Content uploaded by Samy Selim
Author content
All content in this area was uploaded by Samy Selim on Oct 16, 2018
Content may be subject to copyright.
ORIGINAL PAPER
Antagonistic yeasts from a salt-lake region in Egypt:
identification of a taxonomically distinct group
of phylloplane strains related to Sporisorium
Matthias Sipiczki .Samy A. Selim
Received: 12 August 2018 / Accepted: 6 October 2018
ÓSpringer Nature Switzerland AG 2018
Abstract Non-pathogenic yeasts antagonising
microorganisms that cause pre- and postharvest dis-
eases of plants have been found in diverse habitats.
Their practical applicability as biocontrol agents
(BCAs) depends on the strength of their antagonistic
activity and/or spectrum of sensitive target microor-
ganisms. In this study, yeasts were isolated from the
phylloplane and fruits of plants growing in the alkaline
water lake region Wadi El-Natrun, Egypt, and tested
for antifungal and antibacterial activity. All phyllo-
plane yeast isolates belonged to the Basidiomycota
and most of them could antagonise at least certain test
organisms. One group of isolates showing strong
antagonism against almost all fungi and yeasts appears
to represent a hitherto undescribed species distantly
related to the smut genus Sporisorium. This is the first
report of antagonistic activity in Sporisorium. The
isolates assigned to Naganishia and Papiliotrema
were more effective against bacteria. The broadest
range and intensity of antagonism was observed in the
fruit-associated strains belonging to the ascomycetous
species Wickerhamomyces subpelliculosus. The Wick-
erhamomyces strains are good broad-spectrum BCA
candidates, the Sporisorium strains could be used as
efficient antifungal BCAs, whereas the Papiliotrema
isolate can be exploited as an antibacterial biocontrol
agent.
Keywords Antagonistic yeasts Antibacterial
Antifungal BCA Biocontrol Desert Phylloplane
yeasts
Introduction
Stored fruit and vegetables are subject to a variety of
rots caused by a wide variety of fungi. Traditionally,
fungicides have been used in postharvest disease
control. However, the development of resistance in the
postharvest fungal pathogens to the fungicides and the
public concerns over the potentially deleterious effects
of the synthetic antifungal compounds on human
health and environmental safety have resulted in the
desire to seek safer and eco-friendly alternatives for
reducing the decay loss in the harvested commodities.
In order to satisfy this demand, biological strategies
have been developed in which naturally occurring
antagonistic microorganisms are exploited for the
M. Sipiczki (&)
Department of Genetics and Applied Microbiology,
University of Debrecen, Debrecen 4032, Hungary
e-mail: gecela@post.sk
S. A. Selim
Clinical Laboratory Sciences Department, College of
Applied Medical Sciences, Jouf University, Al-Jouf,
Kingdom of Saudi Arabia
S. A. Selim
Faculty of Science, Botany Department, Suez Canal
University, Ismailia, Egypt
123
Antonie van Leeuwenhoek
https://doi.org/10.1007/s10482-018-1184-8(0123456789().,-volV)(0123456789().,-volV)
control of postharvest diseases (for recent reviews, see
Dukare et al. 2018; Leyva Salas et al. 2017; Mari et al.
2014; Usall et al. 2016; Zhimo et al. 2014).
Among the microorganisms applicable to posthar-
vest bioprotection, antagonistic yeasts are more
acceptable to consumers than other microbes due to
their frequent natural association with foods and their
inability to produce toxic secondary metabolites (for a
review, see Liu et al. 2013). Over the past two decades,
many yeast strains have been described which can
inhibit the propagation of destructive fungi and
bacteria (for a recent review, see e.g. Muccilli and
Restuccia 2015). Several modes of action have been
suggested to explain their antagonistic activities such
as competition for nutrients, production and secretion
of growth inhibitors and/or degradative enzymes and
induction of resistance in the plant tissues (for reviews,
see e.g. Dukare et al. 2018; Muccilli and Restuccia
2015; Sharma et al. 2009). However, only a few strains
proved to be suitable for application as biocontrol
agents (BCAs) because most antagonistic strains have
only weak inhibitory effects and can efficiently
antagonise only limited numbers of microbes. Even
if they have strong activity their practical use is
frequently hampered by the difficulties with the
translation of a strong activity in the lab and green-
house to a reliable performance in the field and on
large scale (e.g. Chen et al. 2018; Le Mire et al. 2016;
Pe
´rez-Montan
˜o et al. 2013; Ciancio et al. 2016;
Parvatha Reddy 2016; Gross et al. 2018).
In our previous studies we described a highly
effective mode of antimicrobial antagonism, which
inhibits the germination of conidia and the growth of
hyphae and cells by immobilising the iron essential for
these processes (Sipiczki 2006) and characterised
antagonistic strains of diverse taxonomic affiliations
in the yeast biota of shrivelled grape berries (Sipiczki
2016). In this study we search for novel antagonistic
yeasts in the phylloplane of the lake-side vegetation of
a salt alkaline water lake in Egypt. We show that the
phylloplane yeast communities of the plants are also
rich in species and contain strains that might poten-
tially be applied as BCAs due to their antagonistic
effects on certain moulds, yeasts and/or bacteria. One
group of the isolates showing strong antifungal
activity is distantly related to Sporisorium but appears
to represent a hitherto undescribed species.
Materials and methods
Organisms and media
All yeast strains isolated in this study are listed in
Table 1. The microorganisms used for testing the
isolates for antagonism were Saccharomyces cere-
visiae S288c (Yeast Genetic Stock, Berkeley, Cali-
fornia, USA), Candida zemplinina 10-372
T
(Sipiczki
2003), Metschnikowia pulcherrima CBS 5833
T
(Wes-
terdijk Fungal Biodiversity Institute, Utrecht, The
Netherlands), six yeast strains (Rhodotorula mucilagi-
nosa 4/8, Naganishia albida 5/1, Naganishia sp 16/4,
Papiliotrema laurentii 8/1, Wickerhamomyces subpel-
liculosus 15/22, Sporisorium sp. 16/6) isolated in this
study, Botrytis cinerea 3318 (Sipiczki 2006), Penicil-
lium expansum SZMC 2175, Alternaria alternata
SZMC 16085 (Szeged Microbiology Collection,
Szeged, Hungary), Aspergillus niger ATTC 10575
(American Type Culture Collection, Manassas, Vir-
ginia, USA), Escherichia coli DH5 (Invitrogen) and
Staphylococcus capitis (this study). Fungal and yeast
strains were maintained on yeast extract agar (YEA) or
PDA (Potato Dextrose Agar, Acharlab S.L.) or in yeast
extract liquid (YEL) medium (Sipiczki and Ferenczy
1978). Bacteria were maintained on LB agar (Sam-
brook et al.1989).
Sampling and strain isolation
For yeast isolation, young branches were cut from
plants or fallen fruits were collected. The collected
material was mildly homogenised (macerated with
sterile loop and vortexed several times) in 100 ml of
sterile water, and samples (5 ll) of the homogenates
were spread on YEA plates (5 plates for each sample).
After incubation at 20 °C for 7 days, representatives
of the morphological types of yeast colonies were
isolated.
