Journal of

Fungi
Article
Analysis of Stored Wheat Grain-Associated Microbiota Reveals
Biocontrol Activity among Microorganisms against
Mycotoxigenic Fungi
Manoj Kumar Solanki 1,†,‡ , Ahmed Abdelfattah 2,† , Sudharsan Sadhasivam 1 , Varda Zakin 1 ,
Michael Wisniewski 3 , Samir Droby 1, * and Edward Sionov 1, *

1 Volcani Center, Agricultural Research Organization, Institute of Postharvest and Food Sciences,
Rishon LeZion 7528809, Israel; mkswings321@gmail.com (M.K.S.); sudharsan@volcani.agri.gov.il (S.S.);
veredz@volcani.agri.gov.il (V.Z.)
2 Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12,
A-8010 Graz, Austria; ahmed.abdelfattaah@gmail.com
3 Agricultural Research Service, United States Department of Agriculture (USDA-ARS), 2217 Wiltshire Road,
Kearneysville, WV 25430, USA; Michael.Wisniewski@ars.usda.gov
* Correspondence: samird@volcani.agri.gov.il (S.D.); edwardsio@volcani.agri.gov.il (E.S.)
† These authors contributed equally to this work.
‡ Current address: Plant Cytogenetics and Molecular Biology Group, Faculty of Natural Sciences, Institute of
Biology, Biotechnology and Environmental Protection, University of Silesia in Katowice,
40-032 Katowice, Poland.





 Abstract: Wheat grains are colonized by complex microbial communities that have the potential to
Citation: Solanki, M.K.; Abdelfattah, affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities
A.; Sadhasivam, S.; Zakin, V.; have been shown to protect plants against pathogens through antagonism. We evaluated the role
Wisniewski, M.; Droby, S.; Sionov, E. of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon
Analysis of Stored Wheat sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes
Grain-Associated Microbiota Reveals
differ in their fungal and bacterial diversity and community composition. We then isolated culturable
Biocontrol Activity among
fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi.
Microorganisms against
The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota
Mycotoxigenic Fungi. J. Fungi 2021, 7,
of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic
781. https://doi.org/10.3390/
jof7090781
properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples
of up to a 3.59 log10 CFU/g compared to untreated controls. We also found that a strain of the yeast,
Academic Editor: Kimiko Yabe Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B1 , one of the
most dangerous mycotoxins that negatively affects physiological processes in animals and humans.
Received: 26 August 2021 The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the
Accepted: 17 September 2021 yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are
Published: 20 September 2021 a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential
against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these
Publisher’s Note: MDPI stays neutral antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially
with regard to jurisdictional claims in
replace traditionally used synthetic chemicals.
published maps and institutional affil-
iations.
Keywords: biocontrol; stored wheat grain microbiota; epiphytes; endophytes; mycotoxigenic
fungi; mycotoxins

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
Wheat is one of the major cultivated grain crops, and an important source of calories
distributed under the terms and
conditions of the Creative Commons
and plant-derived protein in the human diet. The maintenance of high-quality and healthy
Attribution (CC BY) license (https://
wheat grains is essential to ensuring the stability of the world’s food supply and providing
creativecommons.org/licenses/by/ global food security. Wheat grains, like seeds of other cereal crops, are colonized by
4.0/). complex epiphytic and endophytic microbial communities that play an important role in

J. Fungi 2021, 7, 781. https://doi.org/10.3390/jof7090781 https://www.mdpi.com/journal/jof

J. Fungi 2021, 7, 781 2 of 16

grain health, quality, and susceptibility to disease [1]. Microorganisms, both bacteria and
fungi, naturally occur in cereal crops without causing any damage, and may influence
host growth and development. Conversely, other microorganisms can cause disease and
spoilage, thereby decreasing crop value, and may also have a harmful effect on human
health. For example, many species of Fusarium, Aspergillus, Penicillium, and Alternaria are
not only recognized as plant pathogens but also as sources of important mycotoxins of
concern to animal and human health [2,3]. Poor postharvest management can lead to
rapid deterioration in grain quality, with severe decreases in germinability and nutritional
value of the stored grain, possibly accompanied by undesirable fungal contamination
and, consequently, toxin production [4]. Mycotoxigenic fungal species may also cause
significant wheat grain yield losses in field and storage facilities due to their ability to
produce mycotoxins and render the crops unsafe for consumption [5–9]. Despite the
major threat posed by these fungi, effective control measures are lacking. Current disease-
management practices are primarily based on the use of synthetic fungicides, which have
proven to be effective against several genera of mycotoxigenic fungi, applying them in the
field before harvest. Increasing awareness of the possible harmful effects of these chemical
compounds on the environment and human health, however, has fostered research to find
effective and less toxic alternative strategies to control fungal infections.
Biological control using microbial antagonists against fungal plant pathogens is one
alternative approach that can be used as part of an integrated management strategy for
the control of mycotoxigenic fungi and mycotoxin contamination in stored wheat. The
protective ability of some bacterial epiphytes and endophytes against fungal pathogens
has been reported in several crops, including wheat [1,10–13]. Nevertheless, one of the
major drawbacks associated with the use of biological control agents is their inconsistent
efficacy over time [14,15]. This can be attributed in part to the fact that most commercially
available biocontrol isolates do not originate from the plants that they are intended to
protect, a factor that may be related to their inconsistent performance in the field and/or
storage warehouses. Exploring the epiphytic and endophytic composition of the stored
wheat grain microbiome may help in identifying novel antagonistic microorganisms with
potential antifungal and antitoxigenic activity. The major objective of the present study
was to use amplicon sequencing to identify and characterize the epiphytic and endophytic
microbiome of stored wheat grain. We then examined the potential of the culturable
microorganisms isolated from stored seeds to inhibit the growth of mycotoxigenic fungi
and degrade their toxins.

2. Materials and Methods

2.1. Wheat Grain Sources
Twenty-seven samples of wheat grain (Triticum aestivum) were collected from three
wheat grain storage facilities located in northern and southern districts of Israel. In each
of the three storage facilities, nine samples (1 kg of grain each), destined for human
consumption, were collected in equal proportions (9 samples × 3 storage sites = a total of
27 samples) from the front face and center, at points located at 1 m horizontal depth within
the grain mass, and from areas close to the walls. Grain temperature and moisture content
were in the ranges of 27–33 ◦ C and 10.5–12.9%, respectively. The collected samples were
kept in sterile plastic bags during transport to the laboratory and stored at 4 ◦ C (for up to
4 weeks) for further analysis.

2.2. DNA Extraction, Amplification, and Sequencing

To isolate DNA from epiphytic microbiota, 10 g of each grain sample was soaked in
45 mL peptone water (10 g/L peptone, 5 g/L NaCl) (Difco; Becton Dickinson, Franklin
Lakes, NJ, USA) containing 0.05% Triton X-100 (Sigma, Saint Louis, MO, USA) in a 250-mL
Erlenmeyer flask and shaken at room temperature on an orbital shaker at 150 rpm for 1 h.
Grains were aseptically removed by filtration through 4 layers of sterile cheesecloth and the
remaining liquid fractions were centrifuged at 4000× g for 15 min. Pellets were collected
J. Fungi 2021, 7, 781 3 of 16

and immediately frozen in liquid nitrogen, freeze-dried, then subjected to DNA extraction.
DNA from endophytic microbiota was isolated after surface sterilization of wheat grains
with 3% sodium hypochlorite for 2 min, followed by 70% ethanol for 2 min and rinsing five
times in sterile distilled water. Surface-sterilized wheat grains (10 g) were frozen in liquid
nitrogen, freeze-dried and milled into a fine powder with a grain grinder. The grain grinder
was cleaned and disinfected with 70% ethanol solution between samples and the powder
was used for DNA isolation. Extraction of both epiphytic and endophytic DNA samples
was performed using a previously described protocol [16]. The purity of the extracted DNA
was assayed with a Nanodrop™ One spectrophotometer (Thermo Scientific, Wilmington,
DE, USA), and the total DNA concentration in each sample was adjusted to 50 ng/µl. The
universal primers 799F/1392R [17] and 5F/86R [18] were used to amplify the 16S and ITS2
rRNA gene regions of bacteria and fungi, respectively (Supplementary Materials Table S1).
The primers were modified to include Illumina adapters (www.illumina.com (accessed
on 7 March 2019)) for subsequent multiplexing. PCR amplification of each sample was
performed in triplicate. The PCR mixture (25 µL) contained 12.5 µL 2× DreamTaq Green
PCR Master Mix (Thermo Scientific, Vilnius, Lithuania), 1 µL of each primer (5 µM), and 1
µL DNA template. Nuclease-free water (Thermo Scientific) replaced the DNA template in
negative controls. All amplicons and amplification mixtures, including negative controls,
were sequenced using Illumina MiSeq V3 (2 × 300 bp) chemistry.

