Abstract

   Alternaria alternata is an important phytopathogen causing fruit black
   rot and also producing a variety of mycotoxins, such as alternariol
   (AOH) and alternariol monomethyl ether (AME) as two main contaminants.
   This could lead to economic losses of agricultural products as well as
   human health risks. In this study, magnolol extracted from the
   traditional Chinese herb, Mangnolia officinalis, exhibited an obvious
   antifungal property and could completely suppress the mycelial growth
   at 100 μM. Morphological differences of A. alternata were observed to
   be significantly shrunk and wrinkled after the exposure to magnolol.
   Furthermore, AOH and AME were no longer produced in response to 50 μM
   of magnolol. To uncover the antifungal and antimycotoxigenic
   mechanisms, the transcriptomic profiles of A. alternata—treated with or
   without magnolol—were evaluated. The clustered genes responsible for
   AOH and AME biosynthesis were obviously less transcribed under magnolol
   stress and this was further confirmed by qRT-PCR. The global regulators
   of carbon and nitrogen utilization, such as CreA and NmrA, were
   significantly down-regulated and this possibly caused the reduction in
   mycotoxins. In addition, fatty acid β-oxidation was regarded to
   contribute to polyketide mycotoxin production for the supply of
   precursor acetyl-CoA while the expression of these related genes was
   inhibited. The response to magnolol led to the marked alteration of
   oxidative stress and the down-expression of the mitogen-activated
   protein kinase (MAPK) signaling pathway from the transcriptome data and
   the determination of peroxidase (POD), superoxide dismutase (SOD) and
   glutathione (GSH) assays. This above might be the very reason for the
   growth supression and mycotoxin production of A. alternata by magnolol.
   This study provides new insights into its potential as an important
   active ingredient for the control of A. alternata and its mycotoxins in
   fruits and their products.

   Keywords: Alternaria alternata, mycotoxin, alternariol, magnolol,
   transcriptome

1. Introduction

   The genus of Alternaria is one of the most important postharvest
   phytopathogens as the causal agent of fruit black rot, resulting in
   agricultural yield losses worldwide. A. alternata is the most common
   species among the genus Alternaria containing a variety of
   host-specific pathogenic strains [[32]1]. In addition to economic
   losses and fruit quality reduction, they can produce multiple
   mycotoxins that are detrimental to the health of humans and animals.
   The Alternaria mycotoxins mainly include alternariol (AOH), alternariol
   monomethyl ether (AME) and tenuazonic acid (TeA) [[33]2]. They
   frequently contaminate a variety of fruits, including tomatoes, apples,
   blueberries, grapes, and dried fruits, under suitable temperature and
   humidity conditions [[34]1,[35]3]. Of these mycotoxins, AOH and AME
   have been reported to show genotoxicity [[36]4,[37]5]. The genetic
   basis of AOH and AME biosynthesis has been elucidated and unraveled
   [[38]6]. A polyketide synthase encoded by pksI is responsible and
   sufficient for AOH formation and an O-methyl transferase (omtI)
   transforms AOH to AME, the methyl ether of AOH. A pathway specific
   regulatory gene, aohR, encoding a Zn(II)2Cys6 transcription factor
   activated the expression of the downstream enzymatic genes for AOH and
   AME production.

   In consideration of pathogenic Alternaria and the mycotoxins, the
   prevention and control is therefore of vital point. A number of plant
   extracts are considered to exert brilliant antifungal activities,
   including some naturally occurring polyphenols. Among the polyphenols,
   magnolol, extracted from the traditional Chinese herb Magnolia
   officinalis, showed strong antifungal activities against Fusarium
   species [[39]7], Candida isolates [[40]8], Magnaporthe grisea [[41]9],
   Penicillium expansum as well as A. alternata [[42]10]. However, little
   is known on its inhibitory effects against Alternaria toxins,
   especially AOH and AME. Accordingly, the deep exploration of antifungal
   and antimycotoxigenic mechanisms in response to magnolol are also still
   unclear in A. alternata.

   Therefore, the antifungal and antimycotoxigenic efficiency of magnolol
   was first observed to be validated in the present work. The potential
   mechanisms were to be investigated by comparative transcriptomic
   profiling between the control and magnolol-stressed A. alternata. Given
   that magnolol was considered safe by various food safety authorities
   over the past years [[43]11], it would be a promising alternative for
   preservatives for the control of Alternaria pathogens and their
   mycotoxins.

2. Results

2.1. Antifungal Effects of Magnolol on A. alternata

   The antifungal effect of magnolol against A. alternata ATCC 66,981 was
   significantly exhibited on the mycelial growth in a dose-dependent
   manner. After 6 days incubation, the extension of A. alternata was
   inhibited by 46.5%, 66.9% and 93.5% with 12.5, 25 and 50 μM of
   magnolol, respectively ([44]Figure 1). Similarly, the accumulation of
   the biomass weight was inhibited from 8.1% to 100% with the increasing
   magnolol ([45]Figure 1). The extending of A. alternata was completely
   inhibited as the minimal inhibitory concentration (MIC) at 100 μM
   during the whole incubation period.

Figure 1.

   [46]Figure 1
   [47]Open in a new tab

   Inhibition of magnolol on the mycelial growth of A. alternata ATCC
   66981. The spores were cultured in the medium plus with serial
   concentration of magnolol at 25 °C for 6 days. The inhibition rates
   were calculated by the colony radius and dry weight.