Taxonomic identification
For taxonomic identification of the yeast isolates, total
genomic DNA was extracted as described previously
(Sipiczki 2003). The isolated DNA was used for the
amplification of the D1/D2 domains of the 26S rRNA
gene and the ITS1-5.8S-ITS2 region of the rDNA
repeats. The primers used were NL-1 and NL-4 for the
D1/D2 domains (O’Donell 1993) and ITS1 and ITS4
123
Antonie van Leeuwenhoek
Table 1 Source and taxonomic affiliation of isolates
Source (host) Isolation
number
rDNA accession
number
I
Type/reference strains with highest sequence similarity Proposed
taxonomic
affiliation
Strain Accession number
I
/SH code
U
Number of
substitutions
or indels
Tamarix nilotica 3/1-o D1/D2: MH196541 Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 4/628 Sporisorium sp.
Sporisorium trachypogonis-plumosi voucher 56635 AY740113 9/628
Pseudozyma hubeiensisCBS 10077
T
DQ008953 11/628
ITS: MH196543 Mycosarcoma (Ustilago) maydis CBS 504.76 AY854090
SH209150.07FU
104/746
Sporisorium trachypogonis-splumosi voucher 56635 AY740060
SH182338.07FU
35/699
Pseudozyma hubeiensis CBS 10077
T
DQ008954
SH182312.07FU
63/776
3/4-o D1/D2: MH196544 Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 4/624 Sporisorium sp.
Sporisorium trachypogonis-splumosi voucher 56635 AY740113 9/624
Pseudozyma hubeiensis CBS 10077
T
DQ008953 11/624
Tamarix amplexicaulis 5/1-o D1/D2: MH198781 Vishniacozyma heimaeyensis CBS 8933
T
DQ000317 0/561 Vishniacozyma heimaeyensis
5/2-o D1/D2: MH196567 Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 4/628 Sporisorium sp.
Sporisorium trachypogonis-splumosi voucher 56635 AY740113 9/628
Pseudozyma hubeiensis CBS 10077
T
DQ008953 11/628
ITS: MH198780 Mycosarcoma (Ustilago) maydis CBS 504.76 AY854090
SH209150.07FU
104/746
Sporisorium trachypogonis-splumosi voucher 56635 AY740060
SH182338.07FU
35/699
Pseudozyma hubeiensis CBS 10077
T
DQ008954
SH182312.07FU
63/776
5/3-o D1/D2: MH204154 Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 6/588 Sporisorium sp.
Sporisorium trachypogonis-splumosi voucher 56635 AY740113 11/588
123
Antonie van Leeuwenhoek
Table 1 continued
Source (host) Isolation
number
rDNA accession
number
I
Type/reference strains with highest sequence similarity Proposed
taxonomic
affiliation
Strain Accession number
I
/SH code
U
Number of
substitutions
or indels
Pseudozyma hubeiensis CBS 10077
T
DQ008953 13/588
ITS: MH204153
R
Mycosarcoma (Ustilago) maydis CBS 504.76 AY854090
SH209150.07FU
104/746
Sporisorium trachypogonis-splumosi voucher 56635 AY740060
SH182338.07FU
35/699
Pseudozyma hubeiensis CBS 10077
T
DQ008954
SH182312.07FU
68/709
Tamarix amplexicaulis 9/1-o D1/D2: MH200629 Naganishia diffluens CBS6436
T
AF181543 0/576 Naganishia diffluens
Zilla spinosa 1/1 D1/D2: MH204156 Naganishia diffluens CBS6436
T
AF181543 0/572 Naganishia diffluens
1/10 D1/D2: MH204155 Naganishia albida CBS 142
T
AF075474 0/571 Naganishia albida
Cynodon dactylon 3/1 D1/D2: MH203021 Filobasidium magnum CBS140
T
KY107722 0/592 Filobasidium magnum
Filobasidium elegans CBS 7640 AF181548 0/585
Filobasidium floriforme CBS 6241
T
KY107703 0/592
ITS: MH203407 Filobasidium magnum CBS140
T
AB032680
SH197623.07FU
0/608
Filobasidium elegans CBS 7640 AF190006
SH197623.07FU
7/538
Filobasidium floriforme CBS 6241
T
AF190007
SH197623.07FU
40/631
3/5 D1/D2: MH197102 Filobasidium magnum CBS140
T
KY107722 2/597 Filobasidium magnum
Filobasidium elegans CBS 7640 AF181548 2/594
Filobasidium floriforme CBS 6241
T
KY107703 2/597
ITS: MH197140 Filobasidium magnum CBS140
T
AB032680
SH197623.07FU
0/608
Filobasidium elegans CBS 7640 AF190006
SH197623.07FU
7/558
Filobasidium floriformeCBS 6241
T
AF190007
SH197623.07FU
41/652
123
Antonie van Leeuwenhoek
Table 1 continued
Source (host) Isolation
number
rDNA accession
number
I
Type/reference strains with highest sequence similarity Proposed
taxonomic
affiliation
Strain Accession number
I
/SH code
U
Number of
substitutions
or indels
Eruca sativa 4/1 D1/D2: MH200634 Naganishia albida CBS 142
T
AF075474 4/579
Y
Naganishia albida
4/8 D1/D2: MH200635 Rhodotorula mucilaginosa CBS 316
T
AF070432 3/568
Z
Rhodotorula mucilaginosa
Medicago sativa 5/1 D1/D2: MH200626 Naganishia albida CBS 142
T
AF075474 0/564 Naganishia albida
5/5 D1/D2: MH200637
Q
Naganishia albida CBS 142
T
AF075474 0/568 Naganishia albida
Vinca rosea 6/1 D1/D2:
Q
Naganishia albida CBS 142
T
AF075474 0/566 Naganishia albida
Rudbeckia grandiflora 8/1 D1/D2: MH201039 Papiliotrema laurentii CBS 139
T
AF075469 1/582 Papiliotrema laurentii
8/12 D1/D2: MH201041 Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 4/583 Sporisorium sp.
Sporisorium trachypogonis-splumosi voucher 56635 AY740113 9/583
Pseudozyma hubeiensis CBS 10077
T
DQ008953 11/583
ITS: MH201043 Sporisorium trachypogonis-splumosi voucher 56635 AY740060
SH182338.07FU
36/699
Pseudozyma hubeiensis CBS 10077
T
DQ008954
SH182312.07FU
68/709
Mycosarcoma (Ustilago) maydis CBS 504.76 AY854090
SH209150.07FU
105/743
Pelargonium graveolens 9/1 D1/D2: MH204099 Naganishia diffluens CBS6436
T
AF181543 0/563 Naganishia diffluens
Verbena officinalis 10/1 D1/D2: MH201171 Naganishia albida CBS 142
T
AF075474 4/570
Y
Naganishia albida
Bougainvillea glabra 11/1 D1/D2:
Q
Naganishia albida CBS 142
T
AF075474 0/605 Naganishia albida
123
Antonie van Leeuwenhoek
Table 1 continued
Source (host) Isolation
number
rDNA accession
number
I
Type/reference strains with highest sequence similarity Proposed
taxonomic
affiliation
Strain Accession number
I
/SH
code
U
Number of
substitutions
or indels
Phoenix dactylifera (fallen
fruits)
15/1 D1/D2:
S
Wickerhamomyces subpelliculosus CBS 5767
T
U74593 1/556 Wickerhamomyces
subpelliculosus
15/2 D1/D2:
MH201323
Metschnikowia pulcherrima CBS 5833
T
U45736 22/494 Metschnikowia sp.
