimentation time and the difficulty of
increasing the volume analyzed.
The alternative total community approaches based on vacuum pressure con-
centrations on polycarbonate filters, i.e. white (0.6-μm-pore-size) and black (0.8-
μm-pore-size) filters, yielded similar quality images of fungal parasites, either
when CFW staining was done before (i.e. in solution) or after (i.e. on filters)
concentrating phytoplankton host cells onto filters. However, substantial differ-
ences were noted depending both on the type of the filter and on the concentration
of the stain. In general, for the two types of filters, high levels of background
noises were obtained when using CFW at final concentrations of 3%, 10%, or
20%, precluding any accurate assessment of numerical and phenotypic charac-
teristics of both host cells and their fungal parasites. Staining with 1% CFW final
concentration substantially improved the viewing of chytrids on filters, with an
increasing contrast from the white DTTP Millipore to the black Whatman filters.
However, none of the membrane-retaining approaches yielded satisfactory
images of morphological and cellular features of the host cells, e.g. presence of
chloroplast and viability of the host cell. Accordingly, the proposed protocol is
based on the size-fractionation approach using 1% to 2.5% vol/vol CFW final
concentration (from the stock solution), which substantially enhanced the obser-
vational results.
2. The approach is efficient since it is based on the concentration of large initial
volumes and size partitioning of samples, a step that we judged necessary in order
to yield good analytic images of infectious sporangia for an accurate diagnosing
and identification of parasites. In addition, this approach yielded satisfactory
images of morphological and cellular features of the host cells, for phytoplankton
identification based on phenotypic features and viability of the host cell, through
the integrity of cell wall and the presence of chloroplasts, which are fundamental
parameters to assess the intensity of the disease. We consider this protocol as an
optimal for the diagnosis and quantitative assessment of phytoplanktonic chytrid
infections in natural samples. Finally, the approach was designed to freeze-
conserved particulate DNA samples for quantifying the propagule stages
(i.e. zoospores) of chytrids via FISH targeting of specific rRNA oligonucleotide
probes (see Chap. 3).
3. To estimate the infectivity parameters of ecological interest in the phytoplankton
population, several algorithms are used according to the formula proposed by
Bush et al. [37]. These parameters include the prevalence of infection (Pr), i.e. the
proportion of individuals in a given the phytoplankton population having one or
more sporangia or rhizoids, expressed as Pr (%) ¼ [(Ni/N) 100], where Ni is
the number of infected host cells and N the total number of host cells. The second
parameter is the mean intensity of infection (I), calculated as I ¼ Np/Ni where Np
is the number of parasites and Ni is the number of infected individuals within a
host population.
4. We propose a third parameter concerning the prevalence of infection of cells in
colonial (or filamentous) species (PrCF). PrCF (%) ¼ [(Ni/N) 100] where Ni is
the number of infected host cells in parasitized colonies (or filaments) and N the
total number of parasitized host colonies (or filaments).
Acknowledgements and Additional Information SR and MG were supported by PhD Fellow-

ships from the French Ministère de la Recherche et de la Technologie (MRT). This study receives
grant-aided support from the French ANR Programme Blanc # ANR 07 BLAN 0370 titled DREP:
Diversity and Roles of Eumycetes in the Pelagos.
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14. Canter HM (1953) Annotated list of British aquatic chytrids. Trans Brit Mycol Soc 36:278–303
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116:351–377
16. Beakes GW, Canter HM, Jaworski GHM (1992) Comparative ultrastructural ontogeny of
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18. Beakes GW, Canter HM, Jaworski GHM (1993) Sporangium differentiation and zoospore fine-
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Chapter 5
Assessment of Host Immune Responses
to Fungal Pathogens
Huilin Su, Chunxiao Li, Jiande Han, Clement K. M. Tsui, and Min Zhu
Contents
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.2.1 Analysis of the Fungal Burden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.2.2 Isolation of the Immune System Organs, Infected Organs, and Immune Cells . 106
5.2.3 Detection and Quantification of Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.2.4 Detection and Quantification of Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
H. Su (*)
Department of Dermatology, The First Affiliated Hospital of Zhongshan University,
Guangzhou, China
Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China
C. Li
Department of Radiation Oncology, Peking University Third Hospital, Beijing, China
J. Han
Department of Dermatology, The First Affiliated Hospital of Zhongshan University,
Guangzhou, China
C. K. M. Tsui (*)
Department of Pathology, Sidra Medicine, Doha, Qatar
Department of Pathology and Laboratory Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar
Division of Infectious Diseases, Faculty of Medicine, University of British Columbia,
Vancouver, Canada
M. Zhu
Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China
104 H. Su et al.
5.1 Introduction
Fungal infections (mycoses) to animals and humans have increased substantially in

the last few decades [1]. Fungi cause not only superficial infections such as
dermatophytosis but also tissue and organ invasions and disseminated infections
[2, 3]. Owing to the rise of immunocompromised patients, the morbidity of invasive
fungal infections has increased remarkably worldwide [2]. The clinical outcome of
fungal infection is linked to the pathogen virulence level, the immune responses of
hosts, as well as the interaction between pathogens and hosts [4–6].
Our immune system is mainly composed of immune organs, immune cells, and
immune active substances, which performs immunological surveillance, defense,
and regulations [7]. The primary immune organs include the bone marrow and the
thymus, which provide the sites for cytogenesis, differentiation, and maturation of
immune cells. Peripheral immune organs like the spleen, lymph nodes, and mucosa-
associated lymphoid tissue are the colonization sites in which immune responses are
induced. Fungi are recognized by cells of the innate immune system (e.g. dendritic
cells and macrophages) that bind to the components of fungal cell walls. Mononu-
clear phagocyte system, granulocytes, lymphocytes, dendritic cells, and mast cells
have different roles in defense against fungal infection [6, 8].
Host defense mechanisms range from innate immunity to adaptive immunity
when exposed to fungi [7, 9]. Innate immunity is considered the first-line defense
and plays an important role in antifungal immunity. The physic barriers, innate
immune cells, and related immune active factors work together to protect the hosts
from fungal invasion [10]. Immune cells such as dendritic cells function as a linkage
between innate immunity and adaptive immunity, inducing the differentiation of
adaptive immune cells. Detecting the immune cells numbers, function, and produc-
tion of related cytokines or chemokines is useful to the understanding of immune
response [11].
Immune response varies with fungal species, morphologies, and the immune
condition of the host. Impaired host factors can lead to increased susceptibility to
unusual, sporadic fungal pathogens, particularly during subcutaneous and systematic
infections. When constructing the infection model in vitro and in vivo, both the host
and pathogen conditions should be taken into consideration for research [12]. As the
infection is a dynamic process, the infection time and host status should be also taken
into account. Here, we will describe a few frequently used methods for investigating
the immune responses to fungal pathogens, particularly to A. fumigatus.
5 Assessment of Host Immune Responses to Fungal Pathogens 105
5.2 Methods
5.2.1 Analysis of the Fungal Burden
Fungal burden is an important parameter that reflects the ability of the host to
eliminate the pathogen. In general, the colony-forming unit (CFU) per unit weight
of infected organ or tissue is calculated; for bronchoalveolar lavage fluid (BALF),
CFU per unit of volume is calculated instead. CFU detection is more suitable to
quantify yeast than filamentous fungi since the number of yeast colonies is simpler to
estimate. Nowadays, the quantitative polymerase chain reaction (qPCR) approach is
commonly used to determine the fungal burden. qPCR assays targeting the 18S
rRNA gene [13] and FKS gene [14] in A. fumigatus have been frequently reported in
recent research. Here are two protocols for determining the fungal burden in infected
animals.
5.2.1.1 CFU
1. Remove the infected organ/tissue from the infected hosts in sterile condition.
Weigh the infected samples in 1.5 ml Eppendorf tubes.
2. Homogenize the tissue in saline (e.g. unilateral lung in 2 ml saline). Sterile mortar
and pestle are conventional tools (precaution: grind the organs first before adding
saline), but homogenizers with glass beads at 4 C in 10–20 Hz/s for 30s in the
5 ml sterile tubes with a screw top are recommended for a larger number of
samples. (precaution: reduce or increase the grinding time according to the
sample types until the tissue is completely homogenized).
3. Dilute the homogenate with saline and plate onto Sabouraud dextrose agar (SDA,
Difco, NJ, US) plates in duplicate. A serial diluting concentration (such as 1:10,
1:100, 1:1000) is recommended (30–300 CFU/plate).
4. Count the CFU on the SDA of different dilution ratios after incubation at 35 C
for 48 h.
5. Calculate the fungal burden in the infected organ/tissue (CFU/g).
5.2.1.2 qPCR13 Targeting the 18S rRNA Gene
1. Weigh the infected organ in sterile Eppendorf tubes after dissection.

2. Add 3.5 times the volume of saline to the tissue in tubes. Homogenize the tissue
with beads (4 C, 10–20 Hz, 30s). Centrifuge at 1500 rpm at 4 C for 5 min.
Collect the homogenate for DNA extraction.
3. Extract the total DNA with DNeasy Blood and Tissue kit (Qiagen, CA, US)
according to the manufacturer’s instructions.
4. Perform the PCR with primers and probes targeting the 18S rDNA of
A. fumigatus in optical 96-well reaction plates (Applied Biosystems, MA). The
106 H. Su et al.
Table 5.1 Sequences of primers and probe targeting the 18S rDNA of A. fumigatus [13]
Primer name Sequence
Sense amplification primer 5’-GGCCCTTAAATAGCCCGGT-3’
Antisense amplification primer 5’-TGAGCCGATAGTCCCCCTAA-3’
Hybridization probe 5’-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3’
Table 5.2 qPCR system [13] DNA sample 5uL

Sense amplification primer 900 nM
Antisense amplification primer 900 nM
Hybridization probe 200 nM
TaqMan universal PCR master mix(2X) 25uL
H2O –
Total volume 50uL
recommended PCR conditions are 10 min at 94 C, 40 cycles of 15 s at 95 C,

1 min at 60 C (Tables 5.1 and 5.2).
5. Generate a standard curve by using various known amounts of A. fumigatus
conidia into naive, uninfected kidneys. Calculate the conidial equivalents
(CE) according to the standard curve, with fungal burden shown as CE/g of
tissue.
5.2.2 Isolation of the Immune System Organs, Infected

Organs, and Immune Cells
The immune system organs consist of the central and peripheral immune organs
[15]. Hematopoietic stem cells proliferate and differentiate into B lymphocyte and T
lymphocyte in the central immune organs and are transferred to the peripheral
immune organs [16, 17]. Peripheral immune organs include blood circulation and
lymphatic circulation, which are interconnected into a network and supply the area
where immune cells aggregate and immune responses occur [18, 19]. To investigate
the immune responses in the hosts, it is important to separate the immune organs and
immune cells from the hosts. The size and status of organs and constitution of
immune cells in the organs could reflect the immune responses in local or general
infection after fungi invasion [20, 21]. If the separated immune cells need to be
cultured for further experiment, all the procedures should be performed in sterile
condition. Here, we use the mouse model as an example to show the process of
immune organs separation.
5.2.2.1 Thymus
The thymus is an organ in the central immune system for cytogenesis, differentia-
tion, and maturation of different T cells [22]. The thymus consists of thymus cells
and thymus stromal cells, and stromal cells include thymus epithelial cells, macro-
phages, dendritic cells, and fibroblasts. Thymus epithelial cells secrete thymosin and
thymulin, which activates the function of immune cells [23].
1. After euthanasia is performed, disinfect the mice’s skin on the chest and
abdomen.
2. After dissection, cut and lift the ribs and sternum. The thymus of mice is located
in the anterior superior thorax. The white lobes above the heart are the thymus.
3. Remove the thymus and put it in the complete medium (DMEM or RPMI1640,
10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin
sulfate) (Gibco, MA, US) or PBS buffer (1%FBS + 2 mM EDTA) (Gibco, MA,
US) for further experiment.
5.2.2.2 Bone Marrow
Bone marrow is the main hematopoietic organ that produces erythrocytes,

granulocytes, monocytes, lymphocytes, and platelets [24]. Bone marrow contains
cells with the ability to differentiate into all blood cell types. Bone marrow-derived
macrophages (BMDM) represent the primary macrophages and have been
established as important inflammation host cells in vitro in the immunological
study [25, 26]. The bone marrow of mice is mainly obtained from the tibia and
femur. The procedure for BMDM should be performed under sterile condition.
1. Disinfect the skin of mice with 75% alcohol. Remove the skin and muscles from
the tibia and femur.
2. Cut the epiphyses of the tibia and femur and wash the bone marrow using an
injection syringe with a complete medium for a few times.
3. Resuspend the bone marrow cells in BMDM medium (Iscove’s modified.
Dulbecco’s medium (IMDM)) supplemented with 10% fetal calf serum,
100 u/mL penicillin, 100 μg/mL streptomycin, or phosphate buffer saline (PBS)
for further experiment.
5.2.2.3 Spleen
The spleen is the largest peripheral immune organ in the body, which accounts for
25% of the total lymphoid tissue in the whole body. It contains a large number of
lymphocytes and macrophages and is the center of cellular immunity and humoral
immunity [27]. It also filters the blood of pathogens and abnormal cells and works as
a hemopoietic organ during embryonic development [28].
108 H. Su et al.
1. Disinfect and cut the skin on the abdomen.

2. The spleen is next to the fundus of the stomach with the shape of a sickle.
3. Remove the spleen and put it in a complete medium or PBS for further
experiment.
5.2.2.4 Lymph Nodes
Lymph nodes (LNs) are filters of lymphatic fluid and supply sites where T and B
lymphocytes migrate, proliferate, and differentiate into immune effector cells after
being stimulated by antigens [29]. In fungal infection, it performs the function of
filtering, killing, and clearing pathogens [30].
The LNs are easy to isolate and commonly used in research and mainly include
cervical, axillary, inguinal, and mesenteric LNs [31, 32]. The superficial cervical
LNs are located on the superficial and lateral sides of the left and right submandib-
ular glands, often surrounded by adipose tissue. Axillary LNs are located in the
adipose tissue of the axilla. Inguinal LNs are located in the left and right gluteus
muscles lacuna. Mesenteric lymph nodes are located in mesenteric adipose tissue.
During infection, the draining lymph nodes of the infected site would be larger than
the normal ones and easy to see and separate. Remove the lymph nodes and put them
in a complete medium or PBS for further experiment.
5.2.2.5 Blood
Blood cells are derived from the hematopoietic pluripotent stem cells in the bone
marrow [33]. Neutrophils, eosinophils, basophils, monocytes, and lymphocytes are
the main immune cells in the blood. Neutrophils play an important role in the
prevention of invasive fungal infections [34]. Peripheral blood mononuclear cells
(PBMCs) are indispensable in immunological diagnostics and research. PBMCs
include lymphocytes (T cells, B cells) and monocytes in blood. A few methods for
isolation of PBMCs are available, among which density centrifugation using a
commercial kit is frequently used. The procedures could be referred to references
or kit instructions [35, 36]. The number and function of these immune cells in blood
determine the results of infection and are considered as the indicator for evaluating
the infectious status of the host. Here, we only describe the protocol for whole blood
cells isolation.
1. Collect blood in tubes containing heparin for cells separation.
2. Centrifuge at 4 C at 500 g for 5 min, and the supernatant is discarded.
3. Collect the cells for erythrocyte lysis following the session 2.8.4–2.8.7.
5.2.2.6 Bronchoalveolar Lavage Fluid (BALF)
The contents of BALF include the immune cells and immunological materials
[37]. During fungal infection in the lung, the constitution and number of immune
cells, level of cytokines, and CFU could reflect the level of immune responses in
infected lung [38]. CFU in BALF could also be used as a sign of fungal burden. CFU
per unit of volume is calculated. Galactomannan in BALF could be used in the
diagnosis of pulmonary aspergillosis [39, 40].
1. The airway contents are recovered by the instillation and retrieval of 1 ml of
sterile PBS through a tracheotomy tube. Cells are collected by a total of 3 lavages
and pooled.
2. Centrifuge at 1500 rpm for 5 min at 4 C. After centrifugation, the supernatant is
used for biochemical and immunological tests for the pulmonary infection model.
3. Resuspend the cells in complete medium. Centrifuge at 1500 rpm at 4 C for
5 min.
4. Perform gentle operations in the whole process. If there are erythrocytes in the
cells, collect the cells for erythrocyte lysis following 2.8.4–2.8.7.
5.2.2.7 Infected Organs
In fungal infection, the immune cells migrate to the infected organs. Analyzing the
immune cells in infected organs or cytokines in tissue homogenate can reflect the
degree of inflammation and the immune responses after infection.
Here, we use a lung tissue sample to illustrate the methods for tissue digestion.
Adjust the constitution of digestion buffer according to the types of tissue or
organ [41].
1. Wash the lung sample with sterile PBS to remove the blood on the surface of the
organ. Dissect the tissue into small pieces with ophthalmic scissors.
2. Transfer the unilateral lung tissue into the tube containing 2 mL digestion buffer
(1.7 ml DMEM+0.2 ml FBS + 100ul 10 mg/ml collagenase IV + 4 μl 0.05 mg/μl
DNase) (ThermoFisher Scientific, MA, US).
3. Incubate the tube for 30 min at 37 C, mixing at 260 r/min in the shaker. The
parameters for enzymatic digestion vary with tissue pieces size and enzymatic
activity.
4. Collect the digested lung tissue and filtrate with 100 μm-pore-size screens.
5. Centrifuge the filtrated cell suspension at 1500 rpm for 5 min.
6. Discard the supernatant. Resuspend the cells in a complete medium for erythro-
cyte lysis.
110 H. Su et al.
5.2.2.8 Separation of Immune Cells and Erythrocyte Lysis
For the immune solid organs isolated in 2.1–2.4, they need to be processed in the
immune cell suspension for further experiment especially in the flow cytometry. For
the immune organs with a rich blood supply, erythrocyte lysis needs to be performed
before further study of the immune cells. Blood cells, BALF cells, and bone marrow
can be directly used for erythrocyte lysis and start from 2.8.4 [42].
1. Weigh the immune organs (spleen, LNs, thymus) in Eppendorf tubes after
dissection of the euthanized animals.
2. Grind the immune organs using the blunt end of the syringe.
3. Filter the cells through a 100 μm-pore-size screen to separate the cells from the
connective tissue.
4. Add Tris-NH4Cl (tris(hydroxymethyl)aminomethane-ammonium chloride)
erythrocyte lysis buffer (3.735 g NH4Cl, 1.3 g Tris in 500 ml ddH2O) into the
cells to lyse the erythrocytes. Volumes are adjusted for different cells (e.g. 5 times
the volume for normal immune organs; 10 times volume for blood cells).
5. Wash the cells with a complete medium, and centrifuge and resuspend the cells in
the complete medium.
6. If the erythrocyte lysis process is incomplete, repeat steps 4 and 5 again.
7. Resuspend the cells in a complete medium for cell counting and flow cytometry.
5.2.2.9 Histology
Histopathology of the infected tissue and immune organs would demonstrate

directly the extent of inflammation. It is helpful to study the inflammation type
and to evaluate the immune condition. The immune organs and infected tissue are
fixed by inflation with 4% paraformaldehyde. After paraffin embedding, 5 μm-thick
sections are cut and stained according to different staining methods for different
objectives. Hematoxylin and eosin (HE) stain is usually used to evaluate the
inflammation in the infected sites and observe the immune cells [43]. Periodic
acid-Schiff (PAS) stain and Gomori’s methenamine silver (GMS) stain are used
for fungi detection. These two methods could dye the fungal pathogen in red and
black, respectively [44, 45]. Also, immunohistochemistry (IHC) using specific
markers is useful to show the different immune cells in tissue and to compare the
relative expression of different cytokines and receptors [46].
5.2.3 Detection and Quantification of Immune Cells
Flow cytometry is commonly used for characterizing different immune cells

[47]. Since different immune cells have different specific markers, choosing suitable
cell surface markers with a fluorescence color scheme is important. For instance,
Table 5.3 Cell markers commonly used in flow cytometry

Marker in mice peripheral Marker in human peripheral
Cells blood and lymphoid organ blood and lymphoid organ
Innate Monocyte CD11b+CD115+Ly6C+ CD14+
immunity Macrophage CD45+ CD11b+ F4/80+ CD11b+CD68+
Neutrophil CD11b+Ly6G++ CD11b+CD16+CD15+CD32+
Adaptive T cell CD3+ CD3+
immunity Th cell CD3+ CD4+ CD3+ CD4+
Treg cell CD3+ CD4+ CD25+FoxP3+ CD3+ CD4+ CD25high PD-1high
Cytotoxic T CD3+CD8a+ CD3+CD8a+
cell
B cell B220+ CD19+ CD20+ CD79a/b+
Plasmacyte B220+CD19+ CD19+ CD20 CD38high
NK cell CD49b+CD3 CD56bright CD16+/CD56dim
CD16+
NKT cell CD1d-tetramer+TCRβ+ CD3+ TCRVα24+
some immune cells have different surface markers in humans and animals. Here is a
list of common markers for innate and adaptive immune cells (Table 5.3).
5.2.3.1 Cell Detection Using Flow Cytometry
1. Choose the suitable surface marker for targeting immune cells and design the
multicolor plan.
2. Measure and add about 106 cells in the Eppendorf tube.
3. Preincubate cell suspension with purified CD16/CD32 mAb ( 1 μg/million cells
in 100 μl) (BD, NJ, US) at 4 C for 5 min or dilute the antibodies in PBS
containing 2% bovine serum albumin (BSA) to reduce Fc receptor-mediated
binding.
4. Add 50 μl antibodies for staining. Cells are stained for 30 min at 4 C and
protected from light.
5. Centrifuge at 1500 rpm for 5 min at 4 C.
6. Wash the cells with PBS, and centrifuge and resuspend the cells in 1 mL PBS
containing 0.1% BSA.
7. Perform the flow cytometry in a few hours according to the protocol published
before [48] or the protocol provided by the flow cytometer.
8. Fix the cells in paraformaldehyde if they are detected in a timely manner. Keep
the samples in the dark after fixation.
9. Analyze the flow cytometry data using FlowJo (BD, New York, USA) or other
software package.
112 H. Su et al.
5.2.3.2 Cell Counting
1. Weigh the immune organs. Prepare blood sample or cell suspension for erythro-
cyte lysis. Resuspend the cells in a complete medium in 2.1.
2. Count the cells using a hemocytometer or automated cell counting instrument.
3. Calculate the number of cells per organ or volume of blood.
4. Calculate the number of specific immune cells using the total number and the
percentages generated from flow cytometry.
5.2.4 Detection and Quantification of Cytokines
Cytokines are small molecular proteins with extensive biological activities, and they
work together as a network in the innate and adaptive immunity systems in fungal
infection [49, 50]. Cytokines are usually protein or polypeptide molecules which
have antigenicity. Based on the high specificity of the antigen-antibody reaction,
immunoassays are the most common methods for detecting cytokines. The common
methods include flow cytometry, enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), enzyme-linked immunospot assay (ELISPOT), and
Western blot.
5.2.4.1 Intracellular Cytokine Detection Based on Flow Cytometry
1. Pipette 2 106 cells in the 48-well cell culture plate.

2. Suspension cells are for stimulation in a complete medium with protein transport
inhibitor according to the cells and cytokines types. 20ug/ml PMA (phorbol
myristate acetate), 1ug/ml ionomycin, and 1ug/ml lipopolysaccharide (LPS) are
commonly used as stimulatosr; 3.0 ug/ml brefeldin A and 2uM monensin are
commonly used as protein transport inhibitors.
3. Collect the cells for surface marker staining (refer to cell detection steps
3.1.1–3.1.5).
4. Add 100 μL Cytofix/Cytoperm solution and incubate at room temperature
protected from light for 30 min.
5. Add 500 μL Perm/Wash solution, and centrifuge at 4 C at 5000 rpm for 5 min.
Wash the cells twice.
6. Dilute the cytokine antibody in 100 mL Perm/Wash solution. Add the antibody in
the tubes. Cells are stained for 30 min at 4 C and protected from light.
7. Add 500 μL Perm/Wash solution, and centrifuge at 4 C at 5000 rpm for 5 min.
8. Resuspend the cells in 1 mL PBS for flow cytometry.
9. Analyze the flow cytometry data using (Flowjo) or other suitable software. The
relative expression of cytokines is shown in percentages.
5.2.4.2 ELISA
ELISA is a qualitative or quantitative test that uses antibodies to bind and measure a
molecule of interest. It remains the method of primary choice and one of the most
widely used methods for detecting cytokines for its specificity and simplicity in
many laboratories.
1. Select the antigen-coated plates and enzyme (take horseradish peroxidase (HRP)
as an example).
2. Add 100 μL of standards and samples (select appropriate dilution ratio) to wells.
Incubate the samples at room temperature for 2 h.
3. Discard the solutions from wells.
4. Wash the plates four times.
5. Add 100 μL HRP enzyme-labeled antibody to wells and incubate at room
temperature for 30 min to 1 h according to the manufacturer’s instructions.
6. Discard the solutions from wells.
7. Wash the plates four times.
8. Add 100 μL chromogenic substrate to wells.
9. Develop the plate at room temperature in the dark for 30 min.
10. Add 100 μL stop solution to wells.
11. Evaluate the results in 30 min after stopping the reaction. Read the plates at
450 nm and 550 nm. Subtract 550 nm values from 450 nm values to correct for
optical imperfections in the microplate.
12. Calculate the results according to the standard curve.
5.2.4.3 Other Methods
There are other methods important in immunological research not covered in this
chapter. Several traditional methods including radioimmunoassay (RIA), enzyme-
linked immunospot assay (ELISPOT), and Western blot are commonly used for
cytokine detection. Most of these methods are based on antigen/antibody reaction;
the antibodies are incorporated with a radioisotope or biotin-labelled secondary
antibody for detection. Molecular methods are also used for cytokine detection
through the gene probes of cytokines to detect the expression level. The detection
methods include Northern blot, real-time PCR, and in situ hybridization.
With the advent of novel technologies in laboratory diagnosis, multiple cytokine
detection methods have been developed recently, such as flow cytometry and
electrochemiluminescence. Based on flow cytometry platform, technologies such
as cytometric bead array (CBA) [51], FlowCytomix, LEGENDplex, and Luminex
Assay have been developed. Briefly, fluorescent microbeads are coated with specific
antibodies. When the beads are mixed with the biological sample, biomolecular
binding interaction occurs between the specific antibodies and cytokines antigens,
the binding activity/analyst can be detected by signals generated by means of flow
cytometry. In the electrochemiluminescence assay, electrochemistry-induced
114 H. Su et al.
specific chemiluminescence reaction is occurring on the surface of the electrode, and

the light signal is detected and used for analysis. These methods have higher
sensitivity and can detect a series of cytokines at the same time with a smaller
sample volume [52, 53].
5.3 Summary
The interaction between the host and fungus pathogens are complicated; human
hosts having different immune status may lead to contrasting clinical outcome. Early
diagnosis and treatment contribute to the prognosis of fungal infections. Better
understanding of antifungal immune response contributes to the improvement of
diagnosis and therapy of fungal infections. The protocols described above are
fundamental and useful in immunological research.
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Chapter 6
Recent Advances in Applications of Support
Vector Machines in Fungal Biology
Sonal Modak, Ashwin Lahorkar, and Jayaraman Valadi
Contents
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6.1.1 Support Vector Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6.2 Domain Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.3 Review of Some Recent Fungal Bioinformatics Applications Using SVM . . . . . . . . . . . . . 120
6.3.1 Haar Wavelets Features for Fungal Disease Detection in Maize Leaves . . . . . . . 120
6.3.2 Quality Detection of Pomegranate Fruit Infected with Fungal Disease . . . . . . . . 121
6.3.3 Support Vector Machines Enabled Fungal Rust Disease Detection in Pea Plant
(Pisam Sativam) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
6.3.4 Volatomic Visualization for Fungal Infection Detection on Storage Jasmine . 122
6.3.5 Hyperspectral Imaging Enabled Electronic Nose for Fungal Contamination
Identification in Strawberries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.3.6 Rapid Discrimination of Fungal Species by the Colony Fingerprinting . . . . . . . 124
6.3.7 SVM Classifier Based Grape Leaf Disease Detection . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.3.8 Hyphae Detection in Fungal Keratitis Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
6.3.9 Plant Diseases Classification Using SVM and ANN (Artificial Neural Network) 126
6.3.10 A Hybrid Combination of Multiple SVM Classifiers for Automatic
Recognition of the Damages and Symptoms on Plant Leaves . . . . . . . . . . . . . . . . . 126
6.3.11 Machine Learning of Protein Interactions in Fungal Secretory Pathways . . . . . . 127
6.3.12 Fungal Adhesins and Adhesin Like Proteins Predictions . . . . . . . . . . . . . . . . . . . . . . . 127
6.3.13 Computational Prediction of Antifungal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
6.3.14 A Novel Method of Annotation of Antifungal Peptides Based on Distributed
Representation of Protein Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.3.15 Identification of Antifungal Using Distributed Representation of Sequences . . 130
S. Modak
Life Sciences and Healthcare Unit, Persistent Systems Inc., Santa Clara, CA, USA
A. Lahorkar
Center for Modelling and Simulation, Savitri Bai Phule Pune University, Pune, India
J. Valadi (*)
Department of Computer Science, Flame University, Pune, India
e-mail: jayaraman.vk@flame.edu.in
118 S. Modak et al.
6.4 Illustration of Use of SVM in Fungal Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

6.5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.1 Introduction
Machine learning has recently been employed successfully in fungal biology. The
methodologies used include both supervised and unsupervised learning. Supervised
learning employs prior domain knowledge to build models. The models built can be
used to derive information for new examples. Classification and regression form
major supervisory learning paradigms used in fungal biology. Unsupervised learn-
ing, on the other hand, is used to discover structured information without prior
domain knowledge. Several classification and regression algorithms are available in
machine learning which is routinely employed for quick and low-cost acquisition of
invaluable information. Support Vector Machines is a valuable classification and
regression tool employed in different fields of science and engineering for high-
performance prediction tasks. Support Vector Machines have been used profusely in
a fungal domain for the identification of plant diseases, characterization of fungal
keratitis, and other fungal infections. Support Vector Machines have also been
successfully used to predict potential antifungal peptides. In this chapter, after a
brief discussion on SVM, we will be discussing various case studies where SVM has
been used in fungal biology. Apart from that, we have also presented our own
unpublished work on the prediction of antifungal peptides. This is based on a recent
method on the distributed representation of protein sequences which has been used
for extracting embedding vectors using unsupervised learning. The informative
embedding vectors are further employed as input attributes to SVM classifier to
find whether a given peptide sequence has antifungal activity or not.
6.1.1 Support Vector Machine
Support Vector Machine is a hyperplane-based classifier [1]. Support Vector

Machine employs a linear maximum margin hyperplane for classifying examples
into two groups. As an illustrative example assume that we are given a set of peptide
sequences with experimental proven antifungal activity. We also have been given
sequences with no known antifungal activity. Support Vector Machine finds a
hyperplane which maximizes the distance between the hyperplane and the closest
sequences belonging to both classes of sequences (Fig. 6.1).
If the sequences are linearly classifiable, the accuracy of classifications will be
high and acceptable. If the classification accuracies are not good, then the sequences
may not be linearly classifiable. For the non-linearly classifiable tasks, SVM
6 Recent Advances in Applications of Support Vector Machines in Fungal Biology 119
Fig. 6.1 Cartoon of SVM as a maximum margin classifier. Examples on the margin are Support
Vectors
transforms the examples to a higher dimensional attribute space. After this, SVM
employs a linear maximum margin classifier to separate these classes of sequences.
Further, it reduces complexity by defining different kernel functions. These include
RBF, Polynomial, Gaussian, and Neural network kernels. These are domain-
dependent kernels like mismatch kernel, Fischer kernel, and string kernels. Detailed
discussions on the working of the SVM algorithm along with diverse types of the
algorithm have been provided in Modak et al. [2].
6.2 Domain Features
Several classes of domain features are extracted for predictions and classifications.
These include sequence-based features, image-based features, and spectral features.
Sequence-based features include (1) amino acid frequencies, (2) dipeptide &
tripeptide, and higher-order K-mer frequencies; (3) Physiochemical properties
(4) Position specific Scoring Matrix (PSSM) profile-based attributes (5) Pseudo-
amino acid composition-based features etc. Image-based features include pixel-
based features, shape descriptors, frequency domain-based features like wavelet
features, and other descriptors extracted from leaf images and solid phase
microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) based
volatility markers. Once the features are extracted, feature selection algorithms can
120 S. Modak et al.
be employed for the identification of the most informative features. Feature selection
algorithms include filter, wrapper, and embedded methods. A detailed discussion of
diverse types of feature selection algorithms has been provided in Modak et al. [2].
6.3 Review of Some Recent Fungal Bioinformatics

Applications Using SVM
In this section, we outline few important problems in bioinformatics where SVM has
been applied with interesting results on many real-world fungal biology case studies.
6.3.1 Haar Wavelets Features for Fungal Disease Detection

in Maize Leaves
Agriculture is the most vital part of the economy of any of the countries in the world.
That is why the diseases which affect the agricultural crops are serious concerns
since they affect the economy of the country. One of the most common crops
cultivated throughout the globe is maize. The importance of this crop is not only
limited to being food for humans and cattle, it is also the source of a lot of
commercial products like baby corn, corn starch, and corn oil. Thus, the research
community has been exploring the avenues to detect such diseases in maize crops in
the preliminary stages to minimize the loss. From the range of the diseases affecting
maize crops, Desai et al., proposed an image-based method to detect diseases in
maize plants [3].
For this study, the authors targeted fungal diseases along with common rust and
northern leaf blight in the maize leaf. The very first step was the collection of images,
which was carefully executed by making sure that all the images collected over the
period are consistent. This was achieved by capturing images; all pictures are taken
at 11:00 AM and by keeping a consistent distance of two feet from the leaves. The
final dataset of images consisted of 200 images, which were distributed in the
following categories (a) 50 healthy, (b) 50 common rust, (c) 50 northern leaf blight,
and (d) 50 images with both diseases present in it. All these images were
preprocessed to make sure that the illumination is uniform throughout all the images.
Once the images were converted to RGB colour space, red, blue, and green channels
were separated for feature extraction. The first-order histogram features were con-
sidered for this study which are estimated properties of individual pixel values by
ignoring the spatial interaction between image pixels [4]. One important aspect of
image analysis is texture analysis, which was incorporated in this work by wavelet
representation. The wavelet transformation for feature extraction involves Haar
transform [5]. Haar wavelet is applied to RGB images to obtain horizontal, vertical,
and diagonal coefficients. Later, Gray-Level Co-occurrence Matrix (GLCM) is
constructed in two directions (0 and 90 ) for each of the coefficients. Finally, k-NN
and SVM classifiers were employed for classification. k-NN yielded the highest
accuracy of 85% for k ¼ 5 and while SVM-based classification yielded 88% of
accuracy.
6.3.2 Quality Detection of Pomegranate Fruit Infected

with Fungal Disease
The hazards of fungal infections are not only restricted to common crops like maize
but also some varieties of fruits that are well known and widely consumed for their
known benefits. Pomegranate fruit is one such example because of its several health-
promoting effects including positive effects on the cardiovascular system, human
immune system, menopausal complaints, and diabetes mellitus [6]. Like most crops,
the harvest and transportation of these fruits involve the risk of contamination of
pathogenic fungi. Alternaria specie or Aspergillus specie is known which infects
pomegranate throughout all its stages of life, from the early stage of ripeness through
its development [7, 8].
Even with current advances in technologies, a lot of time and effort will be needed
to develop a non-destructive quality assessment of Alternaria fungi. To address this
problem, Rafiee S. et al., worked on developing a non-destructive, inexpensive, and
fast technique to evaluate the quality of pomegranate [9]. Metal Oxide Semiconduc-
tor (MOS) biosensors were employed using linear and non-linear methods for
quality assessment. In this elaborative work, sampling was done using 60 Pomegran-
ates of the ‘Robab’ genotype, which were similar in shape and size. These samples
were classified into different amounts of Alternaria specie infection, ranging from
0%, 25%, 50%, 75%, and 100%. The methods which were employed for linear and
non-linear analysis methods for detection were (a.) Back Propagation Neural Net-
work (BPNN), (b.) Discriminant Analysis (LDA), and (c.) Support Vector Machine
(SVM). Finally, the results from each different method were compared with each
other. In this study, authors concluded that 100% detection accuracy between
healthy and infected samples was achieved by the LDA method, by using only
two MOS sensors. Back Propagation Neural Network proved to be the most efficient
prediction method with an accuracy of 100% in the detecting of 0%, 25%, 50%,
75%, and 100% infected pomegranates. In this comparison study, the BPNN method
performed better than LDA and SVM analysis, in terms of accuracy, in detecting
fungal infestation in various stages. The authors concluded that this technique can be
reliably used to effectively evaluate the quality of the pomegranate with high
precision.
122 S. Modak et al.
6.3.3 Support Vector Machines Enabled Fungal Rust Disease

Detection in Pea Plant (Pisam Sativam)
Another prominent example of the dreadful consequences of agricultural diseases is

the fungal rust disease of the Pea plant. Uromyces fabae (Pers.) de Bary fungus cases
Pea rust, which is characterized by mutilation of upper and/or lower surfaces of
leaves, spotting in leaves, and rust coloured spotting on petioles, pods, and stem
[10]. Such plant diseases harm the economy by reducing production. Thus, the
research community has been working towards cost-effective and rapid solutions
where such diseases can be detected as early as possible [11, 12]. There are some
notable studies where Image processing-based tools were effectively used to screen
crops for diseases [13, 14]. Out of all these studies, specific attention has been given
to digital imaging at spore germination or penetration level for fungal diseases
identification [15–17] since the plants can be rarely saved after such infection.
Thus, Kaur P et al. aimed to prove the early detection of rust diseases of Peas at
the microscopic level by using image processing techniques [18]. In this work,
authors tried to develop a technique which helps identification of symptoms at an
early stage of infection of rust disease. Image data was gathered by collecting leaves
of affected plants. Staining techniques and biochemical methods were applied to the
leaves as part of preprocessing [19]. The images obtained were subjected to further
preprocessing, which included (a) image smoothing, (b) noise/background removal,
(c) image enhancing, (d) image format changing, and (e) image segmentation. After
preprocessing, features could be extracted from the region of interest (ROI) for
classification. In this work, the authors employed Discrete Wavelet Transform
which is a technique for 2-D decomposition of the image with respect to Haar
wavelet. Once the features were extracted, an SVM classifier was used to build the
model for leaf disease detection in Pea Plant. Authors were able to reach an accuracy
of 89.60% in disease detection in this study, which could be further explored to
develop efficient, rapid, and cost-effective technology for plant disease
identification.
6.3.4 Volatomic Visualization for Fungal Infection Detection

on Storage Jasmine
Fungal contamination in grains can not only affect the economy but also can cause
danger to human health since some fungal strains such as Aspergillus can produce
harmful mycotoxins [20–24]. Siripatrawan U. et al., with the aim of preventing
infected rice from entering the food chain by detecting the fungal infection in rice at
an early stage [25]. In this study, the detection unit, i.e. the electronic nose when
coupled with chemometrics, proved to be a fast and non-destructive detection
method for fungal contamination in Jasmine brown rice grain. For the prediction
model, the source of data was the artificially infected Jasmine brown rice, which was
achieved by storing Aspergillus infected rice at 30 C and 85% relative humidity.

Solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-
MS) was employed in this study for the identification of volatile markers in the
infected rice.
An electronic nose (described thoroughly in the reference) was used to analyze
the profiles generated from the identification, i.e. Volatomic profiles. Principal
component analysis (PCA) was employed to explore the multivariate electronic
sensor response data and to reduce the data dimension. Machine learning techniques,
SVM and Linear discriminant analysis (LDA) were employed for the classification
of samples, contaminated at a different level over the time series of storage. Finally,
the prediction of fungal growth on brown rice was formulated by implementing the
Partial least squares (PLS) regression model. The PLS regression model combined
with the electronic nose predicted fungal growth precisely. It reached a coefficient of
determination (R2) value of 0.969, and root mean square error (RMSE) value of 0.31
Log CFU/g.
In the work, the authors demonstrated the use of PCA reducing the data, but this
technique was not able to classify the sample groups in different categories, while
both SVM and LDA were not only able to perform well identifying and classifying
fungal infections in samples, but it also detected the level of infection with respect to
storage time. The authors concluded that the electronic nose described in this study
can be effectively used for fast and non-destructive detection of rice grain samples
with fungal infection.
6.3.5 Hyperspectral Imaging Enabled Electronic Nose

for Fungal Contamination Identification
in Strawberries
E-nose coupled with hyperspectral imaging (HSI) was also used by Tu K. et al. to
show a non-destructive method for detection of quality and microbial content of
strawberries during decay [26]. The spectral data and images of strawberries were
acquired using a lab-scale visible/near-infrared HSI system. In the proposed method,
PCA was employed to reduce the dataset dimensionality and for extracting features
from the HSI and E-nose data.
First, the aroma, spatial, and spectral information was acquired by HSI and
E-nose during the decay of strawberries. Multivariate calibration models were
developed using the sensor and spectral signals to detect fungal contaminations in
strawberries. For the development of calibration models, E-nose and HSI data was
integrated for improving the decay detection of strawberries. In this study, one of the
important conclusions was the correlation between the interior compositions of the
fungi-infected strawberries and changes in exterior appearances was highly signif-
icant. Colony-forming units were found with a correlation coefficient value of 0.925
and root mean square error of 0.38. In conclusion, the authors recorded and
124 S. Modak et al.
recommended that the two sensing techniques can be combined for the detection of
the quality of strawberries.
6.3.6 Rapid Discrimination of Fungal Species by the Colony

Fingerprinting
In continuation with series of examples where agricultural goods are affected by

fungal contamination, this example deals with addressing the same problem with
another approach. Tanaka T. et al., used a bioimage informatics approach for fungal
discrimination in the samples [27]. For this work, the concept of ‘colony finger-
printing’ was used which was already proven in earlier studies [28, 29]. Colony
fingerprinting is an imaging technique where the colonies of the microorganism are
visualized using a lens-less imaging system. This was followed by the extraction of
quantitative features having discriminative power needed for processing by machine
learning techniques.
The authors aimed to prove the effectiveness of the colony fingerprinting
approach in fungal specie discrimination since its high performance for bacterial
identification was already reported in earlier studies [29]. In contrast to bacterial
discriminative parameters (e.g. roundness, solidity, doughnut-ness etc.), seven dis-
criminative distinct parameters for fungal specie were extracted as colony finger-
prints. Some of the parameters were as simples as the number of hyphae (fungal
filaments), while other complex characters include the number of branches of
hyphae. The overall idea was to extract the count of hyphae and their branches,
along with information on distributions of intensities on the images. Based on this
approach, authors were able to successfully discriminate five distinct species of fungi
belonging to Fusarium, Eurotium, Alternaria, Aspergillus, and Penicillium genre.
This work was further extended by discriminating six closely related Aspergillus
specie by introducing more parameters in the feature set. The most exciting discov-
ery in the work was the turnover time required for deriving the colony fingerprinting
from generated fungal colonies, which was just 48 h. This time is less than that of
other methods used for discrimination like MALDI-TOF-MS.
6.3.7 SVM Classifier Based Grape Leaf Disease Detection
India is one of the major producers of grapes, which adds up to the agro-economy of
the country. The annually major percentage of this important crop is affected by the
diseases due to microbial infections; thus, technological solutions aiding the increase
in the yield of this crop has been one of the focused areas of the research. Plant
diseases are mostly caused to leaves by attacks of viruses, fungi, and bacteria. As
discussed in the above examples, one simple approach is to find the disease in the
early stage, so that necessary control steps could be taken to minimize the hazard.
There are several successful studies proving the use of image processing to effi-
ciently detect and classify leaf diseases in plants [30]. Yadav et al. worked on the
same approach, using image processing coupled with SVM to detect and classify
diseases in grape leaves [31].
For this study, the images data of healthy leaves were combined and leaves
infected by Powdery Mildew and Downy Mildew. In such a method, it is important
to preprocess the image data to filter any noise in the background and suppress
distortions to make all images consistent. After preprocessing, the authors performed
image segmentation by separating images into homogenous regions as per certain
features. K-means clustering was employed for image segmentation. Clustering
methods group similar data from large sets of data into much smaller clusters.
Once the diseased region in the leaves is detected using segmentation by K-means
clustering, colour, and texture features were extracted. Total nine texture features
were considered for this study, namely; (a) contrast, (b) uniformity, (c) maximum
probability, (d) homogeneity, (e) diagonal variance, (f) difference variance,
(g) entropy, (h) inverse difference, (i) and nine colour features. Finally, Linear
Support Vector Machine (LSVM) was employed for the classification of leaf
diseases. Classification accuracy of 88.89% was achieved for examined diseases.
6.3.8 Hyphae Detection in Fungal Keratitis Images
Some fungal organisms also cause infection in the human cornea and that results in
conditions like Fungal Keratitis [32]. This inflammatory infection can cause blind-
ness in most cases, thus accurately diagnosing Fungal Keratitis is critical [33]. Fungal
Keratitis can be clinically diagnosed by methods like microscopic examination of
cornea scrapings, slit-lamp examination, polymerase chain reaction (PCR), tissue
biopsy, fungal culture, and confocal microscopy [34]. Out of all these techniques,
confocal microscopy is based on the optical imaging approach, where hyphae
detection can be done by analyzing the cornea images from confocal microscopy.
The challenging part is to accurately find the abnormal cornea images with hyphae
from the normal cornea images with nerves. To address this issue, Ren J. et al.,
proposed a novel method to aid physicians in diagnosing this condition by automat-
ically detecting hyphae after processing images from confocal microscopy [35].
The method is composed of two important steps: (a) classification of texture in the
input images and (b) detection of hyphae. Adaptive robust binary pattern (ARBP) is
evolved as a contrast stretching algorithm and is sub-regional in character. This
method is used to extract the texture features from the images, which are eventually
fed into the SVM classification model to classify between abnormal and normal
images. In the downstream pipeline, the targets are further enhanced by binarization,
where hyphae detection was achieved by a line segment detector algorithm. Finally,
the severity of infection is quantitatively evaluated by the hyphal density. The
126 S. Modak et al.
authors were able to show the effectiveness of improvement of the AMBP in the
extraction of texture features from images.
6.3.9 Plant Diseases Classification Using SVM and ANN

(Artificial Neural Network)
With the advent of computational techniques, there has been a sudden increase in its
application in areas of agriculture and horticulture. In simple words, the computa-
tional techniques are explored to develop a decision support system which can be
used as an expert system for decision making for agricultural yield or plant protec-
tion. Byadgi AS. et al. showed a study where colour and texture features were used
to build a classification model to recognize diseases in plants [36]. The method
proposed by authors in this work starts with acquisition of images, followed by
preprocessing the images, features extraction and selection, and finally building the
classification model.
Because most of plant diseases are caused by five factors, namely: (a) viral,
(b) fungal, (c) bacterial, (d) nematodes, and (e) deficiency [37], authors used
900 sample images as test data. This set consisted of 150 images for each category
mentioned above, and 150 images from normal plants which were not affected, thus
resulting in six classification categories. In the work, colour and texture features
were extracted, where colour features were based on RGB and HSI colour models,
while the texture features were based on Gray level cooccurrence matrices (GLCM)
[38, 39]. For classification, authors used studied the behaviour of SVM and ANN in
the context of the suitability of classifiers by employing ANN-based classifiers using
multilayer feed-forward with back propagation and SVM based classifiers for
recognizing plant disease in the images. In this comparative study, SVM yielded
maximum accuracy of 98% using joint colour and texture features, showing that
SVM classifiers are more suitable for the identification and classification of plant
diseases that affect agricultural and horticulture crops.
6.3.10 A Hybrid Combination of Multiple SVM Classifiers

for Automatic Recognition of the Damages
and Symptoms on Plant Leaves
Benazoun A. et al. described in their work how image recognition was addressed to
automatically recognize the disease symptoms and damages in plant leaves using a
hybrid combination of three SVM classifiers [40]. The proposed system was based
on the serial combination of two classifiers, which is used in parallel with an
individual classifier [41].
In the work, the test dataset consisting of images were composed of two catego-
ries; symptoms of three fungal diseases namely, (a) Early blight, (b) Late blight, and
(c) Powdery mildew and damage by three pest insects namely, (d) Leaf miners,
(e) Thrips, and (f) Tuta abosluta. To improve the quality of the data, images were
preprocessed, which included filtering and resizing. This is a crucial step for efficient
segmentation and analysis, which are further steps in the proposed method. In the
feature extraction step, segmented images were represented as the feature vector.
Texture, colour, and shape of plant leaves were features included in this study to
classify damage and symptoms in the image data. The serial architecture handled the
colour-based classification of images. It dealt with the damages and/or symptoms,
taking into consideration the similarity or nearest colour belonging to the same class.
While the second classifier further classified similar colours based on texture and
shape. In this comparative study, an overall recognition rate of 93.9% was achieved,
which was better than other existing methods. In conclusion, the authors recorded
that the proposed method can easily overcome the classification problems by other
methods and it can be strategically used for phytosanitary and diagnosis for recog-
nition of infected plants using image data.
6.3.11 Machine Learning of Protein Interactions in Fungal

Secretory Pathways
Magnaporthe grisea (M.grisea) causes excessive destruction of rice crops. Protein-

protein interactions (PPIs) of rice and M.grisea is the key factor of this disease.
Protein-protein interaction study and machine learning based prediction would be
most useful in finding ways to counter this destruction. Biswajit Karan et al, applied
the machine learning algorithms to address the problem of PPI predictions [42].
From whole rice data base these authors [42] extracted a total 12091 positive
candidates and rest 54062 negative candidates. For the blast Fungi M.Grisia they
filtered 6151 positive examples and 5236 negative examples. For domain informa-
tion they used 20 amino zacid features and seven conjoint triad (CTD) features.
Employing SVM as the classifier they obtained 89% accuracy with CTD features
and equally good accuracy of 88% with amino acid feature.
6.3.12 Fungal Adhesins and Adhesin Like Proteins

Predictions
The cell surface interacts with the outside environment, which is important to carry
out cellular functions. This interaction involves macromolecules such as Adhesins.
They are present in a wide variety of microorganisms such as fungi, bacteria etc. The
key role of adhesins is to help adhesion of pathogens to host cells, which is followed
128 S. Modak et al.
by colonization of the microorganisms in the hosts. One of the most significant roles
played by adhesins includes the formation of a biofilm, which acts as a defensive
shield against hazardous surroundings and aids in prolonging the infection
[43]. Identification and characterization of fungal adhesins have been explored less
in contrast with bacterial adhesins. Insights on sequence characterization of the
fungal adhesins might open new avenues to understand the process where they
play a critical role in pathogenesis. Thus, research efforts are directed towards
exploring computational methods to develop a rapid and effective approach to
characterize and find fungal adhesins.
Nath A. proposed a method to accurately predict the fungal adhesins [44]. His
work also includes the identification of consensus molecular signatures present in
fungal adhesins. Dataset for this study consisted of 75 fungal adhesins and
341 non-adhesin protein sequences, which were processed to extract sequence-
based and evolutionary features. These features include Amino acid composition
(AAC), Dipeptide counts (DPC), Property group composition (PGC), Physicochem-
ical-2-grams (P2G), Atomic composition (ATC), Physicochemical properties (PCP)
and features based on evolutionary information. In this comparative study, different
algorithms were employed to find the best performing machine learning algorithms
which can effectively predict adhesin protein. These methods include (a) Naïve
Bayes, (b) SVM with Sequential minimization optimization implemented with
Poly-K, RBF, and PuK kernels, (c) Bagging with REPTree, (d) Real Adaboost,
and (e) Adaboost with decision stumps. The author noted that the SVM kernel-based
approaches viz., RBF, PuK, and SMO-PolyK, performed better than other algo-
rithms. Based on the leave, one outperformance measure accuracy of 94.9% was
obtained on cross validation procedure and a validation set accuracy was achieved
up to 98.0%.
6.3.13 Computational Prediction of Antifungal Peptides
Most of the living organisms show innate shield immunity gotten during lifecycle
and its strength depends on serval factors. In humans, patients with weak immunity
are prone to fungal infections, which can lead to dreadful consequences. Invasive
fungal infections cause high morbidity and mortalities. Despite tremendous
advances in the field of antibiotics research, Candida, Aspergillus, Pneumocystis,
and Cryptococcus spp. [45] cause more than a million deaths every year [46]. To
combat the twin problems of fungal infections and drug resistance, peptide-based
therapeutics are actively under consideration. Antimicrobial peptide-based therapeu-
tics have gained momentum in recent years with several formulations available in the
market. For fungal infections, more specific actions can be expected from antifungal
peptides. It is thus contingent to focus research on the discovery of novel antifungal
peptides with high activity.
Employing machine learning based methods is a cost-effective way of identifi-
cation of antifungal peptides. Several methods have been developed in the past for
the identification of antifungal peptides; these include template-based methods,

docking simulations, and sequence-based methods. These have been listed in the
work of Agrawal et al. [47]. In sequence-based methods, it is possible to extract
several fixed-length attributes which include amino acid, dipeptide and K-mer
frequencies. Apart from this, several physiochemical properties can be employed
as attributes. Information about Position specific scoring matrix (PSSM) profiles can
also be included in the feature list.
Agrawal et al. [47] have developed different models for the prediction of anti-
fungal peptides. These include amino acid & dipeptide composition features, binary
profile, and terminal residues-based attributes. Their Support Vector Machines
compositional features-based method yielded 88.78% training accuracy and inde-
pendent test accuracy of 83.33%. Further, their terminal residues based binary
patterns features yielded 84.88% and 84.64% accuracies respectively on training
and test sets and validation dataset. They have developed a mobile app, a standalone
and a user-friendly web server.
6.3.14 A Novel Method of Annotation of Antifungal Peptides

Based on Distributed Representation of Protein
Sequences
One of the methods in classical Natural Language Processing (NLP) in text mining
jobs is to convert the text into frequency vectors. While this is quite successful, it has
its own drawbacks. This representation is unable to capture the meaning of the words
under a given context, as all vectors are orthogonal to each other. The introduction of
distributed representation of words has introduced a revolutionary change in text
mining. The main idea being the meaning of a word under a given context is given
by surrounding words that often occur close to it. In this method, a dense represen-
tation of words of a predetermined size is created to capture the meaning of the word
and words with similar meanings will appear closely in a higher dimensional
embedding space. An extremely popular algorithm knowns skip-gram model by
Mikilov et al. [48] is the front runner of the distributed representation of words. The
idea behind skip-gram representation is to predict the context words surrounding it
by using the word as input in a shallow network. Using this unsupervised learning,
embedding vectors of certain dimensions can be created for a large vocabulary of
words. The most informative embedding vectors are obtained the prediction loss.
Asgari and Mofrad [49] first applied the word2vec model for distributed represen-
tations annotating protein sequences and named it as ProtVec (Protein Vectors)
model. They first extracted embedding vectors from the entire SWISSPROT exper-
imentally verified list of sequences using the skip-gram model. They used these
embedding vectors for handling multiple protein annotation tasks. They reported
excellent accuracy. This method uses only sequence information and achieves
superior performance.
130 S. Modak et al.
6.3.15 Identification of Antifungal Using Distributed

Representation of Sequences
As a first step, the complete set of experimentally verified sequences are first
converted to K sets of shifted non-overlapping K-grams. The skip-gram embedding
model is then used to learn the embeddings with a setting like that of Asgari and
Mofrad [49]. We employed word sizes of three and five and a context size of 25 to
build the unsupervised learning model to create embedding vectors; the size created
varied from 100 to 500 vectors. We used the same dataset used by Agrawal et al. [47]
which has 1168 antifungal peptides as positive dataset obtained from the DRAMP
database and 1168 antimicrobial peptides other than antifungal as negative dataset
obtained from the DRAMP database. We used the embedding vectors specific to the
training set as input to the SVM classifier and built the training model. The training
model was used to validate a set of test sequences having 291 antifungal peptides as
positive dataset obtained from DRAMP database and 291 antimicrobial peptides
other than antifungal as negative dataset obtained from DRAMP database.
Our results show that the ProtVec model with a vector size of three hundred,
K-mer word size of three and context vector size of 25 achieved particularly reliable
performance with 86% validation accuracy. Our results show that the distributed
representation of Antifungal peptides is a very efficient way of robust annotation of
antifungal peptides.
6.4 Illustration of Use of SVM in Fungal Bioinformatics
The below table enlists all the examples that illustrate the use of SVM in Fungal
bioinformatics:
Title Method Dataset Performance

Haar wavelet features K-NN classifier and Images of maize leaf k-NN for k ¼ 5
based fungal disease SVM classifier were are captured using yielded 85%accuracy
detection [3] used for classification digital camera keep- and SVM-based clas-
(Published in 2018) Features used: First- ing uniform bright- sification gave 88%
order features ness and a constant
Haar wavelet based distance of two feet.
GLCM features All images were in
PNG format. Totally,
two hundred images
are captured:
50 healthy
50 common rust
50 northern leaf blight
50 images with both
diseases present in it
(continued)

Quality detection of Linear Discriminant Data was collected As a prediction
pomegranate fruit Analysis (LDA), Back after screen 100 pome- method, Back propa-
infected with fungal Propagation Neural granates of ‘Robab’ gation neural net-
disease [9] Network (BPNN) and genotype, similar in work showed higher
(Published in 2020) Support Vector size and shape. Based accuracy of 100% in
Machine (SVM) on the amount of the detection of 0%,
methods were applied infestation samples 25%, 50%, 75%, and
and compared as lin- were divided into five 100% infected
ear and non-linear groups including pomegranates
analysis methods for 0 (healthy samples),
detection 25%, 50%, 75%, and
Features used: 100% infected with
Metal Oxide Semi- Alternaria specie of
conductor (MOS) bio- fungi
sensors data, which
watch and record the
changes in different
food products during
storage time and
assessment of shelf
life and spoilage
Fungal rust disease The fungal disease The leaves samples Finally, support vec-
detection in Pea Plant symptoms were seg- were collected from tor machine classifier
(Pisam sativam) [18] mented using Gauss- the field and prepared yielded an accuracy
(published in 2018) ian filters, long for the dataset of 89.60% to detect
transform and the leaf disease of pea
2D-discrete wavelet plant
transform used for
feature extraction of
infected leaves
Features used:
Discrete wavelet
transform which is a
technique for two
dimensional decom-
position of image with
respect to Haar
wavelet
Volatomic visualiza- Chemometric Fungal infected rice All methods achieved
tion for fungal infec- methods including was used in this study. quite good and robust
tion detection on PCA, LDA, SVM, The number of yeast performance
storage Jasmine [25] and PLS regression and mould in brown
(Published in 2020) were used to analyze rice before inoculation
the complicated data was less than2 log
qualitatively and CFU/g. A
quantitatively from non-pathogenic asper-
the electronic sensor gillus strain was used
array to avoid the occur-
Features used: rence of mycotoxins
Volatomic profiles in the laboratory envi-
and volatile metabolic ronment which can be
(continued)
132 S. Modak et al.

compounds of fungal harmful to students
infected Jasmine and researchers in the
brown rice laboratory
Rapid discrimination Fungal Species classi- Deployment of lens Rapid identification
of fungal species by fication using SVM free imaging system to and fingerprinting
the colony finger- and Random Forest capture micro sized within 2 days and
printing [27] Features used: fungi colonies faster than MALDI-
(Published in 2019) Hyphae and their TOF-MS. and related
branches count and methods
distribution of inten-
sity across the hyphae
on the lens-less
images
SVM classifier based The diseased region is Images data was gath- Classification accu-
grape leaf disease found using segmen- ered of healthy leaves racy of 88.89% was
detection [31] tation by K-means and leaves infected by achieved for exam-
(Published in 2016) clustering, then both Powdery Mildew and ined diseases
colour and texture Downy Milde
features are extracted.
Finally, the classifica-
tion technique is used
to detect the type of
leaf disease
Features used: Nine
texture features-
contrast uniformity
maximum probability
homogeneity diagonal
variance difference
variance entropy
inverse difference and
nine colour features
Hyphae Detection in SVM model was used 183 normal images With SVM classi-
Fungal Keratitis to classify the normal and 195 abnormal fiers, texture analysis
Images [35] and abnormal images images, which were methods can achieve
(Published in 2018) Features used: Adap- collected in vivo using accuracies more than
tive Robust Binary Heidelberg engineer- 90%
Pattern (ARBP) ing HRT-3 confocal
microscopy
Plant Diseases classi- SVM and ANN clas- 900 sample images: SVM yielded maxi-
fication Using SVM sifiers 150 samples of each mum accuracy of
and ANN [36] Features used: Colour class mentioned 98% using colour and
(Published in 2016) features based on below: Fungal, bacte- texture features
RGB, HSI colour rial, viral, nematodes,
models and texture deficiency and normal
features based on (not affected)
Gray level
cooccurrence matrices
(GLCM)
(continued)

A hybrid combination Hybrid combination Images were gathered Overall accuracy of
of multiple SVM of three SVM classi- based on two catego- 93.9% for disease
classifiers for auto- fiers: A serial combi- ries: Symptoms: recognition was seen
matic recognition of nation of two (a) early blight,
the damages and classifiers, which is (b) late blight, and
symptoms on plant used in parallel with (c) powdery mildew
leaves [40] an individual classifier Damages: (d) leaf
(published in 2016) Features used: Tex- miners, (e) Thrips, and
ture, colour, and shape (f) Tuta abosluta
of plant leaves
Hyperspectral imag- Kernel function of The spectral data and Colony forming units
ing enabled electronic SVM was radial basis images of strawberries were effectively
nose for fungal con- function (RBF) and were acquired using a found with a correla-
tamination identifica- important parameters lab-scale visible/near- tion coefficient value
tion in strawberries ‘cost’ and ‘λ’ were infrared HSI system of 0.925
[26] optimized by the grid
(Published in 2019) search procedure
Features used: Spec-
tral profiles
Fungal adhesins and Naïve Bayes (NB), 75 fungal adhesins Obtained robust per-
adhesins like proteins SVM with sequential and 341 non-adhesin formance with leave
prediction [44] minimization optimi- protein sequences one out error 0f 5.9%
(Published in 2019) zation (implemented and test error of only
with Poly-K 2%
(SMO-PolyK), RBF
(SMO-RBF) and PuK
(SMO-PuK) kernels),
Bagging with
REPTree as the base
learner, Real
Adaboosting(RAB)
and Adaboosting
(AB) with decision
stumps as the base
learners
Features used:
Sequence-based and
evolutionary features:
Amino acid composi-
tion (AAC) Dipeptide
counts (DPC) Prop-
erty group composi-
tion (PGC)
Physicochemical-2-
grams (P2G) Atomic
composition (ATC)
Physicochemical
properties (PCP) Fea-
tures based on evolu-
tionary information
134 S. Modak et al.
6.5 Concluding Remarks
In this chapter, we have discussed in detail the utility in the usefulness of SVM in
fungal biology. We have highlighted the robustness of the algorithm in several
predictions and case studies. These include among others recognition of damage
and disease symptoms in plant leaves, detection of fungal infections, and contami-
nations in fruits prediction of antifungal peptides etc. In these recent applications, it
is evident that SVM has performed very well and can be used as a reliable and robust
paradigm not only in fungal biology but also in other bioinformatics applications.
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Chapter 7
Real-Time Quantitative PCR Assay
for the Assessment of Uncultured Zoosporic
Fungi
Télesphore Sime-Ngando and Marlène Jobard
Contents
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.3.1 DNA Extraction and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.3.2 Real-Time qPCR Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
7.1 Introduction
Molecular surveys of microbial eukaryotes have revealed overlooked uncultured

environmental fungi with novel putative functions [1–3], among which zoosporic
forms (i.e., chytrids) are the most important in terms of diversity, abundance, and
functional roles, primarily as infective parasites of phytoplankton [4, 5] and as
valuable food sources for zooplankton via massive zoospore production, particularly
in freshwater lakes [6–8]. However, due to their small size (2–5 μm), their lack of
distinctive morphological features, and their phylogenetic position, traditional
microscopic methods are not sensitive enough to detect fungal zoospores among a
mixed assemblage of microorganisms. Most chytrids occupy the most basal branch
of the kingdom Fungi, a finding consistent with choanoflagellate-like ancestors
[9]. The above reasons may help explain why both infective (i.e., sporanges) and
disseminating (i.e., zoospores) life stages of chytrids have been misidentified in
T. Sime-Ngando (*) · M. Jobard

138 T. Sime-Ngando and M. Jobard
previous studies, respectively, as phagotrophic sessile flagellates (e.g.,

choanoflagellates) and as “small undetermined” cells. These cells often dominate
the abundance of free-living heterotrophic nanoflagellates (HNFs) and are consid-
ered the main bacterivores in aquatic microbial food webs [2, 10]. Their contribution
ranges from 10% to 90% of the total abundance of HNFs in pelagic systems (see
review in [11]). Preliminary data have provided that up to 60% of these unidentified
HNFs can correspond to fungal zoospores [12], establishing the HNF compartment
as a black box in the context of microbial food web dynamics [4]. A simulation
analysis based on Lake Biwa (Japan) inverse model indicated that the presence of
zoosporic fungi leads to (i) an enhancement of the trophic efficiency index, (ii) a
decrease of the ratio detritivory/herbivory, (iii) a decrease of the percentage of
carbon flowing in cyclic pathways, and (iv) an increase in the relative ascendency
(indicates trophic pathways more specialized and less redundant) of the system
[13]. Unfortunately, because a specific methodology for their detection is not
available, quantitative data on zoosporic fungi are missing.
Molecular approaches have profoundly changed our view of eukaryotic microbial
diversity, providing new perspectives for future ecological studies [3]. Among these
perspectives, linking cell identity to abundance and biomass estimates is highly
important for studies on carbon flows and the related biogeochemical cycles in
natural ecosystems [11]. Historically, taxonomic identification and estimation of in
situ abundances of small microorganisms have been difficult. In this context, our
inability to identify and count many of these small species in the natural environment
limits our understanding of their ecological significance. Thus, new tools that
combine both identification and quantification need to be developed. Fluorescent
in situ hybridization (FISH) has been an assay of choice for simultaneous identifi-
cation and quantification of specific microbial populations in natural environments
[14, 15]. However, this technique is limited because of (i) the relatively low number
of samples that can be processed at a time and (ii) its relatively low sensitivity due to
background noise and the potentially low number of target rRNA molecules per cell
in natural environments [16]. In contrast, real-time quantitative PCR (qPCR) which
has been widely used to estimate prokaryotic and eukaryotic population abundances
in natural ecosystems, allows the simultaneous analysis of a high number of samples
with a high degree of sensitivity [15].
format, a qPCR assay for the quantitative assessment of uncultured zoosporic fungi
and other zoosporic microbial eukaryotes in natural environments (cf. [15]), together
with practical advice on how to apply the method. QPCR was recently used to
estimate fungal biomass in a stream during leaf decomposition [17] and in biological
soil crusts [18]. The interpretation of the semiquantitative data obtained in these
studies was relatively difficult because the whole fungal community was targeted
(including unicellular, multicellular, and multinuclear fungal species). Thus, an
estimation of fungal density or even fungal biomass was not possible. In the
following protocol, the primary targets are zoospores in liquid suspensions. Because
zoospores are unicellular, qPCR data could be directly converted into cell density
estimates, i.e., by multiplying semiquantitative data by the number of rDNA copies
7 Real-Time Quantitative PCR Assay for the Assessment of Uncultured. . . 139
per cell. Moreover, we designed primers targeting Rhizophidiales taxon to limit

quantification bias generated by the variability in the number of rDNA copies within
the eukaryotic ribosomal operon.
7.2 Materials
1. Gloves (should be worn when manipulating most of the following materials).

