Host Factors Modulating Ochratoxin a Biosynthesis During Fruit Colonization by Aspergillus Carbonarius

Journal of Fungi

Article Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius

Uriel Maor 1,2, Omer Barda 1, Sudharsan Sadhasivam 1 , Yang Bi 3, Varda Zakin 1, Dov B. Prusky 1,3 and Edward Sionov 1,*

1 Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel; [email protected] (U.M.); [email protected] (O.B.); [email protected] (S.S.); [email protected] (V.Z.); [email protected] (D.B.P.) 2 Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel 3 College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, China; [email protected] * Correspondence: [email protected]; Tel.: +972-3-968-3693

Abstract: Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and consid- ered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact Citation: Maor, U.; Barda, O.; on the capability of A. carbonarius to produce and accumulate OTA in grapes. Sadhasivam, S.; Bi, Y.; Zakin, V.; Prusky, D.B.; Sionov, E. Host Factors Keywords: OTA accumulation; sugar content; organic acids; pH modulation; fungal pathogenicity Modulating Ochratoxin A Biosynthesis in fruits during Fruit Colonization by Aspergillus carbonarius. J. Fungi 2021, 7, 10. https://dx.doi.org/ 10.3390/jof7010010 1. Introduction Grapes are one of the largest produced fruit crops in the world. It is also one of the most Received: 8 December 2020 abundant fruits: while almost 50% of grapes are used to make wine, one third is consumed Accepted: 24 December 2020 as fresh fruit and the rest is dried, consumed as grape juice or stored in the form of grape Published: 28 December 2020 musts [1]. Approximately 40% of table grapes are lost annually due to pathological and physiological deterioration [2], and in particular, because of the lack of efficient methods to Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims prevent decay and senescence [3]. The black aspergilli (Aspergillus section Nigri) are frequently in published maps and institutional responsible for fungal decay of fresh fruit, and the consequent accumulation of mycotoxins. affiliations. Among black aspergilli, A. carbonarius is a major producer of OTA in grapes, where it causes fungal decay and poses a significant threat to human health [4]. OTA contamination of grapes presents a serious health and economic problem, especially in Mediterranean countries [5,6]. OTA is a potent nephrotoxin that is responsible for several adverse effects on animals and Copyright: © 2020 by the authors. Li- humans; it has hepatotoxic, neurotoxic, carcinogenic, immunotoxic, genotoxic and teratogenic censee MDPI, Basel, Switzerland. This properties [7–9]. The International Agency for Research on Cancer (IARC) has classified OTA article is an open access article distributed as a Group 2B possible human carcinogen, based on demonstrated carcinogenicity in animal under the terms and conditions of the studies [10,11]. Our recent study demonstrated that environmental pH plays an important Creative Commons Attribution (CC BY) role in A. carbonarius pathogenicity and OTA production [12]. Secretion of gluconic acid (GLA) license (https://creativecommons.org/ by A. carbonarius caused direct fruit tissue acidification and induced accumulation of OTA licenses/by/4.0/).

J. Fungi 2021, 7, 10. https://dx.doi.org/10.3390/jof7010010 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, 10 2 of 12

in colonized grapes. Moreover, other environmental conditions, such as temperature, water activity and light, have been shown to contribute to OTA biosynthesis [13–17]. Composition of the growth medium has a strong effect on OTA production. Medina et al. [18] demonstrated that accumulation of the mycotoxin significantly altered under various carbon sources. The authors showed that the highest OTA levels produced by three Aspergillus isolates, A. ochraceus, A. carbonarius and A. tubingensis, were found in media supplemented with arabinose and glucose; fructose was the least favorable sugar for OTA production by the isolates. The same study, however, showed no marked difference in OTA production with regard to the nitrogen source. Compared to the extensive research on environmental factors that modulate mycotoxin formation, only a few studies were carried out to understand the effect of fruit host factors on mycotoxin biosynthesis. Fruit nutritional components that undergo changes during fruit maturation and ripening include sugars and organic acids, which may change fruit pH. This raises the question of whether such changes in the host can modulate fungal mycotoxin biosynthesis. Several studies on aflatoxin synthesis suggest that the high content of carbohydrates in figs, dates, citrus fruit and raisins probably enhances mycotoxin production by Aspergillus flavus and Aspergillus parasiticus [19,20]. It has been recently reported that apple host intrinsic factors, such as sucrose, organic acids and phenols, differentially modulate patulin accumulation during fruit colonization by Penicillium expansum [21]. In addition, several studies indicated that different apple and grape cultivars vary in their susceptibility to pathogen attack and mycotoxin accumulation in these fruits [22–25]. In the present work, we investigated the impact of grape intrinsic factors that change during fruit maturation on OTA accumulation in colonized fruits. Our results suggest that fruit host factors, including sugars, organic acids and pH, contribute to the complex activation of the global regulator of fungal secondary metabolism (LaeA) and OTA gene cluster, and may modulate the mycotoxin biosynthesis during fruit colonization by A. carbonarius.