15/22 D1/D2:
MH201176
S
Wickerhamomyces subpelliculosus CBS 5767
T
U74593 0/556 Wickerhamomyces
subpelliculosus
15/23 D1/D2:
MH201178
Papiliotrema terrestris CBS 10810
T
KY108747 3/594 Papiliotrema terrestris
ITS: MH201180 Papiliotrema terrestris CBS 10810
T
KY108747 0/525
15/25a D1/D2:
MH201187
Rhodosporidiobolus fluvialis CBS6568
T
KY108963 7/584 Rhodosporidiobolus sp.
15/25b D1/D2:
MH201189
Wickerhamomyces subpelliculosus CBS 5767
T
U74593 0/556 Wickerhamomyces
subpelliculosus
Oleae uropaea 16/4 D1/D2:
MH201300
Naganishia albidosimilis CBS 7711
T
AF137601 4/573
X
Naganishia sp.
Naganishia liquefaciens CBS968
T
AF181515 4/573
X
16/6 D1/D2:
MH201375
Mycosarcoma (Ustilago) maydis CBS 504.76 AF453938 6/628 Sporisorium sp.
Sporisorium trachypogonis-splumosi voucher
56635
AY740113 11/628
Pseudozyma hubeiensis CBS 10077
T
DQ008953 13/628
ITS:
R
Mycosarcoma (Ustilago) maydis CBS 504.76 AY854090
SH209150.07FU
104/746
Sporisorium trachypogonis-splumosi voucher
56635
AY740060
SH182338.07FU
35/699
Pseudozyma hubeiensis CBS 10077
T
DQ008954
SH182312.07FU
68/709
123
Antonie van Leeuwenhoek
Table 1 continued
Source (host) Isolation number rDNA accession
number
I
Type/reference strains with highest sequence similarity
Proposed
taxonomic
affiliation
Strain Accession number
I
/SH code
U
Number of
substitutions
or indels
Nerium oleander 18/1 D1/D2:
MH201301
Rhodotorula mucilaginosa CBS 316
T
AF070432 1/554 Rhodotorula mucilaginosa
18/2 D1/D2:
MH201299
Naganishia albida CBS 142
T
AF075474 0/564 Naganishia albida
T: type strain
X: N. liquefaciens and N. albidosimilis differ by 8 nucleotides. 16/4 differs from both by 4 substitutions
Y: blast search in INSDC: many N. albida hits with differences 0–4
Z: blast search in INSDC: many R. mucilaginosa CBS hits with differences 1–3
Q: identical sequences
R: identical sequences
S: identical sequences
I: Accession number in International Nucleotide Sequence Databases (e.g. https://www.ncbi.nlm.nih.gov/genbank/)
U: SH code in the UNITE database (https://unite.ut.ee/analysis.php)
123
Antonie van Leeuwenhoek
for the ITS regions (White et al.1990). The amplifi-
cation primers were also used for sequencing. DNA
was isolated from bacterial cells by the method
described in Sambrook et al. (1989). The bacterial
16S rRNA gene was amplified and sequenced with the
primers 515F and 1492R (Lane 1991).
Database sequences similar to those of the isolates
were identified with MEGABLAST similarity search
in the linked databases of the International Nucleotide
Sequence Consortium (INSDC) (https://blast.ncbi.
nlm.nih.gov/Blast.cgi). The sequence differences
from the type strain sequences were determined by
pairwise Blast alignment using the bl2seq algorithm
available in NCBI (https://blast.ncbi.nlm.nih.gov/
Blast.cgi). SH codes were obtained from the UNITE
database (https://unite.ut.ee/analysis.php).
To obtain multiple alignments for phylogenetic
analysis, the D1/D2 and ITS sequences of the isolates
and the type strains of the most similar species were
aligned separately with the Clustal W 1.7 algorithm
(Thompson et al. 1994). As the sequences differed in
length, the overhangs were cut off and the trimmed
ITS and D1/D2 sequences were then concatenated.
The alignments of the concatenated sequences were
analysed with the PhyML 3.0 maximum likelihood
algorithm (Guindon et al. 2010) using the HKY85
nucleotide substitution model and performing non
parametric bootstrap analysis with 1000 replicates.
Antagonism tests
(a) Antagonism between the isolates and moulds. The
isolates were tested for antagonism against filamen-
tous fungi on YEA plates in two ways. In one method
(Sipiczki 2016) the plates were flooded with suspen-
sions of conidia (*10
7
conidia ml
1
). The suspension
of conidia was prepared by washing the surface of
2-week old fungal cultures grown on PDA plates at
room temperature with sterile water. After removing
the rest of the suspension and drying the surface of the
plates, loopful amounts of the cultures (grown on
YEA) of the isolates to be tested were smeared on the
plates to form spots of *5 mm in diameter. The
plates were incubated at 20 °C for 2 weeks and the
effect of the isolates on the growth of the fungi (e.g.
formation of inhibition zone) was examined at regular
time intervals. In the other method four isolates were
smeared on the YEA plate as described above and the
fungus to be tested for sensitivity was inoculated into
the centre of the square. Its growth was monitored at
20 °C for 3 weeks.
(b) Antagonism between the isolates and yeasts and
bacteria. The isolates were tested for antagonism
against yeasts and bacteria on plates flooded with
suspensions of the test organisms as described previ-
ously (Sipiczki 2016). Dense suspensions (*10
8
cells ml
-1
) were prepared in sterile water from 5-day
old cultures of the testers grown on YEA plates. Then
YEA plates were flooded with the suspensions to
obtain homogeneous lawn of cells. The rests of the
suspensions were poured off. After drying the surface
of the plates, loopful amounts of the cultures (grown
on YEA) of the strains to be tested were smeared on
the plates to form spots of *5 mm in diameter. The
plates were then incubated at 20 °C for 7 days and the
growth intensity of the lawn around the colonies of the
isolates was examined at regular time intervals. The
growth intensity of the isolates on the lawn of the
organisms was also evaluated.
Results
Sample collection and yeast isolation
Plant material was collected from randomly selected
plants in the vegetation growing around salt alkaline
water lake Hamra, Wadi El-Natrun valley (Fig. 1).
The lakes are a unique aquatic ecosystem among
saline lakes due to the hyper saline (283–540 g/L) and
alkaline waters (pH values of 8.5–9.5) (Taher 1999).
Characteristic plant species of the valley are Tamarix
nilotica,Tamarix amplexicaule, semi-wild Phoenix
dactylifera, but many other species common in desert
regions grow there as well, such as Cynodon dactylon
and Zilla spinosa (Abd El-Ghani et al. 2017). In this
study, these species and other plants introduced by
human activity were sampled. Yeasts were isolated
from 15 samples (Table 1). 30 isolates, each repre-
senting a different morphotype in the individual
samples were subjected to molecular taxonomic
identification.
Molecular taxonomy
For taxonomic identification, the D1/D2 domain
regions of the rDNA repeats of the isolates were
amplified and sequenced. The sequences were then
123
Antonie van Leeuwenhoek
used for similarity searches in the databases of the
International Nucleotide Sequence Database Collab-
oration. Isolates whose sequences did not differ by
more than 3 substitutions/indels from the sequence of
a type/reference strain of the most similar hits were
considered as conspecific with that strain. 18 isolates
were assigned to 6 species (N. albida, N. diffluens, P.
laurentii, R. mucilaginosa, Vishniacozyma
heimaeyensis, W. subpelliculosus) in this way. The
rest of the isolates could not be assigned unambigu-
ously to species (Table 1).