   Ultrastructural observation by SEM indicated that there were
   significant morphological alterations between the control and
   magnolol-treated A. alternata cells. After exposure to 12.5 μM of
   magnolol, the spores were a little shriveled ([48]Figure 2A,B). A
   greater depression of morphological modifications could be observed as
   the magnolol concentration increased. The exposure to 25 μM of magnolol
   caused an evidently sunken phenomenon and even a major break on the
   surface of the spore with no germ tubes observed ([49]Figure 2C). The
   hyphae of A. alternata became wrinkled and abnormal after exposure to
   12.5 and 25 μM of magnolol ([50]Figure 2D–F).

Figure 2.

   [51]Figure 2
   [52]Open in a new tab

   Scanning electron microscopy images of spores (A–C) and mycelia (D–F)
   of A. alternata ATCC 66,981 exposed to 0 (control), 12.5 and 25.0 μM of
   magnolol. Bar = 10 μm.

2.2. Transcriptomic Profiles of A. alternata in Response to Magnolol

   To understand the potential inhibitory mechanism of magnolol on A.
   alternata, a comparative transcriptome analysis of A. alternata was
   performed between 0 and 25 μM of magnolol treatment. The details of the
   RNA-Seq data are shown in [53]Supplementary Table S1. An average of
   37.00 million and 28.63 million clean reads were separately obtained
   from the control and magnolol-treated group. Based on the mapped reads,
   the expression levels of 13,678 genes were totally quantified by the
   FPKM (fragments per kilobase of exon model per million mapped reads)
   value. The differentially expressed genes (DEGs) in accordance with the
   |log[2] Fold Change| ≥ 1 and false discovery rate (FDR) < 0.05 are
   listed in [54]Supplementary Table S2. Totally, 2981 genes were
   considered to be significantly expressed in response to magnolol at 25
   μM, with 1471 genes up-regulated and 1510 genes down-regulated compared
   to untreated A. alternata samples. The distribution of these DEGs is
   shown in [55]Supplementary Figure S1.

2.3. Functional Analysis of DEGs

   The functional analysis of DEGs was used to understand the inhibitory
   mechanisms at the molecular level. The significant results of GO
   enrichment analysis were apparently different between the up-regulated
   and down-regulated DEGs ([56]Figure 3A,B). The up-regulated DEGs were
   significantly enriched in the processes of ribosome formation and RNA
   and protein processing; such as rRNA processing and metabolic
   processes, ribosome biogenesis, non-coding RNA (ncRNA) processing and
   metabolic processes, and RNA processing and protein refolding, which
   were necessary for the survival in exposure to stress. However, the
   down-regulated DEGs were mostly enriched in biological processes and
   molecular functioning, such as oxidoreductase activity, antibiotic
   metabolic processes and catabolic processes ([57]Figure 3B).

Figure 3.

   [58]Figure 3
   [59]Open in a new tab

   Gene ontology (GO) functional classification (A,B) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (C,D) of
   differentially expressed genes (DEGs). The up-regulated (A,C) and
   down-regulated (B,D) DEGs were separately analyzed and the top 20
   results were listed at the highest enrichment level in the figure.

   KEGG pathway enrichment analysis of the DEGs between magnolol and
   magnolol-free groups was performed to evaluate their underlying
   biological significance. Genes related to the ribosome biogenesis
   showed significantly higher expression in the magnolol treated group
   ([60]Figure 3C). On the other hand, the pathways mainly belonging to
   the peroxisome, nitrogen metabolism, and carbohydrate and energy
   metabolism were suppressed significantly in the magnolol stress samples
   ([61]Figure 3D).

2.4. Genes Involved in Mycotoxin Biosynthesis

   To understand the roles of magnolol on Alternaria mycotoxins
   production, the expression levels of genes involved in AOH and AME
   biosynthesis were analyzed. Based on the transcriptome data, the
   transcripts of pksI and omtI, responsible for AOH and AME biosynthesis,
   were significantly down-regulated by 4.39- and 2.28-fold when A.
   alternata was treated with 25 μM of magnolol, respectively, whereas the
   aohR showed no significant difference in expression ([62]Figure 4A).
   The down-regulation of pksI and omtI may directly cause a reduction in
   AOH and AME biosynthesis. In addition, co-expression of pksI with
   another three enzymatic genes (moxI, sdrI and doxI) was also
   responsible for AOH modification [[63]6]. Therein, the transcripts of
   two genes (moxI and sdrI) were significantly down-regulated in the 25
   μM magnolol treated samples, while the doxI gene expression showed no
   significant differences ([64]Figure 4A). The relative expression levels
   of three genes were down-regulated under magnolol stress, further
   confirming the results of transcriptomic analysis ([65]Supplementary
   Figure S2). Meanwhile, AOH and AME production actually decreased
   dramatically by 86.2% and 98.2% in 25 μM of magnolol treated samples by
   the quantitative analysis ([66]Figure 4B).

Figure 4.

   [67]Figure 4
   [68]Open in a new tab

   Effects of magnolol on the expressions of the clustered genes involved
   in mycotoxin biosynthesis (A), and alternariol (AOH) and alternariol
   monomethyl ether (AME) production (B) of A. alternata ATCC 66981. Each
   column represents the mean of three replicates and the results are
   presented as the mean ± standard errors (p < 0.05).

2.5. Genes Involved in the Primary Metabolism

   Mycotoxin production by A. alternata is influenced by nutrition
   factors, such as carbon and nitrogen sources [[69]12,[70]13]. It is
   noteworthy that the influence of the nitrogen source for AOH and AME
   production is higher than that of the carbon source [[71]12]. KEGG
   metabolic pathway analysis also showed that nitrogen metabolism was
   suppressed to a much stronger degree after magnolol treatment. The
   nitrogen metabolic regulators AreA and NmrA modulate gene expression
   for the utilization of nitrogen sources [[72]14]. The expression of the
   global nitrogen metabolic regulator AreA exhibited no significant
   difference compared with the magnolol-free sample. However, the
   transcription of nmrA (CC77DRAFT_1016967) was significantly
   down-regulated by 2.90-fold in response to magnolol stress ([73]Table
   1). The nitrate assimilation system including nitrite reductase (NiR),
   nitrate reductase (NR) and a nitrate transporter NrtB was responsible
   for non-preferred nitrogen source utilization [[74]15]. These genes
   showed lower expression from 4.31- to 5.19-fold by magnolol treatment
   ([75]Table 1).