2.3. Bioinformatics
Illumina adaptors were clipped and low-quality reads removed by Trimmomatic
0.36 [19] using sliding-window trimming, cutting once the average quality in a window
of 4 bases fell below the quality threshold of 15. Paired-end reads were merged utilizing
PEAR [20] for the 16S rRNA gene, and PANDAseq [21] for the ITS rRNA gene region
sequences with default parameters. Chimeric sequences were identified and removed
using USEARCH [22,23] for the 16S rRNA gene, and VSEARCH 1.4.0 [24] for ITS rRNA
gene region sequences. UCLUST algorithm [22], as implemented in QIIME 1.9.1 [25], was
used to cluster sequences queried against the Greengenes 13_8_97 database for 16S rRNA
genes [26], and for ITS UNITE dynamic database released on 1 December 2017 [27] at
a similarity threshold of 97%, respectively. Sequences that failed to cluster against the
database were de novo clustered using the same algorithm. After removing singletons,
the most abundant sequences in each operational taxonomic unit (OTU) were selected
as representative sequences and used for the taxonomic assignment with the BLAST
algorithm [23,28] as implemented in QIIME 1.9.1. The OTU table was normalized by
rarefaction to an even sequencing depth in order to eliminate sample heterogeneity. The
OTU tables were rarefied to 300 bacterial and 8000 fungal sequences per sample and used to
calculate α-diversity indices, including observed species (Sobs ) and Shannon index. The α-
diversities were compared based on a two-sample t-test using nonparametric (Monte Carlo)
methods and 999 Monte Carlo permutations. Results were visualized in boxplot figures.
MetagenomeSeq’s Cumulative Sum Scaling (CSS) [29] was used as a normalization
method for other downstream analyses. The CSS-normalized OTU table was analyzed
using Bray–Curtis metrics [30] and utilized to evaluate β-diversity and construct principal
coordinates analysis (PCoA) plots using Emperor [31]. The similarity in community com-
position was tested via ANOSIM in QIIME 1.9.1 using 999 permutations. Differential OTU
abundance of the most abundant taxa (≥0.1%) between sample groups was determined
by t-test and Kruskal–Wallis test [32]. In all tests, significance was determined using 999
Monte Carlo permutations; the false discovery rate (FDR) was used to adjust the calculated
p-values and when the FDR p < 0.05, it was considered significant (Tables S2 and S3).

2.4. Isolation and Identification of Microorganisms

To isolate epiphytic microorganisms from wheat grains, 10 g of whole grain samples
were incubated in 45 mL of sterile peptone water (Difco; Becton Dickinson, Franklin Lakes,
NJ, USA) and shaken (150 rpm) at room temperature for 1 h. Then, 100-µL aliquots of
J. Fungi 2021, 7, 781 4 of 16

serial dilutions of the broth were plated on Luria–Bertani (LB) agar plates for isolation
of bacteria, and potato dextrose agar (PDA) plates supplemented with chloramphenicol
(20 µg/mL) for isolation of filamentous fungi and yeasts. To isolate endophytic microbes,
5 g of sterilized, ground wheat grain samples were added to 45 mL of sterile peptone water,
and 100-µL aliquots of serial dilutions were plated on LB agar plates for isolation of bacteria
and PDA plates supplemented with chloramphenicol for isolation of filamentous fungi and
yeasts. Inoculated LB agar plates were incubated at 37 ◦ C for 24–48 h, whereas PDA plates
were incubated at 28 ◦ C for 48–72 h. Bacterial colonies were randomly selected from the
plates and streaked on fresh culture media to obtain pure cultures. Filamentous fungi and
yeasts were transferred singly to PDA plates and subcultured twice to obtain pure cultures.
DNA was extracted from each bacterial strain using the lysozyme lysis method described
by De et al. [33]. Fungal DNA extraction was performed on lyophilized mycelium/yeast
cells using the CTAB-based method as previously described [16]. DNA quality and yield
were determined using the Nanodrop One spectrophotometer. The 16S rRNA gene in
bacteria, ITS rRNA gene region in fungi, and D1/D2 domain of large-subunit ribosomal
DNA (rDNA) in yeast were amplified with universal primers 27F/1492R, ITS1/ITS4, and
NL1/NL4, respectively (Table S1). PCR products were purified and sequenced by standard
Sanger sequencing; sequences were identified via BLAST matches to the NCBI database
(https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 26 June 2021)) and deposited as
accession numbers MZ452446–MZ452604 (bacterial 16S rRNA gene), MZ578164–MZ578238
(fungal ITS rRNA gene region) and MZ452263–MZ452328 (D1/D2 domain of large-subunit
rDNA in yeast).

2.5. In Vitro Antagonism against Mycotoxigenic Fungi

Mycotoxigenic fungal isolates Aspergillus flavus (NRRL3518) and Fusarium proliferatum
(NRRL31866) were obtained from USDA Agricultural Research Service Culture Collection
(Northern Regional Research Laboratory, Peoria, IL, USA); Alternaria infectoria (strain F11)
was isolated from stored wheat grains. Strains were refreshed from −80 ◦ C by subculturing
on PDA plates and maintained at 28 ◦ C before each experiment. Conidia were collected
in sterile saline and the conidial suspension was adjusted to the required concentration
by counting in a hemocytometer. The inoculum of the test strains was verified by plating
on PDA plates for determination of colony forming units (CFU) counts. The possible
antagonistic effects of the obtained bacterial and yeast isolates against the mycotoxigenic
fungi were assessed by dual-culture method. Each bacterial/yeast strain and fungal isolate
were inoculated on a 90-mm PDA plate. Each tested bacterium or yeast was precultured
overnight in LB broth or potato dextrose broth (PDB), respectively, and streaked in a
straight line in the center of the 90-mm Petri dish, then 5 µL of fungal spore suspension
at 107 spores/mL was spotted at opposite edges of the plate. The plates were sealed with
parafilm and incubated at 28 ◦ C until the mycelia in the untreated controls reached the
center of the plates.
The production of volatile metabolites with antifungal activity released by bacterial
and yeast isolates was also investigated. Precultivated overnight cultures of each bacterial
or yeast strain were inoculated on a 55-mm LB or PDA agar plate, respectively, and
incubated at 28 ◦ C for 3 days. Then, the lids of these plates were replaced by the bottom
of a PDA plate inoculated with a fresh fungal mycelial plug. Plates were sealed together
with sticky tape to minimize gas exchange and further incubated for 5 days. Controls were
prepared in a similar manner, but the bottom plate contained no bacteria/yeast. In all
of these experiments, growth inhibition (mm) of the tested fungi was measured with a
ruler and compared with the fungal growth in the untreated control plates. At least three
independent repetitions of each test were conducted.

2.6. Biocontrol Activity of Selected Bacterial Strains and Yeast against Mycotoxigenic Fungi on
Wheat Grains
Surface-disinfected seeds of wheat (10 g) were placed in sterile Petri dishes and
inoculated with 1 mL of a cell suspension of overnight culture (OD600 = 1.00) of each
J. Fungi 2021, 7, 781 5 of 16

bacterial and yeast isolate selected based on their inhibitory effect on fungal growth.
Grains treated with sterile saline instead of bacterial/yeast inoculum were used as negative
controls. After 24 h incubation at 28 ◦ C, wheat grains were inoculated with 1 mL of 105
conidia/mL of each fungal isolate. Untreated wheat grains were also inoculated with
fungal conidia and served as positive controls. Plates were incubated at 28 ◦ C for 7 days
and fungal development was assessed visually. For each bacterial and/or yeast treatment
of wheat grains inoculated with each fungal isolate, at least three independent experiments
were conducted. The antifungal activity of bacterial and/or yeast strains on inoculated
wheat grains was evaluated using the plate counting method and expressed as fungal
colony-forming units (CFUs)/g dry weight of wheat grains. Briefly, grain samples (5 g)
were suspended in 50 mL phosphate-buffered saline containing 0.05% Triton X-100 and
incubated with shaking (160 rpm) at 28 ◦ C for 30 min, then 100 µL of serial dilutions of the
suspension were plated on PDA plates supplemented with chloramphenicol, followed by
incubation at 28◦ C for 4 days, for counting of fungal CFUs.

2.7. Aflatoxin-Degradation Assays

Commercial aflatoxin B1 (AFB1 ) standard was purchased from Fermentek (Jerusalem,
Israel). AFB1 at a concentration of 1 mg/mL was dissolved in methanol and stored at
−20 ◦ C. Based on previous reports indicating that the yeast Rhodotorula glutinis negatively
affects mycotoxin accumulation in stored pome fruit [34], we included R. glutinis strain
TY1, which we isolated from the wheat grains, in our mycotoxin-degradation assay. The
analysis of AFB1 persistence in PDB medium was performed as follows. R. glutinis TY1
was grown overnight in 50 mL PDB with shaking at 28 ◦ C. The culture was centrifuged
for 5 min at 8000× g, the cells were resuspended in PDB, and their concentration was
adjusted to 1 × 108 CFU/ml. A 20-mL aliquot of this cell suspension was incubated on an
orbital shaker for 3 days at 28 ◦ C in a 100-mL flask in the presence of 250 ng/mL of AFB1 .
Controls included PDB medium only with 250 ng/mL of AFB1 and PDB without AFB1
inoculated with R. glutinis TY1 at the same cell concentration. Yeast growth was monitored
on a daily basis by recording OD600 in the Nanodrop One spectrophotometer. At the same
time points, the time course of AFB1 degradation was analyzed by high-performance liquid
chromatography (HPLC).
AFB1 degradation by the R. glutinis TY1 isolate was also examined in stored wheat
grains. Briefly, 10 g of surface-disinfected wheat grains were contaminated with AFB1 (final
concentration 250 ng/g) and inoculated with strain TY1 (final concentration 1 × 108 cells/g)
on Petri dishes. Controls consisted of noninoculated wheat grains with and without AFB1
and TY1-inoculated grains without AFB1 . The plates were prepared in triplicate and kept
at 28 ◦ C for up to 3 days. Then, the grain samples were freeze-dried and milled to a fine
powder with a grain grinder. AFB1 was extracted after 24, 48, and 72 h and samples were
analyzed quantitatively by HPLC. Experiments were performed three times.