The D1/D2 sequence of the isolate 4/1 differed
from that of N. albida CBS 142
T
, the most similar type
strain at 4 positions but many database sequences of
other strains of this species were identical or less
different, so we assigned it to N. albida (Cryptococcus
albidus).
Isolates 15/2 and 15/25 could not be assigned to any
species. 15/2 showed close affinity to pulcherrimin-
producing Metschnikowia species but its D1/D2
sequence differed from those of all species. Its
assignment to one of these species is further hampered
by the high intragenomic diversity of the rDNA
repeats of the type strains of these species. Both the
D1/D2 and ITS sequences of these strains evolve by
the birth-and-death mechanism and interspecific retic-
ulation due to their interfertility (Sipiczki et al. 2018).
The sequence of 15/25 differed at 7 positions from the
sequence of Rhodosporidiobolus fluvialis CBS 6568
T
,
the most similar type strain.
The D1/D2 sequences of the isolates 3/1 and 3/5
were indistinguishable from those of the type strains of
the closely related Filobasidium magnum,F. elegans
and F. floriforme. Therefore, we sequenced their ITS1-
5.8S-ITS2 regions as well. The sequences were 100%
identical with the ITS sequence of the F. magnum type
strain and differed from the corresponding sequences
of the other species by 7 and 40 substitutions.
The D1/D2 sequences of the isolates 3/1-o, 3/4-o,
5/2-o, 5/3-o, 8/12, and 16/6 were most similar to
Ustilago maydis database sequences but differed from
the U. maydis reference strain CBS 504.76 by 4
substitutions. For this species no type strain is
designated. CBS 504.76 serves as a reference strain
(Boekhout 2011). A recent taxonomic revision
changed its name to Mycosarcoma maydis (McTag-
gart et al. 2016). As the differences from CBS 504.76
were higher than the threshold of conspecificity set by
us in this study, we also sequenced the ITS1-5.8S-
ITS2 regions of the six isolates. Their ITS sequences
Alexandria
Cairo
Port Sa id
Suez
Wadi El Natrun
Sadat City
Hamra
Fig. 1 Location of Hamra, the site of sample collection
123
Antonie van Leeuwenhoek
did not reinforce the close relationship with M. maydis
(Table 1). The least different sequence was that of S.
trachypogonis-plumosi 56635. However, even in this
case the number of different positions was 35–36
(5.2%), which is too high for conspecificity. Thus,
these isolates cannot be assigned to any known
species. The maximum-likelihood analysis of the
concatenated ITS and D1/D2 sequences grouped them
to S.trachypogonis-plumosi with high statistical
support (Fig. 2). To reflect this affinity we proposed
the tentative name Sporisorium sp. for this group of
isolates.
When testing the isolates for antifungal antagonism
(see next section), we noticed that the B. cinerea stock
culture was contaminated with a bacterium. We spread
samples of the contaminated culture onto YEA plates
to obtain bacterium-free Botrytis colonies and Botry-
tis-free bacterial colonies. One bacterial colony was
isolated and a segment of its 16S rDNA gene was
sequenced for taxonomic identification. The sequence
(MH266464) showed 100% identity with many S.
capitis sequences in the blast search of the INSDC
databases. This Gram-positive bacterium was previ-
ously found in a wide range of habitats such as the
natural risophere of willow (Weyens et al. 2013), a
halotolerant lignocellulose degrading bacterial com-
munity (Cortes-Tolalpa et al. 2018) and soybean
epiphytes (de Almeida Lopes et al. 2016). This strain
was then also used as a tester in the antagonism tests.
Antagonistic and synergistic interactions
The isolates were tested for interactions with four
filamentous fungi, 9 yeasts and two bacteria (Tables 2,
3). Basically, four types of interactions could be
distinguished on the test plates: inhibition over
distance, contact inhibition, growth promotion and a
dual interaction when the isolate both inhibited and
promoted the growth of the test organism. Inhibition
over distance was manifested as an inhibition zone
(Fig. 3d, m–p) around the colony of the isolate
inoculated on the lawn of conidia or cells of the tester
or as a gap between the colony of the isolate and the
front of the mycelium of the tester fungus expanding
on the plate (Fig. 3e). The inhibition zones were
mostly turbid indicating that some growth could take
place even within the zone (Fig. 3m–o). This might
have been due to growth taking place prior to the
release of effective amounts of the antagonistic agents
by the colonies of the isolates into the medium. Clear
inhibitory zones were rarely observed (Fig. 3p).
Contact inhibition could be detected only with
filamentous fungi which grew over the colonies of
the non-antagonistic isolates (Fig. 3a, e, f, i) but
stopped expanding at the edges of the colonies of the
antagonistic isolates (Fig. 3b, e, g, h, j–l). Contact
inhibition was frequently combined with growth
facilitation around the antagonistic colony (Fig. 3b,
j–l). A similar phenomenon was previously observed
in pulcherrimin-producing Metschnikowia strains and
explained by the diffusion of nutrients (and/or growth
0.01
Moesziomyces bullatus MS220 (AY740153/AY740153)
Ustilago vetiveriae HUV 17954 (AY345011/JN367337)
Sporisorium trachypogonis-plumosi 56635 (AY740060/AY740113)
3/4o (MH196543/MH196544)
3/1o (MH196543/MH196541)
5/2o (MH198780/MH196567)
8/12 (MH201043/MH201041)
16/6 (MH204153/MH201375
5/3o (MH204153/MH204154)
Pseudozyma hubeiensis CBS 10077T (DQ008954/DQ008953)
Mycosarcoma pachycarpus HUV 21891 (JN871718/JN871717)
Pseudozyma prolifica CBS 319.87 (AF294700/AJ235298)
Mycosarcoma maydis CBS 504.76 (AY854090/AF453938)
97
66
53
100
Fig. 2 Maximum likelihood tree of the concatenated ITS-D1/D2 sequences of Sporisorium sp. isolates and related species. Outgroup:
Moesziomyces bullatus MS220. Bar, 0.01 changes per position
123
Antonie van Leeuwenhoek
Table 2 Antagonistic effect of isolates on moulds and bacteria
Isolate Effect on
Moulds Bacteria
q
A. alternata A. niger B. cinerea
q
P. expansum E. coli S.
capitis
Mycelium growth Mycelium growth On lawn Mycelium growth Mycelium growth On
lawn
On
lawn
Isolation
number
Taxonomic
affiliation
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited Onto
yeast
colony
AAB
1/1 Naganishia diffluens c.i. – – ?–?–?c.i. – 3 3 4
1/10 Naganishia albida –?–?–?–?c.i. – 3 2 3
3/1 Filobasidium
magnum
–?–?–?
-
?–?1–
3/5 Filobasidium
magnum
–?–?–?