2. Sterile distilled water.
3. 0.6 μm pore size polycarbonate filters.
4. Filtration columns.
5. Sodium dodecyl sulfate (SDS).
6. Proteinase K.
7. TE buffer – 10 mM Tris–HCl pH 7.5 (25 C), 1 mM EDTA.
8. NucleoSpin Plant kit® (Macherey-Nagel) with silica-membrane columns and
the materials for running the provided protocol from the manufacturer.
9. Molecular-biology-grade agarose.
10. Ethidium bromide: because suspected as a mutagen, particular care should be
taken when handling (consult safety data sheet).
11. Calf thymus DNA (Sigma).
12. Oligonucleotidic primers resuspended in sterile distilled water and stored at
20 C (see Note 1).
13. SYBR Green (Sigma).
14. dNTPs: a mixture of dATP, dCTP, dGTP, and dTTP (10 mM of each), stored at
20 C.
15. Thermostable DNA polymerase and reaction buffer supplied by the manufac-
turer. To avoid nonspecific amplicon, use hot-start (e.g., HotStarTaq, Qiagen).
16. Vortexer.
17. Centrifuge.
18. Water bath.
19. Horizontal electrophoresis machine.
20. TBE buffer: 50 mM Tris, 50 mM boric acid, 1 mM EDTA, diluted when needed
from a 50x stock solution.
21. Spectrophotometer – we use NanoDrop (NanoDrop Technologies, Inc.,
Wilmington, USA).
22. Disposable conical tubes (1.5 ml); PCR tube strips or plate with adhesive film
and cap adapted for real-time quantitative PCR assay.
23. Thermal Cycler – we use e.g., Mastercycler ep realplex detection system
(Eppendorf).
24. UV transilluminator equipped with a camera suitable for photographing
agarose gels.
7.3 Methods
7.3.1 DNA Extraction and Purification
Collect zoosporic organisms onto 0.6 μm pore size polycarbonate filters (after the
removal of the algal host by prefiltration when only zoospores are targeted) (see Note
2).
1. For cell disruption, incubate the filters in 560 μl of a buffer containing 1% SDS
and 1 mg.ml1 proteinase K in TE buffer for 1 h at 37 C in a water bath (see
Notes 3 and 4).
2. For DNA purification, use the silica-membrane columns provided with the
NucleoSpin Plant kit® (Macherey-Nagel), following the instructions from the
manufacturer (see Note 4).
3. Visualized the integrity and yield of the extracted genomic DNA in a 1% agarose
gel stained with 0.3 μg ml1 of ethidium bromide solution (Sigma), using a UV
transilluminator and a photograph. For this, (i) heat (45 s using a microwave
oven) a mixture of agarose in 1x TBE buffer, (ii) leave it to cool on the bench for
5 min down to about 60 C before adding ethidium bromide (i.e., to avoid vapor
formation), (iii) mix and pour into suitable gel grey with a comb and leave to set
for at least 30 min, (iv) remove the comb and submerge the gel to 2–5 mm depth
in an electrophoresis tank containing 1x TBE buffer, (v) transfer DNA sample
aliquots (i.e., 2 volumes of sample and 1 volume of loading buffer), marker, and
the serial dilution of 5–10 ng of calf thymus DNA (Sigma) to the wells of the
agarose gel, and (vi) start the electrophoresis migration for about 30 min at 100 V.
4. Calculate DNA extract concentrations from dilutions of calf thymus DNA
(Sigma), using a standard curve of calf thymus DNA versus band intensity.
7.3.2 Real-Time qPCR Assays
1. PCR mix contained SYBR Green (Sigma), 200 μM of each dNTPs, 10 pM of

each primer, 2.5 units of Taq DNA polymerase, the PCR buffer supplied with the
enzyme, and 1.5 mM MgCl2. Vortex briefly (less than 10 sec) and centrifuge the
mix before distributing aliquots in suitable PCR tubes (strips or plates) and place
on ice.
2. Add variable quantity of DNA (we used 5 ng for our environmental freshwater
samples and 10 ng for DNA from appropriate PCR negative control strains) used
as template in a final volume of 25 μl (see Note 5).
3. Standard curve of Ct (see Note 4) versus DNA copy number required to calculate
target copy numbers (see Note 6) in each reaction is generated using triplicates of
PCR reactions of tenfold dilutions of linear plasmid (containing Rhizophidiales
18S rDNA insert; PFB11AU2004) ranging from 100 to 1 108 copy.μl1
(See Note 7). This number of copies was calculated using the equation:
molecules.μl1 ¼ a / (b x 660) x 6.022.1023, where a is the plasmid DNA

concentration (g.μl1), b the plasmid length in bp, including the vector and the
inserted 18S rDNA fragment, 660 the average molecular weight of one base pair,
and 6.022.1023 the Avogadro constant [15, 19].
4. Place all tubes (i.e., samples, controls, and standards) in the real-time qPCR
cycler and run the appropriate cycling program: initial HotStarTaq activation
at 95 C for 15 min, 35 cycles with denaturation at 95 C for 1 min, annealing at
63.3 C for 30 sec with Fchyt/Rchyt primers pair (See Note 1), elongation at
72 C for 1 min, and a final additional elongation step at 72 C for 10 min.
5. Using SYBR Green molecule, melting curves analysis can be performed imme-
diately following each qPCR assay to check the specificity of amplification
products (to confirm the absence of primer dimers or unspecific PCR products)
by increasing the incubation temperature from 50 to 95 C for 20 min.
6. Analyze the real-time PCR result with suitable software. Check to see if there is
any bimodal dissociation curve or abnormal amplification plot (see Note 5) before
calculating the initial concentration of the targeted uncultured fungal 18S rDNA
(copies.ml1) in the environmental genomic DNA (see Note 6).
7.4 Notes
1. Consensus (universal) primers can be used to amplify regions of fungal ribosomal

RNA genes. For natural waters, we have designed primers specific to chytrids
using a database containing about a hundred 18S rDNA environmental sequences
recovered from surveys conducted in different lakes and sequences belonging to
described fungi (cf. 15). Sequences were aligned using BioEdit software (http://
www.mbio.ncsu.edu/BioEdit/bioedit.html), and the resulting alignment was
corrected manually. A great proportion of the environmental chytrid sequences
recovered from lakes was closely affiliated to the Rhizophidiales. Thus,
Rhizophidiales-specific primers F-Chyt (sequence 50 > 30 : GCAGGCTTACGC
TTGAATAC) and R-Chyt (sequence 50 > 30 : CATAAGGTGCCGAACAAGTC)
were designed in order to fulfill three requirements: (1) a GC content between
40% and 70%, (2) a melting temperature (Tm) similar for both primers and close
to 60 C, and (3) PCR products below 500 bp (i.e., between 304 and 313 bp
depending on the species considered). The absence of potential complementar-
ities (hairpins and dimers) was checked using Netprimer (http://www.
premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html) and confirmed by
inspection of the melting curve following the qPCR assay.
2. For targeting uncultured zoosporic fungi, zoospores are discarded from other
environmental microorganisms by successive prefiltrations through 150, 80, 50,
25, 10, and 5 μm filters before being collected onto 0.6 μm polycarbonate filters.
Filters can be conserved at 80 C until DNA extraction in appropriate tubes
(2 ml).
3. Other enzymes such as lyticase can be used for cell disruption, with no significant
difference compared to proteinase K. However, the one-step proteinase K yields a
higher amount of genomic DNA than the lyticase method and has a better
reproducibility. Physical disruption procedures such as sonication or thermal
shocks (i.e., freezing in liquid nitrogen and thawing) are to be avoided because
of the possible degradation of DNA.
4. A standard phenol–chloroform purification procedure can also be used, but when
the genomic DNA extracts are used as templates in PCR reactions, the DNA
purification method using the commercial kit gave significantly better results
(based on Ct, the threshold cycle during PCR when the level of fluorescence gives
signal over the background and is in the linear portion of the amplified curve) than
the phenol–chloroform method. Consequently, the DNA extraction method using
Proteinase K and the commercial kit was selected and considered the best overall.
5. In the case of novel designed primers (see Note 1) for uncultured fungi, DNA
from both positive and negative plasmids and different mixtures (e.g., 5%, 10%,
25%, and 50% of the positive plasmids) will be used for the optimization of the
conditions (annealing temperature, cycling), cross-reactivity, the detection limit
(using serial tenfold dilutions of the positive plasmids, see Note 7), and the
amplification efficiency of the qPCR essays which should be at least 80%. Poor
primer quality is the leading cause of poor PCR efficiency. In this case, the PCR
amplification curve usually reaches a plateau early, and the final fluorescence
intensity is significantly lower than that of most other PCRs. This problem may be
solved with resynthesized primers.
6. The initial concentration of target 18S rDNA (copies.ml1) in environmental
samples can be calculated using the formula: [(a/b) x c] / d, where a is the 18S
rDNA copy number estimated by qPCR, b is the volume of environmental
genomic DNA added in the qPCR reaction, c is the volume into which the
environmental genomic DNA was resuspended at the end of the DNA extraction,
and d is the volume of sample filtered from which environmental DNA was
extracted.
7. In the absence of cultures, plasmids used in qPCR to construct standard curves
and to optimize qPCR reactions come from genetic libraries constructed during
previous environmental surveys [2]. Briefly, the complete 18S rRNA gene was
amplified from environmental genomic DNA extracts using the universal eukary-
ote primers 1f and 1520r. An aliquot of PCR products was cloned using the
TOPO-TA cloning kit (Invitrogen) following the manufacturer’s recommenda-
tions. The plasmid containing the insert of interest was extracted with
NucleoSpin® plasmid extraction kit (Macherey Nagel) following the manufac-
turer’s recommendations. The 18S rRNA gene was sequenced from plasmid
products by the MWG Biotech services using M13 universal primers [M13rev
(29) and M13uni (21)]. Phylogenetic affiliation of sequences acquired was
established using neighbor joining and Bayesian methods. In our case, positive
plasmids contain insert affiliated to target chytrid (i.e., Rhizophidiales species)
and displaying less than 2 mismatches with primers F-Chyt and R-Chyt
sequences (see Note 1). The plasmid PFB11AU2004 (Genbank accession number
DQ244014) was selected to construct the standard curve required for qPCR.
Linearized plasmids were produced from supercoiled plasmids by digestion
with restriction endonuclease one-time cutting into the vector sequence. Linear
plasmid DNA concentration can be determined by measuring the absorbance at
260 nm (A260) in a spectrophotometer.
Acknowledgements MJ was supported by a Ph.D. Fellowship from the Grand Duché du Luxem-
bourg (Ministry of Culture, High School, and Research). This study receives grant-aided support
from the French ANR Programme Blanc # ANR 07 BLAN 0370 titled DREP: Diversity and Roles
of Eumycetes in the Pelagos.
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Chapter 8
Assays for the Quantification of Antioxidant
Enzymes in Fungi
Konstantinos Grintzalis, Ioannis Papapostolou, and Christos D. Georgiou
Contents
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
8.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
8.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
8.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
8.3.1 Fungal Tissue Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
8.4 Protein Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
8.5 Antioxidant Enzymes Related to the Decomposition of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
8.5.1 Catalase (CAT) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
8.5.2 Peroxidases (Px) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
8.5.3 Superoxide Dismutase (SOD) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
8.6 Thiol Redox State Related Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
8.6.1 Glutathione-S-Transferase (GST) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
8.6.2 Glutathione Reductase (GR) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
8.6.3 Glutathione Peroxidase (GP) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
8.6.4 Glucose-6-Phosphate Dehydrogenase (G6PD) Activity . . . . . . . . . . . . . . . . . . . . . . . . . . 155
8.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
K. Grintzalis (*)
School of Biotechnology, Dublin City University, Dublin, Republic of Ireland
e-mail: konstantinos.gkrintzalis@dcu.ie
I. Papapostolou · C. D. Georgiou
Department of Biology, Section of Genetics, Cell Biology and Development, University of
Patras, Patras, Greece
146 K. Grintzalis et al.
8.1 Introduction
Molecular oxygen (Ο2) was introduced to the anoxic atmosphere as a by-product of

photosynthesis from cyanobacteria. Additionally, Ο2 generation allowed organisms
to settle on the land by providing a protective filter from ultraviolet radiation, which
is known as the ozone layer. In general, organisms employed Ο2 in their metabolism
and oxidative metabolism could be considered an evolutionary advantage of aerobic
life, mainly because it yields a higher amount of ATP per catabolized sugar molecule
via the glycolysis, Kerbs Cycle, and oxidative phosphorylation pathways. However,
there is a detrimental side effect of oxidative metabolism, which is the generation of
reactive oxygen and nitrogen species (ROS and RNS, respectively) [1]. In physio-
logical conditions, the concentration of ROS/RNS is maintained low by endogenous
antioxidants and antioxidant enzymes, while the condition when these defenses fail
to decompose ROS/RNS is referred to as oxidative stress. Oxidative stress has been
implicated in several physiological phenomena such as aging, fungal metamorphosis
[2], and pathological conditions (i.e., neurodegenerative diseases) [3].
Antioxidant enzymes are important parameters of the cellular antioxidant defense
mechanisms, and their role focuses on (i) the removal of ROS, (ii) the detoxification
of xenobiotic substances, and (iii) the repair of cellular damages (i.e., regeneration of
reduced thiols and reducing power). In relation to the elimination of ROS, the
primary component of oxidative stress is superoxide radical (O2˙¯). The enzyme
that specifically scavenges this reactive species, superoxide dismutase (SOD), was
discovered by Fridovich [4]. SODs are highly conserved enzymes that rely on
manganese, zinc, iron, or nickel [5] to catalyze the dismutation reaction of two
O2˙¯ molecules to generate one O2 molecule and one molecule of hydrogen peroxide
(H2O2). Hydrogen peroxide is well known for its signaling role as well as being a
radical initiator via the metal-catalyzed Fenton reaction, which effectively leads to
the generation of hydroxyl radical, another potent ROS. Therefore, scavenging H2O2
is another major task for the enzymatic antioxidant defense, and this is achieved by
the catalase enzyme, which reduces one molecule of H2O2 to water and oxidizes a
second one to O2. Another category of enzymes that decompose H2O2 and organic
hydroperoxides, in general, are the peroxidases, which consume reducing power
from a proton donor. Catalases and peroxidases are also conserved across species,
and their activity relies on metal catalytic centers.
As mentioned earlier, oxidative stress is not only mediated by the generation of
ROS but also by imbalances triggered in relation to redox balances such as the status
of thiol groups. This balance is better known as the thiol redox state, and when
oxidized moieties overpower the reduced forms, antioxidant enzymes may replenish
their reduced forms at the expense of reducing power. Consumption of reduced
thiols (i.e., glutathione, cysteine) may be related to detoxification of toxicants by
glutathione-S-transferases, which inactivate xenobiotics by coupling them with
glutathione or via glutathione peroxidases which consume glutathione as reducing
power to decompose hydrogen and other peroxides. This expense of reduced
glutathione is regenerated by glutathione reductase which consumes NADPH
8 Assays for the Quantification of Antioxidant Enzymes in Fungi 147
.-
2O2 + 2H+ SOD O2 + H2O2
2H2O2 CAT O2 + H2O
H2O2 + Proton donor PX H2O + Proton acceptor
H2O2 + GSH GP H2O + GSSG
NAD(P)H + 2H+ + GSSG GR NAD(P)+ + 2GSH
G6P + NAD(P)+ G6PD NAD(P)H + 2H+ + Lactone
Xenobiotic + GSH GST Xenobiotic-GSH
Fig. 8.1 Reactions catalyzed by antioxidant enzymes. SOD: superoxide dismutase, CAT:
catalase, PX: peroxidase, GP: glutathione peroxidase, GR: glutathione reductase, G6PD: glucose-
6-phosphate dehydrogenase, GST: glutathione-S-transferase
which in turn is regenerated by glucose-6-phosphate dehydrogenase an enzyme of

the pentose phosphate pathway.
All the aforementioned network of enzymatic reactions reveals the complexity
(Fig. 8.1) of enzymatic defense mechanisms and the necessity of specific protocols
to quantify all these parameters. This chapter will present simple biochemical
methods for their accurate quantification and discuss the impact of oxidative stress
in fungal physiology. Additionally, an initial protocol for homogenization and
protein quantification of fungal tissues has also been included.
8.2 Materials and Methods
8.2.1 Materials
3-amino-1,2,4-triazole, ammonium sulfate, bovine serum albumin (BSA),

Coomassie Brilliant Blue G-250 (CBB), cummene hydroperoxide (CumOOH),
glucose-6-phosphate, disodium hydrogen phosphate (Νa2ΗPO4), ethanol,
ethylenediaminetetraacetic acid (EDTA), hydrochloric acid (HCl), horseradish per-
oxidase, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), NADH,
NADPH, NAD+, NADP+, o-dianisidine, oxidized glutathione (GSSG), reduced
glutathione (GSH), riboflavin, phenylmethylsulfonyl fluoride.
8.3 Methods
8.3.1 Fungal Tissue Homogenization
In general, for biochemical assays, it is preferred to avoid substances that may

interfere in the assays to be performed afterward. Therefore, avoiding detergents,
thiol reductants (e.g., β-mercaptoethanol or dithiothreitol), metals, organic solvents,
acids, or keeping their concentrations to the minimum is advised. Using metal
chelators, such as EDTA (at 1 mM) and protease inhibitors (e.g., 0.5 mM
phenylmethylsulfonyl fluoride; PMSF), are also potential precautions for the pres-
ervation of the samples to avoid artificial oxidation. A phosphate (50 mM) buffer,
pH 7.2, supplemented with the aforementioned is a preferable homogenization
buffer. Furthermore, hard fungal tissues contain rigid cell walls and are preferably
first homogenized in liquid nitrogen (e.g., using a mortar and a pestle) to generate a
fine powder form. Following, phosphate buffer is added, and the tissues are homog-
enized on ice using a Potter-Elvehjem type glass-glass or a Teflon homogenizer or
any other mechanical disruption available. The crude homogenate obtained needs to
be cleaned from cellular debris by centrifugation at 20,000 g for 10 min at 4 C, and
the clear supernatant is collected for analysis. At this step, it is possible to concen-
trate proteins by precipitation. In this case, proteins are precipitated in their native
(functional) form with ammonium sulfate by bringing the clear supernatant to 90%
ammonium sulfate (by dissolving solid ammonium sulfate) and incubating overnight
at 0 C. Proteins are precipitated by centrifugation at 20,000 g for 10 min at 4 C and
solubilized in a minimum volume of buffer or water. If needed, ammonium sulfate
can be removed by dialysis in another step.
Homogenization buffer: 50 mM Νa2ΗPO4, 1 mM EDTA, 500 μM PMSF, 0.5%
ethanol, pH 7.2: Dissolve 0.73 g Νa2ΗPO4.2H2O, 0.03 g EDTA, in 80 ml
ddH2O and adjust pH to 7.2. Dissolve 7 mg PMSF in 0.4 ml ethanol and add it
to the phosphate buffer solution.
8.4 Protein Quantification
The assay is based on the electrostatic reaction of proteins with the Coomassie
Brilliant Blue G-250 (CBB) reagent and is presented in a rapid sensitive microplate
method [6].
2 M HCl: Dilute the 37% (or 12 M) HCl six-fold with ddH2O, by mixing 250 ml
ddH2O with 50 ml 12 M HCl under stirring.
CBB: Dissolve 60 mg CBB in 100 ml 2 M HCl. After stirring for 40 min, centrifuge
at 20,000 g for 5 min at room temperature or alternatively, filter under vacuum or
with a syringe to remove any undissolved dye particulates. This solution is stable
for 2 months kept light-protected.
CBB:2 M HCl (1:1): Prepare fresh by mixing 1 volume of CBB with 1 volume of
2 M HCl.
Bovine serum albumin (BSA): Prepare a stock of 1 mg BSA/ml ddH2O and dilute
with ddH2O to 20 μg BSA/ml. Prepare a series of dilutions of standards for the
linear standard curve according to Table 8.1.
Samples (and standards, Table 8.1) are assayed for protein concentration
according to Table 8.2.
Table 8.1 BSA linear standard curve. The volumes are in μl

BSA final μg/ml 2 4 6 8 10 12 14 16 18 20
20 μg BSA/ml 50 100 150 200 250 300 350 400 450 500
ddH2O 450 400 350 300 250 200 150 100 50 0
Table 8.2 Protein quantification assay conditions. The volumes are in μl

Reagents RB S
Sample appropriately diluted in ddH2O or BSA standard in ddH2O – 200
ddH2O 200 –
CBB:2 M HCl 50
Incubate mixtures for 10 minutes at room temperature and measure absorbance at

610 nm. The net absorbance is derived from the absorbance difference of Sample
(S) minus Reagent Blank (RB), and the following is converted to protein (BSA)
concentration equivalents using the corresponding standard curve (Table 8.1).
8.5 Antioxidant Enzymes Related to the Decomposition

of ROS
8.5.1 Catalase (CAT) Activity
The activity of catalases is measured by kinetics for either the decomposition of

hydrogen peroxide photometrically or the production of oxygen using an
oxygenometer [7]. For confirmation that the measured kinetics is a result of only
catalase activity, its specific inhibitor aminotriazole can also be included as an
additional control (not showing any kinetic change) for the oxygenometric measure-
ments (since aminotriazole absorbs).
50 mM Νa2ΗPO4 pH 7.2: Dissolve 0.71 g Νa2ΗPO4 in a final volume of 80 ml and
adjust pH at 7.2.
5xH2O2: Dilute the concentrated 11 M (or 30%) with phosphate buffer appropriately
in order to have an absorbance of ~0.3 when using 50 μl in 250 μl reaction
volume. Use this solution for the photometric version as most spectrophotometers
have a linear absorbance range to perform kinetics to monitor even small changes.
2 mM H2O2: Dilute the concentrated 11 M (or 30%) solution to 2 mM with
phosphate buffer. Use this solution for the oxygenometer version.
0.4 M 3-amino-1,2,4-triazole: Prepare fresh by dissolving 33.6 g aminotriazole in
1 ml phosphate buffer.
For the photometric version, analyze samples by continuous kinetics for the decom-
position of hydrogen peroxide follow Table 8.3.
Table 8.3 Photometric quantification of catalase activity. The volumes are in μl

Sample appropriately diluted in 50 mM phosphate buffer, pH 7.2 200
H2O2 50
Table 8.4 o-dianisidine standards. The volumes are in μl

o-dianisidine final μM 1 2 3 4 5 6 7 8 9 10
μl from 10 μM o-dianisidine 50 100 150 200 250 300 350 400 450 500
stock
50 mM phosphate buffer, 450 400 350 300 250 200 150 100 50 0
pH 7.2
Incubate at room temperature and measure absorbance at 340 nm with continuous

kinetics to calculate the rate. The final result will be expressed as the rate per mg
protein of the sample. When performing the assay photometrically, the production of
oxygen will generate bubbles which will account for absorbance, therefore, only the
linear part of the measurements should be used. Finally, for the oxygenometer
version, catalase activity can be also confirmed by its specific inhibitor aminotriazole
by preincubation with 10 mM aminotriazole before the addition of hydrogen perox-
ide to initiate the reaction and measure oxygen production.
8.5.2 Peroxidases (Px) Activity
Nonspecific peroxidase activity is measured by the production of a colorful oxidized

product resulting from the oxidation of its reduced form upon consumption of
hydrogen peroxide. There are several substrates for peroxidases, and here, we
present a photometric method based on the enzymatic oxidation of o-dianisidine [7].
adjust pH at 7.2.
10 mM o-dianisidine: Prepare fresh by dissolving 3.2 mg o-dianisidine in 1 ml
phosphate buffer. The solution must be kept light protected because o-dianisidine
is photosensitive. Prepare a series of dilutions of standards for the linear standard
curve according to Table 8.4.5 mM H2O2: Dilute the concentrated 11 M (30%)
solution to 5 mM with phosphate buffer.
Horseradish peroxidase: Dissolve 1 mg in 5 ml and use it to create a standard curve
of oxidized o-dianisidine.
Perform the reaction with HRP to generate the standard curve of o-dianisidine as
follows (Table 8.5):
Table 8.5 o-dianisidine linear standard curve. The volumes are in μl

Reagents RB St
50 mM phosphate buffer, pH 7.2 200 –
o-dianisidine standard in 50 mM phosphate buffer, pH 7.2 – 200
Horseradish peroxidase 25
5 mM H2O2 25
Table 8.6 Peroxidase activity assay. The volumes are in μl

5 mM H2O2 25
10 mM o-dianisidine 25
3 O2 1
O2
RF*
O2.-
==SOD
Dred Dox (500 nm)
hv
hv
1
RF RF*
Fig. 8.2 Mechanism of SOD-inhibited reduction of oxidized o-dianisidine and light apparatus used
when volumes are scaled up for cuvettes
Incubate at room temperature for 15 min and measure absorbance at 560 nm.
Express the standard curve in moles of oxidized o-dianisidine acid in 200 μl sample
volume.
For analyzing samples, use end point kinetics (at 15 minutes’ incubation) follow-
ing Table 8.6.
Incubate at room temperature for 15 min and measure absorbance at 560 nm.
Calculate the moles of oxidized o-dianisidine using the corresponding standard
curve and following calculate the rate. The final result will be expressed as a rate
per mg protein of the sample.
8.5.3 Superoxide Dismutase (SOD) Activity
Superoxide dismutase activity assays use a superoxide radical generator and a

scavenger molecule which is used to monitor the SOD-inhibitable scavenging
activity of the sample [8]. The assay presented is based on the SOD-inhibited
reduction of oxidized o-dianisidine. The latter results from the reaction of its reduced
form by the photochemically sensitized riboflavin. This reaction is in competition
with the reaction of the formation of reduced dianisidine from oxidized dianisidine
and superoxide radical generated photochemically by riboflavin (Fig. 8.2) [9]. Thus,
SOD increases the rate of the photooxidation of dianisidine due to the catalytic
Table 8.7 Superoxide dismutase activity assay. The volumes are in μl

Reagents B1 B2 SB S
Sample appropriately diluted in 50 mM phosphate buffer, pH 7.2 200 200
50 mM phosphate buffer, pH 7.2 225 200 25
10 μM riboflavin 25 25 25 25
10 mM o-dianisidine 25 25
scavenging of superoxide radicals that would, otherwise, nullify the overall

dianisidine photooxidation by reducing an intermediate dianisidine oxidation prod-
uct (absorbing at 500 nm).
adjust pH at 7.2.
10 mM o-dianisidine: Prepare fresh by dissolving 3.2 mg o-dianisidine in 1 ml
phosphate buffer. The solution must be kept light protected because o-dianisidine
is photosensitive.
10 μM riboflavin: Prepare fresh by a 60 μM solution by dissolving 1.2 mg
riboflavin in 50 ml phosphate buffer. Dilute the 60 μM solution six-fold with
ddH2O, by mixing 50 ml ddH2O with 10 ml 60 μM riboflavin.
For analyzing samples, use end point kinetics (after incubation for 5 minutes)
following Table 8.7.
Incubate at room temperature under light for 5 min and measure the absorbance of
the samples and blanks at 500 nm. The final sample net absorbance is derived from
the absorbance difference of Sample (S) minus Sample Blank (SB) and subtracted
the difference of Blank 2 (B2) minus Blank 1 (B1), and the following is converted to
moles of oxidized o-dianisidine using the corresponding standard curve (from the
peroxidase assay) and following the rate. The final result will be expressed as a rate
per mg protein of the sample.
8.6 Thiol Redox State Related Enzymes
8.6.1 Glutathione-S-Transferase (GST) Activity
Glutathione-S-transferases are enzymes that detoxify xenobiotic compounds by

coupling them with reduced glutathione (GSH) and their activity can be measured
by continuous kinetics of the product formed (S-DNP-GS) measured at 340 nm
(Fig. 8.3) [10, 11].
adjust pH at 7.2.
1.5 mM 1-chloro-2,4-dinitrobenzene (CDNB): Prepare fresh by dissolving 6.2 mg
CDNB in 1 ml ethanol. Dilute 30-fold to 1.5 mM with phosphate buffer.
Fig. 8.3 Kinetic determination of GST activity
Table 8.8 Glutathione-S-transferase activity assay. The volumes are in μl

750 μM CDNB 1.5 mM GSH 100
Fig. 8.4 Kinetic

determination of GR activity
3 mM glutathione (GSH): Prepare fresh by dissolving 1.9 mg GSH in 1 ml

phosphate buffer. Dilute two-fold to 3 mM with buffer.
0.75 mM CDNB:1.5 mM GSH (1:1): Prepare fresh by mixing equal volumes of
1.5 mM CDNB and 3 mM GSH.
Analyze samples by continuous kinetics for the production of S-DNP-GS fol-
lowing Table 8.8.
kinetics to calculate the rate. The final result will be expressed as a rate per mg
protein of the sample.
8.6.2 Glutathione Reductase (GR) Activity
Glutathione reductase activity is estimated by the decrease of NAD(P)H measured

by absorbance at 340 nm with continuous kinetics (Fig. 8.4) [12, 13].
adjust pH at 7.2.
5 mM oxidized glutathione (GSSG): Prepare fresh by dissolving 3.3 mg GSSG in
1 ml phosphate buffer.
NAD(P)H: Prepare fresh an NADH or NADPH stock in phosphate buffer. Dilute
appropriately in order to have an absorbance of ~0.3 when using 25 μl in 250 μl
reaction volume as most spectrophotometers have a linear absorbance range to
perform kinetics to monitor even small changes.
Analyze samples by continuous kinetics for the decomposition of NAD(P)H
Table 8.9 Glutathione reductase activity assay. The volumes are in μl

1 mM GSSG 25
NAD(P)H 25
Fig. 8.5 Kinetic determination of GR activity

8.6.3 Glutathione Peroxidase (GP) Activity
Glutathione peroxidase activity is measured by the decrease of NAD(P)H photo-

metrically at 340 nm by continuous kinetics. The assay is using as a detection of the
reaction catalyzed by glutathione reductase. Therefore, it is a system of two reactions
coupled together, where the latter is in excess rate, while the reaction catalyzed by
glutathione peroxidase regulates the system via the generation of GSSG by endog-
enous GP (Fig. 8.5) [13]. As there are two categories of enzymes with glutathione
peroxidase activity, those with selenium and those without selenium in their active
site, the assay detects the selenium GP when hydrogen peroxide is used as substrate
and the non-selenium GP when cummene hydroperoxide is used as substrate.
adjust pH at 7.2.
20 mM GSH: Prepare fresh by dissolving 6.3 mg GSH in 1 ml phosphate buffer.
20 mM sodium azide: Prepare fresh by dissolving 1.32 mg sodium azide in 1 ml
ddH2O. Sodium azide can be used to block possible existing catalase (if expected
to be present) in the sample when hydrogen peroxide is used as the substrate of
the reaction, while it is omitted when cummene hydroperoxide is used as
substrate.
1 mM cummene hydroperoxide (CumOOH): Dilute the concentrated solution of
5.5 M with ethanol to 10 mM. Dilute the 10 mM to 1 mM in phosphate buffer.
1 mM H2O2: Dilute the concentrated 11 M (30%) solution to 1 mM with phosphate
buffer.
4 units ml21 GSSG reductase: Prepare fresh by diluting the original stock reagent
100x with phosphate buffer.
NAD(P)H: Prepare fresh an NADH or NADPH stock. Dilute appropriately in order
to have an absorbance of ~0.3 when using 25 μl in 250 μl reaction volume (this is
Table 8.10 Glutathione peroxidase activity assay. The volumes are in μl

20 mM GSH 25
20 mM sodium azide/H2O 20
1 mM H2O2/CumOOH 25
GSSG reductase 30
NAD(P)H 25
Fig. 8.6 Kinetic

determination of G6PD
activity
Table 8.11 Glucose-6-phosphate dehydrogenase activity assay. The volumes are in μl

4 mM NAD(P)+ 25
10 mM glucose-6-phosphate 25
to allow you to perform kinetics and monitor even small changes in the linear part
of your spectrophotometer).
Analyse samples by continuous kinetics for the decomposition of NAD(P)H
8.6.4 Glucose-6-Phosphate Dehydrogenase (G6PD) Activity
Glucose-6-phosphate dehydrogenase activity is quantified by the generation of

reducing power in the form of NAD(P)H by continuous kinetics at 340 nm from
the reduction of NAD(P)+ and glucose-6-phosphate (Fig. 8.6) [14].
adjust pH at 7.2.
4 mM NADP+: Prepare fresh an NAD+ or NADP+ stock.
10 mM glucose-6-phosphate: Prepare fresh by dissolving 3.3 mg glucose-6-phos-
phate (304.31) in 1 ml phosphate buffer.
Analyze samples by continuous kinetics for the production of NAD(P)H follow-
ing Table 8.11.

8.7 Conclusions
The aforementioned protocols are optimized to be universal and can be applied to

any type of homogenate. In relation to fungal physiology, which is the interest of this
topic, we have previously demonstrated and provided substantial bibliography over
the impact of oxidative stress in the physiology of filamentous fungi [3]. In this
section, we discuss results we have obtained thus far based on these methods.
In a recent study, we explored the proliferating and differentiating roles of H2O2
by altering its levels in cultures of Sclerotium rolfsii and Sclerotinia sclerotiorum.
Hydrogen peroxide was administered extracellularly (in the media) or alternatively,
aminotriazole, the specific inhibitor of catalase, was added to the media to inhibit the
intracellular decomposition of H2O2 [15]. Results showed that the externally added
H2O2 caused an increase in endogenous catalase activity, while aminotriazole
administration resulted in a significant decrease of the enzyme. These changes
were also accompanied by an increase in proliferation in both fungal species
(by approximately 50%) and reduced the production of sclerotia, hence,
dedifferentiated the fungi. These results are in accordance with previous studies
where we compared the sclerotiogenic and non-sclerotiogenic mutants of the afore-
mentioned fungal species [16], which implies that a high concentration of intracel-
lular H2O2 is associated with sclerotial differentiation in filamentous fungi. We
verified this conclusion by another study showing that endogenous SOD activity
was higher in non-sclerotiogenic mutants [17] while using SOD mimetics (artifi-
cially increasing superoxide radical dismutation); we also decreased fungal sclerotial
differentiation [18]. In relation to the role of thiol redox state modulating enzymes,
we have extensively studied their impact on filamentous fungal growth. For
Sclerotinia sclerotiorum, we have detected a decrease in glutathione reductase as
the fungus differentiates. This finding was also accompanied by an increase in
oxidized thiols [19]. Additionally, administration of certain thiol redox state modu-
lators resulted in dedifferentiation of the tested fungi and resulted in changes of
certain thiol redox status parameters [20].
Acknowledgments This work was financially supported by the internal funds of Dublin City
University and the Greek Ministry of Education, University of Patras, Greece.
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18. Papapostolou I, Georgiou CD (2010c) Superoxide radical induces sclerotial differentiation in
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Chapter 9
Cellulomics of Live Yeast by Advanced
and Correlative Microscopy
Zinnat Shahina, Supriya V. Bhat, Easter Ndlovu, Taranum Sultana,

André Körnig, Étienne Dague, and Tanya E. S. Dahms
Contents
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
9.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
9.3 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
9.3.1 Preparing Coverslips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
9.3.2 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
9.3.3 Pros and Cons of Immobilisation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
9.4 Imaging Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
9.4.1 Imaging Fixed Cells in QI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
9.4.2 Imaging Live Cells in QI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
9.4.3 Tracking Dynamic Processes in QI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
9.4.4 Correlative AFM-QI-LSCM Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
9.5 AFM QI Mode Image/Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
9.5.1 AFM Image Processing and Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
9.1 Introduction
Atomic force microscopy (AFM) is a fundamental tool to investigate the morphol-

ogy and ultrastructural properties of biomaterials and cells at the micro-nanometer
scale, and to evaluate the real-time relationship between their physicochemical
properties and biological response. AFM is becoming one of the gold standards
for detailing biological interfaces, including the surfaces of microbes.
Z. Shahina · S. V. Bhat · E. Ndlovu · T. Sultana · A. Körnig · É. Dague · T. E. S. Dahms (*)

Department of Chemistry and Biochemistry, University of Regina, Regina, SK, Canada
e-mail: Shahinaz@uregina.ca; supriya.bhat@uregina.ca; ene234@uregina.ca;
taranum.sultana@uregina.ca; andre.koernig@bruker.com; edague@laas.fr;
Tanya.Dahms@uregina.ca
160 Z. Shahina et al.
AFM is capable of acquiring surface images with nanometer (nm) scale resolution
or any type of physical interaction between a sharp tip and a surface, for example,
mapping interaction forces from a few pN to hundreds of nN for probing single
molecule interactions to overall cellular mechanics, respectively. Ultrastructure,
roughness, viscoelasticity and macromolecular interaction forces measured by
AFM are informative parameters that describe biological characteristics and pro-
cesses such as cell morphology, adhesion, transport and motility, and signalling
[1–4].
Traditional modes of AFM data collection are tricky for challenging samples with
steep edges, as well as those that are fragile, soft, sticky, or loosely attached to the
surface [5, 6]. Recently developed AFM operating modes, like Quantitative Imag-
ing™ (QI), enables not only the study of sample surface architecture with nm-scale
resolution, but also the mapping mechanical and adhesive properties of a sample in
situ. QI uses pN forces to quickly image immobilised samples at high resolution,
resulting in force-curve data at each pixel that can be analysed by any model [7]. The
major advantage of QI is that it does not impart any lateral forces on the sample and
provides precise force control during scanning, preserving the integrity of soft
biological samples. QI also allows for kinetic studies on difficult biological samples
and is not limited by either sample geometry or environment. Any standard cantile-
ver can be used for QI. Very sensitive samples (i.e. biological cells) or the detection
of small interaction forces requires a low spring constant (0.01–0.6 N/m) cantilever.
In comparison with AFM imaging, QI can record up to 512 512 (262144) QI force
curves, which contain much more information than only surface topography. The
force curves can be further analysed to obtain additional information, for example,
stiffness, using the Heinrich Hertz formulated theory [8] or any other appropriate
model. Decreasing either the pixel resolution or imaging area reduces the time per
image, which enables the study of dynamic processes.
Recently, correlative AFM-QI and laser scanning confocal microscopy (LSCM)
has been used to develop a multiplexed assay of living cells (bacteria, fungi, human)
in response to a common herbicide, with broad application to other xenobiotics. In
addition to determining surface architecture, AFM in QI mode probes surfaces
biochemistry and cellular mechanics, while LSCM is a window into the cell to
simultaneously track fluorescently tagged macromolecules, as many as can be
optically separated. Thus, correlative AFM-QI-LSCM generates multiplexed data
for the extensive characterisation of specimens ranging from single molecules or
nanoparticles to living cells [9–11].
Sample preparation for AFM is governed by a combination of overall cell
morphology and cell wall surface characteristics. The dimorphic fungus
C. albicans, able to grow both as fission yeast and filamentous (hyphal) cells, is an
opportunistic pathogen, whereas the budding yeast, Saccharomyces cerevisiae, has
been domesticated for culinary uses (i.e. brewing and baking) [12]. We have
previously detailed methods for imaging Aspergillus nidulans hyphal cells [13], so
this chapter will focus on the fission yeast form of C. albicans and budding yeast
form of S. cerevisiae.
9 Cellulomics of Live Yeast by Advanced and Correlative Microscopy 161
S. cerevisiae is one of the most highly studied organisms based on its role in food
production, and the detailed research on its cell wall became the basis for cell wall
models of other yeast such as C. albicans [14]. Similar to S. cerevisiae, the cell wall
of C. albicans consists of three main components: mannoproteins (~ 39%),
β-glucans (~ 59%), and chitin (~ 2%), to which external cell wall proteins (CWPs)
are attached. The cell walls contribute to a highly dynamic molecular architecture,
which is continuously remodelled in response to cell surface interactions. Moreover,
the cell wall surface is decorated with homogenously dispersed mannoproteins
called adhesins that play pivotal roles in cell communication, adhesion and microbial
infection. Various factors such as cell wall stress, temperature variation, exposure to
antifungal agents, host interaction, and biofilm formation can alter the cell’s adhe-
sive properties [15–19].
This chapter will detail the methods required to successfully acquire AFM-QI and
AFM-QI-LSCM data for Candida and S. cerevisiae, including sample preparation
techniques and imaging.
9.2 Materials
Unless otherwise specified, all solvents and acids were reagent grade.
Acetone (HPLC Grade, Fisher Scientific).
Cantilever Probes for QI™-AFM (AppNano Hydra4V-100NG, spring constant
0.039–0.184 N/m, resonance frequency 31–58 kHz; Bruker MLCT probes, spring
constant 0.01–0.6 N/m, resonance frequencies 7–125 kHz; nanosensors qp-BioAC,
spring constant 0.06–0.3 N/m, resonance frequencies 30–90 kHz).
Cell-Tak (corning).
Deionised water (18 MΩ NanoPure water).
Glass coverslips (22 mm x 22 mm, Fisher Scientific for fixed cells; 18 mm x 18 mm,
Carl Zeiss™ cover glasses, high performance for live cells).
Hydrochloric acid (HCl) (EMD Millipore).
Hydrogen peroxide 30 wt % in H2O (H2O2, Sigma-Aldrich).
Kim wipes (Kimberly Clark).
Methanol (HPLC Grade, Fisher Scientific).
PDMS stamps (Laboratory for Analysis and Architecture of Systems) [20].
Petri dish (100 mm x 15 mm, VWR International).
Phosphate Buffered Saline (0.01 M PBS, Sigma Aldrich).
Sodium bicarbonate (Fisher Scientific).
Sodium hydroxide (Caledon laboratories).
Sulphuric acid (H2SO4) (Caledon laboratories).
PLL (Poly-L-lysine) (Sigma Aldrich).
Triton-X 100 (Sigma Aldrich).
YPD (Yeast, Peptone, Dextrose) medium (Sigma Aldrich).
9.3 Experimental Methods
9.3.1 Preparing Coverslips
The methods described below detail the preparation of materials required for
immobilising fixed and live cells, which is crucial for successful AFM imaging.
9.3.1.1 Cleaning Coverslips
Coverslips must be cleaned prior to coating.