2.8. HPLC Analysis

For AFB1 extraction from PDB broth, the samples were centrifuged at 8000× g for
5 min to pellet out the cells, and 2 mL of the supernatant was mixed with 2 mL of chloroform
and vortexed for 15 min. After discarding the upper phase, the chloroform phase was dried
at 50 ◦ C under a stream of nitrogen gas. For the mycotoxin analysis from wheat grains, 2.5 g
of the ground seeds were mixed with 10 mL of 84:16 (v/v) acetonitrile:water and placed in
an orbital shaker at 200 rpm for 1 h at room temperature. After centrifugation at 8500× g
for 15 min, 2 mL of the supernatant was passed through a SupelTM TOX AflaZea column
(Supelco, Bellefonte, PA, USA); then, the collected sample was evaporated by nitrogen
stream at 50 ◦ C.
The dried samples were reconstituted in 900 µL of 10% acetonitrile and 100 µL of
trifluoroacetic acid solution (70% water, 20% trifluoroacetic acid and 10% acetic acid (v/v))
for 15 min at 50 ◦ C. After incubation, the samples were filtered through a 0.22-µm PTFE
membrane filter, and quantitatively analyzed by injection of 30 µL into a reversed phase
J. Fungi 2021, 7, 781 6 of 16

HPLC/UHPLC system (Waters ACQUITY Arc, Milford, MA, USA) with gradient elution
of 70% water, 15% acetonitrile and 15% methanol at 1 ml/min through a Kinetex 3.5 µm XB-
C18 (150 × 4.1 mm) column (Phenomenex, USA). The column temperature was maintained
at 35 ◦ C. Three noncontaminated PDB and/or wheat grain samples were spiked with AFB1
standard solution at three concentrations to construct calibration curves, which were used
for mycotoxin quantification. The AFB1 peak was detected with a fluorescence detector
(excitation at 365 nm and emission at 455 nm) and quantified by comparing with calibration
curves of the mycotoxin standard.

2.9. Statistical Analysis

Data presented in the figures are mean values of three independent experiments.
Standard errors of the mean (SEM) are presented. Differences among treatments were
analyzed by one-way analysis of variance followed by Duncan’s multiple range test to
determine treatment effects, as well as significant differences between the strains (SPSS
16.0 software). AFB1 persistence in the biodegradation assays was analyzed by one-way
analysis of covariance to determine statistically significant differences between treatment
effects on mycotoxin concentration controlling for time (covariance).

3. Results and Discussion

3.1. Composition of Epiphytic and Endophytic Microbiota Associated with Stored Wheat Grain
After quality filtering and removing chimeric, chloroplast and mitochondrial se-
quences, 682,478 16S and 1,044,499 ITS sequences were retained and assigned to 63,108 and
114,849 bacterial and fungal OTUs, respectively.
The taxonomic assignment of 63,108 bacterial OTUs revealed 22 phyla in the epiphytic
and endophytic datasets, including Proteobacteria, the predominant in the datasets (48.1%
and 67.8%), followed by Actinobacteria (10.4% and 18.8%), Firmicutes (13.7% and 10.6%),
Bacteroidetes (9.2% and 2.1%) and Acidobacteria (0.2% and 0.1%), respectively (Figure 1A).
Planctomycetes (7.7%) and Chloroflexi (2.7%) were found only as endophytes. The relative
abundance of the epiphytic and endophytic bacterial microbiota varied at the genus level.
The most abundant endophytic bacterial genera were Pantoea (7.5%), Ohtaekwangia (5.8%),
Bacillus (4.9%), Burkholderia (4.0%), Actinobacteria (3.9%), Enterobacter (3.7%), Pseudomonas
(3.1%), Caulobacter (2.6%), Staphylococcus (2.5%), Sphingomonas (1.9%), Wolbachia (1.8%),
Klebsiella (1.6%), Micrococcus (1.5%), Stenotrophomonas (1.3%), Massilia (1.1%), Paenibacillus
(1.0%), and Ralstonia (1.0%) (Figure 1B). In the epiphytic bacterial community, the most
abundant genera were Pantoea (20.3%), Sphingomonas (10.2%), Pseudomonas (8.1%), Massilia
(8.5%), Curtobacterium (4.3%), Microbacter (4.3%), Paracoccus (3.3%), Agrococcus (2.7%), Enter-
obacter (2.7%), Paenibacillus (2.1%), Enhydrobacter (2.0%), Hymenobacter (1.4%), Anaerococcus
(1.4%), Acinetobacter (1.2%), Brevundimonas (1.1%), and Bacillus (0.8%) (Figure 1B). These
findings support earlier studies on seed-associated microbiota, where the bacterial taxa
Pantoea, Bacillus, Enterobacter, and Pseudomonas were detected as both endophytic and
epiphytic microorganisms [12,13,35,36].
Among all of the identified bacterial taxa, Ruminococcaceae, SG8-4, Anaerolineaceae,
Devosia, Actinobacteria, Diplorickettsiaceae, Pseudoalteromonas, Ohtaekwangia, Comamonas,
and Klebsiella were significantly (p < 0.05) higher in the wheat grain endosphere than in
epiphytic communities. In contrast, bacterial epiphytic taxa such as Microbacteriaceae, Sph-
ingomonas, Agrococcus, Curtobacterium, Massilia, Hymenobacter, Methylobacterium, Paracoccus,
Pseudomonas, Clavibacter, Pantoea, Enterobacteriaceae, and Arthrobacter were significantly
higher (p < 0.05) as epiphytes than as endophytes (Figure 1B). The differentially abun-
dant or unique taxa characterizing the endophytic niche of wheat grains were mostly
unidentified bacterial species. This finding highlights the lack of information that exists on
the microorganisms that inhabit the endosphere of seeds and their role in plant growth,
development, and health.
J. Fungi 2021, 7, x FOR PEER REVIEW 7 of 16
J. Fungi 2021, 7, 781 7 of 16

Figure 1. Bar charts showing the relative abundance of most dominant bacterial phyla (A) and genera (B) among endophytes
and epiphytes detected in stored wheat grain samples using high-throughput sequencing technology.
Figure 1. Bar charts showing the relative abundance of most dominant bacterial phyla (A) and genera (B) among endo-
phytes and epiphytes detected in stored wheat grain samples using high-throughput sequencing technology.
The fungal communities were dominated by a few phyla. Ascomycota was the most
abundant phylum, with a relative abundance of 95.3% and 72.9% in the endophytic and
Among all of the identified bacterial taxa, Ruminococcaceae, SG8-4, Anaerolineaceae,
epiphytic communities, respectively. The relative abundance of Basidiomycota was much
Devosia, Actinobacteria, Diplorickettsiaceae, Pseudoalteromonas, Ohtaekwangia, Comamonas,
higher in the epiphytic fungal community (26.1%) relative to fungal endophytic community
and Klebsiella were significantly (p < 0.05) higher in the wheat grain endosphere than in
(4.3%) (Figure 2A). At the genus level, OTUs of the fungal endophytes were assigned to
epiphytic communities.
Alternaria (76.8%), RussulaIn(3.4%),
contrast, bacterial(11.8%),
Stemphylium epiphytic taxa such
Aspergillus as Microbacteriaceae,
(1.9%), Epicoccum (1.0%),
Sphingomonas, Agrococcus,
Cladosporium (0.9%), Curtobacterium,
Pyrenophora Massilia, Hymenobacter,
(0.5%), Mycosphaerella Methylobacterium,(0.4%),
(0.4%), Paradendryphiella Para-
coccus, Pseudomonas, Clavibacter, Pantoea, Enterobacteriaceae, and Arthrobacter
Sporobolomyces (0.1%) and Filobasidium (0.1%), and epiphytes annotated to Alternaria (50.3%), were signifi-
cantly higher (p (9.5%),
Sporobolomyces < 0.05) Filobasidium
as epiphytes(8.8%),
than asVishniacozyma
endophytes (Figure(4.2%), 1B). The differentially
Mycosphaerella (3.6%),
abundant or unique taxa characterizing the endophytic niche
Stemphylium (3.5%), Cladosporium (10.5%), Epicoccum (1.4%), Dioszegia (1.1%),of wheat grains were mostly
Aspergillus
unidentified bacterial (0.7%),
(0.8%), Aureobasidium species.Paradendryphiella
This finding highlights the lack
(0.2%), Russula of information
(0.1%) that exists
and Pyrenophora (0.1%)
on the microorganisms
(Figure 2B). that inhabit the endosphere of seeds and their role in plant growth,
development,
The Shannon and health.
α-diversity was higher in the epiphytic community of both bacteria
and fungi than communities
The fungal it was in thewere dominated
endophytic by a few (Figure
community phyla. Ascomycota was the most
3). A two-sample t-test
abundant phylum, with a relative abundance of 95.3% and 72.9%
based on the Shannon index revealed a significant difference between the structure in the endophytic andof
epiphytic communities, respectively. The relative abundance of Basidiomycota
the endophytic and epiphytic bacterial (p = 0.001) and fungal (p = 0.001) community was much
higher
(Table in the
S4). epiphytic
PCoA fungal
was used to community
measure the(26.1%) relative
dissimilarity ofto
thefungal endophytic
epiphytic commu-
and endophytic
nity (4.3%) communities
microbial (Figure 2A). At ofthe genus
wheat level,(Figure
grains OTUs of 4).theThe
fungal endophytes
structure of the were assigned
epiphytic and
to Alternaria bacterial
endophytic (76.8%), Russula (3.4%),were
communities Stemphylium (11.8%),asAspergillus
clearly different evidenced(1.9%), Epicoccum
by their evident
(1.0%), Cladosporium
separation in the PCoA(0.9%),
plot,Pyrenophora
and the same (0.5%), Mycosphaerella
was true (0.4%),
for the structure Paradendryphiella
of the epiphytic and
(0.4%), Sporobolomyces (0.1%) and Filobasidium (0.1%), and epiphytes
endophytic fungal communities. The ANOSIM test (nonparametric 999 permutations) annotated to Alter-of
naria (50.3%),
variance analysisSporobolomyces
also indicated(9.5%),
that theFilobasidium
composition(8.8%), Vishniacozyma
of epiphytic (4.2%),bacterial
and endophytic Myco-
sphaerella
and fungal (3.6%), Stemphylium
communities was (3.5%), Cladosporium
significantly (R2 = 0.89,
different(10.5%), Epicoccum
p = 0.001(1.4%),
and R 2 = 0.85,
Dioszegia
(1.1%), Aspergillus
p = 0.001), (0.8%), Aureobasidium (0.7%), Paradendryphiella (0.2%), Russula (0.1%)
respectively.
and Pyrenophora (0.1%) (Figure 2B).
J. J.Fungi
Fungi2021,
2021,7,7,781
x FOR PEER REVIEW 88 ofof1616