2. Materials and Methods 2.1. Fungal Strain and Culture Conditions The A. carbonarius NRRL 368 strain used throughout this study was obtained from USDA Agricultural Research Service Culture Collection (Northern Regional Research Laboratory, Peoria, IL, USA). The isolate was stored in 25% glycerol at −80 ◦C until use and maintained on potato dextrose agar (PDA) plates (BD, Franklin Lakes, NJ, USA). Conidia were harvested with 10 mL of sterile saline and adjusted using an hemocytometer to the indicated concentrations. A 106 fungal spores/mL solution (100 µL) was inoculated onto 55 mm petri dishes with 10 mL of solid synthetic grape juice media (SGM; [26]) containing (per liter): 0.67 g (NH4)2HPO4, 0.67 g KH2PO4, 1.5 g MgSO4·7H2O, 0.15 g NaCl, 0.15 g CaCl2, 0.0015 g CuCl2, 0.021 g FeSO4·7H2O, 0.0075 g ZnSO4·7H2O, different sugar concentrations (10, 50, 100 and 150 g/L, as indicated in each experiment)—glucose-fructose at a ratio of 2.33 (70% D(+)glucose and 30% D(−)fructose), different concentrations of malic acid (10, 20, 30 and 40 g/L), different concentrations of tartaric acid (0, 3.5, 7 and 14 g/L), various concentrations of hydrated catechin (0, 0.025, 0.05 and 0.1 g/L) and 2% agar adjusted to different pH levels (3, 4 and 5) with concentrated KOH. The plates were incubated at 28 ◦C in the dark for 2–8 days as needed for sample collection.

2.2. Fruit Colonization and Pathogenicity Experiments White “Superior” grapes were freshly harvested from two trees in a single vineyard in central Israel (Moshav Pedaya) during two consecutive years, approximately 30 days after fruit set (early harvest). Four additional harvests were made within 10 days intervals. For each harvest, ﬁve bunches containing 20–30 berries in each bunch were collected. The grape berries were homogeneous in size and color, and had no visible damage or symptoms of fungal attack on the skin. Harvested fruits were analyzed for acidity, pH and TSS as described below. On the day of harvest, berries in the same bunch were submerged in 1% sodium hypochlorite solution for 1 min and immediately rinsed twice in sterile distilled water for surface sterilization. The sterilized grapes were aseptically air-dried and inocu- J. Fungi 2021, 7, 10 3 of 12

lated by injection with 10 µL of A. carbonarius conidial suspension (106 spores/mL) onto each grape berry. Following inoculation, the fruits were incubated under high humidity at 28 ◦C for 7 days as needed for symptom monitoring and sample collection, and the diameters of the rotten spots were recorded daily.

2.3. pH Measurements, Organic Acids and OTA Analysis pH was measured directly in the agar cultures with a double pore slim electrode con- nected to a Sartorius PB-11 Basic Meter (Sartorius, Goettingen, Germany). For assessment of GLA production, five 1-cm diameter discs of agar were placed in 5 mL of sterilized water and crushed to homogeneity. A 1 mL aliquot of the solution was sampled in a 1.5 mL mi- crocentrifuge tube and centrifuged for 10 min at 20,800× g. The supernatant was taken for GLA analysis using a test kit applying an enzymatic method for the specific measurement of total D-Gluconic acid content (Megazyme, Wicklow, Ireland) according to the manufacturer’s instructions. To evaluate OTA levels, five 1-cm diameter discs of agar were added to 1.7 mL of HPLC grade methanol (Bio-Lab, Jerusalem, Israel) and crushed to homogeneity. OTA was extracted by shaking for 30 min at 150 RPM on an orbital shaking platform and centrifuged for 10 min at 20,800× g. The supernatant was filtered through a 0.22 µm PTFE syringe filter (Agela Technologies, Tianjin, China) and kept at −20 ◦C prior to HPLC analysis. OTA was quantitatively analyzed by injection of 20 µL into a reverse phase UHPLC system (Waters ACQUITY Arc, FTN-R, Milford, MA, USA). The mobile phase consisted of acetonitrile:water:acetic acid (99:99:2, v/v/v) at 0.5 mL/min through a Kinetex 2.6 µm XB-C18 (100 mm × 2.1 mm) with a security guard column C18 (4 mm × 2 mm) (Phenomenex, Torrance, CA, USA). The column temperature was maintained at 30 ◦C. The OTA peak was detected with a fluorescence detector (excitation at 330 nm and emission at 450 nm) and quantified by comparing with a calibration curve of the standard mycotoxin (Fermentek, Jerusalem, Israel). The pH of grape tissues was measured by inserting a double pore slim electrode directly into the tested area. For assessment of malic acid content in freshly harvested grapes and GLA production in colonized grape tissue, the berries were crushed manually in 50 mL tubes; the juice was recovered after centrifugation and was taken for analysis of these organic acids. GLA and malic acid concentrations in grapes were determined by using enzymatic methods for measurement of total GLA/malic acid content (Megazyme, Wicklow, Ireland) according to the manufacturer’s instructions. The TSS content in grape juice was measured by a digital refractometer (Atago, Tokyo, Japan). For OTA analysis in colonized grapes, 1.7 g of the macerated necrotic area was taken, 1.7 mL of HPLC grade methanol (Bio-Lab, Jerusalem, Israel) was added and the tissues were homogenized. Then, OTA was quantitatively analyzed as described above.