1. Dip coverslips (22 22 mm) in 1 M HCl for 2 min.
2. Wash coverslips in deionised water and let air dry (it is helpful to have a rack that
separates the coverslips for processing many at a time).
3. Soak coverslips in Piranha solution (5 mL of 30% H2O2 + 15 mL of 18 H2SO4)
for 1 h.
4. Remove the coverslip, immerse and wash with a copious amount of deionised
water.
5. Dip coverslip in methanol for 2 min and air dry.
6. Dip coverslip in acetone for 2 min and air dry.
7. Store the clean coverslips in a dust-free container (Petri dish) until ready to coat.
9.3.1.2 Coating Coverslips
Poly-L-Lysine (PLL)
1. Make a 1:10 dilution of the PLL solution (1 part PLL to 9 parts deionised water).
2. Add 200 μl PLL onto the coverslip.
3. Incubate at room temperature for 30 min.
4. Wash twice with 200 μl deionised water.
5. Place the coverslip in a dust-free container and dry in a fume hood for 10 min or
let dry overnight (O/N) if convenient.
Cell-Tak
Coverslips were coated with Cell-Tak with a procedure slightly modified from
Louise Meyer et al. [21]
1. Prepare a solution of Cell-Tak by mixing 3.5 μl of Cell-Tak with 140 μl of buffer
(NaOH (1 M) and NaHCO3 (0.1 M pH 8)).
2. Spread 40 μl of the Cell-Tak solution onto a clean coverslip and incubate for
30 min at room temperature.
3. Wash 3 with deionised water (0.2–0.5 mL, depending on coverslip size) and
air dry.
Coated coverslips can be stored for up to 2 weeks at 4 C in a desiccator.
9.3.2 Sample Preparation
Samples can first be fixed or imaged live, but both require immobilisation.
9.3.2.1 Fixed Samples
1. Culture cells in YPD (Yeast, Peptone, Dextrose) O/N at 30 C in a shaker at

200 rpm (Candida) or 165 rpm (Saccharomyces).
2. Add 200 μl of the culture to PLL-coated coverslips and incubate at room
temperature for at least 30 min, up to 1 h.
3. Wash 3 times with 100 μl of phosphate buffered saline (PBS) and air dry.
4. Add 200 μl of fixative solution (3.7% formaldehyde 0.2% Triton X-100 in 10 mM
phosphate buffer saline) and incubate for 10 min.
5. Wash 5–10 with 100 μl deionised water.
6. Air dry and store in a dust-free container (Petri dish) at 4 C until ready for AFM
imaging.
9.3.2.2 Live Samples
Live cell imaging requires immobilisation, and here, we describe two such methods
using biochemical [20] and physical methods [22], respectively.
1. Culture cells in YPD media overnight at 30 C in a shaker.
2. Dilute the culture to an OD600 of 0.2 in pre-warmed YPD and incubate at 30 C
with shaking until it reaches exponential phase (OD600 ¼ 1).
3. Continue to prepare samples as per 9.3.2.2.1 or 9.3.2.2.2
Physical Immobilisation of Cells with PDMS Stamps
1. Using a scalpel, cut a small section of the PDMS stamp containing the pattern
multi-patterned PDMS stamp and place it onto a clean glass microscope slide
with the pattern side up.
2. Place an O-ring on the slide to secure the PDMS stamp and pipette approximately
100 μL of cell culture onto the PDMS stamp so it covers the entire stamp.
3. To capture cells in the PDMS wells, manually drag a coverslip across the PDMS
stamp, ideally with an advancing and receding contact angle of approximately
96 and 84 degrees respectively [23], pulling the droplet of the cell suspension
along with the stamp several times.
4. Check to see if cells have entered the wells under a bright-field microscope at
400 resolution.
5. Once the wells contain cells, add phosphate buffered saline (PBS) or 1:1 YPD:
PBS to cover the entire stamp and proceed to liquid AFM (Fig. 9.1b).
Biochemical Trapping of Cells with Cell-Tak
A circular hole (18 mm) was cut at the bottom of a 100 X 15 mm polystyrene Petri
dish and sealed with a Zeiss high precision coverslip using epoxy resin. The
coverslip was pre-cleaned [22] and then coated with Cell-Tak as described in
9.3.1.2.2.
1. Add 500 μl of the culture to Cell-Tak coated coverslip attached to the polystyrene
Petri dish, as described above, and incubate at 30 C for 30 min in the dark.
2. Rinse with filtered (0.2 um filter) and pre-warmed 1:1 diluted YPD: PBS (0.01 M,
pH 7) and add 500 μL of the same solution for imaging and maintained at 30 C
prior to use).
4. Place the Petri dish with the sample in a heated AFM sample holder maintained at
30 C.
9.3.3 Pros and Cons of Immobilisation Techniques
Since AFM is a surface scanning technique, live cells must first be immobilised on
solid substrates prior to imaging, using methods such as those outlined above [20].
PLL and Cell-Tak both biochemically immobilise cells onto a surface of choice.
PLL coating effectively immobilises both live and fixed cells of bacteria and fungi,
and it is less expensive than other methods. PLL works through electrostatic
interactions between the cationic (Poly-L-Lys+) thin film on a substrate which
creates a positively charged surface that attracts negatively charged microbes. The
method works well with highly charged cells, but the interaction is easily reversible
by shear forces during sample rinses or liquid imaging [23, 24]. Furthermore, PLL is
antimicrobial and directly hinders bacterial cell division and physiology, especially
when used at higher concentrations [14, 25] which can sometimes induce cell death
[11]. So based on all of the above considerations, we only use PLL for fixed cells.
For live cells, we have found Cell-Tak to be the better alternative since it
non-specifically attaches yeast and bacteria to glass surfaces. Cell-Tak is a formula-
tion of the polyphenolic proteins extracted from the marine mussel Mytilus edulis,
the key components of the glue secreted that allows the mussel to anchor itself to
solid structures in its natural environment [21].
PDMS stamps are an efficient way of physically immobilising cells and other
samples by convective or capillary deposition as described above for live cell
imaging [23]. The surface layer of the PDMS pattern needs to be sufficiently thin
so that cells are not trapped too deeply, which would prevent the AFM tip from
reaching the sample and preclude imaging [18, 26].
Immobilisation techniques are not limited to those we describe above, and other
methods exist including the use of optical tweezers and coating with gelatin [20].
Gelatin-coated surfaces used to immobilize cells can bias AFM images through
adhering to the AFM tips [20] or by coating the sample and changing its topography
[16, 24]. Optical tweezers can be used to accurately trap cells in 3D by focusing a
laser to a single diffraction-limited location [16, 24], however, these cells are easily
detached in comparison to those immobilised on glass surfaces [20, 27]. Furthermore,
the laser used for optical tweezers can damage cells [16, 24].
9.4 Imaging Modes
For all experiments, a JPK NanoWizard 3 or 4 AFM was used in quantitative

imaging (QI) mode. We have found that yeast is best imaged with silicon nitride
cantilevers with calibrated spring constants ranging from 0.01 to 0.6 N/m. For QI
adhesion data, a cantilever with a lower spring constant may be preferable for greater
sensitivity, but QI viscoelasticity data from yeast, which has a relatively rigid cell
wall, requires a cantilever with a slightly higher spring constant. So, choosing an
AFM cantilever can often be a trade-off between the two. Furthermore, the choice of
the cantilever will be governed by whether or not the sample is fixed or live, the latter
requiring a cantilever with a lower spring constant. When imaging at a higher speed
to reduce imaging time, there are shorter cantilevers which combine a low spring
constant with a high resonant frequency.
9.4.1 Imaging Fixed Cells in QI Mode
1. Turn on the AFM, followed by the AFM imaging software on the computer
connected to the AFM.
2. Place the coverslip with the sample to be imaged on the microscope slide and
secure it on both sides using tape, and mount the slide onto the AFM stage
(Fig. 9.1a).
3. Use the CCD camera or optical microscope to locate the specimen.
4. Once the specimen has been located, place the AFM head on the sample stage.
5. Using the z-stepper motors, bring the AFM cantilever within a few hundred
micrometres of the sample surface.
Caution: If the tip hits the sample surface, the cantilever will likely break
Fig. 9.1 Common

immobilisation strategies for
yeast cells to be imaged by
AFM using several different
methods: (a) fixed cells
deposited onto PLL-coated
coverslips, (b) live yeast
cells deposited into a
polydimethylsiloxane
(PDMS) stamp by
convective capillary
deposition, and (c) live yeast
cells firmly immobilised
onto a Cell-Tak coated
coverslip glued to the
bottom of a Petri dish
6. Align the laser onto the cantilever portion directly above the AFM tip and
optimize to get the maximum sum signal (>1 V) on the four-quadrant photode-
tector. In general, a higher sum indicates a good feedback signal from the
cantilever.
7. Adjust the lateral and vertical deflection and if needed, the mirror, to bring the
laser to the centre of the photodiode.
8. Approach the cantilever to the surface, using the automatic approach. Retract the
cantilever by approximately 10–20 μm and calibrate the AFM tip position
against the sample using the direct overlay optical calibration.
Note: Following tip-sample calibration (optical overlay), it is important to
only move the sample in x and y directions, rather than the AFM head, which
would render the calibration void and would require recalibration of the sample
position in relation to the tip
Fig. 9.2 QI™ images of C. albicans cell surface, adhesion and elasticity. (a, b) height images
(10 μm 10 μm, 128 128 pixels) of whole fixed cells deposited on PLL-coated coverslips, for (a)
untreated cells and (b) those treated with cinnamon bark essential (CNB) oils at 0.5 MIC
(62.5 μg/ml). High-resolution QI AFM images recorded (128 128 pixels) on a 1 μm2 area
(white boxed squares) on the top of cells reveal (f) increased surface roughness, (g) altered adhesion
patterns and (h) greater viscoelasticity for treated compared to (b, c, d) untreated cells, respectively
9. Calibrate the tip:

(a) Change to force spectroscopy mode, collect a force curve on an area of the
coverslip with no cells using a z-length of 1 μm. Now, obtain the sensitivity
from the force curve and the spring constant from the thermal noise to
calibrate the tip (thermal noise or contact-based method).
(b) For rectangular cantilevers, the tip calibration can be done without a force
curve using the dimensions of the cantilever and density and viscosity of the
medium (Sader or contact free method).
10. Change the AFM to force mapping mode and bring the tip into feedback.
11. Select a 50 50 μm scan area at low resolution (128 128 pixels), and quickly
collect a force map to determine the precise location of cells.
Note: Areas with bright pixels indicate the presence of cells available for
centering and QI imaging.
12. Select the area around the cell to be imaged by QI, and change the imaging
setting to QI mode.
13. Choose appropriate imaging parameters, such as speed, resolution, Z-length and
set-point.
Note: It is better to start with a lower speed and larger set point (z-length). For
Candida albicans, the starting Z-length was generally in the range of 3.5–6 μm
with a scan speed 100 μm/s. This is then optimised for shorter imaging times
with shorter z-length and higher scan speed (up to 300 μm/s).
14. Choose a specific area of interest on the cell surface for further high-
resolution (512 x 512 pixels) imaging (Fig. 9.2b-d, f-h).
Note: With a raster scanning method, there is a balance between image
resolution and image speed, with very high-resolution images taking longer to
collect.
9.4.2 Imaging Live Cells in QI Mode
1. Insert a Petri dish heater designed to hold a polystyrene Petri dish and connect it
to the controller.
2. Turn on the controller and set it to the desired temperature.
3. Place the polystyrene Petri dish containing the sample onto the AFM stage and
secure both sides using the steel clamps (Fig. 9.1c).
4. Using the CCD camera or optical microscope, locate the specimen using a low
magnification objective such as 10–20 .
5. Slowly and carefully put one drop of the imaging media onto the cantilever glass
block mount, tilt the mount, and allow the drop to slowly cover the cantilever.
6. Place the AFM head into the sample stage.
7. Using the z-stepper motors, carefully lower the AFM cantilever until the drop on
the cantilever surface fuses with the sample imaging media.
Caution: Again, avoid touching the sample surface, which could break the
cantilever
8. Once the cantilever is under liquid and within a few hundred microns of sample
surfaces, align the laser onto the cantilever portion directly above the AFM tip to
optimize the sum (>1).
9. When switching from air to liquid the mirror needs to be adjusted to account for
the refraction of the laser as it passes through the liquid–glass interface.
10. Calibrate the tip position and sample using the optical overlay according
to 9.4.1.
11. Determine the precise location of the cells using fast force mapping (Fig. 9.3),
switch to QI mode, set imaging parameters, and begin imaging (Fig. 9.4) as
described in 9.4.1.
Note: Live cells can be AFM or QI-AFM [26] imaged using cells
immobilised in PDMS stamps (see Sect. 9.3.2.2.2) under PBS, 1:1 PBS/YPD
or YPD media, by following steps 4–11 above. If the exact density and viscosity
of the media are unknown, use water and the Sader method [27] to determine the
spring constant.
Fig. 9.3 AFM images of Candida albicans array on a PDMS stamp. (a) 2-dimensional AFM
height image on 10 10 PDMS wells, corresponding (b) elasticity and (c) adhesion maps. Taken
from [20]
9.4.3 Tracking Dynamic Processes in QI Mode
When the pixel resolution is reduced and z-length and speed are optimised, it is
possible to study dynamic processes on the order of minutes using QI mode. Short
cantilevers, with a low spring constant (0.06–0.3 N/m) and a high resonant fre-
quency (30–90 kHz), allow a high z-speed and reduced acquisition times.
It is imperative that samples are well immobilised, including both the cells and the
substrate, so the PDMS stamp itself was glued to the glass substrate. This particular
method has been used to examine changes to the S. cerevisiae cell wall in response to
enzymatic degradation (Fig. 9.4).
1. Immobilize the PDMS stamp on a glass slide using epoxy resin.
2. Grow and immobilize cells in PDMS stamps according to 9.3.2.2.2.
3. Glue the imaging ring to the glass slide around the PDMS stamp and add 1.5 mL
of the desired medium (see Fig. 9.1b).
4. Follow steps 4–11 in Sect. 9.4.2.
9.4.4 Correlative AFM-QI-LSCM Live Cell Imaging
1. Replace the LSCM stage with the AFM sample stage.

2. Mount the AFM on the inverted LSCM and attach an additional camera onto the
front port of LSCM and connect it to the AFM ECU.
3. Set the AFM-LSCM according to the instrument schematic in Fig. 9.5, and
mount a specimen with a suitable fluorescent label for simultaneous imaging
by LSCM.
4. Mount a cantilever and place the AFM head on the stage as described in 9.4.2 for
live cell imaging.
5. Focus the sample from below using a 20 objective to assess the distance
between the specimen and the cantilever, but avoid bumping the sample with the
objective.
6. Lower the cantilever until its blurry image is visible under the 20 objective.
Caution: Do not lower the cantilever completely into the focal plane, since at
this point it will be too close to the sample with the associated risk of breaking
the cantilever.
7. Align the laser on the cantilever and maximize the sum as described for live cell
imaging (9.4.2).
8. At this point, the objective can be carefully switched to 40 or oil immersion, a
fluorescence image collected and the LSCM gains optimised.
Caution: When imaging in oil immersion, it is important that the sample
focus is not changed while the tip is in feedback. When the objective is in oil
immersion, it is in physical contact with the sample and moving the objective
will cause the AFM tip to crash into the sample.
Fig. 9.4 AFM QI images a S. cerevisiae cell as (a) height, (b) surface topography as pixel
difference, and (c) viscoelasticity at high resolution. The sequence of changes in (d) height, (e)
pixel difference, and (f) elasticity maps at 0, 16, and 29 min exposure to lyticase show dramatic
changes to the surface properties
9. The cantilever should be calibrated on a clean glass surface free of specimen to

obtain a force constant, as previously described for live cell imaging (9.4.2
and 9.4.3).
Fig. 9.5 AFM-QI-LSCM schematic illustration of the inverted LSCM objective focused onto the
sample from below, with the AFM cantilever and tip imaging from above. The live sample is firmly
immobilised to a Cell-Tak coated coverslip mounted in a Petri dish (left). Image of living Candida
albicans AFM-QI™ surface topography (middle), and overlay for AFM-QI™-LSCM confocal data
(right) that were simultaneously collected. In addition to surface ultrastructure, shown on the right is
the localisation of tubulin2-GFP (Tub2-GFP, green) and histone protein B-RFP (Htb-RFP, purple).
(Adapted from [11])
10. Calibrate the AFM tip position in x-y using the direct overlay and bring the
AFM tip into feedback mode (9.4.2).
11. Select a desired location on the sample for imaging and determine the precise
location using force mapping (9.4.2).
Note: It is important to select a cell which is immobile, has a height in an
expected range and shows bright fluorescence signals in the expected regions of
the cell. It is also important to have cells sparsely distributed since overcrowding
negatively affects imaging quality.
12. LSCM imaging parameters need to be adjusted prior to initiation of the
AFM-QI scan.
Note: It is important not to change any confocal parameters that will move
large components such as filters and lasers, which will cause large noise and
could cause the AFM tip to crash into the surface during imaging. The best
practice to collect an LSCM image directly prior to and after the AFM scan to
ensure similar results during simultaneous scanning.
13. For multicolour LSCM imaging, select a suitable excitation wavelength for each
fluorophore that will prevent bleed [11].
14. LSCM imaging is much faster than AFM-QI, therefore it is possible to obtain
multiple LSCM scans during QI imaging, provided LSCM settings are not
changed during imaging.
Note: With the advent of high-speed AFM, it is possible to better match the
data collection speeds of AFM and LSCM.
15. Since LSCM has a much wider field of view than AFM, match, as much as
possible, AFM and LSCM image resolution and size to vastly simplify image
overlay (Fig. 9.5).
9.5 AFM QI Mode Image/Data Processing
1. Using the JPK data processing software retrieve QI force curves at each pixel of
the raster scan.
2. Select a single reference force curve in the middle of the specimen and define the
operations for batch processing such as spring constant, sensitivity, baseline
correction, tip-sample separation, then measure the adhesion and apply the
Hertz or other appropriate model to estimate the Young’s modulus.
3. Apply the same processing parameters for all force curves in the image using the
batch processing module.
4. Batch process all force curves and export to excel, plot the data using Origin and
use the Gaussian distribution to place a fit on the histograms, or use a statistical
analysis package (i.e. GraphPad Prism).
5. Determine the surface roughness by selecting 200 200 nm squares at the centre
of the specimen using the QI™ height images.
9.5.1 AFM Image Processing and Overlay
1. Process images using the data processing software (DPS), ImageJ or photoshop,
for adjusting brightness and contrast, for which the former allows roughness and
subunit measurements.
2. Multi-colour LSCM images should be processed using linear unmixing to sepa-
rate different channels, along with other downstream analyses, including intensity
measurements and particle counts.
3. AFM-QI and LSCM images collected at the same time with similar resolution
parameters for the same specimen at the same position can be manually overlaid
using Photoshop or ImageJ.
Note: In the majority of cases, LSCM images need to be digitally enlarged to
correspond with the higher resolution AFM height image.
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Chapter 10
Molecular Taxonomy and Multigene
Phylogeny of Filamentous Fungi
Nikita Mehta, Reshma Jadhav, and Abhishek Baghela
Contents
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
10.2 Fungal Genomic DNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
10.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
10.2.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
10.3 Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
10.3.1 Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
10.3.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
10.4 Selection of Gene Target and Their Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
10.4.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
10.4.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
10.5 PCR Product Purification and Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
10.6 DNA Sequencing (Sanger Method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
10.6.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
10.6.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
10.7 Perform Sequencing PCR in Thermal Cycler Using Following Conditions . . . . . . . . . . . 198
10.7.1 Purification of Sequencing PCR Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
10.8 Sequence Editing, Analysis, and Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
10.8.1 Basic Local Alignment Search Tool (BLAST) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
10.8.2 MycoBank Database Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
10.8.3 UNITE Database Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
10.8.4 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
N. Mehta · A. Baghela (*)

National Fungal Culture Collection of India (NFCCI), Biodiversity and Palaeobiology Group,
MACS’ Agharkar Research Institute, Pune, India
Savitribai Phule Pune University, Pune, India
e-mail: abhishekbaghela@aripune.org
R. Jadhav
176 N. Mehta et al.
10.9 Molecular Phylogeny and Phylogenetic Tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