Figure 2. Relative abundance of fungal phyla (A) and genera (B) among endophytes and epiphytes detected in grain
samples using high-throughput sequencing technology.

The Shannon -diversity was higher in the epiphytic community of both bacteria and
fungi than it was in the endophytic community (Figure 3). A two-sample t-test based on
the Shannon index revealed a significant difference between the structure of the endo-
phytic and epiphytic bacterial (p = 0.001) and fungal (p = 0.001) community (Table S4).
PCoA was used to measure the dissimilarity of the epiphytic and endophytic microbial
communities of wheat grains (Figure 4). The structure of the epiphytic and endophytic
bacterial communities were clearly different as evidenced by their evident separation in
the PCoA plot, and the same was true for the structure of the epiphytic and endophytic
fungal communities. The ANOSIM test (nonparametric 999 permutations) of variance
Figure2.2.Relative
Figure analysis
Relativeabundance
abundance alsophyla
ofoffungal
fungal indicated
phyla(A) that
(A)and
and the composition
genera
genera(B)
(B)among of epiphytic
amongendophytes
endophytes and and endophytic
andepiphytes detected
epiphytes bacterial
detected grainand
iningrain
samplesusing
samples fungal
usinghigh-throughput communities
high-throughputsequencing was
sequencingtechnology.significantly
technology. different (R2 = 0.89, p = 0.001 and R2 = 0.85, p = 0.001),

respectively.
The Shannon -diversity was higher in the epiphytic community of both bacteria and
fungi than it was in the endophytic community (Figure 3). A two-sample t-test based on
the Shannon index revealed a significant difference between the structure of the endo-
phytic and epiphytic bacterial (p = 0.001) and fungal (p = 0.001) community (Table S4).
PCoA was used to measure the dissimilarity of the epiphytic and endophytic microbial
communities of wheat grains (Figure 4). The structure of the epiphytic and endophytic
bacterial communities were clearly different as evidenced by their evident separation in
the PCoA plot, and the same was true for the structure of the epiphytic and endophytic
fungal communities. The ANOSIM test (nonparametric 999 permutations) of variance
analysis also indicated that the composition of epiphytic and endophytic bacterial and
fungal communities was significantly different (R2 = 0.89, p = 0.001 and R2 = 0.85, p = 0.001),
respectively.

Figure 3. Alpha
Figure 3. Alpha diversity
diversity analysis
analysis (based
(based on
on Shannon
Shannon diversity
diversity index)
index) of
of the
the bacterial
bacterial (A)
(A) and
and fungal
fungal (B) epiphytes and
(B) epiphytes and
endophytes in stored wheat grain samples. The centered square represents the mean, black line inside the box represents
endophytes in stored wheat grain samples. The centered square represents the mean, black line inside the box represents
the median, and circles indicate outliers.
the median, and circles indicate outliers.

Figure 3. Alpha diversity analysis (based on Shannon diversity index) of the bacterial (A) and fungal (B) epiphytes and
endophytes in stored wheat grain samples. The centered square represents the mean, black line inside the box represents
the median, and circles indicate outliers.
J.J. Fungi
Fungi 2021, 7, x781
2021, 7, FOR PEER REVIEW 9 9ofof 16
16

Figure
Figure 4.
4. Principal
Principal coordinate
coordinate analysis
analysis (PCoA)
(PCoA) based
based on
on the
the beta
beta diversity
diversity Bray
Bray Curtis
Curtis dissimilarity
dissimilarity metrics,
metrics, showing
showing the
the
distance in the wheat grain-associated bacterial (A) and fungal (B) communities between endophytic and epiphytic
distance in the wheat grain-associated bacterial (A) and fungal (B) communities between endophytic and epiphytic microbes.mi-
crobes.
Endophytic microbial diversity was significantly lower than that of the epiphytic
Endophytic
microbiota. Thismicrobial diversity to
can be attributed wasthesignificantly
need for seedlower than that to
endophytes of the
passepiphytic
through mi-
the
crobiota. This can be attributed to the need for seed endophytes to pass through
plant ecological, and physiological sieve to become internalized and tolerate harsh seed the plant
ecological,
conditions,and physiological
including sieveof
a high level todesiccation.
become internalized and tolerate harsh seed condi-
tions, including a high level of desiccation.
3.2. Microbe Isolation from Stored Grains and the Antagonistic Activity of Selected Isolates against
Mycotoxigenic
3.2. Fungalfrom
Microbe Isolation Pathogens
Stored Grains and the Antagonistic Activity of Selected Isolates
againstA Mycotoxigenic
total of 159 bacterial
Fungalisolates
Pathogenswere cultured from wheat grains and identified by 16S
rRNA sequencing; 118 of which were
A total of 159 bacterial isolates were isolated from from
cultured the grain
wheatsurface
grainsasand
epiphytes andby
identified 41
as endophytes from the inner tissues of the grains. Bacillus spp. were dominant
16S rRNA sequencing; 118 of which were isolated from the grain surface as epiphytes and among
cultured
41 bacterialfrom
as endophytes epiphytes (53.4%),
the inner tissuesfollowed by Pseudomonas
of the grains. Bacillus spp.spp.
were(10%), Pantoea
dominant spp.
among
(6.8%), Paenibacillus spp. (5%), and Staphylococcus spp. and Enterobacter
cultured bacterial epiphytes (53.4%), followed by Pseudomonas spp. (10%), Pantoea spp. spp. (3.4% for
each genus) (Figure 5A). Diversity among endophytes was lower than for epiphytes at
(6.8%), Paenibacillus spp. (5%), and Staphylococcus spp. and Enterobacter spp. (3.4% for each
the taxonomic level of genus. Similarly to bacterial epiphytes, however, most endophytes
genus) (Figure 5A). Diversity among endophytes was lower than for epiphytes at the tax-
isolated from stored wheat grains belonged to the genera Bacillus (46.3%) and Pseudomonas
onomic level of genus. Similarly to bacterial epiphytes, however, most endophytes iso-
(14.6%) (Figure 5A). These observations are consistent with previous studies of seed-
lated from stored wheat grains belonged to the genera Bacillus (46.3%) and Pseudomonas
associated endophytic bacteria, where Bacillus and Pseudomonas were the genera most
(14.6%) (Figure 5A). These observations are consistent with previous studies of seed-asso-
frequently found in plant seeds [37]. In a study on barley seed endophytes, most endophytic
ciated endophytic bacteria, where Bacillus and Pseudomonas were the genera most fre-
bacteria were assigned to the same genera, with Bacillus and Pseudomonas being retrieved
quently found in plant seeds [37]. In a study on barley seed endophytes, most endophytic
from the seeds placed under selective pressure for nitrogen-fixing microorganisms [38].
bacteria were assigned to the same genera, with Bacillus and Pseudomonas being retrieved
Among mold and yeast strains isolated from stored grains, 104 epiphytes and 37 en-
from the seeds placed under selective pressure for nitrogen-fixing microorganisms [38].
dophytes were identified by ITS and D1/D2 region sequencing. The epiphytic fungal
communities were the richest and most abundant, while richness and diversity in the
endophytes was much lower. Most of the epiphytic and endophytic filamentous fungi
were identified as Alternaria, Aspergillus, Fusarium, and Penicillium species. Filobasidium,
Cryptococcus, Rhodotorula, and Candida species dominated both epiphytic and endophytic
yeast cultures isolated from grains (Figure 5B). In general, the composition of the bacterial
and fungal taxa isolated from wheat grains in our study were in agreement with previous
reports [12,13,36,37,39]. The culturing results were also consistent with the results obtained
by high-throughput sequencing in the present study. Bacterial communities assigned to
Pantoea, Pseudomonas, Bacillus, Enterobacter, and Paenibacillus spp., and fungal microbiota
comprising Alternaria, Aspergillus, Fusarium, and Filobasidium spp. were identified as the
common genera among the members of the shared epiphytic and endophytic microbiota of
stored wheat seeds.
 x FOR PEER REVIEW
J. Fungi 2021, 7, 781 10
10of
of 16