2.4. RNA Extraction and Gene Expression Analysis by qRT-PCR Mycelia from the in vitro experiments were harvested at the appropriate time, weighed, frozen in liquid nitrogen, lyophilized for 24 h and kept at −80 ◦C until used. Total RNA was extracted from 100 mg of ground mycelium of the selected samples using the Hybrid-R RNA isolation kit (GeneAll, Seoul, Korea) according to the manufacturer’s protocol. The DNase and reverse-transcription reactions were performed on 1 µg of total RNA with the Maxima First-Strand cDNA Synthesis Kit (Thermo Scientiﬁc, Waltham, MA, USA) according to the manufacturer’s instructions. The cDNA samples were diluted 1:10 (v/v) with ultrapure water. The quantitative real-time PCR was performed using Fast SYBR green Master Mix (Applied Biosystems, Waltham, MA, USA) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The PCR conditions were as follows: 95 ◦C for 20 s, followed by 40 cycles of 95 ◦C for 3 s and 60 ◦C for 20 s. The samples were normalized using β-tubulin as endogenous control and the relative expression levels were measured using the 2(−∆∆Ct) analysis method. Results were analyzed with StepOne software v2.3. Primer sequences used for qRT-PCR analysis are listed in Table S1. J. Fungi 2021, 7, x FOR PEER REVIEW 4 of 12

were as follows: 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 20 s. The samples were normalized using β-tubulin as endogenous control and the relative expression levels were measured using the 2(–ΔΔCt) analysis method. Results were analyzed J. Fungi 2021, 7, 10 with StepOne software v2.3. Primer sequences used for qRT-PCR analysis are listed4 of 12in Table S1.

2.5. Statistical Analysis 2.5. Statistical Analysis Student’s t-test was performed when data was normally distributed and the sample variancesStudent’s weret -testequal. was For performed multiple compar when dataisons, was one-way normally ANOVA distributed was performed and the sample when variancesthe equal werevariance equal. test For was multiple passed. comparisons, Significance was one-way accepted ANOVA at p < was 0.05. performed All experiments when thedescribed equal variance here are testrepresentative was passed. of Signiﬁcance at least three was independent accepted at experimentsp < 0.05. All with experiments the same describedpattern of here results. are representative of at least three independent experiments with the same pattern of results. 3. Results and Discussion 3. Results and Discussion 3.1.3.1. FruitFruit MaturationMaturation AffectsAffects OTAOTA ProductionProduction by by A. A. carbonarius carbonarius SeveralSeveral physiologicalphysiological parametersparameters ofof freshlyfreshly harvestedharvested whitewhite “Superior”“Superior” grapesgrapes werewere measuredmeasured atat differentdifferent periodsperiods duringduring fruitfruit maturationmaturation(Table (Table1 ).1). A A gradual gradual increase increase in in the the totaltotal solublesoluble solidssolids (TSS)(TSS) ofof grapegrapeberries berries was was observed observed during during fruit fruit maturation. maturation. TSSTSS ofof thethe fruitsfruits fromfrom thethe 5th5th latestlatest harvestharvest time time point point increased increased by by 11.01% 11.01% to to 14.98%, 14.98%, compared compared toto 3.97%3.97% ofof thatthat fromfrom the the 1st 1st “early” “early” grape grape harvest harvest time time point. point. Furthermore, Furthermore, comparison comparison ofof “early”“early” andand “late”“late” maturitymaturity harvestedharvested fruitsfruits showedshowed aa decreasedecrease byby2.08% 2.08%in inmalic malicacid acid (from(from 3.16% 3.16% to to 1.08%), 1.08%), which which is is the the main main organic organic acid acid found found in in grapes, grapes, and and an an increase increase in in pHpH valuesvalues fromfrom 2.962.96 toto 4.61 4.61 in in more more mature mature fruits fruits (Table (Table1). 1).

TableTable 1. 1.Physiological Physiological parameters parameters of of harvested harvested fruits. fruits.

Grape HarvestHarvest TSS TSS (%)(%) Fruit Fruit pH pH Malic AcidAcid (%) (%) 1st 3.97 ± 0.15 (e) 2.96 ± 0.15 (e) 3.16 ± 0.06 (a) 1st 3.97 ± 0.15 (e) 2.96 ± 0.15 (e) 3.16 ± 0.06 (a) 2nd2nd 5.60 5.60± ± 1.291.29 (d)(d) 3.16 3.16± ± 0.140.14 (d) (d) 3.22 3.22± ± 0.180.18 (a) (a) 3rd3rd 7.40 7.40± ± 1.261.26 (c)(c) 3.46 3.46± ± 0.210.21 (c) (c) 2.712.71± ± 0.220.22 (b) (b) 4th4th 13.44 13.44± ± 1.251.25 (b) (b) 4.08 4.08± ± 0.190.19 (b) (b) 1.36 1.36± ± 0.070.07 (c) (c) ± ± ± 5th5th 14.98 14.98 ± 0.780.78 (a) (a) 4.61 4.61 ± 0.250.25 (a) (a) 1.08 1.08 ± 0.050.05 (c) (c) TotalTotal soluble solidssolids (TSS), (TSS), pH pH values values of harvested of harvested grapes gr andapes concentration and concentration of malic of acid malic in grapes acid in grapes at different time points. The harvests were made within a 10 days interval. For each harvest, five at different time points. The harvests were made within a 10 days interval. For each harvest, five bunches containing 20–30 berries in each bunch were collected. Different letters in parentheses bunchesindicate statisticallycontaining significant20–30 berries differences in each atbunchp < 0.05, were as collected. determined Different using the letters Tukey’s in parentheses honest indicatesignificant statistically difference test.significant differences at p < 0.05, as determined using the Tukey’s honest significant difference test. ToTo determinedetermine thethe effecteffect ofof thesethese dynamicdynamic changeschanges onon OTAOTA accumulationaccumulation byby A.A. car-car- bonariusbonarius,, grape grape berries berries were were inoculated inoculated at at different different harvest harvest periods periods during during fruit fruit maturation matura- (Figuretion (Figure1). 1).