Table 1.

   Effects of magnolol treatment on the differential expression of genes
   related to the primary metabolites in A. alternata.
   Gene Log[2] (AaM/AaC) ^1 p-Value Function
   Nitrogen metabolism
   CC77DRAFT_1016967 −1.536 0.001 nmrA
   CC77DRAFT_1034395 −2.647 0.000 2-nitropropane dioxygenase precursor
   CC77DRAFT_1056077 −2.376 0.013 high affinity nitrate transporter NrtB
   CC77DRAFT_937904 −2.108 0.004 nitrite reductase NiR
   CC77DRAFT_937944 −2.159 0.001 nitrate reductase NR
   CC77DRAFT_1011175 −2.026 0.000 cyanate hydratase
   CC77DRAFT_383282 −1.938 0.000 formamidase FmdS
   CC77DRAFT_937213 −1.831 0.000 carbonic anhydrase
   CC77DRAFT_854456 −1.897 0.004 putative oxidoreductase
   CC77DRAFT_201495 −1.541 0.000 carbon-nitrogen hydrolase
   CC77DRAFT_1059060 −1.677 0.000 carbonic anhydrase
   CC77DRAFT_505636 −1.650 0.000 glutamine synthetase
   CC77DRAFT_261749 −1.627 0.000 glutamate synthase
   CC77DRAFT_926595 −1.065 0.000 glutamine synthetase
   Carbon utilization
   CC77DRAFT_1058240 −1.289 0.000 CreA
   CC77DRAFT_318215 −2.128 0.000 C[2]H[2] transcription factor (AmdA)
   CC77DRAFT_947345 −1.766 0.000 acetyl-CoA synthetase-like protein
   CC77DRAFT_1063927 −1.715 0.000 C6 transcription factor FacB
   CC77DRAFT_1020016 −1.526 0.000 pyruvate carboxylase
   CC77DRAFT_30145 −1.177 0.000 mitochondrial uncoupling protein 2
   CC77DRAFT_997221 −1.015 0.000 mitochondrial carrier
   CC77DRAFT_926519 1.412 0.000 isocitrate lyase 2
   Cell wall biogenesis
   CC77DRAFT_598626 −1.007 0.000 Rho GTPase1
   CC77DRAFT_1022845 −1.230 0.000 mannose-6-phosphate isomerase
   CC77DRAFT_1066075 −1.526 0.000 mannose-6-phosphate isomerase
   CC77DRAFT_944335 −1.685 0.000 quinone oxidoreductase putative
   CC77DRAFT_61983 −1.211 0.000 chitinase
   CC77DRAFT_985840 −1.195 0.007 chitin deacetylase 1
   CC77DRAFT_2778 1.515 0.000 chitinase
   CC77DRAFT_1061411 1.247 0.000 nucleotide-diphospho-sugar transferase
   CC77DRAFT_1039824 1.335 0.000 hypothetical protein
   CC77DRAFT_1064369 2.536 0.000 glycoside hydrolase
   CC77DRAFT_906789 −1.612 0.009 glycoside hydrolase
   CC77DRAFT_931389 −1.127 0.004 glycoside hydrolase
   Fatty acid beta-oxidation
   CC77DRAFT_937137 −3.031 0.000 tropinone reductase 1
   CC77DRAFT_1024610 −2.286 0.000 3-ketoacyl-CoA thiolase B
   CC77DRAFT_38276 −2.169 0.000 acyl-CoA dehydrogenase
   CC77DRAFT_1087261 −1.963 0.021 Delta3-Delta2-enoyl-CoA isomerase
   CC77DRAFT_210867 −1.642 0.000 3-oxoacyl-reductase
   [76]Open in a new tab

   ^1 AaC, control; AaM, magnolol treatment. Log[2] (AaM/AaC) ≥ 1
   indicates up-regulated expression and Log[2] (AaM/AaC) ≤ −1 indicates
   down-regulated expression.

   Carbon catabolite repression (CCR) plays an important role in the
   regulation of growth, development and secondary metabolism in mold
   [[77]16,[78]17]. As a principal regulator of CCR, CreA, mediates the
   carbon-utilizing systems. Previous studies have shown that the
   inactivation of CreA induced a marked decrease in aflatoxin
   biosynthesis [[79]18,[80]19]. In this work, the CreA
   (CC77DRAFT_1058240) expression level was significantly down-regulated
   by magnolol treatment, which suggests that CreA could also contribute
   to the reduction in AOH synthesis. In addition, CreA was regarded as a
   modulator of the cell wall integrity (CWI) pathway for the fungal
   resistance against antifungal agents [[81]20], while RHO1—known as a
   small G protein—was considered the master regulator of CWI signaling
   [[82]21]. Our data show that the presence of magnolol caused a
   significant down-expression of the RHO1 gene. Furthermore, some DEGs
   related to cell wall biogenesis markedly fluctuated by exposure to
   magnolol. Of the 11 DEGs, seven DEGs were down-regulated from 2.18- to
   3.22-fold, and the other four DEGs were up-regulated from 2.37- to
   5.82-fold ([83]Table 1).