Figure 5. Relative
Figure 5. Relative abundance
abundance of of bacterial (A), and
bacterial (A), and fungal
fungal (B)
(B) species
species isolated
isolated by
by plating
plating from
from epiphytic
epiphytic and
and endophytic
endophytic
microflora of stored wheat grains. The isolates were identified by sequencing the 16S rRNA gene in bacteria,
microflora of stored wheat grains. The isolates were identified by sequencing the 16S rRNA gene in bacteria, ITS region and
ITS region
and D1/D2
D1/D2 domain
domain of large-subunit
of large-subunit rDNArDNA in filamentous
in filamentous fungifungi
andand yeasts,
yeasts, respectively.
respectively. TheThe sequences
sequences werewere determined
determined via
via BLAST
BLAST matches
matches to the
to the NCBINCBI database.
database.

Among screening
In vitro mold and of microbial
yeast strains isolates
isolatedin dual-culture
from assays
stored grains, 104with Aspergillus
epiphytes and 37 flavus,
en-
Fusarium proliferatum,
dophytes and by
were identified Alternaria
ITS andinfectoria revealed
D1/D2 region only fourThe
sequencing. Bacillus isolates
epiphytic that exhib-
fungal com-
ited considerable
munities were theinhibitory
richest andactivity
mostagainst
abundant,the tested
whilemycotoxigenic fungi (Supplementary
richness and diversity in the endo-
Materialis
phytes wasFigure
much S1). These
lower. Most were twoepiphytic
of the endophytes amyloliquefaciens
and(B.endophytic strain fungi
filamentous EnB28were and
B. amyloliquefaciens
identified strain
as Alternaria, EnB29) and
Aspergillus, two epiphytes
Fusarium, (B. licheniformis
and Penicillium strain EpV5Crypto-
species. Filobasidium, and B.
subtilis Rhodotorula,
coccus, strain EpV29).andThe isolates
Candida appeared
species to secrete
dominated bothantifungal
epiphytic and substances
endophytic that yeast
were
capable of inhibiting fungal growth (Figure S2). The mean values
cultures isolated from grains (Figure 5B). In general, the composition of the bacterial andof pathogen growth
inhibition
fungal taxafluctuated between
isolated from wheat23grains
and 40% in our studyAspergillus
against flavus, 23with
were in agreement and 41%
previousagainst
re-
F. proliferatum,
ports and 38 The
[12,13,36,37,39]. and culturing
54% against Alternaria
results were alsoinfectoria. In contrast,
consistent with thea results
volatileobtained
organic
compound
by (VOC) assay
high-throughput revealed
sequencing inthat none of study.
the present the isolated exhibited
Bacterial inhibitory
communities activity
assigned to
against any of the tested fungi. These results conflict with previous studies
Pantoea, Pseudomonas, Bacillus, Enterobacter, and Paenibacillus spp., and fungal microbiota in which VOC
inhibitory activity
comprising by Bacillus
Alternaria, strains
Aspergillus, against and
Fusarium, Aspergillus, Fusarium,
Filobasidium and Alternaria
spp. were identifiedspecies
as the
has been reported [40–42].
common genera among the members of the shared epiphytic and endophytic microbiota
To validate
of stored our in vitro results on the biocontrol potential of Bacillus isolates, we de-
wheat seeds.
signedIn vitro screening ofsuitable
a simple bioassay microbial forisolates
analyzing antagonistic activity
in dual-culture of the
assays with selected Bacillus
Aspergillus flavus,
strains against mycotoxigenic fungi in stored grains. All of the bacterial
Fusarium proliferatum, and Alternaria infectoria revealed only four Bacillus isolates that isolates tested
ex-
significantly reduced fungal growth in grains compared to untreated
hibited considerable inhibitory activity against the tested mycotoxigenic fungi (Supple- controls (p < 0.05,
Figure 6). The tested Bacillus species exhibited the strongest antagonistic activity against F.
mentary Materialis Figure S1). These were two endophytes (B. amyloliquefaciens strain
proliferatum, resulting in an up to a 3.59 log10 CFU/g reduction in the fungal population
EnB28 and B. amyloliquefaciens strain EnB29) and two epiphytes (B. licheniformis strain
compared to control samples. Additionally, up to a 2.45 and 1.76 log10 CFU/g decrease
EpV5 and B. subtilis strain EpV29). The isolates appeared to secrete antifungal substances
was recorded for Aspergillus flavus and Alternaria infectoria, respectively. Bacillus spp., such
that were capable of inhibiting fungal growth (Figure S2). The mean values of pathogen
as B. amyloliquefaciens and B. subtilis, have been previously identified and characterized
growth inhibition fluctuated between 23 and 40% against Aspergillus flavus, 23 and 41%
as antagonistic toward Fusarium spp. on wheat [14,43–49]. Few studies, however, have
against F. proliferatum, and 38 and 54% against Alternaria infectoria. In contrast, a volatile
been conducted on the antagonistic potential of Bacillus isolates against Aspergillus and/or
organic compound (VOC) assay revealed that none of the isolated exhibited inhibitory
Alternaria pathogens on wheat [50]. Bacillus strains are well known for their ability to
activity against any of the tested fungi. These results conflict with previous studies in
produce a wide range of potent antimicrobial lipopeptides, including iturins, surfactins,
which VOC inhibitory activity by Bacillus strains against Aspergillus, Fusarium, and Alter-
fengycins, polymyxins, and others, that have broad effects on phytopathogens [51–53].
naria species has been reported [40–42].
Although they were not characterized in this study, some of these compounds could be
 B. amyloliquefaciens and B. subtilis, have been previously identified and characterized as
antagonistic toward Fusarium spp. on wheat [14,43–49]. Few studies, however, have been
conducted on the antagonistic potential of Bacillus isolates against Aspergillus and/or Al-
ternaria pathogens on wheat [50]. Bacillus strains are well known for their ability to pro-
J. Fungi 2021, 7, 781 duce a wide range of potent antimicrobial lipopeptides, including iturins, surfactins, 11 of 16
fengycins, polymyxins, and others, that have broad effects on phytopathogens [51–53].
Although they were not characterized in this study, some of these compounds could be
responsible for
responsible for the
thebacterial
bacterialinhibitory
inhibitoryactivity
activityagainst
againstthe tested
the fungal
tested pathogens.
fungal pathogens. In ad-
In
dition to lipopeptides, other antimicrobial compounds produced by Bacillus strains,
addition to lipopeptides, other antimicrobial compounds produced by Bacillus strains, such such
as polyketides
as polyketides andand lithium
lithium enzymes
enzymes [54],
[54], may
may also
also be
be involved
involved inin the
the antifungal
antifungal activity
activity
observed both in vitro and in vivo. Determining the source of the antimicrobial activity
observed both in vitro and in vivo. Determining the source of the antimicrobial activity of of
the Bacillus
the Bacillus isolates
isolates against
against the
the tested
tested fungal
fungal pathogens
pathogens will
will require
require further
further study.
study.