-
?–?1–
4/1 Naganishia albida –?–?–?–?c.i. – 2
4/8 Rhodotorula
mucilaginosa
–?–?c.i. –
-
?c.i. – 2
5/1 Naganishia albida –?–?c.i. – – ?c.i. – 2 2 2
5/5 Naganishia albida –?–?c.i. – – ?c.i. – 2 2 3
6/1 Naganishia albida –?–?–(?)– ?c.i. – 3, 1
t
25
8/1 Papiliotrema
laurentii
–?–?–(?)– ?–?3 0.5 4
8/12 Sporisorium sp. – ??– c.i.; 3
t
–1;5
t
– (c.i.) (?)–
9/1 Naganishia diffluens –?–?c.i. – – ?c.i. – 3 1 4
10/1 Naganishia albida –?–?(c.i.) (?)– ?–?0.5 3
11/1 Naganishia albida –?–?(c.i.) (?)– ?–?214
15/1 Wickerhamomyces
subpelliculosus
c.i. – (c.i.) (?) c.i.; 1
t
– c.i.; 2
t
––?2
t
3–
15/2 Metschnikowia sp. – ?–?c.i.; 3
t
– c.i.; 1
t
––?1–
15/22 Wickerhamomyces
subpelliculosus
c.i. – (c.i.) (?) c.i.; 0.5
t
–1;2
t
– c.i. – 2, 3
t
4–
15/23 Papiliotrema
terrestris
c.i. – – ?(c.i.) (?) c.i. – – ?8, 3
t
58
15/25a c.i. – – ?c.i. – c.i. – c.i. – –
123
Antonie van Leeuwenhoek
Table 2 continued
Isolate Effect on
Moulds Bacteria
q
A. alternata A. niger B. cinerea
q
P. expansum E. coli S.
capitis
Mycelium growth Mycelium growth On lawn Mycelium growth Mycelium growth On
lawn
On
lawn
Isolation
number
Taxonomic
affiliation
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited
x
Onto
yeast
colony
y
Inhibited Onto
yeast
colony
AAB
Rhodosporidiobolus
sp.
15/25b Wickerhamomyces
subpelliculosus
c.i. – – ?c.i.; 2
t
– c.i.; 2
t
– c.i. – 4
t
3
16/4 Naganishia sp. – ?–?–?–?–?2–
16/6 Sporisorium sp. c.i. – ?– c.i.; 3
t
–1;3
t
– c.i. – –
18/1 Rhodotorula
mucilaginosa
–?–?2
t
(?)– ?–?3
18/2 Naganishia albida –?–?–?–?–?1 1.5 4
3/1-o Sporisorium sp. – ??– c.i.; 2
t
– c.i.; 3
t
– (c.i.) (?)3–
3/4-o Sporisorium sp. (c.i.) (?)?– c.i.; 2
t
– c.i.; 2
t
– c.i. – 3 –
5/1-o Vishniacozyma
heimaeyensis
–?–?
-
(?)– ?–?1–
5/2-o Sporisorium sp. – ??– c.i.; 2
t
– c.i.; 2
t
––?–
5/3-o Sporisorium sp. – ??– c.i.; 3
t
– c.i. – – ?3–
9/1-o Naganishia diffluens c.i. – – ?–?–?c.i. – 5 1 –
t: turbid inhibition zone (mm)
A. Plates were flooded with a suspension of optical density 0.1
B. Plates were flooded with 100X diluted suspension of A
x: symbols and numerals in columns marked with ‘‘x’’: (c.i.), contact inhibition; ?, inhibition zone; t, turbid; numeral, width of inhibition zone
y: symbols in columns marked with ‘‘y’’: -, mycelium does not grow on the yeast colony; ?, mycelium grows on the yeast colony; (?), weak growth on the yeast colony
q: numeral: width of inhibition zone (mm)
123
Antonie van Leeuwenhoek
Table 3 Antagonistic effect of isolates on ascomycetous yeast strains and on other isolates
Isolate Effect on
Testers Isolates from this study
S.
cerevisiae
S288c
C.
zeplinina
10-372
T
M..
pulcherrima
CBS 5833
T
4/8 R.
mucilaginosa
5/1 N.
albida
16/4
Naganishia
sp.
8/1 P.
laurentii
15/22 W.
subpelliculosus
16/6
Sporisorium
sp.
Isolation
number
Taxonomic affiliation A B
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
1/1 Naganishia diffluens ??? –??? –?? –??? –??? –?? –?? –?–??? –
1/10 Naganishia albida ?? –??? –?–??? –??? –?? –?–?–??? –
3/1 Filobasidium magnum ?? –??? –?–??? –??? –?? –– –?–??? –
3/5 Filobasidium magnum ?? –??? –?–??? –??? –?? –– –?–??? –
4/1 Naganishia albida ?? –??? –?? –??? –??? –??? –?? –(?)–??? –
4/8 Rhodotorula mucilaginosa ??? –??? 1
t
??? –??? –??? –??? –??? –?? –??? –
5/1 Naganishia albida ??? –?? –?? –??? –??? –??? –?–(?)–??? –
5/5 Naganishia albida ??? –??? –?? –??? –??? –??? –?–(?)–??? –
6/1 Naganishia albida ??? –??? –?? –??? –??? –?? –?? –(?)–??? –
8/1 Papiliotrema laurentii ??? –??? –?? –??? –??? –??? 3??? –?? –??? –
8/12 Sporisorium sp. ??? 2??? 1?? –??? 1
t
??? –??? –?–??? –??? –
9/1 Naganishia diffluens ??? –??? –?? –??? –??? –??? –?? –?–??? –
10/1 Naganishia albida ??? –??? –?? –??? –??? –??? –?–?–??? –
11/1 Naganishia albida ?? –??? –?? –??? –??? –??? –– –?–??? –
15/1 Wickerhamomyces
subpelliculosus
??? 4??? 1??? 2
t
??? 3
t
??? 2
t
??? 3??? 2
t
??? –??? –
15/2 Metschnikowia sp. ??? –??? –??? –??? –??? –??? –?? –??? –??? –
15/22 Wickerhamomyces
subpelliculosus
??? 4??? 5??? 3
t
??? 2
t
??? 2
t
??? 3??? 2
t
??? –??? –
15/23 Papiliotrema terrestris ??? –??? –??? –??? –??? –??? –??? –(?)–??? –
15/25a Rhodosporidiobolus sp. ???
-
??? –???
-
???
-
???
-
??? –???
-
?? –??? –
15/25b Wickerhamomyces
subpelliculosus
??? 5??? 4??? 5
t
??? 2
t
??? 2
t
??? 4??? 2
t
??? –??? –
16/4 Naganishia sp. ??? –??? –?? –??? –??? –??? –?–(?)–??? –
16/6 Sporisorium sp. ??? 2??? 4??? 1
t
??? 2
t
??? –––??? –??? –
18/1 Rhodotorula mucilaginosa ??? –??? –??? –??? –??? –??? –??? –?? –??? –
18/2 Naganishia albida ?? –??? –?? –??? –??? –?–?–(?)–??? –
123
Antonie van Leeuwenhoek
Table 3 continued
Isolate Effect on
Testers Isolates from this study
S.
cerevisiae
S288c
C.
zeplinina
10-372
T
M..
pulcherrima
CBS 5833
T
4/8 R.
mucilaginosa
5/1 N.
albida
16/4
Naganishia
sp.
8/1 P.
laurentii
15/22 W.
subpelliculosus
16/6
Sporisorium
sp.