   On the other hand, fatty acid β-oxidation contributes acetyl-CoA as a
   fundamental substrate for the polyketide mycotoxin production
   [[84]19,[85]22]. As shown in [86]Table 1, all of the DEGs related to
   fatty acid β-oxidation were significantly less transcribed by 3.12- to
   8.17-fold due to magnolol treatment. Fatty acid β-oxidation is carried
   out through the action of enzymes such as acyl-CoA dehydrogenase and
   3-ketoacyl-CoA thiolase [[87]23]. The expression levels of their
   encoding genes were down-regulated by 4.50- and 4.88-fold,
   respectively.

2.6. Genes Related to Stress Response

   To overcome oxidative stress, both enzymatic and non-enzymatic systems
   involving peroxidase (POD), superoxide dismutase (SOD), catalase (CAT)
   and glutathione (GSH) have been developed for scavenging the toxic ROS
   in fungi. SOD catalyzes the dismutation of superoxide radicals (O[2]^−)
   to molecular oxygen (O[2]) and hydrogen peroxide (H[2]O[2]). CAT
   catalyzes the conversion of H[2]O[2] to water and molecular oxygen. GSH
   is important as a cofactor for antioxidant enzymes, as a scavenger of
   ROS, and as a reducing agent for glutaredoxin. In this work, some DEGs
   involved in POD were up-regulated from 2.28- to 27.25-fold.
   Additionally, the expressions of two genes encoding SOD isozymes were
   also increased by 2.94- and 5.16-fold, respectively. However, the CAT
   encoding genes were all significantly down-regulated from 2.80- to
   5.65-fold under the magnolol stress condition ([88]Table 2). This was
   further confirmed by the assays of enzymatic activities, in which SOD
   activity in treated mycelia was enhanced by 25.6% ([89]Figure 5A),
   whereas CAT activity was decreased by 41.4% compared to the control
   ([90]Figure 5B).

Table 2.

   Effects of magnolol treatment on the significantly differential
   expression of the genes encoding for the antioxidant activities in A.
   alternata.
   Gene Log[2] (AaM/AaC) ^1 p-Value Function
   Antioxidant enzyme
   CC77DRAFT_905830 1.554 0.000 Superoxide dismutase (SOD)
   CC77DRAFT_911584 2.368 0.000 SOD
   CC77DRAFT_1050117 −1.644 0.000 SOD
   CC77DRAFT_1021907 −1.374 0.000 SOD
   CC77DRAFT_1036489 −2.497 0.001 Catalase (CAT)
   CC77DRAFT_1013212 −1.485 0.000 CAT
   CC77DRAFT_296007 3.497 0.000 Peroxidase (POD)
   CC77DRAFT_299044 3.421 0.000 POD
   CC77DRAFT_1039208 1.391 0.000 POD
   CC77DRAFT_960122 1.186 0.000 POD
   CC77DRAFT_1021328 4.768 0.000 POD
   CC77DRAFT_227615 1.812 0.000 POD
   CC77DRAFT_1096817 −2.209 0.001 POD
   CC77DRAFT_1051184 −1.353 0.015 POD
   CC77DRAFT_1008747 −1.162 0.000 POD
   Glutathione metabolism
   CC77DRAFT_1032529 −3.152 0.000 glutathione S-transferase II
   CC77DRAFT_687848 −1.445 0.000 glutathione S-transferase II
   CC77DRAFT_1050574 −1.671 0.000 glutamate-cysteine ligase
   CC77DRAFT_1022906 −1.210 0.000 glutamate-cysteine ligase regulatory
   subunit
   CC77DRAFT_77574 −2.371 0.000 thioredoxin-like protein
   CC77DRAFT_1056344 1.282 0.001 glutathione S-transferase
   CC77DRAFT_356794 1.067 0.001 glutathione S-transferase
   Siderophore biosynthesis
   CC77DRAFT_935643 1.464 0.000 tyrosinase
   CC77DRAFT_1061171 2.376 0.000 Non-ribosomal peptide synthase, NPS6
   CC77DRAFT_1031046 2.732 0.000 ABC transporter
   CC77DRAFT_1020276 4.384 0.000 L-ornithine 5-monooxygenase
   CC77DRAFT_69570 2.126 0.000 aerobactin siderophore biosynthesis protein
   iucB
   CC77DRAFT_69691 4.699 0.000 short-chain-fatty-acid-CoA ligase, sidI
   CC77DRAFT_71629 4.417 0.000 siderophore iron transporter, mirB
   CC77DRAFT_69581 −2.210 0.000 MFS transporter
   Sulfur metabolism
   CC77DRAFT_1015694 −3.069 0.000 thiosulfate sulfurtransferas-like
   protein
   CC77DRAFT_1020633 −1.427 0.000 sulfite reductase hemoprotein
   CC77DRAFT_1022955 −3.093 0.000 o-acetylhomoserine ami
   CC77DRAFT_1067622 −2.515 0.000 molybdopterin binding oxidoreductase
   CC77DRAFT_1099663 −1.502 0.000 sulfide: quinone oxidoreductase
   CC77DRAFT_675919 −1.094 0.000 carbohydrate phosphatase
   CC77DRAFT_786633 −1.703 0.000 adenylyl-sulfate kinase
   CC77DRAFT_925110 −2.268 0.000 phosphoadenosine phosphosulfate reductase
   CC77DRAFT_951586 −1.374 0.000 ATP-sulfurylase
   CC77DRAFT_971236 −5.474 0.000 methanesulfonate monooxygenase
   CC77DRAFT_192219 −1.215 0.000 alpha/beta-hydrolase
   CC77DRAFT_240786 −1.437 0.000 homoserine acetyltransferase family
   protein
   [91]Open in a new tab

   ^1 AaC, control; AaM, magnolol treatment. Log[2] (AaM/AaC) ≥ 1
   indicates up-regulated expression and Log[2] (AaM/AaC) ≤ −1 indicates
   down-regulated expression.