Figure 6. Antagonistic activity of four selected bacterial isolates against three mycotoxigenic fungal pathogens in stored
wheat grains. Data are means of at least three independent repetitions ± standard error. One-way ANOVA differences
Figure
were 6. Antagonistic
considered activity
significant whenof pfour selected
< 0.05. bacterial
Different lettersisolates
above against
the errorthree
bars mycotoxigenic fungal
indicate statistically pathogens
significant in stored
differences
wheat grains.
among Datainare
treatments eachmeans
groupofasatdetermined
least three based
independent repetitions
on Duncan’s ± standard
multiple error. One-way ANOVA differences
range test.
were considered significant when p < 0.05. Different letters above the error bars indicate statistically significant differences
among treatments in each group In as determined basedtoonthe
sharp contrast Duncan’s multiple
bacterial strains,range test.isolates obtained from wheat seeds
yeast
exhibited no inhibition of mycotoxigenic fungal growth in vitro in the dual-culture as-
In sharpevaluation
say. Further contrast toofthe
thebacterial strains,
yeast strains yeast isolates
by screening obtained
for activity onfrom wheatwheat
colonized seeds
exhibited
grains no inhibition
revealed of mycotoxigenic
four species fungal growth
that exhibit antagonistic in vitro in
properties the dual-culture
against assay.
the tested fungal
Further evaluation
pathogens. The yeastof the yeast were
isolates strains by screening
Naganishia for activity
albidosimilis on colonized
(strain wheat grains
D1), Naganishia albida
revealed four species that exhibit antagonistic properties against the tested
(strain D34), Cryptococcus albidus (strain D37), and Rhodotorula glutinis (strain TY1). As fungal patho-
gens. The
shown yeast isolates
in Figure were Naganishia
7, the growth albidosimilis
of F. proliferatum (strain seeds
on wheat D1), Naganishia
was markedly albidalimited
(strain
D34), Cryptococcus albidus (strain D37), and Rhodotorula glutinis (strain TY1).
when the grains were pretreated with yeast cell suspensions. Among them, R. glutinis TY1 As shown in
Figure 7, the
displayed growth
a strong of F.
ability to proliferatum on wheat
protect the stored seeds
grains wasF.markedly
against proliferatumlimited when the
by reducing
grains wereofpretreated
population the fungal with
load byyeast
3.11cell
log10suspensions.
CFU/g. ThisAmong them,
strain also R. glutinis
displayed TY1 dis-
the strongest
played a strong
antagonistic ability
activity to protect
against the stored
Aspergillus flavusgrains
with aagainst F. proliferatum
reduction of 1.33 log10byCFU/g
reducing the
in the
fungal load compared to the untreated control. All four yeast isolates, however, had a
relatively weak effect on Alternaria infectoria.
The differential efficacy observed between the in vitro and in vivo screening of yeast
isolates could be explained by the different abilities of the potential biocontrol strains
to produce secondary metabolites in a growth medium a vs. on plant material [12,55].
Therefore, determination of an inhibition zone by dual-culture assay, often considered a
preliminary test for selecting antagonistic strains, can be misleading [12].
 population of the fungal load by 3.11 log10 CFU/g. This strain also displayed the strongest
antagonistic activity against Aspergillus flavus with a reduction of 1.33 log10 CFU/g in the
J. Fungi 2021, 7, 781 12 of 16
fungal load compared to the untreated control. All four yeast isolates, however, had a
relatively weak effect on Alternaria infectoria.

Figure 7. Antagonistic activity of four selected yeast isolates against three mycotoxigenic fungi in stored wheat grains.
Error bars represent standard error of three independent biological replicates. Different letters above the error bars indicate
Figure 7. Antagonistic
statistically significant activity of four
differences selected
among yeast in
treatments isolates against
each group at three mycotoxigenic
p < 0.05, fungi
as determined in stored
based wheat multiple
on Duncan’s grains.
Error bars represent standard error of three independent biological replicates. Different letters above the error bars indi-
range test.
cate statistically significant differences among treatments in each group at p < 0.05, as determined based on Duncan’s
multiple range test.
3.3. Mycotoxin-Degrading Activity of the Yeast Rhodotorula glutinis Strain TY1
In vitro
The growthefficacy
differential of R. glutinis
kineticsobserved TY1 the
between in the absence
in vitro andand presence
in vivo of AFB
screening were
of 1yeast
similar could
isolates and characterized
be explainedbyby exponential
the differentgrowth untilofthe
abilities thethird day ofbiocontrol
potential incubationstrains
(Figureto8).
Subsequent
produce HPLC metabolites
secondary analysis indicated that the
in a growth recovery
medium of on
a vs. AFB 1 was
plant lower when
material R. There-
[12,55]. glutinis
strain
fore, TY1 was incubated
determination in PDB medium
of an inhibition zone byamended
dual-culturewithassay,
250 ng/mL AFB1 . Reductions
often considered a pre-
of 47, 61,
liminary and
test for75% in AFB
selecting 1 recoverystrains,
antagonistic were observed on days 1,[12].
can be misleading 2, and 3 of incubation,
respectively, compared to the control (Figure 8A). In stored grain samples artificially
contaminated
3.3. with AFBActivity
Mycotoxin-Degrading 1 , a decrease in mycotoxin
of the Yeast Rhodotorulalevels wasStrain
glutinis recorded
TY1 throughout the
timeIncourse
vitro growth kinetics of R. glutinis TY1 in the absence and presence ofcontrols.
of the experiment for TY1-treated seeds compared to untreated AFB1 wereThe
presence
similar andofcharacterized
the yeast strainbysignificantly
exponentiallowered mycotoxin
growth until levels
the third day inof
grain samples(Figure
incubation by 26.5,
51, and 65% on days 1, 2, and 3 of the experiment, respectively,
8). Subsequent HPLC analysis indicated that the recovery of AFB1 was lower when R. compared to untreated
controls (Figure 8B).
glutinis strain TY1 was incubated in PDB medium amended with 250 ng/mL AFB1. Reduc-
AFB is a mycotoxin that presents an economic challenge to a wide range of agricul-
tions of 47,1 61, and 75% in AFB1 recovery were observed on days 1, 2, and 3 of incubation,
tural products and food industries worldwide, and a health hazard to consumers. The
respectively, compared to the control (Figure 8A). In stored grain samples artificially con-
reduction of aflatoxin contamination in commodities through the application of antago-
taminated with AFB1, a decrease in mycotoxin levels was recorded throughout the time
nistic microorganisms has long been known, and is still an active field of research [56].
course of the experiment for TY1-treated seeds compared to untreated controls. The pres-
Castoria et al. [34] showed that R. glutinis can decrease patulin in vitro and in apple fruit
ence of the yeast strain significantly lowered mycotoxin levels in grain samples by 26.5,
infected by Penicillium expansum. Their data indicated that R. glutinis yeast cells metabolize
51, and 65% on days 1, 2, and 3 of the experiment, respectively, compared to untreated
patulin and/or negatively affect its accumulation or synthesis. Other studies have reported
controls (Figure 8B).
that the biocontrol yeast Sporobolomyces sp. degrades patulin in vitro under aerobic con-
ditions and converts it into less toxic breakdown products [57,58]. Based on the results
obtained in the current study, it appears that R. glutinis can metabolize and/or degrade
AFB1 in vitro and in stored wheat grains. The mechanism responsible for this activity,
however, still needs to be confirmed. Moreover, characterization of the final breakdown
products of AFB1 and their toxicological properties also need to be investigated, to ensure
the safety of using this yeast isolate as a postharvest application on stored grains.
J.J.Fungi
Fungi2021,
2021,7,7,x781
FOR PEER REVIEW 1313ofof16
16

Figure8.
Figure Effectof
8. Effect R.glutinis
ofR. glutinisTY1
TY1strain
strainon
onAFB1
AFB1degradation
degradationin inPDB
PDBmedium
medium(A)(A)and
andwheat
wheatgrains
grains(B).
(B).The
The time
timecourse
course
ofAFB1
of AFB1degradation
degradationwas wasmonitored
monitoredby byHPLC.
HPLC.Error
Errorbars
barsrepresent
representthe
thestandard
standarderror
errorofofthe
themean
mean(SEM)
(SEM)across
acrossthree
three
independentreplicates.
independent replicates. One-way
One-wayANCOVA
ANCOVAwas wasconducted
conductedto todetermine
determineaa statistically
statistically significant
significant difference
difference between
between
treatment
treatmentand andcontrol
controlononmycotoxin
mycotoxinconcentration
concentrationcontrolling
controllingfor
fortime
time(covariance).
(covariance).Time
Timesignificantly
significantlydiffered
differed in
inits
itseffect
effect
on
ontreatment
treatmentandandcontrol
control(p
(p<<0.0001).
0.0001).Testing
Testingfor
foreach
eachtime
timepoint
pointusing
usingANOVA
ANOVAisisan anevident
evidentthat
thatonly
onlytime
timezero
zerodoes
doesnotnot
differ
differ between
between control
control and
and treatment.
treatment. Different letters above
Different letters above the
the error
error bars
barsindicate
indicatestatistically
statisticallysignificant
significantdifferences
differencesatatp p<
<0.05,
0.05,asasdetermined
determinedusing
usingthetheDuncan’s
Duncan’smultiple
multiplerange
rangetest.
test.