Isolation
number
Taxonomic affiliation A B
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
AB
q
3/1-o Sporisorium sp. ?? 2??? 2
t
??? –??? 1
t
??? –??? 3– –?? –??? –
3/4-o Sporisorium sp. ??? 1??? –??? –??? –??? –??? 3?–?? –??? –
5/1-o Vishniacozyma
heimaeyensis
??? –?? –?? –??? –??? –??? –?? –(?)–??? –
5/2-o Sporisorium sp. ?? 2??? –??? –??? –??? –??? 3?–?? –??? –
5/3-o Sporisorium sp. ?? 2??? –??? –??? 0.5
t
??? –??? 3?–?–??? –
9/1-o Naganishia diffluens ?? –??? –??? –??? –??? –?–?? –?–??? 2
t
A: growth of the colony of the isolate on the lawn of the tester
B: inhibition zone around the colony of the isolate in the lawn of the tester (mm)
T: type strain
t: turbid inhibition zone (mm)
q: numeral: width of inhibition zone (mm)
-: no antagonistic effect
?: antagonistic effect; the intensity is proportional with the number of the symbol
(?): weak antagonistic effect
123
Antonie van Leeuwenhoek
factors) from the ‘‘prohibited’’ parts of the medium
(Sipiczki 2006). The same process might have oper-
ated in the lawn of Sta.capitis, which grew stronger
around the colonies of certain isolates even if the
colony of the isolate formed an inhibition zone
(Fig. 3p). This phenomenon, also referred to as
cross-feeding, was observed previously among mem-
bers of interacting yeast communities of grapes
(Sipiczki 2016). The poor growth of the W. subpel-
liculosus isolates on lawns of most other isolates might
be due to competition for nutrients rather than to a
specific inhibitory mechanism.
The results of the tests are shown in Tables 2and 3.
With the exception of F. magnum,N. albida and
Nagnishia sp., all isolates inhibited the growth of at
least one mould. With the exception of three strains,
3/1
I
8/12
6/1
9/1
8/1
E
5/3-o
D
18/1
B
5/3-o
C
4/1
A
15/1
M
5/3-o
O
8/1
F
15/22
G
3/1-o
H
15/1-o
N
3/4-o
P
15/1
J
15/22
K
16/6
L
Fig. 3 Examples of interactions between the isolates and the
test organisms. The identification codes of the isolates used in
Table 1are shown on their colonies in the photographs. a–dOn
lawn of B. cinerea conidia. eA. niger mycelium growing from
the middle of the plate. f–hP. expansum mycelium growing
from the middle of the plate. i–lA. alternata mycelium growing
from the middle of the plate. mOn S. cerevisiae lawn. n–oon
lawn of the isolate 6/1. pon lawn of bacterial cells. d,m–
ptransilluminated plates
123
Antonie van Leeuwenhoek
all isolates showed antibacterial activities, but only 9
isolates were active against the yeast testers. When
strains isolated in this work were used as testers, none
of the isolates formed inhibition zones in the W.
subpelliculosus 15/22 lawn and only a N. diffluens
isolate formed a turbid zone on the Sporisorium sp.
16/6 background. In spite of not forming inhibition
zones, most isolates inhibited or at least reduced the
growth of the colonies of W. subpelliculosus 15/22
inoculated on their lawns but not the growth of
Sporisorium sp. 16/6. Similar phenomenon was
observed with P. laurentii 8/1, with the remarkable
difference that in its lawn the W. subpelliculosus
isolates formed large inhibition zones. Interestingly,
the formation of inhibition zones in the lawns of the
testers and the inhibition of growth of colonies of the
testers did not correlate. The colonies of the Nagan-
ishia strains grew normally on the lawns of almost all
isolates, but all Wickerhamomyces and Sporisorium
isolates formed inhibition zones in their lawns. Taken
the results of all tests together, two groups, the
ascomycetous Wickerhamomyces and the basid-
iomycetous Sporisorium isolates exhibited strong
antibacterial and antifungal activity. Certain Nagan-
ishia strains and the R. mucilaginosa isolates had
milder inhibitory effect on the growth of B. cinerea.
Metschnikowia sp. 15/2 antagonised only B. cinerea.
The Naganishia and Papiliotrema isolates exhibited
strong antibacterial and only weak or no antifungal
antagonism.
Discussion
The molecular taxonomic analysis identified 9 species
among the isolates and three groups of uncertain
taxonomic position. The latter could only be assigned
to genera. All basidiomycetous species found in this
study have previously been detected in phylloplane
microbial communities (e.g. Kemler et al. 2017;
Limtong and Nasanit 2017). Their presence on plants
growing around a salt alkaline water lake demon-
strates their adaptability to diverse environmental
conditions. The occurrence of V. (Cryptococcus)
heimaeyensis (Tremellales) is somewhat surprising
because this species was originally described from soil
samples collected in Iceland (Vishniac 2002) and
detected later on plants in temperate climates (Yurkov
et al. 2015). The isolates assigned to the ascomycetous
genera Metschnikowia and Wickerhamomyces were
isolated from fallen dates. However, we cannot
exclude the occurrence of these species in the
phylloplane yeast communities. We can only say that
we found Metschnikowia and Wickerhamomyces
strains only in fallen dates.
The taxonomic position of the group tentatively
designated Sporisorium sp. deserves special attention.
Their ITS sequences differ from that of the closest
relative, S. trachypogonis-plumosi by 5.2%. This
difference is much higher than 1.39%, the taxonomic
thresholds proposed by Vu et al. (2016) to discriminate
basidiomycetous yeast species. The determination of
their exact taxonomic affiliation is further hampered
by the uncertain position of S. trachypogonis-plumosi
on the phylogenetic trees. It is distant from the
Sporisorium sensu stricto clade (Stoll et al. 2005) and
close to P. hubeiensis (Wang et al. 2015). Pseudozyma
Bandoni emend. Boekhout is a small genus of
anamorphic yeasts related to species of the teleomor-
phic genera Ustilago and Sporisorium, which are
responsible for serious plant diseases (Wang et al.
2015). The smut genus Sporisorium occurs all over the
world, mainly in regions with warmer climate and its
known species exclusively infect species of Poaceae
(Pipenberg 2003). In contrast to this obligate associ-
ation of the genus with Poaceae, none of the isolates
we designate Sporisorium sp. in this study were
isolated from grasses but from dicots. The high ITS
differences from both Sporisorium and Pseudozyma as
well as the very different hosts indicate that these
isolates may represent a hitherto undescribed species
or even genus.
With the aim of selecting efficient candidate BCAs,
we tested all isolates for antagonism against 4 moulds
causing post-harvest diseases of fruit and vegetables, 3
ascomycetous yeasts frequently occurring on fruit and
2 bacteria. Two major forms of antagonism were
observed: inhibition with diffusible inhibitors result-
ing in inhibition zones around the colony of the
antagonist and inhibition by physical contact between
the colonies of the antagonist and the sensitive
organism. From practical point of view, contact
inhibition has less potential in biocontrol because
much higher doses of the antaganists will have to be
used to ensure direct physical contact with the
pathogen in contrast to the antagonists that inhibit
the growth of the pathogen by secreted diffusible
agents.
123
Antonie van Leeuwenhoek
When tested for interactions with each other, all
isolates proved to be either antagonistic against certain
other isolates or sensitive to the inhibitory effect of
other isolates. Although detected on laboratory culture
media, these interactions can be assumed to take place
also on plants. Similar complex interactions were
observed recently in yeasts colonising grapes and
proposed to shape the structure of their communities
(Sipiczki 2016). None of the isolates examined in this
study could antagonise all isolates belonging to
different species. Thus, the antagonistic activity of
neither isolate can provide competitive advantage over
all potential competitors in the phylloplane
community.