10.9.1 Phylogenetic Tree Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
10.9.2 Phylogenetic Tree Construction Using MEGA Software . . . . . . . . . . . . . . . . . . . . . 201
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
10.1 Introduction
The second largest kingdom “Fungi” includes rusts, yeasts, mildews, molds,
oomycetes, and smuts. They are generally classified into four types:
Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota; however, they
have recently been reclassified as into nine phylum-level clades: Opisthosporidia,
Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota,
Mucoromycota, Glomeromycota, Basidiomycota, and Ascomycota [104]. It has
been reviewed that the estimated number of species comprise of 2.2–3.8 million
with only 3%, named on the basis of data acquired from environmental fungi DNA
sequences [105]. Hence, to identify fungi at species level is the fundamental
approach for researcher to carry any type of study, be it basic or applied. Most of
the studies on plant pathogens start from identification of the pathogen and then to
find its cure as it can have economical effect on commercial scale [57, 98]. The
pathogenic behaviour of fungi is not only limited to plants; they can also infect,
human health. Various fungal infections and diseases require the specific identifica-
tion of causal agent before its treatment [11, 73]. The recognition of fungi from the
positive aspect is also very important as their vast industrial application increases the
need to discover more efficient species for commercial purposes.
The initial morphological basis of fungal classification basically applies only for
the cultivable fungus at laboratory scale. They are observed, macro-morphologically
(colony characteristics), micro-morphologically (spore characteristics) [81] and on
the basis of life cycle [32] (e.g. pleomorphic fungi) which can occur in both forms,
i.e. anamorph (asexual form) and teleomorph (sexual form). These dual life forms of
fungi had followed a dual nomenclature system according to Article 59 of ICBN
(International Code of Botanical Nomenclature), thus creating more confusion while
working on them. The recent amendment in Article 59 (at 18th International
Botanical Conference in Melbourne), implied effectively from 1 January 2013, states
the rule of “one fungus – one name” irrespective of its alternative life forms (mitotic
or meiotic). Then there comes polyphyletic genera (same genus appearing at differ-
ent clades or places in phylogenetic tree) which have either lost some characteristics
which resemble to one ancestor or gained some characters resembling another
ancestor, for example, Mycosphaerella was earlier considered as a genus for 30 dif-
ferent anamorphic genera [23, 38], but the sequence data of 28S nuclear rRNA made
it clear that it is an assembly of various genera belonging to different families
[20, 23]. Thus, as much as it is important to study any fungi residing at vast
environmental conditions, morphologically, it is difficult to completely rely on
10 Molecular Taxonomy and Multigene Phylogeny of Filamentous Fungi 177
Fig. 10.1 A ribosomal RNA (rRNA) gene cluster
them; therefore, the need of molecular approach is utmost for accuracy. Various new
molecular tools have been developed in last few decades, which allow efficient
evolutionary inferences, wherein one or more gene sequences can be used for
accurate fungal species identification. In general, the first level molecular identifi-
cation involves single-gene sequence-based identification, and for a reliable phylo-
genetic placement in fungal tree of life, multigene-based phylogenetic approach is
employed to define and recognize new fungal species.
In the 1980s, the initial molecular approach for phylogenetic study used poly-
merase chain reaction (PCR) of nuclear internal transcribed spacer sequences (ITS)
region of ribosomal DNA (rDNA) [28]. These sequences are consensus regions and
occur in long tandem repeats, and the universal primers could be designed for fungi
[97]. The nuclear ribosomal RNA (rRNA) gene is divided into smaller subunit (18S
or SSU) and larger subunit (28S or LSU) with two internal transcribed spacers (ITS1
and ITS2) between them linked by 5.8S region (Fig. 10.1). The 18S region helps to
narrow down the classification from kingdom level to phyla including phyla, class,
order, and family if the primer pair employed is NS1–NS4 [97], whereas LSU region
resolves intermediate level of taxonomy, i.e. family and genera with primer pair
LROR-LR6 [72, 94]. For species level identification, ITS is the most appropriate
region as this portion, though transcribed it does not code for any protein, hence
more prone to evolutionary mutation. It not only discriminates interspecific but also
helps in detecting intraspecific variations if studied combinatorial with hyper-
variable domains of LSU (D1/D2 domain) in some group of fungi [79].
Buée had found 1000 molecular taxonomic operational units based on approxi-
mately 30,000 reads for ITS1 region from pyrosequencing 4 grams of forest soil
sample [17]. Schoch has also mentioned that approximately 172,000 ITS region
sequences have been deposited to GenBank including 25,000 genera and 15,500
species. These studies highlight the general acceptance and wide utility of ITS region
for fungal identification. Unfortunately, this region has limitation when there are
huge numbers of species for a particular genus (Fusarium, Aspergillus, Penicillium,
etc.) [4, 49, 75, 79, 82, 87] or for cryptic species.
This drawback changes into advantage when we incorporate protein-coding
genes (secondary molecular markers) along with ITS, i.e. multigene approach for
superior resolution. The combined analysis of two or combination of more than two
genes or even whole genome has become very popular in molecular phylogenetic
studies [25]. These approaches yield unequivocal results [47]. Combined gene
analyses, in support with morphological information, resolves the conflicts arose
178 N. Mehta et al.
from single-gene analyses and enhances phylogenetic resolution [30]. The leverage
of using these additional gene markers (1) include faster evolving intronic region
than ITS [28], (2) provide better resolution at the level of higher taxonomy [80],
(3) occur in single copy, therefore prone to less mutations, and (4) include easy
understanding of homology and converging aspect [10, 27]. Now the question arises,
which protein coding genes should be used? Assembling the Fungal Tree of Life, a
National Science Foundation project, which has supported extensive studies on
fungal systematics using protein coding genes and designing molecular phylogeny
in the field of systematic and taxonomy [13, 34, 43, 53, 80]. The genetic markers are
RPB1 and RPB2 (RNA polymerase – largest and second largest subunit) [15, 50, 70,
88], tef1 (translational elongation factor 1-alpha) [71], tub2/BenA (beta-tubulin)
[29, 60], and MCM7 (mini chromosome maintenance protein), which provides
superior resolution in association with other genes, e.g. with LSU for Ascomycota
members [2, 68]. In some lineages of Eurotiales (includes genus Aspergillusand
Penicillium), species level identification involves CaM/cal (calmodulin) gene along
with tef1, RPB2, tub2/BenA, and RPB1. Visagie and colleagues have recommended
the use of RPB2 or CaM genes to avoid any ambiguity based on gene tub2/BenA for
Penicillium spp. [95]. Similarly, for molecular phylogeny of Aspergillus sp., CaM
gene is recommended, but RPB2 and tub2/BenA are also preferred and can be used
for classification [75]. Torbati suggested the use of CaM, tef1, tub2, and RPB2
secondary markers along with ITS and LSU to resolve the phylogenetic study of
Fusarium spp., which exhibit immense morphological plasticity [92]. A detailed list
of commonly used gene targets/markers has been given in Table 10.1, which is an
easy guide to select the right gene targets/markers for some specific fungal genera.
In order to determine phylogenetic relationship, DNA sequences of different
fungi can be aligned and compared to establish variability among themselves and
with ancestors. This data is generally represented as “phylogenetic/relationship tree”
where the branch’s length and position explain the similarity and distant resem-
blance between different fungi. The variation at the intra- and inter-specific levels for
a specific locus can be used for identification among all the closely related species,
thus giving a possibility to make a decisive prediction regarding new species
[74]. However, nowadays a molecular method using several appropriate gene
targets/markers instead of only one, has made the phylogenetic study more authen-
ticated to describe particular unknown species [47]. This method facilitates compar-
isons with already present taxonomic information of related species.
Initially, the use of phylogenetic trees was solely in the field of systematics and
taxonomy to depict the relation among species. But nowadays, the advent of DNA
sequencing technology it can be applied to all the branches of biology. Phylogeny
has also explained the relationships between paralogues of gene family along with
evolutionary and epidemiological dynamics of pathogens and histories of
populations, etc. [100]. The methods required for phylogenetic analysis, includes
parsimony, likelihood distance, and Bayesian methods. The detailed information
about these methods is vast and very exhaustive that cannot be accommodated at one
place; however, some basic information regarding phylogeny has been mentioned in
the pretext of phylogenetic tree construction section of this chapter.
Table 10.1 Gene targets/molecular markers for different fungi and their respective primer pairs and sequences
Genes for Primer name
molecular
S. N. Genus of Fungi phylogeny Forward primer (Primer Sequence 50 -30 ) Reverse primer (Primer Sequence 50 -30 ) References
1. Alternaria ITS V9G: TTACGTCCCTGCCCTTTGTA ITS4: TCCTCCGCTTATTGATATGC [99]
GAPDH gpd1: CAACGGCTTCGGTCGCATTG gpd2: GCCAAGCAGTTGGTTGTGC
RPB2 RPB2-5F2: GGGGWGAYCAGAAGAAGGC fRPB2-7cR: CCCATRGCTTGYTTRCCCAT
OPA10-2 OPA10-2L: TCGCAGTAAGACACATTCTACG OPA10-2R: GATTCGCAGCAGGGAAACTA
Alt a 1 Alt-for: ATGCAGTTCACCACCATCGC Alt-rev: ACGAGGGTGAYGTAGGCGTC
endoPG PG3: TACCATGGTTCTTTCCGA PG2b: GAGAATTCRCARTCRTCYTGRTT
TEF1-α EF1-728F: CATCGAGAAGTTCGAGAAGG EF1-986R: TACTTGAAGGAACCCTTACC
2. Aspergillus β-tubulin Bt2a: GGTAACCAAATCGGTGCTGCTTTC Bt2b: ACCCTCAGTGTAGTGACCCTTGGC [85]
ITS ITS1: TCCGTAGGTGAACCTGCGG ITS4: TCCTCCGCTTATTGATATGC
TEF1-α 983F: GCYCCYGGHCAYCGTGAYTT 2218R: ATGACACCRACRGCRACRGTYTGYAT
Calmodulin CF1L: GCCGACTCTTTGACYGARGAR CF4: TTTYTGCATCATRAGYTGGAC
CF1M: AGGCCGAYTCTYTGACYGA cmd6: TTTYTGCATCATRAGYTGGAC
cmd5: CCGAGTACAAGGAGGCCTTC
RPB2 fRPB2-5F: GAYGAYMGWGATCAYTTYGG fRPB2-7CR: CCCATRGCTTGYTTRCCCAT
3. Bipolaris GAPDH gpd1: CAACGGCTTCGGTCGCATTG gpd2: GCCAAGCAGTTGGTTGTGC [54]
ITS ITS1: TCCGTAGGTGAACCTGCGG ITS4: TCCTCCGCTTATTGATATGC
4. Botryosphaeria TEF1-α EF1-728F: CATCGAGAAGTTCGAGAAGG EF1-986R: TACTTGAAGGAACCCTTACC [45, 67]
ITS ITS5:GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC
5. Botrytis GAPDH G3PDHfor: ATTGACATCGTCGCTGTCAACGA G3PDHrev: ACCCCACTCGTTGTCGTACCA [102]
HSP60 HSP60for: CAACAATTGAGATTTGCCCACAAG HSP60rev: GATGGATCCAGTGGTACCGAGCAT
RPB2 RPB2for: GATGATCGTGATCATTTCGG RPB2rev: CCCATAGCTTGCTTACCCAT
6. Cercospora GAPDH Gpd1-LM: ATTGGCCGCATCGTCTTCCGCAA Gpd2-LM: CCCACTCGTTGTCGTACCA [7]
β-tubulin BT1-F: GTCCWCACCGCCCCTGAT BT1-R: CTTGTTRCCRGAAGCCTRTGS
(continued)
molecular
Calmodulin CAL-228F: GAGTTCAAGGAGGCCTTCTCCC CAL-737R: CATCTTTCTGGCCATCATGG
RPB2 fRPB2-7cF: ATGGGYAARCAAGCYATGGG fRPB2-11aR: GCRTGGATCTTRTCRTCSACC
7. Colletotrichum ApMAT AM-F: TCATTCTACGTATGTGCCCG AM-R: CCAGAAATACACCGAACTTGC [22, 84,
ITS ITS-1F: CTTGGTCATTTAGAGGAAGTAA ITS4: TCCTCCGCTTATTGATATGC 96]
GAPDH GDF: GCCGTCAACGACCCCTTCATTGA GDR: GGGTGGAGTCGTACTTGAGCATGT
Actin ACT-512F: ATGTGCAAGGCCGGTTTCGC ACT-783R: TACGAGTCCTTCTGGCCCAT
HIS-3 CYLH3F: AGGTCCACTGGTGGCAAG CYLH3R: AGCTGGATGTCCTTGGACTG
β-tubulin T1: AACATGCGTGAGATTGTAAGT T2: TAGTGACCCTTGGCCCAGTTG
CHS-1 CHS-79F: TGGGGCAAGGATGCTTGGAAGAAG CHS-345R: TGGAAGAACCATCTGTGAGAGTTG
8. Cordyceps SSU NS1: GTAGTCATATGCTTGTCTC NS4: CTTCCGTCAATTCCTTTAAG [90]
LSU LR0R: ACCCGCTGAACTTAAGC LR5: TCCTGAGGGAAACTTCG
RPB1 CRPB1: CCWGGYTTYATCAAGAARGT RPB1C: CCNGCDATNTCRTTRTCCATRTA
RPB2 fRPB2-5F: GAYGAYMGWGATCAYTTYGG fRPB2-7cR: CCCATRGCTTGYTTRCCCAT
β-tubulin T12: AACAACTGGGCCAAGGGTCAC T22: TCTGGATGTTGTTGGGAATCC
mt ATP6 ATP6C1A: AGAWCAATTYGAARTRAGAG ATPC2A: ACAAAYACTTGWGCTTGKATWAAI
GC
9. Curvularia GAPDH GPD1: CAACGGCTTCGGTCGCATTG GPD2: GCCAAGCAGTTGGTTGTGC [45]
10. Diaporthe ITS ITS5:GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC [39]
HIS CYLH3F: AGGTCCACTGGTGGCAAG H3-1b: GCGGGCGAGCTGGATGTCCTT
β-tubulin Bt2a: GGTAACCAAATCGGTGCTGCTTTC Bt2b: ACCCTCAGTGTAGTGACCCTTGGC
11. Fusarium RPB2 5F2: GGGGWGAYCAGAAGAAGGC 7cR: CCCATRGCTTGYTTRCCCAT [44, 52]
7cF: ATGGGYAARCAAGCYATGGG 11aR: GCRTGGATCTTRTCRTCSACC
RPB1 Fa: CAYAARGARTCYATGATGGGWC G2R: GTCATYTGDGTDGCDGGYTCDCC
Calmodulin Cal228F: GAGTTCAAGGAGGCCTTCTCCC CAL2Rd: TGRTCNGCCTCDCGGATCATCTC
TEF1-α ef1: ATGGGTAAGGA(A/G)GACAAGAC ef2: GGA(G/A)GTACCAGT(G/C)ATCATGTT
β-tubulin T1: AACATGCGTGAGATTGTAAGT CYLTUB1R: AGTTGTCGGGACGGAAGAG
12. Lasiodiplodia ITS ITS5:GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC [44]
β-tubulin Bt2a: GGT AAC CAA ATC GGT GCT GCT TTC Bt2b: ACCCTCAGTGTAGTGACCCTTGGC
13. Macrophomina β-tubulin T1: AACATGCGTGAGATTGTAAGT CYLTUB1R: AGTTGTCGGGACGGAAGAG [44]
TEF1-α EF1-728F: CATCGAGAAGTTCGAGAAGG EF2: GGA(G/A)GTACCAGT(G/C)ATCATGTT
Actin ACT-512F: ATGTGCAAGGCCGGT ACT-783R: TACGAGTCCTTCTGG
14. Magnaporthe ITS ITS5:GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC [103]
LSU LS1: GTACCCGCTGAACTTAAGC LR5: TCCTGAGGGAAACTTCG
SSU SR1R: TACCTGGTTGATQCTGCCAGT SR7: GTTCAACTACGAGCTTTTTAA
MCM7 MCM7-709: ACIMGIGTITCVGAYGTHAARCC MCM7-
1348: GAYTTDGCIACICCIGGRTCWCCCAT
TEF1-α EF1-983F: GCYCCYGGHCAYCGTGAYTT EF1-
2218R: ATGACACCRACRGCRACRGTYTGYAT
RPB1 RPB1-Af: GARTGYCCDGGDCAYTTYGG RPB1-Cr: CCNGCDATNTCRTTRTCCATRTA
15. Metarhizium TEF1-α 983F: GCYCCYGGHCAYCGTGAYTT 2218R: ATGACACCRACRGCRACRGTYTGYAT [46]
RPB1 CRPB1: CCWGGYTTYATCAAGAARGT RPB1C: CCNGCDATNTCRTTRTCCATRTA
β-tubulin T12: AACAACTGGGCCAAGGGTCAC T22: TCTGGATGTTGTTGGGAATCC
16. Mucor ITS ITS1: TCCGTAGGTGAACCTGCGG ITS4: TCCTCCGCTTATTGATATGC [33]
(continued)
molecular
LSU LR0R: ACCCGCTGAACTTAAGC LR5: TCCTGAGGGAAACTTCG
MCM7 Mcm7-709for: ACIMGIGTITCVGAYGTHAARCC Mcm7-1348rev: GAYTTDGCIACICCIGGRTCWC
CCAT
17. Mycospaerella LSU LSU1Fd: GRATCAGGTAGGRATACCCG LR5: TCCTGAGGGAAACTTCG [93]
ITS V9G: TTACGTCCCTGCCCTTTGTA ITS4: TCCTCCGCTTATTGATATGC
18. Penicillium ITS ITS1F: TCCGTAGGTGAACCTGCGG ITS4: TCCTCCGCTTATTGATATGC [95]
Calmodulin CF1: GCCGACTCTTGACTGAA CF4: TTTYTGCATCATRAGYTGGAC
RPB2 RPB2-5F_Eur: GAYGAYCGKGAYCAYTTCGG RPB2-7CR_Eur: CCCATRGCYTGYTTRCCCAT
19. Phytophthora LSU LROR-O: ACCCGCTGAACTYAAGC LR6-O: CGCCAGACGAGCTTACC [39]
LSUFint: CKTTGACGAAATGGAGCGAT LSURint: TTTCCACACCCTAACACTTGC
β-tubulin Btub_F1: GCCAAGTTCTGGGAGGTCATC Btub_R1A: CCTGGTACTGCTGGTAYTCMGA
COX II FM35: CAGAACCTTGGCAATTAGG Phy10b: GCAAAAGCACTAAAAATTAAATATAA
nad9 Nad9-F: TACAACAAGAATTAATGAGAAC Nad9-R: GTTAAAATTTGTACTACTAACAT
20. Pseudocercospora TEF1-α EF1-728F: CATCGAGAAGTTCGAGAAGG EF-2: GG(G/A)GTACCAGT(G/C)ATCATGTT [21]
Actin ACT-512F: ATGTGCAAGGCCGGTTTCGC ACT-2Rd: TACGAGTCCTTCTGGCCCAT
ITS ITS5: GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC
LSU LSU1Fd: GRATCAGGTAGGRATACCCG LR5: TCCTGAGGGAAACTTCG
21. Puccinia LSU Rust28SF: TTTTAAGACCTCAAATCAGGTG LR5: TCCTGAGGGAAACTTCG [3]
SSU RustNS2-F: GGGAGGTAGTGACMATAAATAAC NS6: GCATCACAGACCTGTTATTGCCTC
AATG
COX III CO3F1: TCAGTATGTTATTTTAACGATGTAG CO3R1: TCCTCATCAGTAAACACTAATA
22. Pythium ITS ITS1: TCCGTAGGTGAACCTGCGG ITS4: TCCTCCGCTTATTGATATGC [39]
COX II FM58: CCACAAATTTCACTACATTGA FM66: TAGGATTTCAAGATCCTGC
23. Rhizopus ITS V9G: TTACGTCCCTGCCCTTTGTA LS266: GCATTCCCAAACAACTCGACTC [26]
Actin Act-1: TGGGACGATATGGAIAAIATCTGGCA Act-
4ra: TCITCGTATTCTTGCTTIGAIATCCACAT
TEF1-α MEF-4: ATGACACCRACAGCGACGGTTTG MEF-10: GTTGTCATCGGTCACGTCGATTC
24. Sclerotinia ITS ITS1: TCCGTAGGTGAACCTGCGC ITS4: TCCTCCGCTTATTGATATGC [83]
β-tubulin β-tubulin F: GGTAACCAAATCGGTGCTGCTTTC β-tubulin R: ACCCTCAGTGTAGTGACCCTTGGC
25. Sporothrix LSU LR3: CCG TGT TTC AAG ACG GG LR5: TCCTGAGGGAAACTTCG [24]
ITS ITS1F: CTTGGTCATTTAGAGGAAGTAA ITS4: TCCTCCGCTTATTGATATGC
β-tubulin T10: ACGATAGGTTCACCTCCAGAC Bt2b: ACCCTCAGTGTAGTGACCCTTGGC
Calmodulin CL1: GA(GA)T(AT)CAAGGAGGCCTTCTC CL2a: TTTTTGCATCATGAGTTGGAC
26. Talaromyces RPB2 fRPB2-5f: GAYGAYMGWGATCAYTTYGG fRPB2-7cR: CCCATRGCTTGYTTRCCCAT [101]
ITS V9G: TTACGTCCCTGCCCTTTGTA LS266: GCATTCCCAAACAACTCGACTC
27. Trichoderma RPB2 fRPB2-5f: GAYGAYMGWGATCAYTTYGG fRPB2-7cR: CCCATRGCTTGYTTRCCCAT [42]
TEF1-α EF1-728F: CATCGAGAAGTTCGAGAAGG TEF1LLErev: AACTTGCAGGCAATGTGG
acl1 acl1-230up: AGCCCGATCAGCTCATCAAG acl1-1220low: CCTGGCAGCAAGATCVAGGAAG
T
28. Verticilium Actin VActF: TAATTCACAATGGAGGGTAGG VActR: GTAAGGATACCACGCTTGG [40, 44]
TEF1-α VEFf: AACGTCGTCGTCATCGGCCACG VEFr: CCACGCTCACGCTCGGCCTT
GAPDH VGPDf2: GGCATCAACGGTTTCGGCC VGPDr: GTAGGAGTGGACGGTGGTCATGAG
TS VTs3f: GCGCTGCAAGGCCGAGAAC VTs3r: GCGGAACGAGACGGCCTCC
ITS ITS5: GGAAGTAAAAGTCGTAACAAGG ITS4: TCCTCCGCTTATTGATATGC
184 N. Mehta et al.
There has always been a requirement of putting all the essential information and
protocols for fungal molecular taxonomy and phylogeny in one place; therefore, we
assembled all the required protocols starting from fungal DNA isolation, list of
recommended gene targets for fungal identification and phylogeny, their primer
names, sequences and PCR cycling conditions, PCR product purification, and
DNA sequencing in this chapter. Considering the vast number of fungi, we have
enlisted the gene targets/markers for limited number of fungi, which are the common
and important fungal pathogens. Many times, it becomes difficult to get the correct
PCR cycling conditions for different gene targets/markers and for which one has to
check multiple back references; therefore, we have also enlisted the primer pair
names, their sequences, and their PCR cycling conditions. We have also briefed
about sequence editing and analysis. Sometimes, erroneous DNA sequences in
NCBI nucleotide database lead to the misidentification of fungi; therefore, to
cross-check and confirm the identity of the fungi, some additional well authenticated
databases are also described, which can be used for molecular identification of fungi
in addition to NCBI BLASTn. An outline about the construction of phylogenetic tree
using MEGA7 has been described. Though there are many advanced tools like
RAxML and Mr. Bayes available for constructing the phylogenetic trees, however,
MEGA7 has been given because of ease of constructing a phylogenetic tree.
Especially for the beginners in the field, a chart for the entire work flow has also
been given for easy understanding of all the different steps required to perform the
phylogenetic analysis of fungi (Fig. 10.2). Further, the methods, materials, and
protocols vary among the different laboratories; therefore, the same can be twisted
or modified depending upon the availability of reagents, chemicals, and equipment.
10.2 Fungal Genomic DNA Extraction
There are various extraction methods available for fungi, and the choice of method
used for DNA extraction depends on the availability of equipments and nature of
fungal samples from which DNA is to be isolated. Total genomic DNA can be
isolated from fungal specimen by various physical methods like crushing in liquid
nitrogen, bead beating in homogenizer, and use of some cell wall degrading enzymes
with detergents like sodium dodecyl sulphate (SDS) and Cetyltrimethyl ammonium
bromide (CTAB). Unfortunately, sometimes few methods can lead to shearing of
DNA or can be hazardous and laborious (grinding pathogenic fungi in an open
mortar and pestle, or handling multiple fungal isolates). Therefore, a closed DNA
isolation system is always preferred, wherein one can isolate DNA from multiple
fungi simultaneously, thereby enabling high throughput DNA extraction. One such
method has been outlined in the following protocol section. Researchers also use
DNA extraction kits. Most popular kits are DNeasy 96 Plant Kit (Qiagen), Qiagen
MOBIO Laboratories, FastDNA SPIN Kit (MP Biomedicals), and Epicentre’s
MasterPure™ Yeast DNA Purification Kit to isolate genomic DNA from a broad
range of fungi. Specific protocols are provided with each kit for researcher.
Fig. 10.2 A flow-chart of various steps towards molecular phylogenetic analysis of fungi
186 N. Mehta et al.
The basic criteria for the selection of a suitable DNA isolation method include
(1) efficient DNA extraction, (2) good quantity of DNA, (3) removal of contami-
nants, (4) purity and quality of DNA, and (5) possibility of high throughput.
Ultraviolet absorbance is mostly used to determine the purity of DNA. For a good-
quality DNA, the quantitative ratio of absorbance at 260 nm and 280 nm (A260/
A280) must be 1.8. If this ratio is less than 1.8, it indicates the DNA sample has
protein contamination or has impurity of an organic solvent such as phenol, and if
the ratio is more than 1.8, it indicates the DNA sample has RNA contamination. The
quantification of dsDNA should be assessed by Qubit fluorometer analysis, while
A260/A280 can be determined by NanoDrop spectrophotometer. Quality of DNA
can also be assessed by visualization on agarose gel. A rapid and high throughput
fungal DNA extraction protocol is mentioned below [1].
10.2.1 Materials
1. Source material – fungal culture of interest

2. DNA extraction vials containing ceramic pestle
3. Glass beads (425–600 μM, Sigma)
4. 10 ml syringe
5. Glass wool
6. Scalpel
7. Micropipettes
8. Micro centrifuge tubes/eppendorf tubes (1.5 ml)
9. Lysis buffer (100 mM Tris HCl [pH 8.0], 50 mM EDTA, 3% SDS)
10. Phenol: Chloroform: Isoamyl alcohol (25:24:1)
11. Isopropanol
12. 70% ethanol
13. 1XTE buffer (10 μl of 1 M Tris, 2 μl of 0.5 M EDTA and 988 μl dH2O)
14. RNaseA (Sigma Aldrich, USA)
15. FastPrep®-24 tissue homogenizer (MP Biomedicals, USA)
16. Centrifuge 5415 R (Eppendorf)
17. Deep freezer (20 C)
18. Incubator set at 37 C
19. NanoDrop 1000 spectrophotometer
20. Qubit fluorometer
10.2.2 Method
1. Scrape 300 mg of mycelia from agar plate or from the culture broth by filtering it
through a 10–15 ml syringe containing some glass wool that will allow the
fungal mass retaining in syringe and let the broth to pass through, and then
transfer the fungal mass into a screw capped tube containing ceramic pestle and
some fine glass beads.
2. Add 1 ml lysis buffer and homogenize twice for 60s at 6 M/S.
3. Centrifuge the homogenized mycelia at 12,000 rpm for 15 min at room temper-
ature (RT), and then transfer the supernatant in a fresh 1.5 ml Eppendorf tube.
4. Add equal volume of phenol-chloroform-isoamyl alcohol (PCI) and shake well
(at least 30 times). Centrifuge for 10 min at 10,000 rpm at RT; separate upper
aqueous layer in new 1.5 ml Eppendorf tube.
5. If any haziness or pigment persists then repeat the PCI treatment.
6. Add equal volume of Isopropanol and keep at 20 C for 20 min.
7. Centrifuge for 10 min at 10,000 rpm at 4 C. Remove supernatant and wash the
pellet using 500 μL of 70% ethanol.
8. Again centrifuge at 10,000 rpm for 5 min at 4 C. Remove supernatant and
air dry the pellet.
9. Dissolve the pellet in 50–70 μL of 1X TE buffer.
10. Add 1 μL of RNaseA (10 mg/ml) and incubate it at 37 C for 30 min.
11. 2 μL of genomic DNA is subjected to 0.8% agarose gel electrophoresis (refer
agarose gel electrophoresis protocol).
12. Observe the gel under UV trans-illuminator gel documentation system.
13. Determination of quantity and quality of the isolated DNA can be done by
NanoDrop spectrophotometer and Qubit fluorometer.
10.3 Agarose Gel Electrophoresis
Agarose gel electrophoresis is used for separation of nucleic acid (DNA/RNA).

DNA or RNA molecules are separated based on their size and molecular weight
under an electrical field where, negatively charged molecules move toward positive
pole known as anode. The migration flow is decided entirely according to the size
where low molecular weight molecules migrate more rapidly than higher ones. The
below mentioned table denotes the relationship between the Agarose gel percentage
and effective DNA size separation.
Concentration of agarose gel (%) Range of linear DNA Molecules (kb) for separation
0.6 1–20
0.8 0.5–10
1.2 0.4–6
1.5 0.2–3
2.0 0.1–2
188 N. Mehta et al.
10.3.1 Material
1. Gel electrophoretic unit including buffer tank, casting tray, gel cassette and comb
2. Voltage supplier
3. Running Buffer:
(a) TAE (1 L – 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA
of pH 8)
(b) TBE (1 L – 242 g Tris base, 137.5 ml boric acid, 100 ml 0.5 M EDTA of
pH 8)
4. Agarose
5. Conical flask
6. Ethidium bromide
7. Hot plate or microwave oven
8. Gel loading dye (6X- 1.5 g Ficoll400, 25 mg Xylene cyanol FF, 25 mg
bromophenol blue – 10 ml dH2O)
9. Gel documentation system (make Syngene, Biorad, etc.)
10.3.2 Method
1. Prepare 100 ml 0.5X Tris Borate EDTA/Tris Acetate EDTA (TBE/TAE) buffer
of pH 8 in distilled water in a conical flask.
2. Weigh 0.8gm (for DNA, i.e. 0.8%)/1.2–1.5 gm (for PCR product, i.e. 1.2–1.5%)
of agarose and add 100 ml of 0.5 X TBE/TAE buffer.
3. Keep it in microwave oven or on hot plate for 5–10 min to melt the gel.
4. Take the gel solution from oven or hot plate and allow it to cool (~60 C).
5. Add 2–4 μL ethidium bromide (EtBr) of 10 mg/mL concentration and mix
properly (EtBr is a well-known and widely used fluorescent dye used to
visualize the DNA/RNA under UV light).
6. Assemble the gel caster with casting tray, comb and pour the gel. Let the agarose
gel solidify for approx. 20–30 min.
7. Place the gel casting tray in electrophoretic chamber filled with running buffer
(0.5X TBE/TAE, same as the gel preparation buffer).
8. Remove the comb and place the gel along with casting tray in the electrophoretic
chamber.
9. Connect both the electrodes, switch on the current and allow the gel to run at
70–100 V for 20–30 min.
10. After 30 min, take out the gel from the electrophoretic chamber and observe it
under UV transilluminator or in a gel documentation system.
Note: If the DNA bands are bright and sharp, it indicates that the quantity and
quality of DNA is good, but if a smear has been seen on gel, it indicates
degradation of DNA (quantity and quality of DNA is poor).
Table 10.2 PCR conditions of different gene targets and their respective primer pairs
10
No.
Name of gene Initial Final of
S. N. target Primer pair/set denaturation Denaturation Annealing Extension extension cycles References
1. Internal V9G/ITS4 94 C – 5 min 94 C – 30 s 48 C – 30 s 72 C – 90 s 72 C – 7 min 35 [37, 97]
Transcribed ITS1/ITS4 95 C – 5 min 95 C – 30 s 50 C – 30 s 72 C – 1 min 72 C – 7 min 30
Spacer region ITS5/ITS4 95 C – 5 min 95 C – 30 s 50 C – 30 s 72 C – 1 min 72 C – 7 min 30
of the rRNA
ITS-1F/ITS4 95 C – 5 min 95 C – 30 s 50 C – 30 s 72 C – 1 min 72 C – 7 min 30
(ITS)
2. Large Subunit LROR/LR6 95 C – 3 min 95 C – 1 min 53 C – 1 min 72 C – 2 min 72 C – 7 min 35 [94]
of the rRNA LROR/LR5 94 C – 5 min 94 C – 1 min 52 C – 30s 72 C – 1 min 72 C – 7 min 35
(LSU, 28S) LSUFint/ 94 C – 5 min 94 C – 1 min 52 C – 30s 72 C – 1 min 72 C – 7 min 35 [14]
LSURint
LSU1Fd/ LR5 94 C – 3 min 94 C – 30 s 52 C – 30s 72 C – 45 s 72 C – 5 min 35 [23, 94]
LS1/LR5 95 C – 2 min 95 C – 1 min 57 C – 1 min 72 C – 1 min 72 C – 10 min 35 [72]
LR3/LR5 94 C – 5 min 94 C – 1 min 52 C – 30s 72 C – 1 min 72 C – 7 min 35 [94]
Rust28SF/ 94 C – 5 min 94 C – 1 min 55 C – 30s 72 C – 1 min 72 C – 7 min 35 [8]
LR5
NL1/NL4 94 C – 5 min 94 C – 1 min 52 C – 30s 72 C – 1 min 72 C – 7 min 35 [48]
3. Small Subunit NS1/NS4 94 C – 5 min 94 C – 1 min 51 C – 30s 72 C – 1 min 72 C – 7 min 35 [89]
of the rRNA RustNS2-F/ 94 C – 5 min 94 C – 1 min 55 C – 30s 72 C – 1 min 72 C – 7 min 35 [8]
(SSU, 18S) NS6
SR1R/SR7 35 [94]
Molecular Taxonomy and Multigene Phylogeny of Filamentous Fungi
95 C – 2 min 95 C – 1 min 57 C – 1 min 72 C – 1 min 72 C – 10 min

4. RNA polymer- CRPB1/ 94 C – 5 min 94 C – 1 min 37 C – 35 s (1 72 C – 1 min 72 C – 10 min 4 [19]
ase II subunit RPB1Ca 94 C – 35 s every 4 s to 72 ) 72 C – 1 min 29
1 (RPB1) 45 C – 55 s(1
every 4 s to 72 )
(continued)
189
190
No.
Fa/G2R 94 C – 5 min 94 C – 1 min 45 C – 30 s 72 C – 1 min 72 C – 7 min 35 [65]
RPB1-Af/ 95 C – 2 min 95 C – 1 min 57 C – 1 min 72 C – 1 min 72 C – 10 min 35 [15, 88]
RPB1-Cr
RBP1-F1843/ 94 C – 5 min 94 C – 30 s 51 C – 30 s 72 C – 1 min 72 C – 10 min 5 [76]
RPB1-R3096a 94 C – 30 s 49 C – 30 s 72 C – 1 min 5
94 C – 30 s 47 C – 30 s 72 C – 1 min 30
5. Second largest RPB2-5F2/ 94 C – 90 s 94 C – 30 s 55 C – 90 s 68 C – 90 s 68 C – 5 min 40 [64]
subunit of RNA RPB2-7cR
polymerase II RPB2for/ 94 C – 5 min 94 C – 30 s 55 C – 30 s 72 C – 90 s 72 C – 10 min 35 [86]
(RPB2) RPB2rev
RPB2-7cF/ 94 C – 5 min 94 C – 1 min 37 C – 35 s (1 72 C-1 min 72 C – 10 min 30 [51]
RPB2-11aRa every 5 s to 72 )
bRPB2-6F/ 94 C – 4 min 94 C – 1 min 51 C – 1 min 72 C – 1 min 72 C – 8 min 36
gRPB2-6R
6. Beta-tubulin Bt2a/Bt2b 94 C – 5 min 94 C – 1 min 58 C-1 min 72 C-1 min 72 C – 7 min 32 [29]
(β-tub) BT1-F/BT1-R 94 C – 3 min 94 C – 30 s 56 C – 30 s 72 C – 45 s 72 C – 5 min 40 [7]
T1/T2 95 C – 4 min 95 C – 30 s 55 C – 30 s 72 C – 45 s 72 C – 7 min 40 [61]
T12/T22 94 C – 5 min 94 C – 1 min 52 C – 30 s 72 C – 2 min 72 C – 7 min 35 [90]
T1/ 94 C – 2 min 94 C – 1 min 55 C – 30 s 72 C – 1 min 72 C – 10 min 35 [22, 61]
CYLTUB1R
Btub_F1/ 95 C – 3 min 95 C – 1 min 60 C – 1 min 72 C – 2 min 72 C – 5 min 35 [14]
Btub_R1A
T10/Bt2b 96 C – 30s 94 C – 1 min 58 C – 1 min 72 C – 1 min 72 C – 5 min 32 [29, 61]
N. Mehta et al.
10
7. Translation EF1-728F/ 94 C – 5 min 94 C – 30s 52 C – 30 s 72 C – 45 s 72 C – 7 min 40 [18]

elongation EF1-986R
factor 1-alpha EF1-983F/ 94 C – 4 min 94 C – 1 min 56–62 C – 1 min 72 C – 1 min 72 C – 8 min 40 [71]
(TEF1-α) EF1-2218Ra (1 increase in
every cycle till
7 cycle)
EF1/EF2a 94 C – 4 min 94 C – 1 min 62–52 C –30 s 72 C – 1 min 72 C – 8 min 30 [61]
(1 decrease in
every cycle till
9 cycle)
EF1-728F/ 94 C – 2 min 94 C – 1 min 52 C – 30 s 72 C – 1 min 72 C –10 min 35 [18, 61]
EF2
EF1-1577F/ 94 C – 4 min 94 C – 1 min 56–62 C –1 min 72 C – 1 min 72 C – 8 min 40 [71]
EF1-1567Ra (1 increase in
every cycle till
7 cycle)
EF1-728F/ 94 C – 2 min 94 C – 1 min 52 C – 30s 72 C – 1 min 72 C – 10 min 35 [18, 41]
TEF1LLErev
MEF-4/MEF- 94 C – 2 min 94 C –15 s 55 C – 30s 72 C – 1 min 72 C – 5 min 35 [62]
10
VEFf/VEFr 94 C – 2 min 94 C – 10s 60 C – 20s 72 C – 1 min 72 C –7 min 32 [40]
8. Actin ACT-512F/ 94 C – 5 min 94 C – 30s 58 C – 30s 72 C – 45 s 72 C – 7 min 35 [18]
ACT-783R
Act1/Act4a 95 C – 5 min 95 C –1 min 58 C-1 min 72 C – 2 min 72 C – 7 min 35 [5]

VActF/ 94 C – 2 min 94 C – 10s 48 C – 20s 72 C – 1 min 72 C – 7 min 32 [40]
VActR
9. Sixth subunit ATP6C1A/ 94 C – 5 min 94 C – 1 min 37 C – 35 s (1 72 C – 1 min 72 C – 10 min 4 [19]
of ATP ATP6C2Aa 94 C – 35 s every 4 s to 72 ) 72 C – 1 min 29
synthase (ATP6) 45 C – 55 s(1
every 4 s to 72 )
191
(continued)
192
No.
10. HSP60 HSP60for/ 94 C – 5 min 94 C – 30s 55 C – 30 s 72 C – 90 s 72 C – 10 min 35 [86]
HSP60rev
11. GAPDH GPD1/GPD2 94 C – 5 min 94 C – 30s 48 C – 30s 72 C – 90s 72 C – 7 min 35 [9]
G3PDHfor/ 94 C – 5 min 94 C – 30s 64 C – 30s 72 C – 90s 72 C – 10 min 35 [86]
G3PDHrev
Gpd1-LM/ 94 C – 5 min 94 C – 45 s 53 C – 45 s 72 C – 90s 72 C – 5 min 40 [7]
Gpd2-LM
GDF/GDR 95 C – 4 min 95 C – 30 s 60 C – 30 s 72 C – 45 s 72 C – 7 min 35 [91]
VGPDf2/ 94 C – 2 min 94 C – 10 s 56 C – 20 s 72 C – 1 min 72 C – 7 min 32 [40]
VGPDr
12. Calmodulin CF1/CF4 94 C – 5 min 94 C – 45 s 55 C – 45 s 72 C-1 min 72 C – 7 min 35 [66]
(CaM) CF1L/CF4 96 C – 2 min 96 C – 30 s 51 C – 30 s 72 C – 90s 72 C – 5 min 42
CF1M/CF4 96 C – 2 min 96 C – 30 s 51 C – 30 s 72 C – 90s 72 C – 5 min 42
Cal228F/ 95 C – 8 min 95 C – 15 s 55 C – 20 s 72 C-1 min 72 C – 5 min 35 [18]
CAL2Rd
cmd5/cmd6 96 C – 2 min 96 C – 30 s 51 C – 30 s 72 C – 90s 72 C – 5 min 42 [35]
Cal228F/ 94 C – 2 min 94 C – 1 min 55 C – 30 s 72 C-1 min 72 C – 10 min 35 [18]
Cal737R
CL1/CL2a 95 C – 5 min 95 C – 30 s 55 C – 30 s 72 C-1 min 72 C – 8 min 35 [63]
13. Mini chromo- MCM7–709/ 94 C – 94 C – 45 s 56 C – 50 s 72 C-1 min 72 C – 5 min 38 [78]
some mainte- MCM7–1348 10 min
nance
protein(MCM7)
N. Mehta et al.
10
14. CHS-1 CHS-79F/ 95 C – 8 min 95 C – 15 s 55 C – 20 s 72 C-1 min 72 C – 7 min 32 [18]

CHS-345R
15. Histone-3 (HIS3) CYLH3F/ 96 C – 5 min 96 C – 30s 52 C – 30 s 72 C – 1 min 72 C – 5 min 30 [22, 29]
CYLH3R
CYLH3F/H3- 94 C – 5 min 94 C – 30s 58 C – 30 s 72 C – 1 min 72 C – 5 min 40
1b
16. Cox2 FM35/ 94 C – 3 min 94 C – 1 min 54 C – 1 min 72 C – 2 min 72 C – 5 min 35 [56]
Phy10b
FM58/ FM66 94 C – 5 min 94 C – 1 min 52 C – 1 min 72 C – 2 min 72 C – 7 min 32 [55]
17. Cox3 CO3F1/ 94 C – 5 min 94 C – 1 min 50 C – 1 min 72 C – 1 min 72 C – 7 min 35 [8]
CO3R1
18. Nad9 Nad9-F/ 94 C – 3 min 94 C – 1 min 61 C – 1 min 72 C – 2 min 72 C – 5 min 35 [56]
Nad9-R
19. Acl1 acl1-230up/ 95 C – 3 min 95 C – 45 s 64 C – 45 s 72 C – 2 min 72 C – 8 min 5 [31]
acl1-1220lowa 95 C – 45 s 62 C – 45 s 72 C – 2 min 5
95 C – 45 s 56 C – 45 s 72 C – 2 min 30
20. TS VTs3f/VTs3r 94 C – 2 min 94 C – 10s 59 C – 20s 72 C – 1 min 72 C – 7 min 32 [40]
21. ApMAT AM-F/AM-R 94 C – 3 min 94 C – 45 s 62 C – 45 s 72 C – 1 min 72 C – 7 min 30 [84]
22. Cytochrome B E1M4/E2mr3 94 C – 4 min 94 C – 1 min 46 C – 1 min 72 C – 1 min 72 C – 4 min 36 [12]
(CYTB)
23. Alt a1 Alt-for/Alt- 94 C – 4 min 94 C – 30s 57 C – 30 s 72 C – 1 min 72 C – 7 min 35 [36]
rev
24. OPA1–10 OPA10-2L/ 94 C – 5 min 94 C – 30s 62 C – 30 s 72 C – 45 s 72 C – 7 min 36 [6]
OPA10-2R
25. Endo PG PG3/PG2b 94 C – 5 min 94 C – 30s 50 C – 30 s 72 C – 30 s 72 C – 7 min 40
a
Touchdown PCR conditions
193
194 N. Mehta et al.
10.4 Selection of Gene Target and Their Amplification
Selection of gene target is very crucial step in the fungal molecular taxonomy and
phylogeny. Most common DNA markers proposed for molecular identification and
phylogeny of fungi are the nuclear ITS region of the ribosomal RNA gene. We have
enlisted the suitable gene targets/markers which can be used for specific fungal
genera and/or species level identification, their respective primer pair, primer
sequences, and PCR cycling conditions (Tables 10.1 and 10.2). We have provided
this information for few selected fungi, and for the non-listed fungi, the researchers
are advised to find out the recommended gene targets/markers by following the
recent literature on the same.
10.4.1 Materials
1. Taq DNA polymerase and 10X DNA polymerase buffer (Sigma)