(a) (b)

Figure 1. Cont.

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(c)

FigureFigure 1. Disease 1. Disease development development in in freshly freshly harvested harvested fruits at at different different time time points points following following the inoculation the inoculation of “Superior” of “Superior” grapegrape berries berries by A. by carbonarius A. carbonarius. The. The grape grape berries berries werewere inoculatedinoculated immediately immediately after after harvest. harvest. (a) Histogram (a) Histogram showing showing the the decay area of the rotten tissue on infected grape berries. (b) GLA and (c) OTA accumulations were measured in infected decayfruits area at of day the 7 rotten post-inoculation. tissue on infected Error bars grape represent berries. standard (b) GLA error and of three (c) OTA inde accumulationspendent biological were replicates. measured Different in infected fruitsletters at day above 7 post-inoculation. the columns indicate Error statistically bars represent significant standard differences error at of p three < 0.05, independent as determined biological using the Tukey’s replicates. honest Different letterssignificant above the difference columns test. indicate statistically significant differences at p < 0.05, as determined using the Tukey’s honest significant difference test. Fungal infection caused a significant increase in the rotten colonized area of the late harvestedFungal fruits infection (4th and caused 5th harvests) a significant relative increaseto that of the in theearly rotten harvested colonized grape berries area of the late(Figure harvested 1a). Disease fruits development (4th and 5th was harvests) accompanied relative by to accumulation that of the earlyof relatively harvested high grape berriesamounts (Figure of GLA1a). in Disease more mature development grape berries was (Figure accompanied 1b). One byof the accumulation factors that contrib- of relatively ute to the pathogenicity of A. carbonarius is its ability to produce GLA [12,27]. GLA accu- high amounts of GLA in more mature grape berries (Figure1b). One of the factors that mulation by A. carbonarius is carried out by glucose oxidase that catalyzes the oxidation contributeof glucose to to the GLA. pathogenicity Recently we ofhaveA. provided carbonarius evidenceis its ability that deletion to produce of the GLA glucose [12 oxi-,27]. GLA accumulationdase-encoding by gene,A. carbonarius gox, in A. carbonariusis carried outled to by a glucosereduction oxidase in virulence thatcatalyzes toward nectarine the oxidation ofand glucose grape to fruits, GLA. further Recently indicating we have that provided GOX is evidence a virulence that factor deletion of A. of carbonarius the glucose [28]. oxidase- encodingInterestingly, gene, angox opposite, in A. trend carbonarius was observedled to aregarding reduction the in accumulation virulence toward of OTA nectarine in andgrapes grape harvested fruits, furtherat different indicating time points. that A GOX high is amount a virulence of OTA factor was found of A. carbonariusto be pro- [28]. Interestingly,duced by A. ancarbonarius opposite in trend early washarvested observed low sugar regarding grapes the (1st accumulation harvest, 7084 ng of OTA per in grapes harvestedgram of grape at different berries), time compared points. to significantly A high amount lower ofconcentrations OTA was found of the mycotoxin to be produced byaccumulatedA. carbonarius in grapein early berries harvested with increased low sugarmaturity grapes at 4th and (1st 5th harvest, harvests 7084 (241.1 ng ng/g OTA per gramand of71.3 grape ng/g, berries),respectively) compared (Figure to1c). significantly Several host factors lower may concentrations influence the of mycotoxin the mycotoxin accumulatedbiosynthesis inby grape the fungus berries during with the increased early period maturity of grape at 4th berry and growth. 5th harvests A relatively (241.1 ng/g high concentration of malic acid may trigger excessive OTA production by the fungus in and 71.3 ng/g, respectively) (Figure1c). Several host factors may influence the mycotoxin early harvested grape berries. Malic acid levels decrease during grape ripening. A reduc- biosynthesistion in malic by acid the content fungus was during also theobserved early periodin late maturity of grape harvested berry growth. apples A compared relatively high concentrationto early harvested of malic fruits acid [21]. may In this trigger study, excessive however, OTA a relatively production high amount by the of fungus patulin in early harvestedwas observed grape in berries.more mature Malic apples acid colonized levels decrease by P. expansum during, indicating grape ripening. complex A host– reduction inpathogen malic acid interactions content wasin different also observed fruits. in late maturity harvested apples compared to early harvested fruits [21]. In this study, however, a relatively high amount of patulin was3.2. observed Association in of more Sugar mature Content applesand pH with colonized OTA Biosynthesis by P. expansum , indicating complex host– pathogenConsidering interactions the high in different concentration fruits. of OTA produced in early harvested grapes and a reduced amount of the mycotoxin in more mature fruits with increasing TSS and pH 3.2.values Association (Table 1, of Figure Sugar 1), Content we sought and pHto de withtermine OTA the Biosynthesis relationship between sugar concentration,Considering pH and OTA the highproduction. concentration A. carbonarius of OTA was grown produced on solid in earlyartificial harvested SGM me- grapes anddium a reduced amended amount with increasing of the mycotoxin sugar concentrat in moreions mature(from 1 to fruits 15%) with of glucose–fructose increasing TSS and pHcombination, values (Table under1, Figuredifferent1), pH we levels sought (3, 4 toand determine 5). At day 6 the post-inoculation, relationship betweenOTA levels sugar were maximal at 1% sugar concentration under initial pH 4.0, reaching 104 ng/g agar. This concentration, pH and OTA production. A. carbonarius was grown on solid artificial SGM amount decreased with increasing sugar concentration, reaching a minimum of 2.6 ng/g medium amended with increasing sugar concentrations (from 1 to 15%) of glucose–fructose combination, under different pH levels (3, 4 and 5). At day 6 post-inoculation, OTA levels were maximal at 1% sugar concentration under initial pH 4.0, reaching 104 ng/g agar. This amount decreased with increasing sugar concentration, reaching a minimum of 2.6 ng/g in the presence of 15% sugar (Figure2). These results point out to the same trend of OTA accumulation observed in vivo during grape berries maturation and ripening. J. Fungi 2021, 7, x FOR PEER REVIEW 6 of 12