All Sporisorium sp. isolates strongly inhibited the
growth of almost all fungi and yeasts. Antimicrobial
activity has not been reported for this genus yet, but
antagonistic activity has been noticed in certain
species of the related genus Pseudozyma (e.g. Avis
and Be
´langer 2002; Buxdorf et al. 2013; Hajlaou
et al.1994; Jarvis et al. 1989; Lee et al. 2017). The
extensively characterised Pseudozyma flocculosa
(syn: Sporothrix flocculosa) produces flocculosin, a
membrane-active cellobiose lipid with antifungal and
antibacterial activities (Mimee et al. 2005,2009). The
phylogenetic relatedness with Pseudozyma species
and the taxonomic diversity of the sensitive testers
suggest that a similar mechanism may account for the
antagonistic activity of the Sporisorium isolates.
The Naganishia isolates showed stronger antibac-
terial than antifungal activity. The taxonomic analysis
identified them as N. albida or N. diffluens (formerly
C. albidus and C. diffluens). So far, only N. albida
strains have been reported to antagonise postharvest
pathogenic fungi and proposed for application as
bioprotection agents (e.g. Calvo et al. 2003; Chan and
Tian 2005; Fan and Tian 2001). However, those
strains were not identified with molecular tools and
thus their taxonomic position is uncertain and they
may not be conspecific with the antibacterial isolates
of this study.
W. subpelliculosus was described in an early report
to have killer activity (Young and Yagiu 1978) but the
mode of action has not been explored. As killer toxins
are specific agents (generally proteins or glycopro-
teins) that are able to kill susceptible cells belonging to
the same or congeneric species (Golubev 2006; El-
Banna et al. 2011; Liu et al. 2015), the antagonistic
activity of these isolates against moulds, non-related
yeasts and bacteria cannot be attributed to a killer
factor but a less specific agent.
The Metschnikowia isolate showed significant
antagonism only against B. cinerea. The similarity of
its D1/D2 sequence to those of strains belonging to the
group of pulcherrimin-producing species of the genus,
suggests that its antagonism might also be due to the
exhaustion of free iron in the medium by complexing it
with pulcherrimin. The shortage of free iron inhibits
the germination of conidia and the growth of hyphae
and cells (Sipiczki 2006).
In conclusion, some of the yeasts colonising plants
in the Wadi El-Natrun valley have strong antagonistic
effects. All isolates inhibited or at least reduced the
growth of at least certain test organisms in the
laboratory tests. The isolates with weak Sporisorium
affinity exhibited strong antifungal activity but had
almost no effect on the growth of bacteria. In contrast,
the Naganishia and Papiliotrema isolates were more
effective against bacteria than against fungi and
yeasts. The broadest range of antagonism was
observed in the fruit-associated W. subpelliculosus
strains which inhibited the growth of all testers with
the exception of the Sporisorium sp. strains. The
Sporisorium sp. and the W. subpelliculosus isolates are
good broad-spectrum BCA candidates, whereas P.
terrestris 15/23 could be exploited as an antibacterial
agent.
Acknowledgements Samy A. Selim thanks Tempus Public
Foundation, Hungary (Grant No. 20004) for supporting his
postdoctoral research in Hungary. We thank prof. Gamal El-Din
for the taxonomic identification of plants and Anita Csabai-Olah
for excellent technical assistance.
Author contribution MS Conceived and designed study. MS,
SAS Performed research. MS Analysed data. MS Wrote the
paper.
Conflict of interest No conflict of interest to declare.
References
Abd El-Ghani MM, Huerta-Martı
´nez FM, Hongyan L, Qureshi
R (2017) Plant responses to hyperarid desert environments.
Springer, Cham
Avis TJ, Be
´langer RR (2002) Mechanisms and means of
detection of biocontrol activity of Pseudozyma yeasts
against plant-pathogenic fungi. FEMS Yeast Res 2:5–8
Boekhout T (2011) Pseudozyma Bandoni emend. Boekhout
(1985) and a comparison with the yeast state of Ustilago
123
Antonie van Leeuwenhoek
maydis (De Candolle) Corda (1842). In: Kurtzman CP, Fell
JW, Boekhout T (eds) The yeasts, a taxonomic study.
Elsevier, Amsterdam, pp 1857–1868
Buxdorf K, Rahat I, Gafni A, Levy M (2013) The epiphytic
fungus Pseudozyma aphidis induces jasmonic acid- and
salicylic acid/nonexpressor of PR1-independent local and
systemic resistance. Plant Physiol 161:2014–2022
Calvo J, Calvente VDE, Orellano M, Benazzi D, Tosetti M
(2003) Improvement in the biocontrol of postharvest dis-
eases of apple with the use of yeast mixture. Biocontrol
48:579–593
Chan Z, Tian S (2005) Interaction of antagonistic yeasts against
postharvest pathogens of apple fruit and possible mode of
action. Postharvest Biol Technol 36:215–223
Chen L, Heng J, Qin S, Bian K (2018) A comprehensive
understanding of the biocontrol potential of Bacillus
velezensis LM2303 against Fusarium head blight. PLoS
ONE 13:e0198560
Ciancio A, Pieterse CM, Mercado-Blanco J (2016) Editorial:
harnessing useful rhizosphere microorganisms for patho-
gen and pest biocontrol. Front Microbiol 19:1620
Cortes-Tolalpa L, Norder J, van Elsas JD, Falcao Salles J (2018)
Halotolerant microbial consortia able to degrade highly
recalcitrant plant biomass substrate. Appl Microbiol
Biotechnol 102:2913–2927
de Almeida Lopes KB, Carpentieri-Pipolo V, Oro TH, Stefani
Pagliosa E, Degrassi G (2016) Culturable endophytic
bacterial communities associated with field-grown soy-
bean. J Appl Microbiol 120:740–755
Dukare AS, Sangeeta P, Nambi VE, Gupta R, Sharma K,
Vishwakarma RK (2018) Exploitation of microbial
antagonists for the control of postharvest diseases of fruits:
a review. Crit Rev Food Sci Nutr 16:1–16
El-Banna AA, Malak AE, Shehata MG (2011) Yeasts producing
killer toxins: an overview. Alex J Food Sci Technol
8:41–53
Fan Q, Tian SP (2001) Postharvest biological control of grey
mold and blue mold on apple by Cryptococcus albidus
(Saito) Skinner. Postharvest Biol Technol 21:341–350
Golubev WI (2006) Antagonistic interactions among yeast. In:
Rosa C, Peter G (eds) Biodiversity and ecophysiology of
yeasts. Springer, Berlin, pp 197–219
Gross S, Kunz L, Mu
¨ller DC, Santos Kron A, Freimoser FM
(2018) Characterization of antagonistic yeasts for biocon-
trol applications on apples or in soil by quantitative anal-
yses of synthetic yeast communities. Yeast. https://doi.org/
10.1002/yea.3321
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W,
Gascuel O (2010) New algorithms and methods to estimate
maximum-likelihood phylogenies: assessing the perfor-
mance of PhyML 3.0. System Biol 59:307–321
Hajlaou MR, Traquair JA, Jarvis WR, Belanger RR (1994)
Antifungal activity of extracellular metabolites produced
by Sporothrix flocculosa. Biocontrol Sci Technol
4:229–237
Jarvis WR, Shaw LA, Traquair JA (1989) Factors affecting
antagonism of cucumber powdery mildew by
Stephanoascus flocculosus and S. rugulosus. Mycol Res
92:162–165
Kemler M, Witfeld F, Begerow D, Yurkov A (2017) Phyllo-
plane yeasts in temperate climates. In: Buzzini P, Lachance
M-A, Yurkov A (eds) Yeasts in natural ecosystems:
diversity. Springer, Cham, pp 171–198
Lane DJ (1991) 16S/23S rRNA sequencing. In: Goodfellow M,
Stackebrandt E (eds) Nucleic acid techniques in bacterial
systematics. Wiley, New York, pp 115–175
Le Mire G, Nguyen ML, Fassotte B, du Jardin P, Verheggen F,
Delaplace P, Jijakli MH (2016) Review: implementing
plant biostimulants and biocontrol strategies in the agroe-
cological management of cultivated ecosystems. Biotech-
nol Agron Soc Environ 20(S):299–313
Lee G, Lee SH, Kim KM, Ryu CM (2017) Foliar application of
the leaf-colonising yeast Pseudozyma churashimaensis
elicits systemic defense of pepper against bacterial and
viral pathogens. Sci Rep 10:39432
Leyva Salas M, Mounier J, Valence F, Coton M, Thierry A,
Coton E (2017) Antifungal microbial agents for food bio-
preservation—a review. Microorganisms 5:37
Limtong S, Nasanit R (2017) Phylloplane yeasts in tropical cli-
mates. In: Buzzini P, Lachance M-A, Yurkov A (eds) Yeasts
in natural ecosystems: diversity. Springer, Cham, pp 199–224
Liu J, Sui Y, Wisniewski M, Droby S, Liu Y (2013) Review:
utilization of antagonistic yeasts to manage postharvest
fungal diseases of fruit. Int J Food Microbiol 167:153–160
Liu GL, Chi Z, Wang GY, Wang ZP, Li Y, Chi ZM (2015) Yeast
killer toxins, molecular mechanisms of their action and
their applications. Crit Rev Biotechnol 35:222–234
Mari M, Di Francesco A, Bertolini P (2014) Control of fruit
postharvest diseases: old issues and innovative approaches.