2. Forward and reverse primers
3. dNTPs mix (Sigma)
4. DNA template
5. Sterile distilled water
6. 0.2 ml PCR tubes
7. Micropipettes
8. PCR tube stand
9. Thermal cycler
10.4.2 Method
The steps for PCR amplification are as described below. The total volume can either
be 25 or 50 μL.
1. Thaw all the PCR reagents completely on ice before use. (Make sure the reagents
were kept at 20 C).
2. Prepare PCR reaction mix in a thin-walled 0.2 mL PCR tubes for 25/50 μL
reaction volume.
3. All the PCR reagents can be added in the following order: water, buffer, dNTPs,
forward and reverse primers, Taq DNA polymerase and DNA template, or if one
has PCR master-mix, then the order should be water, master-mix
(e.g. EmeraldAmp GT PCR Master Mix, Takara), both primers and DNA
template.
Reagents Volume Final concentration

PCR buffer (10X)* 2.5 μl 5 μl 1X
dNTPs mixture 2 μl 4 μl 200 mm
Forward primer 0.5 μl 1 μl 10pM/μl
Reverse primer 0.5 μl 1 μl 10pM/ μl
Taq DNA polymerase 0.25 μl 0.25 μl 1unit/μl
Template 1 μl 1 μl 10 ng/μl
dH2O 18.25 μl 37.75 μl
Total 25 μl 50 μl
*10x PCR buffer can be used either (Mg2+ free), or MgCl2 solution shall be used with 10x Buffer, if
necessary
4. Gently pipette the reaction mixture to allow good mixing and give short spin to
settle down the reagents.
5. Prepare positive and negative control reactions with and without template DNA
of known size, respectively along with test sample. (Note: positive control
confirms that all PCR reagents are working properly, whereas negative control
confirms that there is contamination in reagents which will result into false band).
Note: If one wants to perform a greater number of PCR reactions, then it is
recommended to first prepare a PCR master mix, except the reagents which are to
be varied. (e.g., DNA template). For all the PCR related information (Primer
name and sequences, PCR conditions), refer Tables 10.1 and 10.2.
6. 2 μL of PCR product is subjected to 1.2% agarose gel electrophoresis (refer
agarose gel electrophoresis protocol).
7. Observe the gel under UV transilluminator gel documentation system.
10.5 PCR Product Purification and Quantification
Purification of PCR product is essential before proceeding with DNA sequencing,

which aids the removal of remaining enzymes, primers, nucleotides, and buffer
components. Traditionally, purification was done by organic extraction methods
like phenol chloroform treatment, followed by ethanol precipitation. Nowadays,
there are various rapid and less hazardous PCR clean-up kits are available. These
kits utilize spin columns having a matrix of silica on which DNA is bound selec-
tively. Finally, elution of the DNA occurs from the column with elution buffer or
milli-Q water and is ready for DNA sequencing. The DNA fragment of interest can
be purified by gel extraction method (gel extraction kits can be used) if the PCR
product is contaminated with any non-specific amplification product and primer
dimers. The most commonly used kits are GenElute PCR Clean-Up Kit Sigma-
Aldrich, FavorPrep™ GEL/PCR Purification Kit – Favorgen, QIAquick PCR Puri-
fication Kit – Qiagen, etc. (each kit has its own protocol; one can follow the
manufacturer’s guidelines provided with the kits).
196 N. Mehta et al.
After PCR product purification, quality and quantity of amplicons must be

checked before proceeding with DNA sequencing. To check the quality and size
of PCR product, they are analyzed on agarose gel electrophoresis along with
appropriate DNA marker/ladder (see agarose gel electrophoresis protocol). For
quantity and quality check, NanoDrop spectrophotometer and Qubit fluorometer
can be used. Details and basic principle of quantification of DNA/PCR product is
given in section High throughput DNA extraction protocol.
10.6 DNA Sequencing (Sanger Method)
DNA sequencing is the process to determine the order of nucleotides in a given DNA
molecule. The first-generation sequencing approach emerged in the 1970s, which
included the Maxam-Gilbert method and the Sanger method (or dideoxy method),
discovered by English biochemist Frederick Sanger. Eventually, Sanger method
became the more commonly employed technique among these two approaches
because it is a rapid and easy method for DNA sequencing. This conventional
chain-termination approach requires a single-stranded DNA template, a primer,
DNA polymerase, dNTPs, and modified di-deoxynucleotide triphosphates
(ddNTPs), the latter of which plays a key role in terminating DNA strand elongation.
These chain-terminating ddNTPs lack a 3’-OH group, which is required for
phosphodiester bond formation between two consecutive nucleotides; hence, the
moment it gets incorporated into newly synthesised strand it halts the extension of
DNA [77]. The ddNTPs are fluorescently labelled for detection in automated
sequencing machines. The protocol mentioned here is of Applied Biosystems –
BigDye™ Terminator v3.1 cycle sequencing kit.
The direct approach of cycle sequencing of amplified and purified PCR products
depends on two factors, i.e. source material (type and amount) and contamination
like proteins, RNA or chromosomal DNA, excess primers, dNTPs, enzyme, residual
salts along with some organic chemicals like phenol and chloroform. Before pro-
ceeding with sequencing, the critical requirement is to quantify the purified DNA
and calculate A260/280 ratios (A260/280 ¼ 1.8–2.0). The optimum concentrations
of DNA template are mentioned below:
Recommended DNA Quantities
DNA template (purified PCR product) Quantity

100–200 bp 1–3 ng
200–500 bp 3–10 ng
500–1000 bp 5–20 ng
1000–2000 bp 10–40 ng
>2000 bp 20–50 ng
10.6.1 Materials
1. BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific)

2. Distilled water
3. Hi-Di formamide (Thermo Fisher Scientific)
Note: Not required for BigDye XTerminator purification kit (Thermo Fisher
Scientific)
4. Primers of interest
5. MicroAmp clear adhesive film (Thermofisher)
6. MicroAmp optical 96-well reaction plate (Thermo Fisher Scientific)
7. Vortex machine
8. Centrifuge with swinging bucket (with PCR plate adapter)
9. BigDye XTerminator purification kit (Thermo Fisher Scientific)
10. EDTA (125 mM)
11. 100% and 70% ethanol
12. Micropipettes
13. 0.2 ml PCR tubes
14. PCR tube stand
10.6.2 Method
1. Thaw all the reagents of the BigDye Terminator v3.1 cycle sequencing kit and
primers completely (place on ice).
Note: Prevent dye terminators to light exposure.
2. Vortex and centrifuge briefly the tubes for 3–4 s to collect all tube contents at
bottom.
3. Add components in 0.2 ml thin-walled tube to prepare reaction mix as
indicated below:
S. N. Reagents Volume
1. 5X sequencing buffer 2 μL
2. Primer (primer of interest) 0.6 μL
3. BigDye terminator 3.1 1.2 μL
4. Template-purified PCR product (10-50 ng depend on length of template, refer 1 μL
recommended DNA quantities)
5. Sterile dH2O 1 μL
6. Total 10 μL
Note: One can use BDT 3.1 ready mix (3–4 μL) that has sequencing buffer also, so no need to add
sequencing buffer separately
198 N. Mehta et al.
10.7 Perform Sequencing PCR in Thermal Cycler Using

Following Conditions
Parameter Temperature Time Cycle

Initial denaturation 96 C 1 min 1
Denaturation 96 C 30 s 25
Annealing 55 C 15 s
Extension 60 C 4 min
Final extension 60 C 2 min 1
Hold 4 C Hold
10.7.1 Purification of Sequencing PCR Product
1. Add 2 μL of freshly prepared EDTA (125 mM) to sequencing PCR product.

2. Add 50 μL of 95–100% ethanol to the tube, mix well by pipetting.
3. Incubate it for 15 min at RT and centrifuge at 12,000 rpm for 20 min at 4 C.
4. Decant the supernatant and add 200 μL of 70% ethanol, and centrifuge at
12,000 rpm for 10 min at 4 C.
5. Repeat 70% ethanol wash and air dry the pellet.
6. Add 10–12 μL of Hi-Di formamide.
Note: One can use BigDye XTerminator™ purification kit (catalog num-
ber: 4376486) for the purification of sequencing PCR product using manufac-
turers’ instruction and protocol.
7. Place the purified sequencing PCR product in MicroAmp optical 96-well reac-
tion plate.
8. Seal the plate with MicroAmp clear adhesive film.
9. Vortex it for 3–4 s, then give short spin in a swinging bucket centrifuge, to
collect all the plate contents at bottom of the wells for 8–10 s.
10. Put the plate in a thermal cycler and denature the DNA at 96 C for 5 min.
11. The plate containing DNA samples is then subjected to appropriate Genetic
Analyser or DNA Sequencer (e.g. ABI PRISM ® 3100-Avant Genetic
Analyzer).
10.8 Sequence Editing, Analysis, and Identification
Once the DNA sequences are generated, the most important task is to ensure the
quality of the DNA sequences. Different tools like Finch TV and Chromas Lite can
be used to visualize the DNA sequence in the form of electropherogram. Both these
tools are freely available to download. Another important point is that one should try
to get full length DNA sequence of any given gene target/marker. If the sequence
length is short, one should sequence both the strands of the DNA and make a
consensus sequence to get the full-length sequence of the gene. Usually the quality
scores of the initial and last few bases (20–30) in a given DNA sequence are not
always up to the mark; therefore, while performing similarity searches, these few
bases should not be considered.
10.8.1 Basic Local Alignment Search Tool (BLAST)
BLAST is an algorithm for comparing primary biological sequence information,

such as the amino acid or nucleotide sequences of proteins and DNA/RNA, respec-
tively. It has been the most widely used portal for comparison of query sequences
with a library or database of sequences, to identify the resembling sequences above a
certain threshold value. There are different tools which can be employed according
to ones’ need for example BLAST (blastn) program (DNA/RNA), in which given
DNA query is compared with the most similar DNA sequences from the DNA
database as per user specifications. Pairwise sequence alignment allows you to
compare the sequence of interest with all known sequences in a database so that
all homologous sequences are attained. Basically, they show most closely related
sequences first, followed by sequences with diminishing similarity. These matches
are generally reported to assess the statistical significance like an Expectation-value.
The important point to be considered while working with fungal query sequences
using BLAST search is its results against GenBank database because approximately
27% of GenBank fungal ITS sequences were deposited with inadequate taxonomic
identification [59] and 20% of fungal sequences are incorrectly annotated, but this
varies according to taxonomic group [16, 69].
10.8.2 MycoBank Database Search
MycoBank is a recognized nomenclature repository for all published fungal novel-

ties since 2004, which provides an open access database to mycologists [22]. It also
allows online pairwise sequence alignment and identification (www.mycobank.org/
biolomicssequences.aspx) against other well-curated databases such as CBS collec-
tions websites, Q-bank, EUBOLD system, ITS Database for Human and Animal
Pathogenic Fungi (ISHAM), Fungal Barcoding, or UNITE. The unknown DNA
sequences are compared against all the required reference databases either at the
same time or against individual database, on the basis of which the results are shown
centrally, as a unique matching list of fungi. This particular database identifies new
species in reference with information based on morphological, physiological, and
molecular data.
200 N. Mehta et al.
10.8.3 UNITE Database Search
UNITE (https://unite.ut.ee/) is an online database managing large number of

sequences which is useful for the fungal molecular identification. It targets the
nuclear rDNA-ITS region and offers 10,00,000 public fungal ITS reference
sequences. There are 4,59,000 species hypothesized cluster to which digital object
identifiers (DOIs) are assigned to avoid ambiguous referencing across studies. In the
past 15 years, the curation and annotation of sequence by a third party have made
approximately 2,75,000 improvements, both in-house and web-based. UNITE in
collaboration with other community resources and fungal sequence database pro-
vides a large amount of data for a number of meta-barcoding software pipelines [58].
10.8.4 Method
1. Obtain sequences from sequencer in the form of an electropherogram and edit

either manually using Chromas Lite software or by BioEdit software for
inconsistencies.
2. Align with sequences in the NCBI BLASTn tool for identification.
3. Cases where no substantial similarity is found with sequences in the databases
confirm the identity with the help of sequences from other loci of same strain or
sequences from additional strains.
Note: A large part of the known fungal diversity is not sequenced at all; hence, one
will be unable to identify an unknown fungus, as the sequence information will not
be available in the database. Moreover, few of the sequences deposited in the
databases might be having errors; therefore, one has to be cautious in identification
and drawing any phylogenetic conclusion based purely on NCBI database compar-
isons. In such situations, one can also try using other databases, e.g., MycoBank and
UNITE.
10.9 Molecular Phylogeny and Phylogenetic Tree
Phylogenetics is the branch of Science that estimates and evaluates evolutionary

relationships among individuals/organisms for their identification. In order to deter-
mine phylogenetic relationship, alignment and comparison of different fungal
sequences can be done and on the basis of their physical and genetic features a
number of differences can be determined. This relationship is represented in the form
of “phylogenetic tree” in which the branch’s position and length represent the
relation between different fungi. Closely related fungi will exhibit lesser sequence
differences than distantly related fungi.
10.9.1 Phylogenetic Tree Construction
Phylogenetic trees can be constructed using DNA sequences of closely related fungi
and various computational phylogenetic methods. The basic and simple method of
tree construction is distance-based method including neighbour joining and
UPGMA method, which performs multiple sequence alignment using ClustalW
method and calculate genetic distances. The most common tree construction method
is character-based: include maximum parsimony that involves an inherent model of
evolution (i.e. parsimony), and maximum likelihood which is an advanced
method that uses Bayesian scheme and applies a precise open model of evolution.
Identification of a most favourable phylogenetic tree is non-parametric; therefore,
the optimization and combination of these methods are required to identify a best
possible and optimal phylogenetic tree, while building trees bootstrapping is
followed. A bootstrap is a statistical method for assessing phylogenetic tree where
the bootstrap value represents that out of 100 how many times the same specific
branch is observed when repeating the phylogenetic tree reconstruction from the
same set of data.
10.9.2 Phylogenetic Tree Construction Using MEGA

Software
1. A number of taxonomical target DNA sequences are retrieved from BLAST

searches and GenBank.
2. The DNA sequence of test strain and all other relevant DNA sequences are saved
in FASTA file format.
3. The FASTA file format is converted to .fas file format.
4. The .fas file is opened in MEGA 7 or X software and subjected to multiple
sequence alignment (MSA) using ClustalW or MUSCLE.
5. The alignment file can be saved for further analysis as MAS file.
6. The MAS file is opened in MEGA 7/X software.
7. The MSA data is subjected to phylogenetic tree construction using suitable
method like maximum parsimony, maximum likelihood, NJ, and UPGMA.
8. Different parameter needs to be instructed to the program to construct a
phylogenetic tree.
9. An example is shown in below mentioned table.
Analysis Phylogenetic tree construction

Statistical method Maximum parsimony
Test of phylogeny Bootstrap method
Number of bootstraps 1000
Model Kimura-2 parameter model
Branch swap filter Very strong
Tree inference option Neighbour joining
202 N. Mehta et al.
10. Once the tree is constructed the session of tree can be saved as MTS file.
11. Post-construction, various modifications can be done in the tree.
12. The tree can be obtained as JPEG image/PDF file along with the caption.
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Chapter 11
Fluorochrome-Based Methods for Fungal
Sample Examination
Silvino Intra Moreira, Lucas Fidelis Pereira, Elaine Aparecida de Souza,

and Eduardo Alves
Contents
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
11.2 Sample Preparation to Observe Fungi Nuclei and Chromosome . . . . . . . . . . . . . . . . . . . . . . 211
11.3 DAPI Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
11.3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
11.3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
11.3.3 Culture Block on Inverted Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
11.3.4 Slide and Coverslip Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
11.4 Culture Block on Inverted Microscope and Propidium Iodide in Nuclei Marking . . . 215
11.4.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
11.4.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
11.5 Sample Preparation to Observe Fungi Chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
11.5.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
11.5.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
11.6 Sample Preparation to Observe Fungi Cell-Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
11.7 Calcofluor White Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
11.7.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
11.8 AlexaFluor 488® WGA Conjugate Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
11.8.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
11.8.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
11.9 Reactive Oxygen Species (ROS) in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
11.9.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
11.9.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
11.10 Fungi Cellular Death Studies with Propidium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
S. I. Moreira · E. Alves (*)

Department of Plant Pathology, Electron Microscopy, and Ultrastructural Analysis Lab, Federal
University of Lavras (UFLA), Lavras, Minas Gerais, Brazil
e-mail: ealves@ufla.br
L. F. Pereira · E. A. de Souza
Department of Biology, Plant Disease Resistance Lab, Federal University of Lavras (UFLA),
Lavras, Minas Gerais, Brazil
e-mail: easouza@ufla.br
210 S. I. Moreira et al.
11.10.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

11.10.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
11.10.3 Conidia or Yeast Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
11.11 Live-Dead Test for Fungi Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
11.11.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
11.11.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
11.12 Sample Preparation to Observe Fungi-Plant Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
11.12.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
11.12.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
11.13 Important Plant Structures Defence Against Phytopathogenic Fungi . . . . . . . . . . . . . . . . . 228
11.14 Callose Deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
11.14.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
11.14.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
11.15 Lignin Localization in Plant-Fungi Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
11.15.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
11.15.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
11.16 Sample Preparation to Observe Autofluorescent Fungi and Specific Structures . . . . . . 230
11.17 Autofluorescent Rust Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
11.17.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
11.17.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
11.18 Autofluorescent Cercosporin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
11.18.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
11.18.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
11.1 Introduction
Fungi are eukaryotic, usually filamentous, spore-producing organisms and can be

obligate parasites, nonobligate parasites, or biotrophs, developing several interac-
tions with plants, animals, or the environment and can be used to produce food and
enzymes for industrial processes [27]. Most fungi species have microscopic struc-
tures and studies on this organism group depend on various microscopy techniques
types.
Stokes related the fluorescence phenomenon in 1852, describing a photon molec-
ular absorption generating the emission of another photon with greater wavelength,
the principle from which it was possible to develop techniques of fluorescence
microscopy [20]. Therefore, natural or induced fluorescence characteristics have
been explored for organisms and macromolecules localization explaining several
types of biological phenomena, mainly by techniques of epi-fluorescence or laser
confocal microscopy.
These techniques enable many morphological and physiological analyzes in cells
and tissues, locating cellular components, interaction with plants, nuclear dynamics,
reactive oxygen species accumulation, and cellular death.
Fluorescence microscopy studies may include analyzes of fluorescence or
autofluorescent samples. Some fungi like Basidiomycota are autofluorescent, and
11 Fluorochrome-Based Methods for Fungal Sample Examination 211
others such as Cercospora spp. produce fluorescent phytotoxins. Other studies types
are conducted by inducing fluorescence in the samples. This may occur through the
use of fluorochromes, immunofluorescence techniques, nucleic acids hybridization,
and molecular markers. Fluorochromes are molecules capable of specifically binding
to cellular components by inducing their fluorescence under the excitation of certain
wavelengths. These components can be the fungal cell wall, nuclei, chromosomes,
mitochondria, and others. Other fluorochromes may indicate physiological aspects,
such as cell death, accumulation of reactive oxygen species, or evidence of defense
reactions in plant tissues colonized by fungi. Immunofluorescence, also called
immunostaining, is a technique where fluorescent molecules are attached to anti-
bodies corresponding to antigens to which they will be located. Nucleic acid
hybridization, called FISH (fluorescent in situ hybridization), allows the use of
nucleic acid probes attached to fluorescent molecules. These probes are complemen-
tary to target sequences, which can identify specific regions on chromosomes, the
expression of certain genes by mRNAs, or specific groups of organisms using
regions of phylogenetically important DNA. One of the most commonly used
molecular markers is fluorescent proteins such as GFP (green fluorescent protein).
By genetic transformation, the fluorescent markers genes are associated with the
genes whose products will be localized. Thus, the expressed proteins are localized
through the fluorescent protein anchored.
In this chapter, protocols for fluorescence microscopy will be discussed in studies
on fungi using only fluorochromes or autofluorescence of structures for localization
techniques.
All following procedures were done at the Electron Microscopy and Ultrastruc-
tural Analysis Lab at Federal University of Lavras, using an inverted
Epi-Fluorescence Zeiss Axio Z.1 and an inverted Laser Confocal Zeiss LSM780
Observer Z.1 and Zen 2012 software.
11.2 Sample Preparation to Observe Fungi Nuclei

and Chromosome
Ascomycota fungi have the characteristic of maintaining monocariotic state but may
present some dicariotic cells throughout the intermediary processes of their repro-
duction, such as plasmogamy and karyogamy. In the case of Basidiomycota, they
may have different phases throughout their life cycle, monocariotic or even
dicariotic (Fig. 11.1e). Basidiomycota may present multinucleate cells, as basidia
before nuclei migration to the basidiospores, and the in certain Rhizoctonia hyphae
(Fig. 11.1f). These nuclear dynamics processes can be studied using fluorescence
markers, such as DAPI, one of the most used. Often, DAPI is used associated with
immunofluorescence and hybridization techniques, for proteins or DNA sequences
specific localization in nuclei. Other fluorescent nuclei dyes can be used, such as
SyBrGreen and Propidium Iodide (in the case of living cells).
Fig. 11.1 Fungi nuclei and chromosome observations by fluorochrome-based methods. (a) Posi-
tioning of DAPI drops above coverslip coupled in an inverted microscope. (b) Sampling of
Rhizoctonia solani colony growth in SNA media. (c) Transferring of colony fragment to the dye.
(d) Vacuum pump linked to a hermetically sealed recipient. Laser scanning confocal micrographs of
Rhizoctonia solani from binucleated (e) and multinucleated lineages (f) marked with DAPI;
Rhizoctonia sp. nuclei marked with Propidium Iodide (g); and Colletotrichum chromosome at
metaphase observation by germ tube burst method using DAPI (h)
11.3 DAPI Methods
Materials to prepare fungi for EFM or LSM observation. The necessary materials
can vary depending on the protocol used.
11.3.1 Materials
1. Nucleus marker DAPI (40 6-Diamidine-20 -phenylindole dihydrochloride)

(VectaShield® H-1200 Vector®) mounting medium.
2. Calcofluor White (Fluorescent brightener 28, Sigma, CAS-4404-43-7) 0.01%
(p/v) prepared in 0.01 M potassium phosphate buffer solution (PBS) pH 7.2.
3. Fungi colony growth within a clear media, such as Synthetic Nutrient-poor Agar
(SNA) [4], with living mycelia, or fixed fungi fragments.
4. Kolle handle with a needle
5. Scalpel.
6. Tweezer.
7. Bunsen burner.
8. Alcohol.
9. Automatic pipette.
10. Slides and regular coverslips (e.g. 20 20 mm).
11. Large coverslips (e.g. 24 40 mm).

12. Nail polish or other coverslip sealants.
13. Vacuum pump linked to a hermetically sealed container.
14. Stereomicroscope.
15. EFM or LSM, vertical or inverted.
16. For EFM, a filter cube that works near 405 ηm excitation and 450 ηm emission
(ex. Filter cube Zeiss #49).
17. For LSM, excitation with Diode 405 ηm laser line and emission filter with
420–480 ηm range. An EC Plan-Neofluar 40/1.30 Oil DIC M27 objective was
used, with 1024 1024 resolution, and around fourfold zoom. The bright-field
image was acquired using the TPM-T detector.
11.3.2 Methods
The methods presented below are for marking filamentous fungi nuclei, and varia-
tions will be presented and discussed.
11.3.3 Culture Block on Inverted Microscope
Firstly, we will show a quick preparation to observe nuclei in fungi (Fig. 11.1e, f)
using an inverted microscope (EFM or LSM) as made by Melo et al. [17].
1. Put a previously cleaned large coverslip (e.g. 24 40 mm) on an inverted
microscope stage. In case of immersion objective using, drop firstly the appro-
priated immersion liquid on the objective lens before that.
2. Put 10 μL of DAPI-VectaShield mounting media above the coverslip
(Fig. 11.1a). In case of many samples working, use a marker pen to delimitate
and identify spots with around 0.5 0.5 cm where each sample will be placed.
Avoid the coverslip corners, where the objective lens does not reach. Develop it
in a room with diffuse light, or as dark as possible.
3. Take colony fragments with around 0.5 0.5 cm with a scalpel (Fig. 11.1b),
choosing the region of interest using a stereomicroscope, if needed. The use of
clear media like SNA allows better light transmission. Besides, it favors the fungi
sporulation and reduces the aerial mycelia growth rate. The colony fragments
may be fresh or fixed.
4. Transfer the upside-down colony fragment above the DAPI drop. The mycelia
should be in contact with the dye (Fig. 11.1c).
5. Leave it in the dark for 5–20 min. The incubation time will depend on the fungus
species. The thinner cell wall species as well as the younger mycelia usually need
less time to mark the nuclei. Some thicker cell wall species demand incubation
with vacuum for dye infiltration. This can be done using a vacuum pump linked to
a hermetically sealed container (Fig. 11.1d). In these cases, it is recommended to
use younger cultures and colony border regions.
6. Observe in inverted EFM or LSM.
If the LSM is equipped with a detector like T-PMT, the bright-field images
acquired overlaid with fluorescent nuclei may dispense the need of other fluoro-
chromes for fungi cell walls marking, such as Calcofluor White. Sometimes, DAPI
colors well the cell walls also. Once the LSM is not equipped for bright field images,
the Calcofluor White use is recommended. For this, the DAPI needs to be applied
before and directly in the culture fragment. After this, 10 μL of the 0.01% (p/v)
Calcofluor White (Fluorescent brightener 28, Sigma, CAS-4404-43-7) prepared in
0.01 M potassium phosphate buffer solution (PBS) pH 7.2 are positioned above
coverslip, as described in step 2. Follow, the colony fragment DAPI-treated should
be positioned upside-down above the Calcofluor drop and incubate for around
20 min in darkness before observation. The excitation/emission parameters are the
same used for DAPI in EFM or LSM.
11.3.4 Slide and Coverslip Preparation
Below is a procedure to observe fungi nuclei using slide and coverslip (vertical or
inverted microscope, EFM or LSM):
1. Put 10 μL of DAPI- VectaShield mounting media above a previously cleaned
slide.
2. Sample fungi structures from fresh or fixed cultures using a needle previously
cleaned with alcohol and flamed. Choose the region of interest using a stereo-
scopic microscope, if needed. In this case, the DAPI fluorescence signal in slides
prepared with fixed structures may persist for weeks, if slides kept in the
refrigerator, in darkness.
3. Transfer the specimens to DAPI drop.
4. Cover the drop with specimen with coverslip and seal with nail polish or other
coverslip sealants.
5. Leave it in the dark for 5–20 min. The time may vary, as discussed above. A
vacuum step may be required.
6. Observe in a vertical or inverted microscope, EFM or LSM.
11.4 Culture Block on Inverted Microscope and Propidium

Iodide in Nuclei Marking
Propidium Iodide can be used as a fungi nuclei marker in the case of living cells
(Fig. 11.1g). After the cell death, the fluorochrome passes through the nuclear
envelope and occupies the cytosol, marking the entire cell. Thus, this marker is
important in cellular death studies, as will be discussed later.
11.4.1 Materials
1. Propidium Iodide (Sigma-Aldrich, CAS-11348639001) work solution 1.0 μg.

mL1 prepared in 0.01 M PBS pH 7.2.
2. Fungi colony growth within a clear media, such as SNA, with living mycelia.
3. Scalpel.
4. Tweezer.
6. Large coverslips (ex. 24 40 mm).
7. Stereomicroscope.
8. Inverted EFM or LSM.
(e.g. Filter cube Chroma TxRed#39004).
10. For LSM, excitation with Argon 514 ηm laser line and emission filter with
used, with 1024 1024 resolution, and around fourfold zoom. The bright-field
image was acquired using the TPM-T detector.
11.4.2 Method

2. Put 10 μL of Propidium Iodide work solution (PIWS) above the coverslip. In case
of many samples working, use a marker pen to delimitate and identify spots with
around 0.5 0.5 cm where each sample will be placed. Avoid the coverslip
corners, where the objective lens does not reach. Develop it in a room with diffuse
light, or as dark as possible. It is recommended to test PIWS dilutions, as it is
possible that concentrations as 0.1 and 0.01 μg.mL1 may provide good results
depending on the case.
3. Take colony fragments with around 0.5 0.5 cm with a scalpel, choosing the
region of interest using a stereoscopic microscope, if needed. The colony frag-
ments should be fresh.
4. Transfer the upside-down colony fragment above the PIWS drop. The mycelia
should be in contact with the dye.
5. Leave it in the dark for 15–20 min.
11.5 Sample Preparation to Observe Fungi Chromosome
Karyotyping involves characterize the number, morphology, and size of chromo-

somes of a species, and the main methods used for fungi are pulsed-field gel
electrophoresis (PFGE) and germ tube burst method (GTBM) [28]. The techniques
are important to determine the polymorphisms in the size and number of chromo-
somes between strains from the same species, or even extra chromosome presence.
The study of these chromosomes is very important, since they may contain
pathogenicity-essential genes of fungi to cause plant diseases [9]. Following, the
GTBM method for Colletotrichum chromosome observation (Fig. 11.1h).
11.5.1 Materials
1. Petri dishes with PDA culture media.

2. PD liquid culture media.
3. Sterilized 40 μm miracloth filter.
4. Autoclaved 1.5 mL microtubes.
5. Centrifuge for microtubes.
6. Neubauer chamber.
7. Bunsen burner.
8. Tweezers.
9. Mili-Q water.
10. Sterilized distilled water.
11. Autoclaved slides.
12. Coverslips.
13. Poly-L-lysine.
14. Rubber glue.
16. Wet chamber.
17. Incubator adjusted for 22 C.
18. Bright field microscope.
19. Thiabendazole (TBZ) solution 50 μg.mL1.
20. Methanol.
21. Glacial acetic acid.

22. DAPI 1 μm.mL1 and VectaShield mounting medium.
23. Propidium Iodide 1 μm.mL1.
24. Nail polish.
25. EFM or LSM.
(e.g. Filter cube Zeiss #49). (A 100 objective was used on an inverted EFM).
420–480 ηm range.
11.5.2 Method
The following method was refined by Gonçalves [11], adapted from Taga et al. [24],
to observe chromosomes from Colletotrichum species.
1. Growth of fungi in PDA at 22 C in darkness. After sporulation, the conidia
suspension is acquired with mili-Q water and scraping and collected with an
automatic pipette.
2. The suspension is filtered with miracloth 40 μm filter and centrifuged within
microtubes at 3000–3500 g for 5 min.
3. After centrifugation, the supernatant should be discarded and the pellet washed
with sterile water twice. Resuspend the pellet in a nutrient medium and adjust
the concentration to 2 to 3 106 conidia.mL1 using a Neubauer chamber.
4. The previously autoclaved slides are treated with poly-L-lysine solution and
marking a rectangle on the slide with the rubber glue.
5. Pipetting 450–600 μL conidia solution into the rectangle marked with the rubber
glue on the face of the poly-L-lysine slide.
6. Place the slides in a humid camera and incubate them in the dark at 22 C.
Monitor the germination after 6 h of incubation using a light microscope,
prolong the incubation time if necessary.
7. Remove the liquid excess from the slide surface without drying out completely.
8. Add 400–600 μL of the nutrient medium containing thiabendazole (TBZ) at the
final concentration of 50 μg.mL1 to stop mitosis during metaphase.
9. Incubate the slide at 22 C in the dark for 2–3 h, then remove the TBZ solution
using an automatic pipette and remove the rubber glue using tweezers.
10. Slowly immerse the slide in mili-Q water to wash off excess TBZ that may still
be present on the blade and remove excess water using a filter paper, but still
leaving the slide moist.
11. Dry the slide by passing it over a flame quickly, without letting it dry completely
or overheating the slide.
12. Add 20–25 μL of the propidium iodide 1 μg.mL1 on the slide surface and wait
15 min. After, immerse the slide in sterile water to remove the dye excess.
13. Add 15–20 μL of the VectaShield mounting medium with DAPI and incubate in
the dark for 10–15 min and then seal the slide with coverslip and nail polish.
14. Observe in EFM or LSM.
The DAPI-VectaShield mounting media may be used instead of the DAPI and
VectaShield mounting media separated.
11.6 Sample Preparation to Observe Fungi Cell-Wall
The fungi cell wall staining with fluorochromes is very important in many studies,
such as during infection processes in plant tissues, delimitating the septa during the
nuclei dynamic studies, and associated with other methods, such as immunolabeling
or hybridization. Calcofluor White is a commonly used dye for glucans, as the fungi
chitin and the plant cellulose. On the other hand, AlexaFluor 488® WGA Conjugate
(Alexa488-WGA) marks exclusively fungi. Thus, fungi-plant interaction can be
studied with both used together, Alexa488-WGA to dye fungi and Calcofluor
marking plant tissues, as will be discussed later.
11.7 Calcofluor White Staining
This fluorochrome dyes the fungi cell wall (Fig. 11.2a) usually with a short time
incubation, around 10–30 min, depending on species and cell type. The procedure
below was performed with a 7-days-old Pyricularia oryzae colony.
11.7.1 Materials
The necessary materials can vary depending on the protocol used. The samples can
be mounted in slide-coverslip with fungal structures in 0.01 mg.mL1 CalcoFluor in
a vertical microscope or with culture block facing-dawn above 10 μL of 0.01 mg.
mL1 CalcoFluor in large coverslip on an inverted microscope, as discussed in
DAPI methods. The excitation and emission conditions are also similar.
11.8 AlexaFluor 488® WGA Conjugate Staining
Alexa488-WGA labels the fungi cell wall (Fig. 11.2b) and the time incubation may
vary depending on species and cell type. Another very important point is that some
fungi with thick conidia cell walls such as Pyricularia and Alternaria require a
Fig. 11.2 Fungi structures were fluorochrome-stained. (a–b) Fungi cell wall dyed with CalcoFluor
White (a) and AlexaFluor488–WGA (b). (c) Reactive oxygen species labeled with DCF-DA. (d–e)
Cellular-death observation using Iodide Propidium for conidia of filamentous fungi (d) and yeasts
(e). (f) Live-dead test for yeasts and bacteria using Iodide Propidium for dead (red) and Syto9 for
living cells (green)
vacuum-infiltration step for 20 min to efficient dyeing [13]. Fungus with thin cell
walls such as Fusarium can be marked without vacuum step. The method shown was
made with common bean leaves infected with fungus.
11.8.1 Materials
1. Wheat Germ Agglutinin (WGA) AlexaFluor 488® Conjugate (Alexa488-WGA)

(ThermoFischer, CAT-W11261) 10 μg.mL1 work solution prepared in 0.01 M
PBS pH 7.2.
2. 0.01 M PBS pH 7.2.
3. Scalpel, tweezers, and scissors.
4. Sterilized 96-well ELISA plates.
6. Clean coverslips.
7. Glass piece (2 2 1 cm).
8. Aluminum paper.
10. Clean tips.