J. Fungi 2021, 7, 10 6 of 12 in the presence of 15% sugar (Figure 2). These results point out to the same trend of OTA accumulation observed in vivo during grape berries maturation and ripening.

Figure 2. OTA biosynthesis is modulated by sugar and pH levels. Solid synthetic grape juice media (SGM) was supple- Figure 2. OTA biosynthesis is modulated by sugar and pH levels. Solid synthetic grape juice me- 6 mented withdia different (SGM) was sugar supplemented concentrations with (glucose/fructose different sugar concentrations at a ratio of 2.33), (glucose/fructose and inoculated at witha ratioA. of carbonarius (10 spores/mL).2.33), OTA and accumulation inoculated with was A. evaluated carbonarius at day(106 6spores post-inoculation./mL). OTA accumulation Error bars represent was evaluated standard at day error of three independent6 biologicalpost-inoculation. replicates. Error Different bars represent letters above standard the columns error of indicatethree independent statistically biological significant replicates. differences at p < 0.05, as determinedDifferent using theletters Tukey’s above honest the columns significant indicate difference statistically test. significant differences at p < 0.05, as determined using the Tukey’s honest significant difference test. To investigate the influence of sugar concentration and ambient pH upon OTA produc- To investigatetion atthe the influence molecular of level,sugar weconcentration analyzed the and differential ambient expressionpH upon OTA of the pro-laeA gene, encod- duction at the ingmolecular a methyltransferase level, we analyzed involved the in differential global regulation expression of fungal of the secondary laeA gene, metabolism, and encoding a methyltransferaseOTA biosynthetic involved cluster in genes global in A.regulation carbonarius of fungalunder differentsecondary physiological metabo- conditions. lism, and OTAAs biosynthetic shown in Figure cluster3, genes the expression in A. carbonarius levels of under the global different regulator physiologicallaeA and all five genes conditions. As involvedshown in in Figure OTA biosynthesis3, the expression correlated levels withof the mycotoxin global regulator production laeA and under all different sugar five genes involvedconcentrations. in OTA biosynthesis The increased correlated OTA accumulation with mycotoxin that occurredproduction under under limited dif- sugar concen- ferent sugar concentrations.trations of 1% The and increased 5% was accompanied OTA accumulation with laeA thatand occurred almost under all OTA limited biosynthesis cluster sugar concentrationsgenes beingof 1% upregulated,and 5% was accompanied while a reduction with oflaeA the and mycotoxin almost all production OTA bio- accompanied synthesis clusterwith genes decreased being upregulated, relative expression while a ofreduction the genes of the was mycotoxi observedn production in the presence of higher accompanied withsugar decreased concentrations relative (Figures expression2 and 3of). the genes was observed in the presence of higher sugarOur concentrations findings are consistent(Figures 2 withand 3). a previous study of Kumar et al. [21], which reported Our findingsrepression are consistent in relative with expressiona previous study of laeA of, patulinKumar et biosynthesis al. [21], which cluster reported genes and patulin repression in relproductionative expression by P. expansum of laeA, patulinin the presencebiosynthesis of increased cluster genes sucrose and concentrations patulin in syn- production by theticP. expansum growth in medium, the presence whereas of increased maximal sucrose gene expression concentrations and patulin in synthetic accumulation were growth medium,obtained whereas when maximal the fungus gene wasexpression grown atand a limitedpatulin sucrose accumulation level. were ob- Another factor that might affect OTA accumulation during fruit maturation is pH tained when the fungus was grown at a limited sucrose level. change as it was found that pH levels in the fruit increased with grape berries maturation Another factor that might affect OTA accumulation during fruit maturation is pH from 2.96 to 4.61 (Table1). A number of studies have reported that OTA biosynthesis change as it was found that pH levels in the fruit increased with grape berries maturation increases in Aspergillus ochraceus when the pH drops below 6.0 [29,30]. Recently, we from 2.96 to 4.61 (Table 1). A number of studies have reported that OTA biosynthesis in- found that OTA production by A. carbonarius is also strongly influenced by ambient pH creases in Aspergillus ochraceus when the pH drops below 6.0 [29,30]. Recently, we found with higher amounts of the mycotoxin being produced under acidic pH (3.0–4.0) than at that OTA production by A. carbonarius is also strongly influenced by ambient pH with neutral or alkaline conditions [12,27]. Our in vitro experiments confirmed that an acidic higher amounts of the mycotoxin being produced under acidic pH (3.0–4.0) than at neutral environment is favorable for OTA production, with increased mycotoxin synthesis observed or alkaline conditions [12,27]. Our in vitro experiments confirmed that an acidic environ- at pH 3 and 4, but only under a limited sugar content (Figure2). pH changes in the presence ment is favorable for OTA production, with increased mycotoxin synthesis observed at of increased sugar concentrations from 1 to 15% also affected laeA and OTA biosynthesis pH 3 and 4, but only under a limited sugar content (Figure 2). pH changes in the presence gene expression (Figure3). of increased sugar concentrations from 1 to 15% also affected laeA and OTA biosynthesis gene expression (Figure 3).