Stewart Postharvest Rev 1:1–4
McTaggart AR, Shivas RG, Boekhout T, Oberwinkler F, Va
´nky
K, Pennycook SR et al (2016) Mycosarcoma (Ustilagi-
naceae), a resurrected generic name for corn smut (Usti-
lago maydis) and its close relatives with hypertrophied,
tubular sori. IMA Fungus 7:309–315
Mimee B, Labbe
´C, Pelletier R, Be
´langer RR (2005) Antifungal
activity of flocculosin, a novel glycolipid isolated from
Pseudozyma flocculosa. Antimicrob Agents Chemother
49:1597–1599
Mimee B, Pelletier R, Be
´langer RR (2009) In vitro antibacterial
activity and antifungal mode of action of flocculosin, a
membrane-active cellobiose lipid. J Appl Microbiol
107:989–996
Muccilli S, Restuccia C (2015) Bioprotective role of yeasts.
Microorganisms 3:588–611
O’Donell K (1993) Fusarium and its near relatives. In: Reynolds
DR, Taylor JW (eds) The fungal holomorph: mitotic,
meiotic and pleomorphic speciation in fungal systematics.
CAB International, Wallingford, pp 225–233
Parvatha Reddy P (2016) Sustainable crop protection under
protected cultivation. Springer, Singapore
Pe
´rez-Montan
˜o F, Alı
´as-Villegas C, Bellogı
´n RA, del Cerro P,
Espuny MR, Jime
´nez-Guerrero I, Lo
´pez-Baena FJ, Ollero
FJ, Cubo T (2013) Plant growth promotion in cereal and
leguminous agricultural important plants: from microor-
ganism capacities to crop production. Microbiol Res
169:325–336
Pipenberg M (2003) Smut fungi (Ustilaginomycetes p.p. and
Microbotryales, Basidiomycota). Flora Neotrop 86:1–291
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor
123
Antonie van Leeuwenhoek
Sharma RR, Singh D, Singh R (2009) Biological control of
postharvest diseases of fruits and vegetables by microbial
antagonists: a review. Biol Control 50:205–221
Sipiczki M (2003) Candida zemplinina sp. nov., an osmotoler-
ant and psychrotolerant yeast that ferments sweet botry-
tised wines. Int J System Evol Microbiol 53:2079–2083
Sipiczki M (2006) Metschnikowia strains isolated from botry-
tized grapes antagonise fungal and bacterial growth by iron
depletion. Appl Environ Microbiol 72:6716–6724
Sipiczki M (2016) Overwintering of vineyard yeasts: survival of
interacting yeast communities in grapes mummified on
vines. Front Microbiol 7:212
Sipiczki M, Ferenczy L (1978) Enzymic methods for enrich-
ment of fungal mutants. I. Enrichment of Schizosaccha-
romyces pombe mutants. Mutat Res 50:153–173
Sipiczki M, Horvath E, Pfliegler PW (2018) Birth-and-death
evolution and reticulation of ITS segments of Metschni-
kowia andauensis and Metschnikowia fructicola rDNA
repeats. Front Microbiol 9:1193
Stoll M, Begerow D, Oberwinkler F (2005) Molecular phy-
logeny of Ustilago,Sporisorium, and related taxa based on
combined analyses of rDNA sequences. Mycol Res
109:342–356
Taher AG (1999) Inland saline lakes of Wadi El Natrun
depression, Egypt. Int J Salt Lake Res 8:149–169
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-specific
gap penalties and weight matrix choice. Nucleic Acids Res
22:4673–4680
Usall J, Torres R, Teixido N (2016) Biological control of
postharvest diseases on fruit: a suitable alternative? Cur
Opin Food Sci 11:51–55
Vishniac HS (2002) Cryptococcus tephrensis, sp.nov., and
Cryptococcus heimaeyensis, sp.nov.; new anamorphic
basidiomycetous yeast species from Iceland. Can J
Microbiol 48:463–467
Vu D, Groenewald M, Szo
¨ke S, Cardinali G, Eberhardt U,
Stielow B et al (2016) DNA barcoding analysis of more
than 9 000 yeast isolates contributes to quantitative
thresholds for yeast species and genera delimitation. Stud
Mycol 85:91–105
Wang Q-M, Begerow D, Groenewald M, Liu X-Z, Theelen B,
Bai F-Y et al (2015) Multigene phylogeny and taxonomic
revision of yeasts and related fungi in the Ustilaginomy-
cotina. Stud Mycol 81:55–83
Weyens N, Beckers B, Schellingen K, Ceulemans R, Croes S,
Janssen J et al (2013) Plant-associated bacteria and their
role in the success or failure of metal phytoextraction
projects: first observations of a field-related experiment.
Microbial Biotechnol 6:288–299
White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification
and direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis MA, Gelfand D, Sninsky JS, White
TJ (eds) PCR protocols: a guide to methods and applica-
tions. Academic Press, San Diego, pp 315–322
Young TW, Yagiu M (1978) A comparison of the killer char-
acter in different yeasts and its classification. Antonie Van
Leeuwenhoek 44:59–77
Yurkov A, Ina
´cio J, Chernov IY, Fonseca A
´(2015) Yeast bio-
geography and the effects of species recognition approa-
ches: the case study of widespread basidiomycetous
species from birch forests in Russia. Cur Microbiol
70:587–601
Zhimo VY, Bhutia DD, Saha J, Panja B (2014) Exploitation of
yeasts as an alternative strategy to control post harvest
diseases of fruits. World Appl Sci J 31:785–793
123
Antonie van Leeuwenhoek
A preview of this full-text is provided by Springer Nature.
Content available from Antonie van Leeuwenhoek
This content is subject to copyright. Terms and conditions apply.