13. For EFM, a filter cube that works near 488 ηm excitation and 520 emission
(ex. Filter cube Zeiss #38HE).
500–550 ηm range, for Alexa 488. A Plan-Apochromat 63/1.40 Oil DIC M27
objective was used, with 1024 1024 resolution.
11.8.2 Method
1. Sampling of common bean leaves infected with fungus and cutting in 4 4 mm

pieces.
2. After, the leaves fragments are place within Elisa plates with 10 μg.mL1
Alexa488-WGA.
3. Wrap the Elisa plate with aluminum foil and keep it at vacuum (Fig. 11.1d) for
1 h.
4. Wash with PBS.
5. Put a leaf fragment above the previously cleaned large coverslip on an inverted
6. Put the leaf fragment above the coverslip with the region of interest facing down.
7. Place a glass piece above the sample (to minimize the irregular topography of the
sample).
11.9 Reactive Oxygen Species (ROS) in Fungi
Reactive oxygen species (ROS) are ubiquitous in fungi living cells, with high
damaging potential but are also essential for gene expression signaling and devel-
opment in several biological processes [14] such as the programmed cellular death
[10]. One way to study the ROS dynamics is using the 20 ,70 -Dichlorofluorescein
diacetate (DCF-DA) fluorochrome. DCF-DA dyes several ROS, as hydrogen per-
oxide (H2O2), peroxyl (ROO•), hydroxyl (HO•), peroxynitrite anion (ONOO-) and
nitric oxide (•NO), and it’s useful for assessing various stress types in plants, such as
osmotic, thermal, and by pathogen infection [2, 13, 19, 29]. Another fluorochrome
variant, H2DCFDA, was important to understanding the ROS role during rice blast
pathogenesis [6]. The strobilurin resistance in Alternaria alternata is caused by a
non-synonymous mutation that changes the cytochrome b product, replacing a
glycine by an alanine at the condon 143 (G143A mutation) [5]. Resistant strains
remain with low DCF-DA signal when cultivated in PDA media plus high concen-
tration fungicide (Fig. 11.2e), while the wild type presents a high signal (not shown).
11.9.1 Materials
1. 20 ,70 -Dichlorofluorescein diacetate (DCF-DA) (Sigma, CAS-4091-99-0) 10 μM

in filtered dimethyl sulfoxide (DMSO).
2. Conidia suspension without fixation.
3. Water-Agar 1.5% in thin layer on a Petri dish.
4. Kolle handle with a needle.
5. Scalpel.
6. Tweezer.
10. Nail polish or other coverslip sealant.
used, with 1024 1024 resolution. The bright-field image was acquired using
the TPM-T detector.
11.9.2 Methods
11.9.2.1 Conidia Suspension
1. Cut water-agar (WA) in 0.5 0.5 cm block.

2. Carry 10 μL of conidia suspension above the WA block.
3. Wait around 10 min for decanting.
4. Apply 10 μL of DCF-DA 10 μM above the WA block, and leave for 5 min in
darkness.
6. In case of many samples working, use a marker pen to delimitate and identify
spots with around 0.5 0.5 cm where each sample will be placed in the coverslip.
Avoid the coverslip corners, where the objective lens does not reach.
7. Take the WA block with a dyed sample using a scalpel and transfer the upside-
down block above the coverslip.
This procedure may be done for observation in a vertical microscope, using slide-
coverslip preparing, mixing 5 μL sample suspension and 5 μL DCF-DA. In this case,
the fluorochrome concentration will be diluted, and maybe a higher concentration

may be necessary.
11.10 Fungi Cellular Death Studies with Propidium Iodide
Experiments with cellular death marking of fungi can help to characterize pheno-
types in many contexts, such as confirming the fungicide activity of a control
product, instead fungistaticity. Another possible subject is the the programmed cell
death (PCD), which may occur during the sexual and asexual reproduction, in some
infection processes for phytopathogenic fungi, and in the non-self-recognition
mechanism heterokaryon incompatibility (HI) [10]. The strobilurin resistance
of Alternaria alternata is caused by the G143A change in cytochrome b [5], and
low mortality of conidia when cultivated in PDA media plus high fungicide con-
centration (Fig. 11.2d). Other assay was for yeast mortality observation during a
fermentation process (Fig. 11.2f). The cell-death marker used was Propidium
iodide [8].
11.10.1 Materials
1. Propidium iodide (Sigma, CAS-25535-16-4) 1.0 μg.mL1 prepared in 0.01 M

potassium phosphate buffer solution (PBS) pH 7.2.
2. Fungi conidia suspension without fixation.
3. Yeast suspension from a fermentation process.
4. Water-Agar 1.5% in a thin layer on a Petri dish.
6. Scalpel.
7. Tweezer.
(ex. Filter cube Zeiss #45). A 100 objective was used on an inverted EFM –
yeast suspension.
14. For LSM, excitation with HeNe 543 ηm laser line and emission filter with
625–665 ηm range. An EC Plan-Neofluar 40/1.30 Oil DIC M27 objective
was used, with 1024 1024 resolution – Alternaria alternata conidia suspen-
sion. The bright-field image was acquired using the TPM-T detector.
11.10.2 Methods
The methods presented below are for filamentous fungi or yeast cell-death marking,
and variations will be considered.
11.10.3 Conidia or Yeast Suspension

2. Carry 10 μL of conidia or yeast suspension above the WA block.
4. Apply 10 μL of Propidium iodide 1.0 μg.mL1 above the WA block, and leave
for 5 min in darkness.
7. Take the WA block with dyed sample using a scalpel and transfer the upside-
This procedure may be done for observation in vertical microscope, using slide-
coverslip preparing, mixing 5 μL sample suspension and 5 μL Propidium iodide. In
this case, the fluorochrome concentration will be diluted, and maybe higher concen-
tration may be necessary.
11.11 Live-Dead Test for Fungi Cells
The methods presented below are for yeasts and bacteria, and variations will be
considered. In Fig. 11.2f, observation of yeasts and bacteria from a probiotic product
was analyzed. The following protocol was developed based on Stiefel et al. [23] and
Batista et al. [1].
11.11.1 Materials
1. Propidium iodide (Sigma, CAS-25535-16-4) 1.0 μg.mL1 in 0.01 M potassium

phosphate buffer solution (PBS) pH 7.2.
2. Syto9 (Thermo Fischer-S34854) 20 μM in filtered DMSO.
3. Probiotic yeasts and bacteria suspension.

4. Water-Agar 1.5% in a thin layer on a Petri dish.
6. Scalpel.
7. Tweezer.
(ex. Filter cube Zeiss #45) in Propidium iodide localization; filter cube to works
near 488 ηm excitation and 535 emission (ex. Filter cube Zeiss #38HE) in Syto9
working.
14. For LSM, excitation with HeNe 543 ηm laser line and emission filter with
625–665 ηm range in Propidium iodide localization; excitation with Argon
488 ηm laser line and emission filter with 520–550 ηm range in Syto9 observa-
tion. (Plan-Apochromat 63/1.40 Oil DIC M27 objective was used, with
1024 1024 resolution, and around twofold zoom).
11.11.2 Method

2. Carry 5 μL of yeast suspension above the WA block.
4. Above the same surface apply 5 μL of Syto9 20 μM and leave for 40 min in
darkness.
5. After, apply 10 μL of Propidium iodide 1.0 μg.mL1 above the WA block, and
leave for 5 min in darkness.
8. Take the WA block with a dyed sample using a scalpel and transfer the upside-
This method may be adapted also for filamentous fungi and other organisms,
adjusting the concentration and the time incubation. The Syto9 fluorochrome dye
all cells and the Propidium iodide the dead cells only. Thus, the merged green and
red channels will show the living cells green and the dead cells in red color
(Fig. 11.2f). If the Syto9 is not available, it may be done using only the Propidium
iodide, merging the red channel with the bright field image (Fig. 11.2d).
11.12 Sample Preparation to Observe Fungi-Plant

Interactions
Below, a protocol for staining plant tissues with Calcofluor White and fungi with
Alexa488 WGA. Before the staining steps, a clarification protocol was develop
based on Warner et al. [26] and Minker et al. [18]. Common bean leaves infected
with phytopathogenic fungus were used (Fig. 11.3).
11.12.1 Materials
1. CalcoFluor White (Fluorescent brightener 28, Sigma, CAS-4404-43-7) 0.01 mg.

mL1 work solution prepared in 0.01 M PBS pH 7.2.
2. Wheat Germ Agglutinin (WGA) AlexaFluor 488® Conjugate (Alexa488-WGA)
(Thermo Fischer, CAT-W11261) 10 μg.mL1 work solution prepared in 0.01 M
PBS pH 7.2.
3. Clarifying solution (6 M Urea, 30% Glicerol, 0.01% Tween 20).
4. 0.01 M potassium phosphate buffer solution (PBS) pH 7.2.
5. Potassium hydroxide (KOH) solution 10% (p/v).
6. Scalpel, tweezers and scissor.
11. Aluminum paper.
12. Automatic pipette and clean tips.
14. Incubator.
15. Inverted fluorescence microscope.
(ex. Filter cube Zeiss #49) to CalcoFluor, and filter cube to works near 488 ηm
excitation and 520 emission (ex. Filter cube Zeiss #38HE) to Alexa 488.
420–480 ηm range, for CalcoFluor; excitation with Argon 488 ηm laser line and
emission filter with 500–550 ηm range, for Alexa 488. A Plan-Apochromat
63x/1.40 Oil DIC M27 objective was used, with 1024 1024 resolution.
Fig. 11.3 Fungi-Plant interactions. Common bean leaf surface before (a) and after (b) clarifying
treatment. (c–g) Common bean colonized superficially by fungi. Plant tissue dyed with CalcoFluor
and fungi marked with AlexaFluor 488 – WGA. (c) 2D overlay of blue and green channels from
98 focal plans within 30 μm range in Z-axis using the maximum intensity projection method. (d–e)
2D of separated channels. (f–g) 3D software reconstruction from focal plans acquired
11.12.2 Methods
1. Sampling of common bean leaves infected with phytopathogenic fungus and

cutting in 4 4 mm pieces.
2. After, the leaves fragments are placing within Elisa plates well containing KOH
10%, following incubation at 10 C for 4 days in darkness.
3. Wash with PBS and replace for a new KOH 10% solution.
4. Other incubation steps at the same conditions.
5. PBS wash and transfer fragments to Elisa wells with clarifying mix [Urea 6 M,
Glycerol 30% (v/v) and Tween 20 0.05% (v/v)].
6. After 4 days in clarifying mix at 10 C and darkness, wash with PBS, and
incubate in a new clarifying mix for 4 days.
7. Wash with PBS, and add 100 μL Alexa488–WGA 10 μg.mL1.
8. Wrap the Elisa plate with aluminum foil and keep it at vacuum (Fig. 11.1d) for
1 h.
9. Add 80 μL of CalcoFluor 0.01 mg.mL1 and incubate for 10 min at the
darkness.
10. Put a previously cleaned large coverslip on an inverted microscope stage. In case
of immersion objective using, drop firstly the appropriated immersion liquid on
the objective lens before that.
11. Put the leaf fragment above the coverslip with the region of interesting
facing down.
12. Place a glass piece above the sample (to minimize the irregular topography of
the sample).
13. Observe in inverted LSM.
14. In this assay, the z-stack method was used to capture 98 focal plans within
30 μm ranges in Z-axis, to obtain the 2D with the maximum intensity method
and the 3D reconstruction, using the Zen software (Carl Zeiss).
The clarifying protocol showed was for leaf tissues. Adjustments are needed
depending on the tissue type and the plant species, changing the incubation time. For
example, the common bean pods can be clarified with 20 days in KOH 10% and
15 days in clarifying mix, changing the solutions every 5 days of incubation.
The importance of the clarification step is the high amount of autofluorescent
compounds such as cell wall components, phenols, and alkaloids [21]. Thus, many
protocols have been developing to minimize these fluorescence signals and optimize
the specific fluorescent label [18, 25, 26]. The transparency increasing in plant
tissues improves the three-dimensional structures imaging quality, especially valu-
able during the investigation of plant–fungi interactions, once it provides better
fluorochrome infiltration, photon and or laser penetration, and so high quality of
fluorescence signal [18]. Other clarification protocol with very satisfactory results
was developed by Ursage et al. [25] which uses the ClearSee solution [xylitol 10%
(w/v); sodium deoxycholate 15% (w/v); urea 25% (w/v)] in the clarification and for
fluorochromes preparing. The ClearSee method also has the advantage of allowing
the fluorescent proteins such as GFP and m-Cherry observation in plant tissues even
after treatment. In cases where the sample preparation evolves alcohol gradients, for
embedding and microtome cutting, clarification is not necessary. And, for sure, for
non-autofluorescent samples.
Fungi-plant interactions fluorochrome-based studies can be made using other
dyes combinations. Ha et al. [13] observed the wheat tissues colonization by
Pyricularia graminis-tritici using Alexa488-WGA and Fusarium graminearum
genetically transformed for GFP expression. In both cases, the fixed plant tissues
were dyed with Propidium iodide. The plant cell wall marker, pontamine fast scarlet
(S4B), is specific for cellulose and may be used combined with other fluorochromes,
such as aniline blue, which label callose [7].
11.13 Important Plant Structures Defence Against

Phytopathogenic Fungi
The plant resistance factors against phytopathogens are divided into biochemical and
structural, preformed or postformed. Through histochemical analysis, some struc-
tural resistance components such as callose, lignin, and cuticle, and biochemical, as
ROS, can be evaluated by microscopy techniques. Then, callose and lignin protocols
are shown.
11.14 Callose Deposition
The callose polymer (β-1,3-glucan) is a plant cell wall component and may plant
defense responses, and their deposition may increase in response to infections or
resistance-inducing agents [7, 16]. Aniline blue is the fluorochrome used in callose
deposition studies, such as in papillae observation [7]. Figure 11.4a shown callose
deposition marked with aniline blue in tangerine leaf.
11.14.1 Materials
1. Aniline blue diammonium (Sigma, CAS-415049) 0.1 mg.mL1 in PBS 0.01 M

pH 7.2.
2. Tangerine leaves fragments with around 0.5 0.5 cm were previously fixed in
4% paraformaldehyde and clarified as seen before for common bean leaves.
Fig. 11.4 (a) Callose deposition in tangerine leaf observed with Aniline blue fluorochrome. (b)
Lignin localization in Eucalyptus cambial region using Auramine-O
3. Scalpel, tweezers, and scissors.

8. Aluminum paper.
9. Automatic pipette and clean tips.
11. Inverted fluorescence microscope.
420–480 ηm range. The 40 objective was used with zoom resource.
11.14.2 Method
1. Take the clarified leaf fragment and incubate samples within Elisa plate wells in
80 μL Aniline blue 0.1 mg.mL1.
2. Wrap the Elisa plate with aluminum foil and keep it at vacuum for 1 h.
3. Incubate overnight at around 25 C.
4. Wash with PBS.
5. Transfer the abaxial side to a large coverslip supported in the microscope stage.
6. Place the glass piece on a sample.
7. Observe with EFM or LSM the leaf surface (In the case of LSM, paradermic
examination is possible).
11.15 Lignin Localization in Plant-Fungi Interactions
Lignification has an important role in plant defense, in resistance to fungi mechanical

penetration, and participating in gene expression signaling and metabolic pathways
of the plant resistance [3]. The usual fluorescent markers in lignin labeling are
Auramine-O [22], which dyes cutin also, and Basic fucsin [25], which dyes lignin
only. In Fig. 11.4b, a cross section of Eucalyptus cambial region with lignin
localization using the Auramine-O.
11.15.1 Materials
1. Auramine-O (Merck, CAS-2465-27-2) 1.0 mg.mL1 work solution.

2. Eucalyptus cross sections of 7 μm thickness prepared in a microtome, in pith to
bark direction.
3. Slides and coverslips.
5. Automatic pipette and tips.
6. EFM or LSM.
7. For EFM, a filter cube that works near 488 ηm excitation and 520 emission
500–550 ηm range. An EC Plan-Neofluar 10x/0.30 M27 was used.
11.15.2 Method
1. Take the slides with sections and apply around 5 μL of Auramine-O 1.0 mg.mL1
to cover all sample areas.
2. Incubate in darkness for 20–40 min.
3. Wash gentlly with PBS.
4. Cover with coverslip and seal.
5. Observe in an inverted or vertical microscope, EFM or LSM.
11.16 Sample Preparation to Observe Autofluorescent

Fungi and Specific Structures
11.17 Autofluorescent Rust Fungi
Several Basidiomycota presents autofluorescence such as some mushroom pro-

ducers and rust fungi [30, 31]. Its feature permits localization in the environment
and plants. For Hemileia vastatrix and many other rust fungi, autofluorescence can
be observed at the green or blue emission spectra. Here, we observed the
autofluorescence of H. vastatrix pustules in coffee leaves (Fig. 11.5a).
11.17.1 Materials
1. Coffee leaf with rust fungi Hemileia vastatrix pustules.

2. Incubator at 20 C.
Fig. 11.5 (a) Autofluorescence of Hemileia vastatrix pustules in coffee leaves. (b)
Autofluorescence of cercosporin crystals produced by Cercoscopa coffeicola in MEA
3. Dissecting scissor.
4. Tweezer.
(ex. Filter cube Zeiss #38 HE).
8. For LSM, excitation with 488 ηm laser line and emission filter with 490–560 ηm
range. An LCI Plan-Neofluar 25/0.8 Imm Korr DIC M27 objective was used,
with 512 512 resolution.
11.17.2 Method
1. Put a previously cleaned large coverslip (ex. 24 40 mm) on an inverted

2. Positioning a leaf fragment with pustules directed to an objective lens (upside-
down). Small fragments, as 1 1 cm are desirable to minimize the irregular
topography of the sample, as well as avoid the midrib.
3. Use a piece of glass as a weight to minimize the irregular topography of the
sample.
11.18 Autofluorescent Cercosporin
The autofluorescence may be useful information to detect certain fungi metabolites

and other endogenous fluorophores [15]. The cercosporin crystals produced by
Pseudocercosporella capsellae are detectable at 561 nm excitation condition using
a laser confocal microscope [12]. In the method described below will be shown the
location of cercosporin crystals produced by Cercoscopa coffeicola in vitro
(Fig. 11.5b).
11.18.1 Materials
1. Cercoscopa coffeicola colony growth in malt extract agar (MEA) 3 weeks at

20 C.
3. Scalpel.
4. Tweezer.
6. For LSM, excitation with 543 ηm laser line and emission filter with 600–710 ηm
range An EC Plan-Neofluar 40/1.30 Oil DIC M27 objective was used, with
1024 1024 resolution, and around three-fold zoom. The bright-field image was
acquired using the TPM-T detector.
11.18.2 Method

2. Take colony fragments with around 0.5 0.5 cm with a scalpel, choosing the
region of interest using a stereoscopic microscope, if needed.
3. Transfer the upside-down colony fragment above the coverslip.
Acknowledgements The authors gratefully acknowledge CNPq (Conselho Nacional de

Desenvolvimento Científico e Tecnológico), CAPES (Coordenadoria de Aperfeiçoamento de
Pessoal de Nível Superior), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas
Gerais—Brazil) and FINEP (Financiadora de Estudos e Projetos) for financial support. We thanks
also Aline Ferreira and Elisa Castro, technicians at the Electron Microscopy and Ultrastructural
Analysis lab at UFLA, Brazil.
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Chapter 12
Yeast Isolation Methods from Specialized
Habitats
Rameshwar Avchar, Snigdha Tiwari, and Abhishek Baghela
Contents
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
12.2 Common Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
12.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
12.4 Yeast Isolation Protocols from Natural Habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
12.4.1 Rumen Fluid/Digesta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
12.4.2 Insect Gut (Termite/Beetle) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
12.4.3 Hot Spring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
12.4.4 Flower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
12.4.5 Nectar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
12.4.6 Rotten Wood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
12.5 Yeast Isolation Protocols from Anthropogenic Habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
12.5.1 Compost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
12.5.2 Molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
12.5.3 Press Mud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
12.5.4 Distillery Effluent/Spent Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
R. Avchar · S. Tiwari · A. Baghela (*)

Savitribai Phule Pune University, Pune, India
e-mail: abhishekbaghela@aripune.org
236 R. Avchar et al.
12.1 Introduction
Yeasts are a unicellular heterogeneous group of fungi which were first observed
under a microscope by Antonie Van Leeuwenhoek in 1680 and successfully isolated
for the first time by Louis Pasteur in the late 1860s. Since 1865, yeast research has
undergone significant progress with respect to their distribution, isolation and
characterization. Currently, more than 1500 yeast species have been reported from
all over the world, which comprise of only 1% of the actual diversity while the
remaining still needs to be explored [18]. Yeasts are ubiquitous in nature and have
been isolated from aquatic, atmospheric and terrestrial habitats. While some species
can be found in great numbers in different habitats, others may be restricted
geographically to only a few specialized habitats; suggesting that the overall yeast
distribution is not uniform.
It would seem that the distribution of yeasts would not be affected by geograph-
ical barriers since they can be dispersed by air currents [34], organismal vectors,
plant material as epiphytes or endophytes. But actually, distinct yeast species occur
in different regions and seasons. Studies on yeasts associated with beetles,
drosophilids, bees, and ephemeral flowers disprove the fact that yeasts are ubiquitous
[13, 19, 34]. The yeast diversity of common habitats like soil, plants and other
terrestrial habitats has been thoroughly explored since the isolation techniques are
easily available and well described [29, 34]. In contrast, the isolation techniques for
the less exploited niches (Fig. 12.1) like rumen, insect gut, hot spring, rotten wood,
flowers, nectar and other anthropogenic habitats like compost, molasses, distillery
wastes etc. are rather cumbersome and not elaborately outlined, since the procedures
vary greatly depending on the yeast density, the volume and shape of the source
[25]. The correlation between yeasts and the habitats that they occupy is determined
by the overall intrinsic factors (chemical, physical and physiological), availability of
nutrients, beneficial interactions with other organisms, and the presence of
competitors.
In the past decade, several novel yeasts have been isolated from natural habitats
which are not fully exploited; one such niche is insect gut [1, 36, 37]. The insect gut
has become increasingly recognized as an important source for the isolation of new
ascomycetous and basidiomycetous yeast taxa. The major purpose for the yeast-
insect association is that the yeasts provide essential amino acids, vitamins, sterols
and allelochemicals to attract the insect dispersers for targeted dispersal to a fresh
environment [4]. Such associations are believed to have promoted fungal diversity
and the expansion of insects into nutrient-poor substrates [37]. Several novel yeasts
have been isolated from the gut of beetles. Likewise, the gut of termites has also been
explored for novel yeasts, which produce valuable enzymes and can degrade xylan
and/or ferment xylose [1] to ethanol. These properties can be exploited in industries
for the sustainable production of ethanol or valuable enzymes. The gut of termites
and other wood-feeding insects can be considered as natural mini bioreactors with
the understudied microbiome, therefore, we have provided a detailed protocol for
isolation of such industrially important yeasts from the termite gut.
12 Yeast Isolation Methods from Specialized Habitats 237
Fig. 12.1 Specialized habitats for isolation of yeasts
In a similar manner, yeasts play a mutualistic role in the rumen of cattle and other
ruminants. The rumen is an anaerobic, cellulose and hemicellulose-rich habitat,
which harbours many microbes including bacteria, protozoa, filamentous fungi
and yeasts [10]. Yeasts isolated from rumen digesta can grow at elevated tempera-
tures since the temperature of rumen itself is 39.6 1 C. There are few reports of
yeasts isolated from the rumen of sheep, bovine, cattle and musk oxen [8, 21,
22]. The exact role of yeasts in the rumen is unclear but it is proposed that they
act as probiotics to prevent various diseases in ruminants and stimulate the growth
and activity of various fibrolytic ruminal bacteria by providing proteins, vitamins
and other growth factors [24, 33]. Since the rumen is also a less explored niche, a
protocol for rumen yeast isolation is given in this chapter.
There are some ‘wood-feeding yeasts’ found on rotting wood, which is a nutrient-
rich substrate for yeast populations. This niche has recently received attention after
isolation of xylose-fermenting yeasts from rotting wood in Brazilian forests and a
novel yeast capable of arabitol production was reported [6, 17]. The hemicellulosic
fraction of wood is broken down to simpler monomers by the yeasts’ enzymes. The
hydrolytic abilities of the enzymes secreted by these yeasts are of great significance
to biotechnological industries, therefore. a brief protocol for yeast isolation from
rotting wood has also been given.
Another interesting niche for obtaining novel or rare yeasts is the flower or floral
nectar. There are many publications on the properties and characteristics of nectar,
but very little is known regarding its role as a natural habitat for micro-organisms,
especially yeasts. The presence of yeasts in the nectar of flowers is well known but
very little effort has been taken for the isolation of yeasts and studying their role in
compositional changes of nectar fluid [16, 43]. It is possible that the yeasts
inhabiting nectars of angiosperms play a significant role as intermediate agents in
plant-pollinator signalling by the production of volatile compounds by fermentation
of available sugars [16, 43]. It is suggested that some osmophilic species can be
found in nectar since it contains high levels of sugars, and this trait can be beneficial
from a biotechnological point of view.
Some extreme environments have been established as niches for various species
of yeasts capable of enduring harsh conditions [34]. One such naturally occurring
habitat is the hot spring which harbours thermotolerant and thermophilic yeasts.
Novel thermotolerant yeasts have been reported from hot springs [38] which can be
used for high-temperature fermentations to produce ethanol. Enzymes like
β-galactosidase, cellulase and lipases produced by thermotolerant/thermophilic
yeasts are expected to have thermal tolerance, which is one of the most desirable
properties of an enzyme to be used for industrial applications [12]. A detailed
protocol for the isolation of yeasts from hot springs has been provided in this
chapter.
Besides the naturally existing habitats, there are a number of anthropogenic
sources from which yeasts can be recovered. Urbanization and human activities
have created multiple ecological or functional niches for specific yeast populations.
One such artificial habitat is compost. Composting is an aerobic process which
involves the microbial conversion of complex organic compounds into their simpler
forms [31]. As a habitat rich in carbohydrates and phenolics, composts harbour a
variety of microbes including yeasts that have the ability to utilize C5 and C6 sugars
and are tolerant to high temperatures and low pH [9, 14]. In the past decade, most
research on composts has been focused on their ecological and functional biodiver-
sity. Very few studies have been undertaken to explore yeast diversity from com-
posts despite being one of the rarest and unexplored habitats [9]. Yeasts isolated
from composts might be possible candidates for fermentation in biofuel or
bioethanol industries.
The yeast biota of carbon-rich substrates like residual juice, molasses and press
mud from sugar cane/beet in sugar industries has not been delved into completely.
Press mud is also a type of waste which is rich in lignocellulosic material, obtained
after ethanol production from molasses and is usually burnt or discarded by most
distilleries. Few yeasts of the genera Saccharomyces, Schizosaccharomyces and
Torulaspora have been reported from this niche [5, 39, 40]. Molasses from sugar
cane and agave are used as the starting material for the production of ethanol or other
distilled spirits [11, 20] and the waste generated (residual molasses and press mud)
can possibly harbour high sugar-tolerant or osmophilic microbes, especially yeasts
[15, 26, 40]. Isolation of such indigenous yeasts and their employment in fermen-
tation industries can increase the efficiency of ethanol production from molasses
[7]. Protocols for yeast isolation from press mud and molasses have been provided.
Much like molasses, distillery waste also constitutes a very rich ecosystem in
which varied yeast species can flourish and carry out fermentation spontaneously.
The yeast community thrives on residual sugars and other by-products generated
after the distillation process to produce ethanol from grape pomace. Very few
surveys have been carried out to study the yeast diversity from distillery waste
[41, 42]. More recently, distillery waste has also been found to be a potential reserve
for novel thermotolerant yeasts [3]. Such yeasts once successfully isolated and
characterised can be of interest in the field of biotechnology for efficient production
of ethanol from lignocellulosic biomass [27].
The above mentioned natural or anthropogenic ecosystems are all reservoirs of
rare and novel yeasts that can significantly contribute to the sustainable advancement
of biotechnological research across the globe. Yeast diversity of such specialised
habitats has not been completely exploited as a consequence of insufficient knowl-
edge or unavailability of detailed step-by-step isolation protocols. The current
chapter presents a comprehensive compilation of available methods that have been
suitably modified to successfully isolate yeasts from specialized and less explored
habitats like rumen, rotting wood, insect gut, flower/floral nectar, hot spring, com-
post, molasses/press mud and distillery waste (Fig. 12.1). We aim to provide a
complete set of methods as a reference for researchers who are interested in
exploring yeast biota from rare ecosystems.
12.2 Common Materials
1. 0.9% saline solution

2. Autoclave
3. Chloramphenicol (HiMedia)
4. Cover-slips
5. Cryo-Vials (2 ml)
6. Deep freezer (20 C & 80 C)
7. Differential Interference Contrast (DIC) Microscope
8. Distilled water
9. Erlenmeyer flasks (250 ml).
10. Ethanol (70%)
11. Falcon tubes (50 ml)
12. Glass slides
13. Glass spreader
14. Glycerol
15. Hand gloves
16. Incubator
17. Laminar air flow cabinet
18. Measuring cylinders (500 ml
19. Micropipettes (P20, P200 & P1000)
20. Milli-Q water
21. Penicillin (HiMedia)
22. Petri-plates (90 mm)
23. pH meter,
24. Plastic bags (600 1000 or 1000 1600 size)
25. Plastic containers (500 and 1000 ml)
26. Refrigerated centrifuge
27. Refrigerator (4 C)
28. Screw-cap glass bottles (500 ml & 1000 ml)
29. Shaker Incubator
30. Streptomycin (HiMedia)
31. Weighing balance
32. Yeast Extract Peptone Dextrose (YEPD) agar plates, (10 g l1 yeast extract, 20 g
l1 mycological peptone, 20 g l1 dextrose, 20 g l1 agar; pH 5.0)
12.3 Methods
12.4 Yeast Isolation Protocols from Natural Habitats
12.4.1 Rumen Fluid/Digesta

12.4.1.1 Materials
(a) Common materials

(b) Specific materials:
(i) Whirl-pak® bags
(ii) BagMixer® CC
(iii) Vortex
(iv) Muslin cloth
(v) Manifold machine (anaerobic media preparation)
(vi) Nitrogen cylinder
(vii) Carbon dioxide cylinder
(viii) Serum bottles (120 ml)
(ix) Resazurin (0.1%)
(x) L-Cysteine-hydrochloride (L-Cys-HCl)
(xi) Dispenser (5–50 ml)
(xii) Sealer and de-sealer machines
(xiii) Sterile syringes (1 ml and 10 ml)

(xiv) Syringe needles (gauge 16)
(xv) Potato Dextrose (PD) agar (200 g l1 potato infusion, 20 g l1 dextrose,
25 g l1 agar; pH 5.00.5)
12.4.1.2 Protocol
1. Collect approximately 100 g of rumen digesta from buffalo/goat/sheep in sterile

whirl-pak® bags (triplicates) from a slaughterhouse or any other source and
transfer them to the laboratory immediately.
2. Store all rumen samples at 39.6 1 C in an incubator until further processing.
(Note: It is recommended to process the sample as soon as possible to obtain
maximum diversity of yeasts. Samples can be stored at 4 C temporarily).
3. Pool the samples and equilibrate with CO2 gas using a manifold machine. Take
10 g of this rumen digesta and suspend it into 100 ml of 0.9% saline solution.
4. Homogenize the above solution using BagMixer® CC for 30 seconds or filter
through a muslin cloth to obtain a uniform suspension of rumen digesta before it
is used for yeast enrichment and isolation processes.
5. Aerobic isolation on solid media:
(a) Spread 100 μl of appropriately diluted suspension (103, 104 and 105) on
YEPD and PD agar plates containing antibiotics (200 μg ml1 streptomycin,
200 μg ml1 ampicillin and 25 μg ml1 chloramphenicol).
(b) Incubate the plates at 39.6 1 C to obtain thermotolerant yeasts, and
30 1 C for 48–72 h to obtain the complete diversity of yeasts.
6. Aerobic isolation in liquid media (enrichment):
(a) Inoculate 10 ml of homogenized rumen suspension into 100 ml of YEPD and
Potato Dextrose broth containing antibiotics.
(b) Incubate the flasks at 39.6 C and 30 C for 12–24 h at 150 rpm to enrich the
indigenous yeast flora.
(c) After observing considerable growth, spread 100 μl of the above culture broth
on YEPD plates and incubate at 39.6 1 C and 30 1 C for 24–72 h to
obtain yeast colonies.
7. Anaerobic isolation on solid media: serum roll bottle method [23].
(a) Add 0.5 ml of appropriately diluted rumen suspension (usually 103 or 104)
in 10 ml YEPD and PDA media prepared anaerobically in 120 ml glass serum
bottles containing 0.1 ml of resazurin (0.1%), 1 g l1 of L-Cys-HCl and
antibiotics (200 μg ml1 streptomycin, 200 μg ml1 ampicillin and 25 μg ml1
chloramphenicol).
(b) Incubate all bottles at 39.6 1 C and 30 1 C separately for 24–72 h and
inspect regularly for the development of yeast colonies.
(c) Select different yeast colonies after observation of morphology under a DIC
microscope and pick them anaerobically in the presence of N2 gas.
(d) Subculture these colonies on aerobic (YEPD plate) and anaerobic media
(serum roll bottles).
8. Anaerobic isolation on liquid media (enrichment):
(a) Inoculate 3 ml of homogenized rumen suspension in a 120 ml glass serum
bottle containing 30 ml of YEPD and PD broth with 0.1 ml resazurin (0.1%),
1 g l1 L-Cys-HCl and antibiotics as mentioned above.
(b) Incubate all serum bottles at 39.6 1 C and 30 1 C separately for
12–24 h at 150 rpm or until considerable growth has been obtained.
(c) Inoculate 100 μl of the above culture broth into serum bottles containing
YEPD media with antibiotics.
(d) Incubate all serum bottles at 39.6 1 C and 30 1 C separately for
24–72 h.
(e) Select morphologically distinct yeasts by observing under microscope and
subculture on aerobic (YEPD plate) and anaerobic media (roll bottles).
9. Preserve purified yeasts in 15% glycerol at 80 C and in liquid nitrogen
(196 C) until further use.
12.4.2 Insect Gut (Termite/Beetle)
12.4.2.1 Materials

(b) Special materials:
(i) Forceps
(ii) Xylose
(iii) Stereomicroscope
(iv) Ethanol (90%)
(v) Sterile syringe (5 ml)
(vi) Yeast Nitrogen Base (YNB) + 1% xylose (pH 5.0)
12.4.2.2 Protocol
( [35] with few modifications)
1. Collect 15–30 live termites/beetles from rotting wood logs, soil, bark of trees and
transfer them to the laboratory at ambient conditions.
(Note: Dead termites/beetles are difficult to dissect and affect the gut micro-flora,
therefore, it is important to keep the insects alive until dissection)
2. Surface-disinfect the insect body using 95% ethanol for 2 min and then rinse with
sterile distilled water.
3. Dissect and remove the gut from the insect under a stereomicroscope using
dissection needles/forceps and transfer the gut into a separate tube for crushing
in 0.9% sterile saline solution.
4. Pass the gut suspension through a 2 or 5 ml syringe twice to macerate the gut
contents.
5. Direct isolation on solid media:
(a) Spread 100 μl of the crushed suspension onto YEPD and YNB + xylose plate
containing antibiotics (100 μg ml1 streptomycin, 100 μg ml1 ampicillin
and 25 μg ml1chloramphenicol).
(Note: Do not autoclave YNB+ xylose medium; sterilize by filtration)
(b) Incubate all plates at 25–27 C for 24–96 h and observe frequently for growth
of yeast colonies.
6. Enrichment in liquid media:
(a) Inoculate 1 ml of gut suspension into 10 ml of YEPD and YNB + xylose
media in 50 ml flasks, each containing antibiotics (100 μg ml1 streptomycin,
100 μg ml1 ampicillin and 25 μg ml1chloramphenicol).
(b) Incubate all flasks at 25–30 C for 24–48 h at 150 rpm.
(c) After observing considerable growth, spread 100 μl of the culture broth on
YEPD and YNB + xylose plates containing antibiotics and incubate at
25–30 C for 24–72 h.
7. Select well-separated, morphologically different yeast colonies and streak on agar
plates (YEPD and YNB + xylose) to obtain purified cultures.
12.4.3 Hot Spring