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Figure 3. laeAlaeA andand OTAOTA gene gene cluster cluster relative relative expression expression as affectedas affected by sugarby su concentrationsgar concentrations and pHand levels. pH levels. Solid SGMSolid mediaSGM 6 mediawas amended was amended with different with different sugar concentrations. sugar concentrations. The media The wasmedia inoculated was inoculated with 100 withµL of100 a µL 10 ofspore/mL a 106 spore suspension/mL sus- pensionand the and relative the relative expression expression of laeA, ofbZIP laeA, nrps, bZIP, p450, nrps, ,hal p450and, halpks andgenes pks genes were comparedwere compared at different at different conditions. conditions. RNA RNA was wasextracted extracted from from mycelia mycelia at day at day 6 post-inoculation. 6 post-inoculation. Relative Relative expression expression was was normalized normalized using usingβ β-tubulin-tubulin asas an internal control. The The expression expression of each gene under sugar concentration of 1% at pH 3 was normalized a ass 1.0. Error Error bars bars represent represent standard error ofof threethree independentindependent biological biological replicates. replicates. Different Different letters letters above above the the columns columns indicate indicate statistically statistically signiﬁcant signifi- cantdifferences differences at p < at 0.05, p < 0 as.05, determined as determined using using the Tukey’s the Tukey honest’s honest signiﬁcant significant difference difference test. test.

TheThe g growthrowth substratesubstrate is is one one of of the the most most important important factors factors influencing influencing mycotoxin mycotoxin biosyn- bi- osynthesis.thesis. High High sucrose sucrose content content as as a carbona carbon source source in in defined defined media media (such(such as YES YES—yeast—yeast eextractxtract sucrose)sucrose) stronglystrongly influences influences OTA OTA production production in inA. A. ochraceus ochraceus, Aspergillus, Aspergillus sclerotiorum scleroti-, orAspergillusum, Aspergillus sulphurus sulphurus, Penicillium, Penicillium verrucosum verrucosumand Penicillium and Penicillium nordicum [31 nordicum,32]. YES medium[31,32]. YES con- mediumtaining 15% containing sucrose was15% shown sucrose to was be highly shown supportive to be highly for OTAsupportive biosynthesis for OTA by A.biosynthesis carbobarius byunder A. carbobarius acidic pH [under12]. This acidic increase pH [12]. in OTA This was increase accompanied in OTA bywas induction accompanied of the by transcript induc- tionlevels of of thelaeA transcriptand OTA levels biosynthesis of laeA and genes OTA under biosynthesis high sucrose genes concentration under high compared sucrose withcon- centrationthat in the compared sucrose-limited with that medium. in the Interestingly, sucrose-limited OTA medium. biosynthesis Interestingly, is strongly OTA inhibited biosyn- in thesismedia is containing strongly glucoseinhibited as in sole media carbon containing source [33 glucose]. P. expansum as soleproduced carbon source less patulin [33]. P. when ex- pansumgrown inproduced the presence less ofpatulin glucose when and grown fructose in compared the presence to sucrose-amended of glucose and fructose medium com- [34]. paredAccording to sucrose to the- resultsamended obtained medium in the[34]. current According study, to this the seems results to obtained be the case in wherethe current high study,concentrations this seems of to glucose be the and case fructose, where high which concentrations are the main of sugars glucose in grapeand fru berries,ctose, inhibitwhich areOTA the production. main sugars Secondary in grape metaboliteberries, inhibit biosynthesis OTA production. in many filamentous Secondary fungi,metabolite including bio- synthesisA. carbonarius in many, is tightly filamentous regulated fungi, by both including the pathway-specific A. carbonarius transcription, is tightly regulated factor embedded by both thein mycotoxin pathway-specific biosynthesis transcription gene cluster factor (e.g., embedded bZIP transcription in mycotoxin factor biosynthesis in the OTA gene cluster) clus- terand (e.g. by, global bZIP transcription regulatory factors, factor including in the OTA the cluster) global regulator and by global of secondary regulatory metabolites factors, including(LaeA), the the pH global regulatory regulator factor of PacC secondary and the metabolites carbon catabolite (LaeA), repressor the pH regulatory (CreA) [27, 35factor–37]. PacCPrevious and studies the carbon have reported catabolite that repressor involvement (CreA) of global [27,35 carbon–37]. Previouscatabolite regulatorstudies have CreA re- in portedsecondary that metabolism involvement may of global reflect thecarbon differences catabolite seen regulator in metabolite CreA production in secondary when metabo- fungi lismare grown may reflect in different the differences carbon sources seen[ 38in ,39metabolite]. In the present production study, when a significant fungi are upregulation grown in differentof creA gene carbon was sources found under[38,39]. high In the sugar present content study (Figure, a significant S1), suggesting upregulation that CreA of iscreA in-

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gene was found under high sugar content (Figure S1), suggesting that CreA is involved volvedin OTA in synthesis, OTA synthesis, possibly possibly through through the negative the negative regulation regulation of ofLaeA LaeA and and the the OTA OTA gene cluster. TheseThese resultsresults are are supported supported by by some some previous previous studies studies that that found found negative negative correlation corre- lationbetween between transcript transcript level of level secondary of secondary metabolites metabolites biosynthesis biosynthesis genes and genescreA expressionand creA ex- in Penicilliumpression in chrysogenum Penicillium ,chrysogenumPenicillium oxalicum, Penicilliumand P.oxalicum expansum and[34 P.,39 expansum,40]. [34,39,40].

