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Department of Botany & Plant Pathology, Purdue University, 915 State Street, West Lafayette, Indiana 47907-2054
Brad L. Reuhs
Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907-2009
Charles P. Woloshuk 1
Department of Botany & Plant Pathology, Purdue University, 915 State Street, West Lafayette, Indiana 47907-2054
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Wicklow 1995). When harvested grain is placed in a storage facility, it is vulnerable to insect and microbial activity. Without intervention, the grain will be degraded by a succession of competing species. To minimize the biodegradation, microbial growth is managed by drying the grain and storing it in the driest environment that is economically feasible. The dry environment gives an advantage to xerophilic and xerotolerant fungi, including a number of Aspergillus species (Dehoff et al 1984, Ayerst 1986, Marin et al 1998, Wicklow et al 1998) with Eurotium Link:Fr teleomorphs. This group of fungi also spoils other dried foods, including jams, dried salted fish and sponge cake (Splittstoesser et al 1989, Wheeler and Hocking 1993, Abellana et al 1999).
Many experimental approaches have been used to study the effect of water availability on the growth of stored grain fungi (Ayerst 1969, Pitt and Hocking 1977, Marin et al 1998, Gock et al 2003). Although growth optima indicate that these fungi have an advantage in dry environments, the maintenance of internal water potential appears to be aided by the internal accumulation of polyhydroxyl alcohols (polyols), especially glycerol, that lower the osmotic potential across the fungal cell membrane (Ramos et al 1999, Nesci et al 2004). To date no study has measured gene expression in this group of fungi in stored grain or under conditions that mimic the moisture equilibrium conditions that occur in a grain bin. We were intrigued by a glass-fiber filter disk assay developed by Norton (1995) to study the effect of different nutrients on aflatoxin production in Aspergillus flavus Link. Norton grew the fungus on a glass-fiber disk suspended in a closed scintillation vial, and a damp environment was maintained by the presence of water at the bottom of the vial. The study presented here addressed the question of whether Norton’s disk-method could be used to measure growth (ergosterol content and conidia production), polyol content and gene expression in response to various water activities. E. rubrum König, Spieck. & Bremer (anamorph: Aspergillus rubrobrunneus Samson & Gams, synonym: A. ruber [König, Spieck. & Bremer] Thom & Church) was grown on disks in equilibrium with various salt solutions. Data obtained from E. rubrum were compared with A. flavus and A. nidulans (Eidam) Wint. (teleomorph: Emericella nidulans (Eidam) Vuill). The results indicate that the disk method is ideal to study gene expression and polyol production in Eurotium rubrum during growth in dry environments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and a match to the internal transcribed spacer region/rDNA sequence (GenBank Number AF455528
[GenBank]
). The E. rubrum isolate was maintained on an asparagine minimal medium (AMM, consisting of 1 g/L KH2PO4, 0.5 g/L MgSO4, 0.5 g/L KCl, 1.6 g/L L-asparagine; pH adjusted to 6.5 with KOH with the addition of 330 g/L sucrose and 15 g/L agar (AMM33 agar). A. flavus NRRL 3357 originated from the collection of USDA National Center for Agricultural Utilization Research, Peoria, Illinois, and was maintained on AMMG5 agar medium (AMM medium plus 50 g/L glucose and 15 g/L agar). A. nidulans A850 was obtained from the collection of J-R Xu of Purdue University and maintained on potato-dextrose agar (PDA; B&D, Sparks, Maryland). Conidia were harvested in 0.01% Triton-X 100 (Sigma, St Louis, Missouri). Conidial suspensions of 6000 conidia/mL were prepared in AMMG1 medium (AMM medium plus 10 g/L glucose).
Growth conditions.— –
Fungi were grown on glass-fiber filter disks (13 mm; Grade GF/B, Pall Life Sciences, Ann Arbor, Michigan) suspended from the caps of scintillation vials (20 mL) with stainless steel insect pins (37 mm; BioQuip Products, Gardena, California) as described by Norton (1995). Water activities were attained with 1.8 mL of these solutions in the bottom of the vials: saturated potassium sulfate (0.98 aw), 3.8 M sodium chloride (0.86 aw), 4.5 M sodium chloride (0.83 aw), and saturated ammonium chloride (0.78 aw) (Robinson and Stokes 1959). Disks were inoculated with 115 µL of conidial suspension, with 57.5 µL applied to the top and bottom of the disk. Vials were incubated at 28 C, and disks were collected at various times for analysis of ergosterol, polyols and conidial production. Experiments were repeated at least twice, and consistent results were obtained.
Conidial production.— – To quantify conidial production, each disk was placed in a test tube containing 2.5 mL of 0.01% Triton-X 100. The tube was vortexed 10 s, and conidia were counted with a hemacytometer.