Figure 5.

   [92]Figure 5
   [93]Open in a new tab

   Effects of magnolol on the superoxide dismutase (SOD) and catalase
   (CAT) activities (A,B), and glutathione (GSH) content (C) of A.
   alternata ATCC 66981. SOD catalyzes the dismutation of superoxide
   radicals (O[2]^−) to molecular oxygen (O[2]) and hydrogen peroxide
   (H[2]O[2]). CAT catalyzes the conversion of H[2]O[2] to water and
   molecular oxygen. GSH is important as a cofactor for antioxidant
   enzymes, as a scavenger of ROS, and as a reducing agent for
   glutaredoxin. Each column represents the mean of three replicates.
   Results are presented as the mean ± standard error (p < 0.05).

   Glutathione is the principal non-enzymatic compound protecting cells
   from oxidative stress [[94]24]. As shown in [95]Table 2, the DEGs
   related to GSH metabolism were down-regulated from KEGG analysis. Some
   glutathione S-transferases, which could catalyze the conjugation
   between GSH and many xenobiotic compounds for the purpose of
   detoxification, were less expressed by 2.72- and 8.89-fold.
   Correspondingly, the GSH contents were reduced by 20.7% in the magnolol
   treatment, compared to the control ([96]Figure 5C). Moreover,
   sulfur-containing defense compounds involved in sulfur metabolism,
   including GSH and various sulfur-rich proteins, are crucial for fungal
   resistance against abiotic stress [[97]25,[98]26]. Our results showed
   that the expressions of 12 DEGs related to sulfur metabolism were
   totally repressed by magnolol.

   In A. alternata, several oxidative stress-related proteins, mainly
   including the redox-responsive Yap1, the high osmolarity glycerol 1
   (Hog1) mitogen-activated protein (MAP) kinase, the Skn7 response
   regulator and the non-ribosomal peptide synthetase (NPS6), have
   demonstrated to be involved in ROS modulation [[99]27]. The
   transcriptomic profile showed that the expression of Hog1
   (CC77DRAFT_164856) was significantly down regulated by magnolol
   treatment. Conversely, an increase of 5.19-fold in the expression of
   NPS6 (CC77DRAFT_1061171) was observed. NSP6, catalyzing the
   biosynthesis of siderophores, may assist the fungus to chelate iron and
   play a critical role for antioxidant activities and ROS detoxification
   [[100]28]. Of the eight DEGs related to siderophore biosynthesis,
   except the major facility superfamily (MFS) transporter encoding gene,
   seven DEGs were overexpressed from 2.76- to 25.97-fold in response to
   the magnolol stress ([101]Table 2). It is worth noting that the
   transcription factors Yap1 and Skn7 were demonstrated as having no
   significant change compared to the control.

3. Discussion

   Magnolol is known as one of major bioactive compounds from Magnolia
   officinalis and has been reported to have biological functions such as
   anti-inflammatory, antifungal, anti-cancer and antioxidant activities
   [[102]9,[103]10]. Previous studies have showed that even at quite low
   concentrations magnolol exhibited high efficacy and strong potency
   against a broad spectrum of toxigenic fungi, including Alternaria spp.
   [[104]9,[105]10] and Fusarium spp. [[106]7,[107]9,[108]29]. The
   mycelial extending and biomass weight of A. alternata in our work could
   be strongly inhibited by up to 93.5% and 88.3% with the treatment of
   magnolol only at 25 μM. Moreover, the MIC value of magnolol on A.
   alternata was much lower than that of other natural phenolics including
   p-coumaric, ferulic and caffeic acids [[109]30,[110]31]. Therefore,
   magnolol could be a potential alternative for controlling Alternaria
   decay. Chemical fungicides could possibly lead to fungal resistance and
   environmental problems. For this reason, many countries have
   established legislation to restrict the overuse of the chemical agents.
   The plant-derived magnolol is promising because of its non-phytotoxic,
   readily biodegradable, and environmentally safe properties. The major
   limitation of magnolol lies in its poor water solubility. Magnolol
   could be developed to be emulsified for antifungal efficiency.
   Additionally, the nanoparticle formulation of magnolol is recognized to
   be worthy to be explored for the improvement of its water solubility,
   such as the therapeutic formulation of magnolol nanoparticles
   [[111]32].

   Besides the pathogenic Alternaria fruits and their products, it is
   necessary to evaluate the magnolol effects on mycotoxins because little
   has been revealed. Of these mycotoxins, AOH and AME were demonstrated
   to be two of the most frequently contaminated mycotoxins
   [[112]26,[113]33]. To our knowledge, magnolol was first reported to
   have intense inhibitory effects on AOH and AME production in the
   present work, and 50 µM of magnolol could completely suppress mycotoxin
   biosynthesis. Similarly, type B trichothecene production by F. culmorum
   was significantly inhibited by magnolol treatment probably due to the
   presence of the free phenolic hydroxyl group on magnolol [[114]29].
   Actually, our previous studies showed that phenolics could be potent
   inhibitors against mycotoxin production in A. alternata [[115]31].
   Moreover, phenolics showed strong inhibition of aflatoxin and
   ochratoxin A biosynthesis by Aspergillus [[116]19,[117]34] and
   trichothecenes production by Fusarium [[118]29,[119]35]. Based on these
   results, the antifungal and antimycotoxigenic mechanism of magnolol on
   A. alternata growth and mycotoxin production was further investigated
   by transcriptomic analysis.