3.3. Malic Acid Modulates laeA Expression and OTA BiosynthesisBiosynthesis Given that the presence of high concentration of malic acid in early harvested grape berries is correlated with increased OTA production during colonization by A. carbonarius carbonarius, we examined the influence inﬂuence of this organic acid on on OTA OTA biosy biosynthesisnthesis at the the molecular molecular level. level. Figure4 4 shows shows thatthat increasingincreasing thethe concentrationsconcentrations ofof malicmalic acidacid fromfrom 11 toto 4%4% inin thethe media,media, led to a 3.5-fold3.5-fold upregulation of laeA and up to a 13-fold13-fold increase in OTA clustercluster genegene expression, accompanied by an elevation in OTAOTA biosynthesisbiosynthesis fromfrom 5454 toto 150150 ng/gng/g agar.

Figure 4. EffectEffect of of malic malic acid acid on on OTA OTA synthesis synthesis by by A. A.carbonarius carbonarius in SGMin SGM media. media. Solid Solid SGM SGM-media-media was wassupplemented supplemented with with1–4% 1–4% concentrations concentrations of malic of malicacid and acid inoculated and inoculated with 100 with µL 100 of aµ L10 of6 spore a 106/mL.spore/mL. OTA accumulatio OTA accumulationn and relative and relativeexpres- expressionsion of laeA of andlaeA OTAand gene OTA genecluster cluster were wereevaluated evaluated 6 days 6 days post post-inoculation.-inoculation. Relative Relative expression expression was was normalized normalized using using β- βtubulin-tubulin as asan an internal internal control. control. Error Error bars bars represent represent a astandard standard error error of of three three independent independent biological replicates. DifferentDifferent lettersletters aboveabove thethe columnscolumns indicateindicate statisticallystatistically signiﬁcantsignificant differences at p < 0.05,0.05, as determined using the Tukey’sTukey’s honesthonest signiﬁcantsignificant differencedifference test.test.

These findings findings align well with a previous study, wherewhere increasingincreasing concentrationsconcentrations of malic acid e enhancednhanced activation of of laeAlaeA andand patulin patulin synthesis synthesis by by P.P. expansum expansum inin vitro vitro [21].[21]. The activation activation of of the the global global regulator regulator LaeA LaeA and and OTA OTA gene gene cluster, cluster, and and consequently consequently in- creasedincreased OTA OTA accumulation, accumulation, was was probably probably caused caused due dueto the to acidification the acidification process process induced in- byduced malic by malic acid. acid. Previous Previous findings findings indicate indicate that that acidification acidification of of the the host host environment through accumulation of organic acids enhancedenhanced fruit maceration and colonization by fungal pathogenspathogens [[12,27,28,4112,27,28,41––4343].]. TheseThese studiesstudies pointpoint out out the the importance importance of of the the acidifica- acidifi- cationtion process process driven driven by by the the accumulation accumulation of of another another organic organic acid acid produced produced by byP. expansumP. expan- sumand A.and carbonarius A. carbonarius, GLA,, GLA which, which contributes contributes to enhanced to enhanced fungal fungal pathogenicity pathogenicityin vivo in vivoand mycotoxin accumulation. Indeed, accumulation of high amounts of GLA was found in

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and mycotoxin accumulation. Indeed, accumulation of high amounts of GLA was found thein the colonized colonized grape grape berries berries at aat late a late maturity maturity stage stage (Figure (Figure1b) 1b with) with high high TSS TSS content content and relativelyand relatively higher higher pH levels pH levels compared compared to those to those parameters parameters of the of early the early harvested harvested grapes (Tablegrapes1 ).(Table These 1). results These results correlate correlate with our within our vitro in experiments,vitro experiments, where where a signiﬁcant a significant upreg- ulationupregulation of the ofgox thegene gox gene expression expression accompanied accompanied by increasedby increased production production of of GLA, GLA was, observedwas observed under under high high sugar sugar contents contents at rising at rising pH levelspH levels (Figure (Figure5a,b). 5a, However,b). However, mycotoxin my- productioncotoxin production was minimal was minimal under these under conditions these conditions both in both vitroin andvitro during and during colonization coloni- of latezation harvested of late harvested grapes that grapes contain that lowercontain amounts lower amounts of malic of acid. malic There acid. There is no clearis no explana-clear tionexplanation on the importance on the import of malicance of acid malic on theacid regulation on the regulation of OTA. of Malic OTA. acid Malic belongs acid belongs to a group oftoC a4 groupdicarboxylic of C4 dicarboxylic acids that are acids produced that are by produced many microorganisms by many microorganisms as key intermediates as key ofintermediates the tricarboxylic of the acid tricarboxylic (TCA) cycle acid in (TCA) primary cycle metabolism. in primary metabolism. While degradation While degra- of malic aciddation during of malic grape acid ripening during isgrape well ripeningknown, itis is well still known, not clear it howis still this not process clear how modulates this anyprocess biochemical modulates pathways any biochemical that may pathways affect OTA that synthesis. may affect OTA synthesis.