12.4.3.1 Materials
(a) Common materials.

(i) Hand shovel
(ii) Ice bucket
(iii) Membrane filter (0.45 μm)
(iv) PD medium (pH 5.0)
12.4.3.2 Protocol
( [2] with few modifications)

1. Collect approximately 1000 ml of water and 500 ml of wet sediments from hot
springs in sterile plastic containers.
2. Store the samples at 4 C while transferring to the laboratory and process
immediately.
3. Concentrate the hot spring water sample by passing through a membrane filter
and resuspend the membrane filter into 10 ml of 0.9% saline solution to obtain a
uniform suspension.
4. Mix wet sediment samples thoroughly before isolation of yeasts.
(a) Spread 100 and 200 μl from the concentrated hot spring water suspension /
wet sediments onto YEPD and PD agar plates with antibiotics (100 μg ml1
streptomycin, 100 μg ml1 ampicillin and 25 μg ml1chloramphenicol).
(b) Incubate all plates at 30, 35, 40 and 45 C separately for 24–96 h or until yeast
growth is observed.
(Note: High incubation temperatures are employed for isolation of
thermotolerant/thermophilic yeasts)
(a) Inoculate 2 ml of concentrated hot spring water suspension and wet sediment
separately into 20 ml of YEPD and PD media with antibiotics (100 μg ml1
(b) Incubate all flasks at 30, 35, 40 and 45 C separately for 12–96 h at 150 rpm.
(c) Take 5 ml culture broth from the above flask and inoculate into 100 ml YEPD
medium with antibiotics (100 μg ml1 streptomycin, 100 μg ml1 ampicillin
(d) Incubate each flask at 30, 35, 40 and 45 C separately for 12–18 h at 150 rpm.
(e) After observing considerable growth, spread 100 μl of culture broth on YEPD
plates containing antibiotics and incubate at 30, 35, 40 and 45 C separately
for 24–72 h.
7. Examine yeast morphology microscopically; select different yeasts and streak on
YEPD/PD agar plates to obtain purified cultures.
8. Preserve all yeasts in 15% glycerol at 80 C and in liquid nitrogen (196 C)
until further use.
12.4.4 Flower
12.4.4.1 Materials

(i) Mortar and pestle
(ii) Muslin cloth
(iii) PD medium (pH 5.0)
12.4.4.2 Protocol
( [32] with slight modifications)

1. Collect fresh flowers and transfer them to the laboratory for processing.
2. Rinse the flower with sterile distilled water for 10 min to remove adhered dust
particles and other contaminants.
3. Dry the flower and surface-sterilize by transferring it into 70% ethanol for 2 min.
4. Rinse the flower again in sterile distilled water for 5 min and air dry maintaining
sterile conditions.
5. Take 1 g of surface-sterilized flower in mortar and pestle and crush thoroughly
in 3 ml sterile water.
6. Add 7 ml sterile water to this slurry, mix well and filter through muslin cloth to
obtain a uniform suspension.
7. Spread 100 μl of the appropriately diluted (104 and 105) suspension on YEPD
and PD agar plates containing antibiotics (100 μg ml1 streptomycin,
8. Incubate the plates at 25 to 30 C for 24–96 h and observe frequently for yeast
colonies.
9. Select well-separated yeast colonies and observe under a microscope for mor-
phological differences; subculture on YEPD/PD agar plates to obtain purified
colonies.
12.4.5 Nectar
12.4.5.1 Materials

(i) Eppendorf tube (1.5 ml and 2 ml)

(ii) Sterile syringe (1 ml)
(iii) Yeast Maintenance (YMA) media (3 g l1 yeast extract, 3 g l1 malt
extract, 5 g l1 mycological peptone, 10 g l1 glucose, 25 g l1 agar;
pH 5.5)
12.4.5.2 Protocol
([43] with apt modifications)

1. Collect sufficient amount of nectar (~1 ml) from inflorescences with a sterile
syringe into a 2 ml tube, transfer to the laboratory as soon as possible and store at
4 C until further processing. (Note: The volume of nectar collected will vary
from flower to flowerFlowers; some flowers may have as little as 5 μl of nectar
fluid)
2. Dilute 100 μl of nectar with 900 μl of sterile distilled water and spread 100 μl of
diluted nectar on YMA and YEPD plates containing antibiotics (100 μg ml1
streptomycin, 100 μg ml1 ampicillin and 25 μg ml1 chloramphenicol). (Note:
In case of overgrowth of yeast colonies, prepare further dilutions to obtain well-
separated yeast colonies)
3. Incubate all plates at 25 to 30 C for 5 days and observe intermittently for yeast
colonies.
4. Select well-separated yeast colonies and streak on agar plates (YMA and YEPD)
to obtain purified cultures.
5. Microscopically examine yeast cells to determine their morphology and select
different yeasts.
12.4.6 Rotten Wood

12.4.6.1 Materials

(i) Membrane filter (0.45 μm)
(ii) Sterile syringe (50 ml)
(iii) Yeast Nitrogen Base (YNB) + 1% xylose (pH 5.0)
(iv) YMA medium (pH 5.0)
12.4.6.2 Protocol
([6] with modifications)

1. Collect 50 g of rotten wood in a sterile plastic bag, transfer immediately to the
laboratory and process the sample for double enrichment. (Note: Xylanolytic
and xylose utilizing yeasts can be isolated through double enrichment as they are
present in less numbers)
2. Add 1 g of rotten wood separately into 20 ml of YMA, YEPD and YNB + xylose
media each containing antibiotics (100 μg ml1 streptomycin, 100 μg ml1
ampicillin and 25 μg ml1chloramphenicol).
3. Incubate all flasks at 25 C for 3–10 days at 150 rpm on a reciprocal shaker.
4. Inoculate 5 ml of the above culture broth into 100 ml of YMA, YEPD and
YNB + xylose media containing antibiotics.
5. Incubate all flasks at 25 C for 12–24 h at 150 rpm on a rotary shaker.
6. Spread 100 μl of culture broth on YMA, YEPD and YNB + xylose plates
containing antibiotics.
7. Incubate all the plates at 25 C for 5 days or until observation of yeast colonies.
8. Select well-separated yeast colonies and streak them on agar plates (YEPD and
YNB + xylose) to obtain purified cultures.
9. Select morphologically different yeasts by microscopic observation and streak
on YEPD/YNB + xylose agar plates.
for long-term maintenance.
12.5 Yeast Isolation Protocols from Anthropogenic

Habitats
12.5.1 Compost
12.5.1.1 Materials

(i) Hand shovel
(ii) Acidified YEPD medium (pH 3.5)
(iii) YEPD 5 (5% dextrose) medium (pH 3.5)
(iv) YEPD 10 (10% dextrose) medium (pH 3.5)
(v) Glass wool
(vi) Glass funnel
12.5.1.2 Protocol
([9] with modifications)

1. Collect approximately 100 g of compost from composting heaps (at least 90 days
old) after excavating at a depth of 1 m from the surface and transfer immediately
to the laboratory under aseptic conditions.
(Note: Compost samples can also be stored at 4 C until further processing, but
not for more than 2 weeks)
2. Inoculate 10 g of compost into 100 ml of 0.9% saline solution, to obtain a uniform
suspension prior to enrichment and isolation experiments.
[Note: In order to avoid mould growth, enrich the samples in an acidified
liquid broth (pH 3.5) using sulphuric acid, as recommended by [28]. Filter the
enriched culture broth with sterile glass wool and use this filtrate for further
isolation experiments]
(a) Spread 100 μl of appropriately diluted suspension on acidified YEPD, YEPD
5 and YEPD 10 agar plates containing antibiotics (200 μg ml1streptomycin,
[Note: Acid hydrolysis of agar at low pH while autoclaving can be avoided
by adding the acid to sterilized molten agar (45–50 C) and mixing gently to
avoid air bubbles]
(b) Incubate the plates at 30, 35, 40 and 45 C separately for 24–96 h to obtain
yeast colonies.
(Note: High incubation temperatures are employed for isolation of
thermotolerant/thermophilic yeasts)
(a) Inoculate 10 ml of suspension into 100 ml of acidified YEPD, YEPD 5 and
YPD 10 media with antibiotics (200 μg ml1 streptomycin, 200 μg ml1
ampicillin and 25 μg ml1chloramphenicol).
(b) Incubate each flask at 30, 35, 40 and 45 C separately for 12–24 h at 150 rpm.
(c) After considerable growth has been observed, take 5 ml culture broth from the
above flasks and inoculate into 100 ml YEPD, YEPD 5 and YPD 10 media
with antibiotics.
(d) Incubate all flasks at 30, 35, 40 and 45 C separately for 12–18 h at 150 rpm.
(e) After observing considerable growth in the flasks, spread 100 μl of culture
broth on YEPD and YEPD 10 agar plates containing antibiotics (200 μg ml1
streptomycin, 200 μg ml1 ampicillin and 25 μg ml1 chloramphenicol).
(Note: Dilute the culture broth before inoculation in case there is excessive
yeast growth)
5. Select well-separated yeast colonies and subculture on YEPD media plates to
obtain pure yeast cultures.

12.5.2 Molasses
12.5.2.1 Materials

(i) YMA medium (pH 5.5)
(ii) Malt Extract Peptone Dextrose (MEA) agar plates, (20 g l1 malt extract,
6 g l1 mycological peptone, 20 g l1 dextrose, 20 g l1 agar; pH 5.0)
(iii) Sugarcane Blackstrap Molasses (SCBM) medium (2.6% molasses, 0.3%
yeast extract, 0.2% KH2PO4, 0.1% (NH4)2SO4, 0.1% MgSO4.7H2O, 2%
agar; pH 5.0)
12.5.2.2 Protocol
([15] with appropriate modifications)

1. Collect 100 ml of molasses from sugar industries in 500 ml plastic containers in
triplicates; transfer to the laboratory immediately and store at 4 C until further
processing.
2. Dilute the molasses sample ten times with 0.9% saline solution prior to enrich-
ment and isolation experiments.
(a) Spread 100 μl of the appropriately diluted suspension on YMA, YEPD and
SCBMM agar plates containing antibiotics (100 μg ml1 streptomycin,
100 μg ml1 ampicillin and 25 μg ml1chloramphenicol). (Note: Dilute
further if there is excessive yeast growth, e.g. 103 or 104)
(b) Incubate the plates at 30 C for 24–96 h to obtain yeast colonies.
(a) Inoculate 10 ml of appropriately diluted molasses suspension into 100 ml of
YMA, YEPD and SCBMM broth, each containing antibiotics.
(b) Incubate the flasks at 30 C for 12–18 h at 150 rpm.
(c) After observing considerable growth, spread 100 μl of culture broth on YMA,
YEPD and SCBMM plates and incubate at 30 C for 24–72 h to obtain yeast
colonies.
(Note: Dilute the culture broth suitably before plating in case of over-growth
of yeasts)
5. Select well-separated yeast colonies; observe under a DIC microscope to select

morphologically distinct yeasts and streak on YMA, YEPD, and SCBMM agar
plates to obtain purified cultures.
until further use.
12.5.3 Press Mud

12.5.3.1 Materials

(i) Hand shovel
(ii) YMA medium (pH 4.0)
(iii) SCBM medium (pH 4.0)
12.5.3.2 Protocol
([30] with suitable modifications)

1. Collect 100 g to 1 kg press mud from sugar-cane composting heaps in sterile
plastic containers and transfer to the laboratory immediately or store at 4 C until
further processing.
2. Suspend 100 g of press mud in 1000 ml of 0.9% saline solution to obtain a
uniform suspension prior to enrichment and isolation experiments.
(a) Spread 100 μl of the appropriately diluted suspension on YMA, YEPD and
SCBMM acidified agar plates (pH 3.5) containing antibiotics (200 μg ml1
(b) Incubate the plates at 30 C for 24–96 h to obtain yeast colonies.
(Note: Prepare serial dilutions (e.g. 104, 105) in case of overgrowth of
yeasts and repeat direct isolation)
(a) Inoculate 10 ml of suspension into 100 ml of YMA, YEPD and SCBMM
media supplemented with antibiotics.
(c) After considerable growth has been observed, spread 100 μl of the above
culture broth on YMA, YEPD and SCBMM plates and incubate at 30 C for
24–72 h to obtain yeast colonies.
7. Examine well-separated yeasts under the microscope and subculture them on agar
plates (YMA, YEPD, and SCBMM) to obtain purified cultures.
12.5.4 Distillery Effluent/Spent Wash

12.5.4.1 Materials

(i) YMA medium (pH 4.5)
(ii) SCBM medium (pH 4.5)
12.5.4.2 Protocol
[3]
1. Collect approximately 500 ml of effluent from distillery units associated with
sugar industries and transfer it to the laboratory immediately under aseptic
conditions.
2. Dilute the distillery effluent ten times with 0.9% saline solution prior to enrich-
ment and isolation experiments.
(a) Spread 100 μl of the diluted suspension on YMA and YEPD agar plates
containing antibiotics (100 μg ml1 streptomycin, 100 μg ml1 ampicillin
(b) Incubate the plates at 30 C for 24–96 h or until observation of yeast colonies.
(Note: Prepare serial dilutions (e.g. 104, 105) in case of overgrowth of
yeasts and repeat direct isolation)
(a) Inoculate 10 ml of diluted distillery effluent suspension into 100 ml of YMA
and YEPD media containing antibiotics (200 μg ml1 streptomycin,
200 μg ml1 ampicillin and 25 μg ml1chloramphenicol).
(c) Spread 100 μl of the above culture broth on YEPD plates and incubate at
30 C for 24–72 h.
5. Pick well-separated yeast colonies and subculture on YMA and YEPD media
plates to obtain pure yeast cultures.
6. Select morphologically different yeasts and streak them on YMA and YEPD agar
plates.
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Index
A imaging modes
Adaptive robust binary pattern (ARBP), 125 fixed cells, QI mode, 165–167
Agarose gel electrophoresis, 187, 188 live cells, QI mode, 168
AlexaFluor 488® WGA Conjugate Staining, quantitative imaging (QI) mode, 165
218–220 tracking dynamic processes, QI mode,
Amino acid composition (AAC), 128 169, 170
Antifungal peptides, 118, 128, 130 immobilisation techniques, 164, 165
Antioxidant enzymes LSCM, 160
cellular antioxidant defense materials, 161
mechanisms, 146 morphology and ultrastructural properties,
and endogenous antioxidants, 146 159
quantification in fungi (see Quantification of operating modes, 160
antioxidant enzymes) physical interaction, 160
scavenging H2O2, 146 physicochemical properties, 159
Artificial neural network (ANN), 126 QI mode image/data processing, 172
Ascomycetes, 19 S. cerevisiae, 161
Aspergillus, 122 sample preparation
Aspergillus brasiliensis, 2 biochemical trapping, 164, 166
Aspergillus nidulans, 160 fixed samples, 163
Atomic composition (ATC), 128 live cell imaging, 163
Atomic force microscopy (AFM) PDMS stamp, 163, 164
AFM-QI, 160 Autofluorescence, 230, 231
AFM-QI-LSCM live cell imaging, 169,
171, 172
biological characteristics and processes, 160 B
biological interfaces, 159 Back propagation neural network (BPNN), 121
biological response, 159 Basic Local Alignment Search Tool
C. albicans, 161 (BLAST), 200
cell morphology, 160 Basidiomycota, 210
cellular mechanics, 160 Basophils, 108
cell wall surface, 161 Bayesian methods, 142
experimental methods Biosafety level one (BSL-1), 84
coating coverslips, 162, 163 Bird seed agar
preparing coverslips, 162 glucose, 79
factors, 161 history, 79
Biology, https://doi.org/10.1007/978-3-030-83749-5
256 Index
Bird seed agar (cont.) Clustering methods, 125

ingredients, 80 Coccidioides immitis, 83
method of inoculation and incubation, 81 Colony-forming unit (CFU), 105
methods of preparation, 80 Compost, 236, 238, 239, 247, 248, 250
microbes, 79 Coomassie Brilliant Blue, 148
Blood, 108 Cryptococcus neoformans, 79, 81
Bone marrow, 107 Culture collections, 1
Bone marrow-derived macrophages Cummene hydroperoxide, 154
(BMDM), 107 Cunninghamella japonica, 2
Bovine serum albumin (BSA), 148, 149 Cycloheximide, 73
Bronchoalveolar lavage fluid (BALF), 105, 109 Cytokines
electrochemiluminescence, 113
ELISA, 113
C flow cytometry, 112, 113
Calcofluor white (CFW), 96, 97, 212, 214, 218, methods, 112
219, 225 molecular methods, 113
Callose deposition, 228 protein/polypeptide molecules, 112
Callose polymer, 228 technologies, 113
CaM gene, 178
Candida albicans, 167
Catalase (CAT), 146, 147, 149, 150, 154, 156 D
Catalyzed reporter deposition-FISH (CARD- DAPI (4’6-Diamidine-2’-phenylindole
FISH), 88–90, 92 dihydrochloride), 212–214
Cell-Tak, 162 Dermatophyte Test Medium (DTM), 81–83
Cell wall proteins (CWPs), 161 Dipeptide counts (DPC), 128
Cell wall surface characteristics, 160 Distillery wastes, 236, 238, 239, 251, 252
Chemokines, 104 DNA extraction, 184, 186, 197
Chloramphenicol, 73 DNA sequencing, 197
Chytridiomycota, 95 conventional chain-termination
Chytrids approach, 197
approaches, 96 cycle sequencing, amplified and purified
chitinous fruit, 95 PCR products, 197
conventional microscopy, 96 editing, analysis and identification
electron microscopy, 96 BLAST, 200
environmental factors, 96 MycoBank, 200
epifluorescence microscopy, 96 tools, 199
fungal infections, 95 UNITE, 201
host cells, 99 first-generation sequencing approach, 197
infection of cells, 100 materials, 198
materials, 97 method, 198
methods, 98 purification of sequencing PCR product,
microbial communities, 96 199
phase contrast light microscopy, 96 recommended DNA quantities, 197
phytoplankton, 95 Sanger method, 197
phytoplanktonic chytrid infections, 100 in thermal cycler, 199
phytoplanktonic communities, 97
size-fractionated community approach, 99
total community approach, 99 E
types of filters, 99 Electropherogram, 199, 201
Utermöhl method, 99 Environmental genomic DNA, 141, 142
UV light, 96 Enzyme-linked immunospot assay
zoosporic fungi, 96 (ELISPOT), 113
ClearSee method, 227 Eosinophils, 108
Index 257
Epi-fluorescence microscopy (EFM), 213–218, autofluorescent cercosporin, 232

220–225, 229–232 autofluorescent rust fungi, sample
Ethylenediaminetetraacetic acid (EDTA), 148 preparation, 230, 231
Eukaryotic microbial diversity, 138 callose deposition in tangerine leaf, 228
lignin localization, 229, 230
sample preparation, 225
F clarification step, 227
Filamentous fungi common bean leaf surface, 226
cryopreservation, 2, 3 fluorochrome-based studies, 227
freeze-drying, 3, 4 materials, 225
silica gel, 4, 5, 16 methods, 226, 227
sterile soil, 4
Flow cytometry, 110
Flowers, 236, 238, 245 G
Fluorescein isothiocyanate (FITC), 96 Gelatin-coated surfaces, 165
Fluorescence in situ hybridization (FISH), 88, Gene markers, 178
138, 211 Gene targets/molecular markers, 179–183
Fluorescence microscopy Genetic markers, 178
fluorescence/autofluorescent samples, 210 Germ tube burst method (GTBM), 216
Fluorescent phytotoxins, 211 Glucose-6-phosphate, 155
Fluorochromes, 211 Glucose-6-phosphate dehydrogenase
Formaldehyde, 89 (G6PD), 147, 155
Fungal biology, 118, 120, 134 Glutathione peroxidase (GP), 146, 147, 154,
Fungal genomic DNA extraction 155
closed DNA isolation system, 184 Glutathione reductase (GR), 146, 147, 153,
extraction methods, 184 154, 156
materials, 186 Glutathione-S-transferases (GST), 146, 147,
method, 187 152, 153
physical methods, 184 Glycolysis, 146
selection of suitable DNA isolation Gomori’s methenamine silver (GMS), 110
method, 186 Gram-positive and gram-negative bacteria, 73
Fungal sample preparation Gray level cooccurrence matrices (GLCM),
to fungi cell-wall, 218 120, 126
AlexaFluor 488® WGA Conjugate Green fluorescent protein (GFP), 211
Staining, 218–220 Guizotia abyssinica, 79, 80
Calcofluor White Staining, 218
ROS in fungi, 220, 221
to fungi chromosome H
karyotyping, 216 Hematopoietic stem cells, 106
materials, 216, 217 Hematoxylin and eosin (HE), 110
method, 217, 218 Heterotrophic nanoflagellates (HNFs), 88, 138
to fungi nuclei and chromosome Histoplasma capsulatum, 74
Basidiomycota, 211 Horseradish peroxidase (HRP), 88, 147,
DAPI, 211–214 150, 151
Propidium iodide, 215, 216 Host immune responses
Fungal tissue homogenization, 147–148 antifungal immunity, 104
Fungi BALF, 109
eukaryotic, 210 blood, 108
morphological basis, classification, 176 blood circulation, 106
polyphyletic genera, 176 bone marrow, 104, 107
types, 176 cell counting, 112
Fungi-plant interaction cell detection, 111
Alexa488-WGA, 218 dermatophytosis, 104
258 Index
Host immune responses (cont.) M

dynamic process, 104 Machine learning, 118
erythrocyte lysis, 110 MEGA 7 software, 184
flow cytometry, 110, 111 MEGA 7/X software, 202
fungal burden Metal Oxide Semiconductor (MOS), 121
CFU, 105 Metarhizium anisopliae, 5
qPCR assays, 105 Microbial cultures, 3
qPCR system, 106 Microorganisms
18S rRNA gene, 105 Ascomycetes, 19
fungal infections, 104 conservation technique, 1
hematopoietic stem cells, 106 fields attributes, 22
histology, 110 filamentous fungi, 1, 2
immune cells, 104 fungal cultures, 1
infected organs, 109 fungal strains, 1
innate immune system, 104 industry, 2
LNs, 108 liquid nitrogen, 1
lymphatic circulation, 106 medicine, 2
mechanisms, 104 microbial resources, 1
peripheral immune organs, 104 preservation methods, 2
spleen, 107 protocol, 19, 21
subcutaneous and systematic infections, 104 silica gel, 6, 8, 9
thymus, 104, 107 storage time, 2, 6–8, 21
Hyperplane equation, 118 temperatures, 17
Hyperspectral imaging (HSI), 123 VKM fungal collection, 2
VKM fungal species, 22–25, 27–30, 32–36,
38–40, 42–46, 48–50, 52, 53, 55–59,
I 61–64
Immune cells, 104, 106, 108–112 zygomycetous fungi, 19
Immunofluorescence, 211 Molasses, 236, 238, 239, 249
Immunohistochemistry (IHC), 110 Molecular markers, 177, 179–183, 211
Immunostaining, 211 Molecular oxygen (Ο2), 146
Insect gut, 236, 239, 242, 243 Molecular phylogenetic analysis, 185
Iscove’s modified, 107 Molecular phylogeny, 178
Molecular taxonomy, filamentous fungi, 177,
178, 184
K agarose gel electrophoresis, 187, 188
Karyotyping, 216 selection of gene target, 195
Kerbs Cycle, 146 Molecular tools, 177
Kernel functions, 119, 133 Monocytes, 108
Multigene phylogeny, filamentous fungi, 178,
189
L Multivariate calibration models, 123
Laser scanning confocal microscopy (LSCM), MycoBank Database Search, 200
160 Mycosphaerella, 176
Laser scanning microscopy (LSM), 212–214 Mytilus edulis, 164
for 3D-fungi imaging, 226, 227
Linear discriminant analysis (LDA), 123
Lipopolysaccharide (LPS), 112 N
Lymph nodes (LNs), 108 Natural Language Processing (NLP), 129
Lymphocytes, 108 Nectar, 236, 238, 239, 245, 246
Lyophilization, 1, 21 Netprimer, 141
Lyticase, 142 Neutrophils, 108
Index 259
Nuclear internal transcribed spacer sequences yeasts, 78

(ITS) region, 177, 178 Press mud, 238, 239, 250
Nuclear ribosomal RNA gene, 177 Principal component analysis (PCA), 123
Nucleic acid hybridization, 211 Programmed cell death (PCD), 222
Property group composition (PGC), 128
Propidium iodide, 211, 212, 215, 216,
O 222–224, 227
Oxidative phosphorylation pathways, 146 Protein-protein interaction (PPI), 127
Oxidative stress, 146, 147, 156 Protein quantification, 147–149
Protein sequences
antifungal, 130
P embedding vectors, 129
Parasitism, 96 skip-gram model, 129
Partial least squares (PLS) regression model, unsupervised learning, 129
123 Protein vectors (ProtVec) model, 129
PCR cycling conditions, 184 Pulsed-field gel electrophoresis (PFGE), 216
PCR product purification and quantification, Purification of PCR product, 196, 197
196, 197
Pea plant (Pisam sativam), 122
Penicillium, 2 Q
Periodic acid-Schiff (PAS), 110 Quantification of antioxidant enzymes
Peripheral blood mononuclear cells fungal tissue homogenization, 147, 148
(PBMCs), 108 materials, 147
Peroxidase (PX), 146, 147, 150–152 protein quantification, 148, 149
Phagotrophic sessile flagellates, 138 reactions catalyzed, 147
Phosphate buffer saline (PBS), 107, 163 to ROS decomposition
Photon molecular absorption, 210 CAT activity, 149, 150
Phylogenetics, 201 PX activity, 150, 151
Phylogenetic tree SOD activity, 151, 152
construction using MEGA Software, 202, thiol redox state related enzymes
203 G6PD activity, 155
“phylogenetic/relationship tree”, 178 GP activity, 154
use, 178 GR activity, 153, 154
Physicochemical-2-grams (P2G), 128 GST activity, 152, 153
Physicochemical properties (PCP), 128 Quantitative ImagingTM (QI), 160
Phytopathogens, 228 Quantitative polymerase chain reaction (qPCR)
Plant diseases, 124 approach, 105
Plasmid PFB11AU2004, 140, 142
Plasmids, 142
Podosphaera fusca, 5 R
Poly-L-lysine (PLL), 162 Radioimmunoassay (RIA), 113
Position specific scoring matrix (PSSM), 129 Reactive nitrogen species (RNS), 146
Potato Dextrose Agar (PDA) Reactive oxygen species (ROS), 146, 220, 221
antimicrobial and additives, 76 Real-time quantitative PCR (qPCR) assay, 138
carbohydrate source, 76 amplification efficiency, 142
dermatophytes, 76 fungal biomass, 138
foods and dairy products, 76 for quantitative assessment of uncultured
history, 75 zoosporic fungi, 138
infusion and dextrose, 76 DNA extraction and purification, 140
ingredients, 77 materials, 139
methods of preparation, 76, 78 procedure, 140, 141
molds, 79 Region of interest (ROI), 122
tartaric acid, 76 Rhizophidiales-specific primers F-Chyt, 141
260 Index
Ribosomal RNA (rRNA) gene cluster, 177 Sodium dodecyl sulphate (SDS), 89
Root mean square error (RMSE), 123 Solid phase microextraction/gas
Rotten wood, 236, 246, 247 chromatography-mass spectrometry
Rumen, 236, 237, 239–242 (SPME/GC-MS), 119, 123
Spleen, 107
Sporangia, 87–90, 96, 97, 99, 100
S 18S rDNA, 142
Sabouraud agar 18S rRNA gene, 142
additives, 72 Standard phenol–chloroform purification
antibiotics/antimicrobials, 70 procedure, 142
antimicrobials, 72, 74 Sterile soil, 2, 4, 21
casein, 71 Streptomycin, 107
chloramphenicol, 74 Superoxide dismutase (SOD), 146, 147, 151,
commercial preparations, 75 152, 156
cycloheximide, 74 Supervised learning, 118
dermatophytes, 70 Support vector machines (SVM)
DTM, 81, 83 adhesins, 127, 128
fungal isolation, 85 ANN, 126
fungal media, 70 antifungal peptides, 118, 128, 129
fungal medium, 84 classification, 118
fungi, 84 colony fingerprinting, 124
glucose, 71 domain features, 119
growth media, 85 electronic nose, 123
growth of bacteria, 70 fungal adhesins, 127, 128
growth of fungi, 70 fungal bioinformatics, 130–133
ingredients, 71 fungal disease, 121
Malassezia species, 70 fungal infection detection, 122, 123
materials, 71, 72 fungal Keratitis images, 125
method grape leaf disease detection, 124, 125
incubation, 74 kernel functions, 119
inoculation, 74 machine learning, 118
modification preparation, 73 maize leaf, 120
standard preparation, 72, 73 maximum margin classifier, 119
variations, 73, 74 Pea Plant (Pisam sativam), 122
microbes, 75 peptide sequences, 118
modification, 70 plant leaves, 126, 127
mold morphology, 71, 75 protein interactions, 127
peptones, 71 quality detection, 121
plates, 84 regression algorithms, 118
Saccharomyces cerevisiae, 160 supervised learning, 118
Sanger method, 197 unsupervised learning, 118
Scavenging H2O2, 146
Selection of gene target
DNA markers, 195 T
in fungal molecular taxonomy and Thymus, 107
phylogeny, 195 Trichophyton species, 74
gene targets/molecular markers, 179–183
materials, 195
methods, 195, 196 U
PCR conditions and their respective primer Uncultured environmental fungi, 137
pairs, 189–194 Uncultured zoosporic fungi
steps for PCR amplification, 195 molecular approaches, 138
Silica gel, 4, 5, 16 plasmids, 142
Index 261
quantitative assessment research, 236

DNA extraction and purification, 140 in rumen, 237
materials, 139 specialized habitats for yeasts
real-time qPCR assay, 138, 140, 141 isolation, 237
zoospores, 141 wood-feeding yeasts, 237
UNITE Database Search, 201 yeast diversity, 236
Unsupervised learning, 118
Z
W Zoospores, 137, 138, 141
Western blot, 113 Zoosporic forms, 137
Wood-feeding yeasts, 237 Zoosporic fungi, 138
CARD-FISH protocol, 92
CARD-FISH resolution, 92
Y Chyt1061, 91
Yeast, 160, 161, 165, 166 Chytridiales, 91
Yeast isolation methods Chytridiomycota, 91
from anthropogenic habitats chytrids, 87
compost, 247, 248 environmental samples, 91, 92
distillery effluent/spent wash, 251, 252 Escherichia coli, 91
molasses, 249 fluorescein, 92
press mud, 250 fluorochromes, 92
carbon-rich substrates, 238 hybridization method, 88
common materials, 239, 240 hybridization temperature, 91
composting, 238 identification and quantitative
diversity, 239 assessment, 88
enzymes, 238 materials, 89
flower, 238 methods, 89, 90
geographical barriers, 236 microbial food web dynamics, 88
insect gut, 236 microorganisms, 87
natural/anthropogenic ecosystems, 239 molecular surveys, 87
from natural habitats organisms, 91
flower, 245 phytoplankton, 87, 92
hot spring, 243, 244 protocol, 92
insect gut, 242, 243 rRNA gene, 91
nectar, 245, 246 simulation analysis, 88
rotten wood, 246, 247 sporangia, 88
rumen fluid/digesta, 240–242 Zygomycetous fungi, 3, 19

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Laboratory Protocols in Fungal Biology

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Fungal Biology

Vijai Kumar Gupta

More information about this series at http://www.springer.com/series/11224

ISSN 2198-7777 ISSN 2198-7785 (electronic)

1 Various Methods of Long-Term Preservation of Fungal

8 Assays for the Quantiﬁcation of Antioxidant Enzymes

Svetlana M. Ozerskaya, Nataliya E. Ivanushkina, Galina A. Kochkina,

S. M. Ozerskaya (*) · N. E. Ivanushkina · G. A. Kochkina · A. A. Danilogorskaya ·

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

1.2 Cryopreservation of Filamentous Fungi

1.3 Freeze-Drying of Filamentous Fungi

Currently, freeze-drying is used to preserve approximately 85% of ﬁlamentous fungi

1.4 Drying in Sterile Soil of Filamentous Fungi

A. raphani), Fusarium oxysporum, and the species of Septoria (S. avenae, S.

1.5 Drying of Filamentous Fungi on Silica Gel

Immobilized cells of microorganisms retain viability and biological activity at action

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

material by transferring a few granules on appropriate culture medium, as well as the

Protocols of cryopreservation, freeze-drying, and drying in sterile soil were

1.7 Protocol of Drying on Silica Gel

1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol

• Pour 5 mL of 10% glycerol into 12 mL glass tubes.

1.7.3 Preparation of Cultures

1.7.4 Silica Gel Inoculation

1.7.5 Filling of Vials

1.7.6 Control of Viability

• Place ampoule in a special metal container, thermostatic inside by expanded

Annex 1: Fields Attributes in the Table «Database Preservation

Annex 2: Maximal Preservation Times for VKM Fungal

Tankeshwar Acharya and Janelle Hare

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 69

2.1 Sabouraud Dextrose Agar

Sabouraud agar medium was developed by the French dermatologist Raymond J. A.

bacterial growth (and sometimes saprophytic fungi, depending on the particular

Table 2.2 Antimicrobial and other additives to Sabouraud agar

1. Deionized, distilled water.

2.1.4 Method of Sabouraud Agar Preparation

2.1.4.1 Standard Preparation

1. Combine all ingredients, except any antimicrobials to be used, in ~900 mL of

2.1.4.2 Method of Sabouraud Agar, Emmons Modiﬁcation Preparation

1. Combine all ingredients, except any antimicrobials to be used, in ~900 mL of

2.1.4.3 Variations on Standard Sabouraud Agar

2.1.5 Methods of Inoculation and Incubation

Depending on the antimicrobials used, different types of microorganisms and groups

2.2 Potato Dextrose Agar

Potato Dextrose Agar (PDA) contains dextrose as a carbohydrate source which

2.2.3 Methods of Preparation

See Sect. 2.1.3 for general comments regarding needed materials.

Table 2.4 Antimicrobial and other additives to Potato Dextrose Agar

A survey of 10 companies’ media found that ﬁve media sources contained

2.2.4 Methods of Inoculation and Incubation

2.2.4.1 Quality Control

2.2.4.2 Inoculation and Incubation

2.3 Bird Seed Agar

In 1962, Staib et al. described that incorporation of an extract of Guizotia abyssinica

recommended PCR conditions are 10 min at 94 C, 40 cycles of 15 s at 95 C,