   In A. alternata, the biosynthesis of AOH and its derivatives is managed
   by a polyketide gene cluster in which aohR serves as a positive
   transcriptional factor for mycotoxin production [[120]6]. Our RNA-Seq
   data have shown that the clustered enzyme genes involved in AOH and AME
   biosynthesis were less transcribed, but for the expression of the
   regulatory gene aohR there was no significant decrease after the
   magnolol treatment. PksI was proven to be sufficient for AOH formation
   in A. alternata, while omtI was responsible for AME formation from the
   methyl ether of AOH [[121]6]. Our previous study also found that the
   expression of pksI and omtI was strongly down-regulated by the
   essential oil citral, although the regulator aohR showed no significant
   change [[122]26]. A similar result was reported by Zhao and his
   colleagues [[123]19]. They found that most structural genes in the
   aflatoxin biosynthetic gene cluster were down-regulated while
   regulators aflR and aflS were not significantly affected by gallic acid
   [[124]19]. Taken together, these finding indicated that magnolol
   inhibited mycotoxin production by lowering the expression of pksI and
   omtI.

   Acetyl-CoA is the precursor for AOH formation [[125]36], and it is
   mainly produced from fatty acid β-oxidation and pyruvate
   decarboxylation [[126]19]. It was reported that gallic acid and
   cinnamaldehyde inhibited aflatoxin formation via the lower expression
   of fatty acid β-oxidation genes [[127]19,[128]37]. Our results also
   showed that all of the DEGs involved in fatty acid β-oxidation were
   highly repressed after exposure to magnolol. The last step of the
   β-oxidation cycle requires A 3-ketoacyl-CoA thiolase (KAT) enzyme which
   is essential for the last step of the β-oxidation cycle catalyzing
   fatty ketoacyl-CoA to produce one acetyl-CoA molecule [[129]38]. It is
   worth noting that the expression of KAT was significantly
   down-regulated by 4.88-fold under magnolol stress. Therefore, the
   suppression of the DEGs responsible for the fatty acid β-oxidation
   pathway by magnolol would likely inhibit mycotoxin biosynthesis due to
   lack of precursors.

   The growth and mycotoxin production in fungi is also influenced by the
   available carbon and nitrogen sources and the corresponding regulators
   [[130]20,[131]39,[132]40]. The transcription factors CreA, AreA and
   NmrA play important roles in modulating the expression of genes for
   utilizing these nutrient sources [[133]14,[134]16,[135]41].
   Furthermore, CreA is essential for controlling secondary metabolism,
   either directly by interacting with the consensus sequences on the
   promoter region of mycotoxin biosynthetic genes or through
   transcriptional cascades and disruption of nutrient utilization
   [[136]19]. Indeed, the deletion of CreA in A. flavus caused the
   significant repression of gene expression in aflatoxin biosynthesis and
   almost completely inhibited mycotoxin production [[137]19,[138]20].
   Therefore, the downregulation of CreA could possibly lead to the
   decrease in AOH and AME production in A. alternata under the magnolol
   stress. In the nitrogen metabolic regulation, although there was no
   obvious significant difference in AreA expression, NmrA demonstrated
   lower levels of transcription in A. alternata with magnolol treatment.
   NmrA appears to affect gene expression through physical interaction
   with AreA [[139]42], and also be involved in the mycotoxin biosynthesis
   and virulence of pathogens [[140]18,[141]40]. The NmrA deletion
   partially restricted the production of aflatoxin [[142]40] and
   deoxynivalenol [[143]43] in some minimal media with one amino acid as
   the sole nitrogen resource. These results suggest that the
   downregulation of CreA and NmrA under the magnolol stress may
   contribute to the significant reduction in AOH and AME production. It
   is noteworthy that CreA may be involved in nitrogen catabolite
   repression [[144]41], while NmrA was considered to regulate carbon
   metabolism [[145]44].

   In addition to the nutritive factors, oxidative stress induced by
   environmental stimuli plays a crucial role in mycotoxin biosynthesis in
   fungi [[146]24,[147]45]. Oxidative stress was reported to stimulate the
   production of mycotoxins, including deoxynivalenol by F. graminearum
   [[148]46], ochratoxin A by A. ochraceus [[149]45] and aflatoxin by A.
   flavus [[150]37] and A. parasiticus [[151]47]. Likewise, fungi can cope
   with ROS toxicity by activating enzymatic and nonenzymatic defense
   systems, since excessive accumulation of ROS can lead to cellular
   dysfunction and even be lethal to cells. Several studies have reported
   that antioxidants could inhibit mycotoxin production by positive
   regulation of the antioxidant system and reducing the ROS in fungi
   [[152]19,[153]48]. However, a variety of inhibitors may act on the
   different types of antioxidant system. For example, gallic acid
   inhibited aflatoxin biosynthesis and stayed oxidative stress
   homeostasis in A. flavus by activating the nonenzymatic antioxidant
   system [[154]19], while piperine depressed aflatoxin production
   accompanied with enhancement of CAT activity [[155]48]. In this work,
   we found that exposure to magnolol led to the up-regulation of
   antioxidant enzymes in A. alternata, such as SOD and POD, while the CAT
   activity showed a sharp decrease. Similar trends were also observed by
   previous studies on the inhibitory effects of essential oils on the
   biosynthesis of patulin [[156]49] and aflatoxin B[1] [[157]50]. They
   stated that essential oils suppressed mycotoxin production through
   enhancing SOD activity. In addition, siderophore-mediated iron uptake
   plays an important role in resistance to the toxic ROS which could be
   detoxified by SOD and CAT in A. alternata [[158]28]. Our results show
   that almost all genes involved in siderophore biosynthesis were highly
   up-regulated after magnolol treatment. Meanwhile, iron present in
   excess could detoxify ROS via a non-enzymic system [[159]28]. Taken
   together, it seems that the enhancement of SOD and POD activities and
   siderophore biosynthesis in A. alternata play important roles in the
   elimination of ROS and the inhibition of mycotoxin production by
   magnolol stress.