(a)

(b)

FigureFigure 5. 5.Effect Effect of of different different sugar sugar concentrations concentrations and pH changes changes on on GLA GLA accumulation accumulation and and relative relative expression expression of Acgox of Acgox gene in vitro. Solid SGM media was supplemented with different sugar concentrations (glucose/fructose at a ratio of 2.33), gene in vitro. Solid SGM media was supplemented with different sugar concentrations (glucose/fructose at a ratio of 2.33), and inoculated with 100 µL of a 106 spores/mL suspension. (a) Acgox relative expression, and (b) GLA accumulation were µ 6 andanalyzed inoculated 6 days with post 100-inoculation.L of a 10 Relativespores/mL expression suspension. was normalized (a) Acgox relative using β- expression,tubulin as an and internal (b) GLA control. accumulation The expres- were analyzedsion of 6gox daysgene post-inoculation. under a sugar concentrati Relative expressionon of 1% at was pH normalized 3 was normalized using β as-tubulin 1.0. Error as anbars internal represent control. a standard The expression error of goxof threegene independent under a sugar biological concentration replicates. of 1%Different at pH 3letters was normalizedabove the columns as 1.0. Errorindicate bars statistically represent significant a standard difference error of threes independentat p < 0.05, asbiological determined replicates. using the Different Tukey’s letters honest above significant the columns difference indicate test. statistically significant differences at p < 0.05, as determined using the Tukey’s honest significant difference test. 3.4. The Role of Other Grape Nutrition Factors in OTA Biosynthesis The influence of another two key nutrient factors in grapes, tartaric acid and catechin, 3.4.on OTA The Role synthesis of Other was Grape examined Nutrition in the Factors current in OTAstudy. Biosynthesis Grapes are one of the few fruits to The influence of another two key nutrient factors in grapes, tartaric acid and catechin, on OTA synthesis was examined in the current study. Grapes are one of the few fruits to contain tartaric acid. Tartaric acid accumulates in grape berries along with malic acid J. Fungi 2021, 7, 10 10 of 12

during the ﬁrst period of the grapes’ growth; these acids are responsible for the acidity of the future wine. During the ripening stage, malic acid is metabolized and used as an energy source; its proportion decreases toward the tartaric acid production, which remains almost unchanged [44]. The concentration range of tartaric acid found in grapes is 1.28–7.45 g/L [45]. In our in vitro experiments, no signiﬁcant change in OTA accumulation was observed in the presence of increasing tartaric acid concentrations in solid media from 0 to 14 g/L (Figure S2a). These results suggest that, unlike malic acid, tartaric acid is not associated with OTA biosynthesis. Catechin is one of the main phenolic compounds in grape seeds and skins, with amounts ranging between 74 and 205 mg/100 g of dry matter [46]. This compound is proven to be a potent antioxidant and to possess important biological, pharmacological and medicinal properties [47]. Similarly to the experiment with tartaric acid, supplementation of different concentrations of catechin to the SGM media did not affect mycotoxin production by A. carbonarius (Figure S2b), suggesting that there is no evidence for the involvement of this phenolic compound in the modulation of OTA synthesis. These results are in contrast with those reported previously by Kumar et al. [21], where phenolic compounds like chlorogenic acid and epicatechin, were shown to differentially modulate laeA expression and patulin synthesis in P. expansum. Moreover, other studies demonstrated antifungal and antimycotoxigenic activities of phenolic compounds in several agricultural commodities [48,49]. We assume that, in general, one would expect large differences in the response of different fungal pathogens to phenolic compounds, mostly determined by the fungal genus and/or specific host.

4. Conclusions This study was important in improving our basic understanding of the mechanisms by which a number of the grape key host factors may affect mycotoxin accumulation during fruit colonization. Our ﬁndings strongly suggest that a series of intrinsic critical host factors that change during fruit ripening, including sugars, organic acids and pH, have a signiﬁcant impact on A. carbonarius capability to produce OTA in grapes. A strong in vivo–in vitro correlation was observed throughout the study, where limited sugar and low pH levels but high malic acid concentrations activated OTA biosynthesis by the fungus both in synthetic media and during fruit colonization through modulation of laeA and OTA gene cluster expression. Regulation of secondary metabolism by fruit host factors could serve as a model system for understanding the pH-regulating processes in fungal pathogens. In addition, modulation of fruit sugar content and/or fruit tissue pH during disease development might enable designing control measures that will focus on mitigating the consequences of the pathogen invasion.

Supplementary Materials: The following are available online at https://www.mdpi.com/2309-6 08X/7/1/10/s1, Table S1: Primer sequences used for qRT-PCR analysis; Figure S1: Effect of sugar concentrations and pH changes on the relative expression creA gene; Figure S2: Effect of tartaric acid and catechin on OTA accumulation in vitro. Author Contributions: Conceptualization, D.B.P., Y.B. and E.S.; designed the experiments, U.M., O.B., D.B.P. and E.S.; performed the experiments, U.M., O.B., S.S., and V.Z.; analyzed the data, U.M., O.B. and S.S.; wrote the manuscript, D.B.P., O.B. and E.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a grant (No. 2432/18) from the Joint Israel Science Foundation (ISF) and the National Natural Science Foundation of China (NSFC) Research Program. Acknowledgments: The authors would like to thank Amnon Lichter for his assistance and advice on physiological aspects of grapes. Conﬂicts of Interest: The authors declare no conﬂict of interest. J. Fungi 2021, 7, 10 11 of 12

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