AFB1 is a mycotoxin that presents an economic challenge to a wide range of agricul-

4. Conclusions
tural products and food industries worldwide, and a health hazard to consumers. The
reduction of aflatoxin
In summary, contamination
we identified in commodities
the composition through
of stored wheatthe application ofepiphytic
grain-associated antago-
nistic microorganisms has long been known, and is still an active
and endophytic microbiota using high-throughput sequencing of amplicons. Large num-field of research [56].
Castoria et al. [34] showedforming
bers of microorganisms that R. glutinis
epiphyticcanand
decrease patulincommunities
endophytic in vitro and in apple
often fruit
possess
infected
beneficial bycharacteristics,
Penicillium expansum.
includingTheir data indicated
antagonistic thatagainst
activity R. glutinis yeast
fungal cells metabolize
pathogens. Indeed,
patulin
we found and/or
that negatively affect
stored wheat its accumulation
seeds harbor bacterialor and
synthesis. Other studieswith
yeast communities haveantagonis-
reported
that the biocontrol
tic potential yeast Sporobolomyces
and biodegradation abilitysp. degrades
against patulin in vitro
mycotoxigenic fungiunder aerobic
and their condi-
respective
toxins.
tions and Application
converts itofinto
such microbes,
less which areproducts
toxic breakdown adapted to their host
[57,58]. Basedplant, may
on the represent
results ob-
an efficient fungal disease control strategy for stored grains, as well as other
tained in the current study, it appears that R. glutinis can metabolize and/or degrade AFB1 agricultural
commodities.
in A better
vitro and in stored understanding
wheat grains. Theofmechanism
the antagonistic mechanism
responsible ofactivity,
for this these microorgan-
however,
isms,
still and the
needs to berole they playMoreover,
confirmed. in the microbiome of seeds,of
characterization maytheassist
final in the development
breakdown productsof
novel
of AFBantifungal biocontrol approaches
1 and their toxicological propertiestoalso
replace
needtraditionally used synthetic
to be investigated, to ensurefungicides.
the safety
of using this yeast isolate as a postharvest application on stored grains.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
4..3390/jof7090781/s1,
Conclusions Figure S1: Graphical representation of antagonistic activity of selected bacterial
isolates against mycotoxigenic fungal pathogens in vitro; Figure S2: Antagonistic activity of Bacillus
In summary, we identified the composition of stored wheat grain-associated epi-
amyloliquefaciens (strain EnB28) against three mycotoxigenic fungal pathogens in vitro; Table S1: List
phytic and endophytic microbiota using high-throughput sequencing of amplicons. Large
of primers used in the study; Table S2: List of bacterial taxa with significant differential abundance
numbers
according of microorganisms
to Kruskal–Wallis test;forming
Table S3: epiphytic and
List of fungal endophytic
taxa communities
with significant differentialoften pos-
abundance
sess beneficial characteristics, including antagonistic activity
according to Kruskal–Wallis test; Table S4: Summary of Shannon index data.against fungal pathogens.
Indeed, we found that stored wheat seeds harbor bacterial and yeast communities with
Author Contributions: Conceptualization, M.K.S., M.W., E.S. and S.D.; methodology, M.K.S., A.A.,
antagonistic potential and biodegradation ability against mycotoxigenic fungi and their
V.Z. and S.S.; software, A.A.; validation, M.K.S., A.A. and S.S.; formal analysis, M.K.S., S.S. and
respective toxins. Application of such microbes, which are adapted to their host plant,
A.A.; investigation, M.K.S., A.A., S.D. and E.S.; data curation, M.K.S. and A.A.; writing—original
may represent an efficient fungal disease control strategy for stored grains, as well as other
draft preparation, M.K.S., A.A., S.D. and E.S.; writing—review and editing, M.W.; supervision, S.D.
agricultural commodities.
and E.S.; project A better
administration, understanding
V.Z. All of the
authors have read antagonistic
and mechanism
agreed to the of these
published version of
microorganisms,
the manuscript. and the role they play in the microbiome of seeds, may assist in the de-
velopment of novel antifungal biocontrol approaches to replace traditionally used syn-
Funding: This research was funded by the Chief Scientist of the Israeli Ministry of Agriculture and
thetic fungicides.
Rural Development, grant number 20–06–0055.
Supplementary
Data AvailabilityMaterials: The following
Statement: are available
The raw sequence filesonline at www.mdpi.com/xxx/s1,
supporting the findings of this Figure
article S1:
are
Graphical representation
available in of antagonistic
the NCBI Sequence activity
Read Archive of selected
(SRA) under bacterial isolates
the BioProject IDagainst mycotoxigenic
PRJNA756939.
fungal pathogens in vitro; Figure S2: Antagonistic activity of Bacillus amyloliquefaciens (strain EnB28)
J. Fungi 2021, 7, 781 14 of 16

Acknowledgments: We thank Elazar Quinn for his assistance during the wheat grains samples
collection from storage facilities.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Links, M.G.; Demeke, T.; Gräfenhan, T.; Hill, J.E.; Hemmingsen, S.M.; Dumonceaux, T.J. Simultaneous profiling of seed-associated
bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum
and Brassica seeds. New Phytol. 2014, 202, 542–553. [CrossRef]
2. Placinta, C.M.; D’Mello, C.P.F.; MacDonald, A.M.C. A review of worldwide contamination of cereal grains and animal feed with
Fusarium mycotoxins. Anim. Feed Sci. Technol. 1999, 78, 21–37. [CrossRef]
3. Magan, N.; Aldred, D.; Mylona, K.; Lambert, R.J.W. Limiting mycotoxins in stored wheat. Food Addit. Contam. Part. A 2010, 27,
644–650. [CrossRef] [PubMed]
4. Magan, N.; Hope, R.; Cairns, V.; Aldred, D. Post-harvest fungal ecology: Impact of fungal growth and mycotoxin accumulation in
stored grain. Eur. J. Plant. Pathol. 2003, 109, 723–730. [CrossRef]
5. Ferrigo, D.; Raiola, A.; Causin, R. Fusarium toxins in cereals: Occurrence, legislation, factors promoting the appearance and their
management. Molecules 2016, 21, 627. [CrossRef] [PubMed]
6. McCormick, S.P.; Stanley, A.M.; Stover, N.A.; Alexander, N.J. Trichothecenes: From simple to complex mycotoxins. Toxins 2011, 3,
802–814. [CrossRef] [PubMed]
7. Lee, H.J.; Ryu, D. Worldwide occurrence of mycotoxins in cereals and cereal-derived food products: Public health perspectives of
their co-occurrence. J. Agric. Food Chem. 2017, 65, 7034–7051. [CrossRef] [PubMed]
8. Streit, E.; Naehrer, K.; Rodrigues, I.; Schatzmayr, G. Mycotoxin occurrence in feed and feed raw materials worldwide: Long-term
analysis with special focus on Europe and Asia. J. Sci. Food Agric. 2013, 93, 2892–2899. [CrossRef]
9. Chen, Y.; Kistler, H.C.; Ma, Z. Fusarium graminearum trichothecene mycotoxins: Biosynthesis, regulation, and management. Annu.
Rev. Phytopathol. 2019, 57, 15–39. [CrossRef]
10. Gdanetz, K.; Trail, F. The wheat microbiome under four management strategies, and potential for endophytes in disease protection.
Phytobiomes 2017, 1, 158–168. [CrossRef]
11. Mousa, W.K.; Shearer, C.; Limay-Rios, V.; Ettinger, C.L.; Eisen, J.A.; Raizada, M.N. Root-hair endophyte stacking in finger millet
creates a physicochemical barrier to trap the fungal pathogen Fusarium graminearum. Nat. Microbiol. 2016, 1, 16167. [CrossRef]
12. Comby, M.; Gacoin, M.; Robineau, M.; Rabenoelina, F.; Ptas, S.; Dupont, J.; Profizi, C.; Baillieul, F. Screening of wheat endophytes as
biological control agents against Fusarium head blight using two different in vitro tests. Microbiol. Res. 2017, 202, 11–20. [CrossRef]
13. Díaz Herrera, S.; Grossi, C.; Zawoznik, M.; Groppa, M.D. Wheat seeds harbour bacterial endophytes with potential as plant
growth promoters and biocontrol agents of Fusarium graminearum. Microbiol. Res. 2016, 186, 37–43. [CrossRef]
14. Baffoni, L.; Gaggia, F.; Dalanaj, N.; Prodi, A.; Nipoti, P.; Pisi, A.; Biavati, B.; Di Gioia, D. Microbial inoculants for the biocontrol of
Fusarium spp. in durum wheat. Bmc Microbiol. 2015, 15, 1–10. [CrossRef]
15. Leah Musyimi, S.; Wanjohi Muthomi, J.; Devi Narla, R.; Maina Wagacha, J. Efficacy of Biological Control and Cultivar Resistance
on Fusarium Head Blight and T-2 Toxin Contamination in Wheat. Am. J. Plant Sci. 2012, 3, 599–607. [CrossRef]
16. Sadhasivam, S.; Britzi, M.; Zakin, V.; Kostyukovsky, M.; Trostanetsky, A.; Quinn, E.; Sionov, E. Rapid detection and identification
of mycotoxigenic fungi and mycotoxins in stored wheat grain. Toxins 2017, 9, 302. [CrossRef] [PubMed]
17. Hanshew, A.S.; Mason, C.J.; Raffa, K.F.; Currie, C.R. Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing
of insect herbivore bacterial communities. J. Microbiol. Methods 2013, 95, 149–155. [CrossRef]
18. Scibetta, S.; Schena, L.; Abdelfattah, A.; Pangallo, S.; Cacciola, S.O. Selection and experimental evaluation of universal primers to
study the fungal microbiome of higher plants. Phytobiomes J. 2018, 2, 225–236. [CrossRef]
19. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120.
[CrossRef] [PubMed]
20. Zhang, J.; Kobert, K.; Flouri, T.; Stamatakis, A. PEAR: A fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 2014,
30, 614–620. [CrossRef] [PubMed]
21. Masella, A.P.; Bartram, A.K.; Truszkowski, J.M.; Brown, D.G.; Neufeld, J.D. PANDAseq: Paired-end assembler for Illumina
sequences. Bmc Bioinform. 2012, 13, 31. [CrossRef] [PubMed]
22. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010, 26, 2460–2461. [CrossRef]
23. McDonald, D.; Price, M.N.; Goodrich, J.; Nawrocki, E.P.; DeSantis, T.Z.; Probst, A.; Andersen, G.L.; Knight, R.; Hugenholtz, P. An
improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J.
2012, 6, 610–618. [CrossRef] [PubMed]
24. Rognes, T.; Flouri, T.; Nichols, B.; Quince, C.; Mahé, F. VSEARCH: A versatile open source tool for metagenomics. PeerJ 2016, 4,
e2584. [CrossRef]
25. Caporaso, J.G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Peña, A.G.; Goodrich, J.K.;
Gordon, J.I.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335–336.
[CrossRef]
J. Fungi 2021, 7, 781 15 of 16

26. DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E.L.; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Env Microbiol. 2006, 72,
5069–5072. [CrossRef]
27. Abarenkov, K.; Henrik Nilsson, R.; Larsson, K.-H.; Alexander, I.J.; Eberhardt, U.; Erland, S.; Høiland, K.; Kjøller, R.; Larsson, E.;
Pennanen, T.; et al. The UNITE database for molecular identification of fungi-recent updates and future perspectives. New Phytol.
2010, 186, 281–285. [CrossRef]
28. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410.
[CrossRef]
29. Paulson, J.N.; Stine, O.C.; Bravo, H.C.; Pop, M. Differential abundance analysis for microbial marker-gene surveys. Nat. Methods
2013, 10, 1200–1202. [CrossRef]
30. Bray, J.R.; Curtis, J.T. An ordination of the upland forest communities of southern Wisconsin. Ecol. Monogr. 1957, 27, 325–349.
[CrossRef]
31. Lozupone, C.; Knight, R. UniFrac: A new phylogenetic method for comparing microbial communities. Appl. Env Microbiol. 2005,
71, 8228–8235. [CrossRef]
32. Kruskal, W.H.; Wallis, W.A. Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 1952, 47, 583–621. [CrossRef]
33. De, S.; Kaur, G.; Roy, A.; Dogra, G.; Kaushik, R.; Yadav, P.; Singh, R.; Datta, T.K.; Goswami, S.L. A simple method for the efficient
isolation of genomic DNA from Lactobacilli isolated from traditional indian fermented milk (dahi). Indian J. Microbiol. 2010, 50,
412–418. [CrossRef] [PubMed]
34. Castoria, R.; Morena, V.; Caputo, L.; Panfili, G.; De Curtis, F.; De Cicco, V. Effect of the biocontrol yeast Rhodotorula glutinis strain
LS11 on patulin accumulation in stored apples. Phytopathology 2005, 95, 1271–1278. [CrossRef] [PubMed]
35. Johnston-Monje, D.; Raizada, M.N. Conservation and diversity of seed associated endophytes in zea across boundaries of
evolution, ethnography and ecology. PLoS ONE 2011, 6, e20396. [CrossRef] [PubMed]
36. Solanki, M.K.; Abdelfattah, A.; Britzi, M.; Zakin, V.; Wisniewski, M.; Droby, S.; Sionov, E. Shifts in the composition of the
microbiota of stored wheat grains in response to fumigation. Front. Microbiol. 2019, 10, 1098. [CrossRef] [PubMed]
37. Truyens, S.; Weyens, N.; Cuypers, A.; Vangronsveld, J. Bacterial seed endophytes: Genera, vertical transmission and interaction
with plants. Env Microbiol. Rep. 2015, 7, 40–50. [CrossRef]
38. Zawoznik, M.S.; Vázquez, S.C.; Herrera, S.M.D.; Groppa, M.D. Search for endophytic diazotrophs in barley seeds. Braz. J.
Microbiol. 2014, 45, 621–625. [CrossRef]
39. Karlsson, I.; Friberg, H.; Kolseth, A.-K.; Steinberg, C.; Persson, P. Organic farming increases richness of fungal taxa in the wheat
phyllosphere. Mol. Ecol. 2017, 26, 3424–3436. [CrossRef]
40. Khan, N.; Martínez-Hidalgo, P.; Ice, T.A.; Maymon, M.; Humm, E.A.; Nejat, N.; Sanders, E.R.; Kaplan, D.; Hirsch, A.M. Antifungal
activity of Bacillus species against Fusarium and analysis of the potential mechanisms used in biocontrol. Front. Microbiol. 2018, 9,
2363. [CrossRef]
41. Zhang, D.; Yu, S.; Yang, Y.; Zhang, J.; Zhao, D.; Pan, Y.; Fan, S.; Yang, Z.; Zhu, J. Antifungal effects of volatiles produced by Bacillus
subtilis against Alternaria solani in Potato. Front. Microbiol. 2020, 11, 1196. [CrossRef]
42. Mannaa, M.; Kim, K.D. Biocontrol Activity of volatile-producing Bacillus megaterium and Pseudomonas protegens against Aspergillus
and Penicillium spp. predominant in stored rice grains: Study II. Mycobiology 2018, 46, 52–63. [CrossRef]
43. Palazzini, J.M.; Alberione, E.; Torres, A.; Donat, C.; Köhl, J.; Chulze, S. Biological control of Fusarium graminearum sensu stricto,
causal agent of Fusarium head blight of wheat, using formulated antagonists under field conditions in Argentina. Biol. Control
2016, 94, 56–61. [CrossRef]
44. Palazzini, J.M.; Ramirez, M.L.; Alberione, E.J.; Torres, A.M.; Chulze, S.N. Osmotic stress adaptation, compatible solutes
accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat. Biol. Control 2009, 51,
370–376. [CrossRef]
45. Zalila-Kolsi, I.; Ben Mahmoud, A.; Ali, H.; Sellami, S.; Nasfi, Z.; Tounsi, S.; Jamoussi, K. Antagonist effects of Bacillus spp. strains
against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum). Microbiol. Res. 2016, 192,
148–158. [CrossRef] [PubMed]
46. Crane, J.M.; Bergstrom, G.C. Spatial distribution and antifungal interactions of a Bacillus biological control agent on wheat
surfaces. Biol. Control 2014, 78, 23–32. [CrossRef]
47. Shi, C.; Yan, P.; Li, J.; Wu, H.; Li, Q.; Guan, S. Biocontrol of Fusarium graminearum growth and deoxynivalenol production in wheat
kernels with bacterial antagonists. Int. J. Env Res. Public Health 2014, 11, 1094–1105. [CrossRef]
48. Khan, N.I.; Schisler, D.A.; Boehm, M.J.; Slininger, P.J.; Bothast, R.J. Selection and evaluation of microorganisms for biocontrol of
Fusarium head blight of wheat incited by Gibberella zeae. Plant Dis. 2001, 85, 1253–1258. [CrossRef]
49. Zhao, Y.; Selvaraj, J.N.; Xing, F.; Zhou, L.; Wang, Y.; Song, H.; Tan, X.; Sun, L.; Sangare, L.; Folly, Y.M.E.; et al. Antagonistic action
of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS ONE 2014, 9, 92486. [CrossRef]
50. Siddiqui, Z.A. Biocontrol of Alternaria triticina by plant growth promoting rhizobacteria on wheat. Arch. Phytopathol. Plant Prot.
2007, 40, 301–308. [CrossRef]
51. Liu, Q.; Li, W.; Feng, Y.; Tao, C. Efficacy and safety of polymyxins for the treatment of Acinectobacter baumannii infection: A
systematic review and meta-analysis. PLoS ONE 2014, 9, e98091. [CrossRef] [PubMed]
J. Fungi 2021, 7, 781 16 of 16

52. Ongena, M.; Jacques, P. Bacillus lipopeptides: Versatile weapons for plant disease biocontrol. Trends Microbiol. 2008, 16, 115–125.
[CrossRef] [PubMed]
53. Cochrane, S.A.; Vederas, J.C. Lipopeptides from Bacillus and Paenibacillus spp.: A Gold Mine of Antibiotic Candidates. Med. Res.
Rev. 2016, 36, 4–31. [CrossRef] [PubMed]
54. Caulier, S.; Nannan, C.; Gillis, A.; Licciardi, F.; Bragard, C.; Mahillon, J. Overview of the antimicrobial compounds produced by
members of the Bacillus subtilis group. Front. Microbiol. 2019, 10, 302. [CrossRef] [PubMed]
55. Kusari, S.; Hertweck, C.; Spiteller, M. Chemical ecology of endophytic fungi: Origins of secondary metabolites. Chem. Biol. 2012,
19, 792–798. [CrossRef] [PubMed]
56. Peles, F.; Sipos, P.; Kovács, S.; Gyori, Z.; Pócsi, I.; Pusztahelyi, T. Biological control and mitigation of aflatoxin contamination in
commodities. Toxins 2021, 13, 104. [CrossRef] [PubMed]
57. Ianiri, G.; Idnurm, A.; Wright, S.A.I.; Durán-Patrón, R.; Mannina, L.; Ferracane, R.; Ritieni, A.; Castoria, R. Searching for genes
responsible for patulin degradation in a biocontrol yeast provides insight into the basis for resistance to this mycotoxin. Appl. Env
Microbiol. 2013, 79, 3101–3115. [CrossRef] [PubMed]
58. Ianiri, G.; Pinedo, C.; Fratianni, A.; Panfili, G.; Castoria, R. Patulin degradation by the biocontrol yeast Sporobolomyces sp. Is an
inducible process. Toxins 2017, 9, 61. [CrossRef]