   Oxidative stress leads to the response of gene transcription regulated
   by specific transcription factors and protein phosphorylation signaling
   pathways in fungi [[160]24]. However, transcription factors like Yap
   and SKN7, which are responsible for regulating the oxidative response
   and ROS detoxification in A. alternata [[161]27], did not show any
   significant difference under the magnolol condition. The other type is
   reported to be the modulation of the mitogen-activated protein kinase
   (MAPK) pathways [[162]24]. Two MAPK pathways, the HOG and CWI pathways,
   are so vital for the adaptation of the cell to stress [[163]51]. Our
   results showed that Hog1, the key MAPK of the HOG pathway, was
   significantly downregulated by magnolol. In fact, the Hog deletion
   mutant of A. alternata was almost unable to produce AOH, indicating
   that AOH biosynthesis required a phosphorylated HOG [[164]52].
   Additionally, Hog1 also modulates the production of several mycotoxins,
   such as fumonisin B[1] [[165]53] and ochratoxin A [[166]54]. The CWI
   pathway is responsible for the maintenance of the cell wall, which is a
   common target for antifungal compounds [[167]55,[168]56]. This pathway
   is regulated by the RHO family of GTPases, of which RHO1 is considered
   the center regulator [[169]21]. Previous studies have demonstrated that
   an increase in the transcription levels of RHO1 corresponded to a
   higher accumulation of AOH and AME, suggesting that the mycotoxin
   biosynthesis might be related to the CWI pathway [[170]21,[171]55].
   Indeed, a significant inhibition of the RHO1 expression exposure to
   magnolol treatment was observed, indicating that the fungus was unable
   to activate the CWI pathway to overcome the stress. Meanwhile, the
   results from SEM microscopy also showed that magnolol destroyed cell
   wall integrity and permeability, which was consistent with the previous
   SEM observations on A. alternata treated with ethyl p-coumarate
   [[172]30]. Taking the above into account, magnolol displayed strong
   antifungal and antimycotoxigenic activities against A. alternata by
   repressing the HOG and CWI signaling pathways and affecting cell wall
   integrity.

4. Conclusions

   Magnolol exerts strong inhibitory effects on the growth and mycotoxin
   biosynthesis of A. alternata in this study, and the results provide a
   new insight into the global transcriptional alterations of A. alternata
   in response to magnolol stress. Based on the transcriptional profile,
   our results demonstrate in [173]Figure 6 that: (1) magnolol inhibits
   the AOH and AME biosynthesis by down-expression of the polyketide
   clustered genes, including pksI and omtI; (2) the suppression of fatty
   acid β-oxidation after magnolol treatment leads to the marked reduction
   in mycotoxin precursor acetyl-CoA; (3) the down-regulation of nutritive
   repression regulators CreA and NmrA in response to magnolol may
   contribute to the significant decline in AOH and AME production; (4)
   magnolol enhances the expression of genes encoding antioxidant defense
   including SOD, POD and siderophore biosynthesis, together with the
   inhibition of the HOG and CWI signaling pathways, suggesting that
   magnolol inhibits AOH and AME production of A. alternata via the
   perturbation of the oxidative stress balance. In summary, magnolol
   could be a potential alternative to the traditional fungicides for
   controlling Alternaria decay and reducing mycotoxin contamination.

Figure 6.

   [174]Figure 6
   [175]Open in a new tab

   The schematic diagram demonstrating the inhibitory effects of magnolol
   against A. alternata and its mycotoxins. HOG: high osmolarity glycerol
   signaling pathway; CWI: cell wall integrity signaling pathway.

5. Materials and Methods

5.1. Strain and Culture Condition

   A. alternata ATCC 66,981 was grown on potato dextrose agar (PDA; Becton
   Dickinson, Franklin Lakes, NJ, USA) at 25 °C for 7 days in order for
   the preparation of spore suspensions as the previous study [[176]57].
   The spore concentration was finally adjusted to 1 × 10^5 spores/mL
   using a hemocytometer for the following assays.

5.2. Antifungal Effects of Magnolol on A. alternata

   Magnolol, purchased from Sigma-Aldrich (St. Louis, MO, USA), was
   dissolved with 50% aqueous ethanol (v/v) to prepare for the stock
   solution. It was mixed with the medium to obtain the different
   concentrations (0, 12.5, 25, 50, 100 and 200 μM). Antifungal trials of
   magnolol were performed as our previous work with slight modifications
   [[177]31]. A total of 5 μL aliquot of spore suspension was inoculated
   on PDA medium with serial concentrations of magnolol and cultured at 25
   °C for 6 days for diameter determination. In addition, liquid culture
   was carried out using potato dextrose broth (PDB; Becton Dickinson,
   Franklin Lakes, NJ, USA) at the final concentration of 10^2 spores/mL
   with shake flask culture in an incubator at 25 °C, 180 r/min. The
   minimum inhibitory concentration (MIC) was recognized as the lowest
   concentration of magnolol without visible fungal extending. After
   incubation for 6 d, the culture was filtered with Whatman filter paper.
   The supernatant was used for the determination of AOH and AME contents,
   while the mycelium was freeze-dried for the biomass analysis or quickly
   frozen with liquid nitrogen for the following RNA extraction. Each test
   was performed in triplicate. The inhibition ratio of the mycelial
   growth and biomass weight was separately calculated.

5.3. Scanning Electron Microscopy (SEM) Analysis

   The mycelia of A. alternata were collected and washed three times using
   phosphate buffered saline (PBS; pH 7.4) after 72 h incubation. The
   mycelia were mixed with PDB medium with 0, 12.5 or 25 μM of magnolol
   (mycelial weight: the volume of the medium = 1:10).

   For the analysis of spore morphological changes, spore suspension was
   mixed with the antifungal agent to get the magnolol final
   concentrations of 0, 12.5 and 25 μM. After incubation at 25 °C, 180
   r/min for 12 h, both mycelia and spores were centrifuged for 5 min at
   10,000× g, resuspended with PBS to remove the residue of medium and
   magnolol. The samples were then followed by serial pretreatments for
   the microscope observation based on Wang et al. [[178]26]. Finally, the
   morphological alteration was compared using a scanning electron
   microscope (S-3400N, Hitachi, Tokyo, Japan).

5.4. AOH and AME Analysis

   The extraction of AOH and AME was carried out following by the method
   of Meena et al. [[179]58] with minor modification. The culture was
   added with equal volume of acetonitrile containing 0.1% (v/v) formic
   acid, vortexed for 3 min and then kept at 4 °C for 24 h. After the
   centrifugation, the supernatant was processed and AOH and AME
   accumulation was quantified under the direction of Wang et al.
   [[180]26].

5.5. RNA Extraction, cDNA Library Construction, and RNA-Seq Analysis

   RNA was extracted from 6 days cultured mycelia from A. alternata ATCC
   66981 grown in PDB medium with or without 25 μM of magnolol using
   TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the
   manufacturer’s instructions. Each group has three biological
   replications. Extracted RNA was evaluated with the agarose gel
   electrophoresis and the Qubit RNA Assay Kit (Invitrogen, Carlsbad, CA,
   USA). The cDNA libraries construction and RNA-seq was performed at
   Beijing Geek Gene Technology Co., Ltd. (Beijing, China).

   The reads adapters, low quality reads, and reads with more than 5% N
   were to be trimmed from the raw reads. The remaining reads were
   conducted to be bioinformatically analyzed based on the genome
   annotation of A. alternata (genome assembly: GCF_001642055.1)
   [[181]27]. The genes of significantly differential expression between
   these compared samples were analyzed. Finally, gene ontology (GO)
   functional annotation and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway enrichment analysis were carried out to understand the
   functions of the differentially expressed genes (DEGs). To further
   confirm the reliability of the results from the gene expression
   analysis by RNA-Seq, quantitative reverse transcription polymerase
   chain reaction (qRT-PCR) was conducted for the clustered genes that
   were involved in AOH and AME biosynthesis by the reference of Wang et
   al. [[182]26].

5.6. Antioxidant Enzymatic Activities and Glutathione Measurement

   To evaluate the impact of magnolol on the antioxidant system activities
   in A. alternata, the analyses of superoxide dismutase (SOD), catalase
   (CAT) and glutathione (GSH) were performed following the same culture
   conditions of RNA extraction. The enzymatic activities of SOD and CAT,
   and the content of GSH were determined by the respective detection kits
   (Solarbio, Beijing, China) following the instructions.

5.7. Statistical Analysis

   Statistical analyses of all the results were completed by Microsoft
   Excel 2016. The significant difference of each treatment was compared
   by one-way analysis of variance (ANOVA) with the least significant
   differences’ test (LSD test) at p < 0.05 using IBM SPSS Statistics 21.0
   (SPSS Inc., Chicago, IL, USA).

Supplementary Materials

   The following are available online at
   [183]https://www.mdpi.com/2072-6651/12/10/665/s1. Figure S1: Volcano
   plot of these differentially expressed genes (DEGs) of A. alternata
   treated with 25 μM of magnolol vs. control, Figure S2: Comparative
   analysis of gene expression of A. alternata in 0, 12.5, 25 μM of
   magnolol by quantitative reverse transcription PCR (qRT-PCR), Table S1:
   Summary of the RNA-Seq data in the control (AaC) and 25.0 μM magnolol
   treated (AaM) groups of A. alternata ATCC 66981, Table S2:
   Differentially expressed genes (DEGs) between the control and magnolol
   stressed samples of A. alternata.
   [184]Click here for additional data file.^ (471.1KB, zip)

Author Contributions

   Conceptualization, L.W. and M.W.; methodology, L.W. and D.W.;
   validation, L.W. and M.W.; investigation, L.W. and D.W.; resources,
   L.W. and D.W.; writing—original draft preparation, L.W. and M.W.;
   writing—review and editing, S.Y. and X.F.; funding acquisition, L.W.
   and M.W. All authors have read and agreed to the published version of
   the manuscript.

Funding

   This research was funded by the National Natural Science Foundation of
   China (grant no. 31701709; 31801648), and the Innovation and
   Capacity-building Projects by Beijing Academy of Agriculture and
   Forestry Sciences (grant no. KJCX20180408).

Conflicts of Interest

   The authors declare no conflict of interest.

Key Contribution

   Magnolol; extracted from a traditional Chinese herb; Mangnolia
   officinalis; exhibits brilliant suppression against Alternaria
   alternata and its producing mycotoxins including AOH and AME;
   especially compared to other natural phenolics. The action mode
   probably lies in the lower transcription of the mycotoxin biosynthetic
   genes and the regulatory genes of carbon and nitrogen utilization (CreA
   and NmrA), and also the disturbance of the supply of the precursor
   acetyl-CoA and oxidative stress.

Footnotes

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   claims in published maps and institutional affiliations.

References