Polyol analysis.— –
For extraction of polyols, disks were placed in 2.0 mL microcentrifuge tubes that contained 1.5 mL of ethanol, incubated at 65 C for 10 min, and stored at –20 C until analysis (Hocking 1986). Before analysis, xylitol (10 µg) was added to 200 µL of sample extract as an internal standard and the extract was dried under air. Polyols were dissolved in 0.1 mL of Tri-Sil Reagent (Pierce, Rockford, Illinois) and incubated 30 min at 80 C for derivitization according to the supplier’s methodology. After derivization, the polyols were separated by gas chromatography (GC) with a Hewlett-Packard 5890 Series II Gas Chromatograph equipped with a DB-1 fused silica column (I.D.: 0.25 mm; length 150 m; Agilent Technologies, Palo Alto, California) and a flame ionization detector. With a helium flow rate of 2 mL/min, the column-temperature program was 2 min at 114 C, increased to 180 C at a rate of 3 C/min, then to 250 C at a rate of 4 C/min, and 3.5 min at 250 C. These methods were as described by Madaj et al (1993), except helium was used here as the carrier gas. The detection limit was 10 pg. Polyol quantification was based on a xylitol standard curve and individual polyol standards. Glucose was not measured in this study; however, it was present in the polyol extracts and recorded on the chromatographs.
Ergosterol analysis.— –
Ergosterol was extracted from disks (two disks per sample) overnight with 5 mL chloroform: methanol (2:1 v/v) at room temperature and with mild agitation (Woloshuk et al 1979). Extracts were dried, and the residue was dissolved in 500 µL of methanol. Ergosterol was analyzed with a Beckman 126 HPLC (Beckman Coulter, Fullerton, California) equipped with a 250 mm x 4.6 mm ODS (octadecylsilane) column and a UV detector. The solvent system was methanol (1 mL/min), and ergosterol was detected at 282 nm. Quantities were calculated based on a dilution curve for an ergosterol standard. The detection limit was 100 ng.
Gene expression.— – E. rubrum was grown on glass disks at 0.86 aw and 0.98 aw as described above. After 6 d disks were placed in sterile 1.5 mL microcentrifuge tubes, frozen in liquid nitrogen and stored at –80 C. RNA was extracted from the disks with the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, California), and the cDNA template was generated from the RNA as described by Pirtilla et al (2004). Six E. rubrum genes were selected for expression analysis. Five genes were identified in the sequence analysis of an E. rubrum cDNA library. These genes were HYD1 (GenBank accession number DN738668 [GenBank] ), a putative hydrophobin; PMA1 (GenBank accession number DN738669 [GenBank] ), a putative plasma-membrane proton-ATPase; TAG1 (Gen-Bank accession number DN738670 [GenBank] ), a putative triacylglycerol lipase; MST1 (GenBank accession number DN738671 [GenBank] ), a putative monosaccharide transporter; and UBI1 (GenBank accession number DN738672 [GenBank] ), a homolog of ubiquitin. A putative mannitol-1-phosphate dehydrogenase (MPD1, GenBank accession number AY987026 [GenBank] ) was isolated from E. rubrum by PCR with these degenerate primers: MPDdgF (5'-CATCATGGGWAARAAGGCTATCCAG-3') and MPDdgR (5'-GGTCCAATGAACCGTTCCTTG-3').
For quantitative real-time PCR (qPCR) analysis, these primers were obtained from Integrated DNA Technologies (Coralville, Iowa): HYDRTF (5'-CTTGGTCTCTTCGACGAGTGCTC-3'), HYDRTR (5'-TGAGACCACCCTTAGATTCGCTG-3'), PMARTF (5'-GATGATGAGTAGACGCTGCGCT-3'), PMARTR (5'-GCGCACGCATGTATGCTCAAG-3'), TAGRTF (5'-GTACGTTCCACGGCAGCGA-3'), TAGRTR (5'-TCGTACACAAAGCTGAGGTAATACGAG-3'), MSTRTF (5'-CACCCTCGAGGAACTCGACT-3'), MSTRTR (5'-GCGTGGTGTCGTATGCAATCTC-3'), UBIRTF (5'-CTAATGACTCGTCTATGGGTTGAATGACC-3'), UBIRTR (5'-GTATGCCCGTTAAGCCGTGTCA-3'), MPDRTF (5' AGCCCCTTTGGCTCCGTT-3'), and MPDRTR (5'-GTGTTGACAGTGTAGAGCTTGGG-3'). Reactions were performed in an ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, California), and data were collected with Sequence Detector Software version 1.7 (Applied Biosystems). Each reaction consisted of: 10 µL of QuantiTect SYBR®-green PCR Master mix (QIAGEN, Valencia, California), forward and reverse primers (500 nM of each), cDNA template, and nuclease-free water (Sigma) to a final volume of 20 µL. PCR cycling conditions consisted of 2 min at 50 C (1 cycle); 10 min at 95 C (1 cycle); 15 s at 95 C followed by 1 min at 60 C (40 cycles). Three biological replicates for each water activity were analyzed, and expression of each gene was measured in triplicate. To verify that the efficiencies of the target and reference (UBI1) reactions were approximately equal, reactions were performed with the primers for each EST or UBI1 with serial dilutions of cDNA as template. After verifying that the efficiencies of the primers were acceptable, the expression levels of the selected sequences were calculated by the comparative Ct method (Applied Biosystems) with UBI1 as the endogenous reference for normalization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Conidial production at the two water activities increased over the course of the experiment, although the numbers produced at 0.86 aw were lower at 8 d (FIG. 2). Cleistothecia were produced only at 0.98 aw; however, mature ascospores were not detected until after 10 d.
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The A. nidulans and A. flavus strains grew only at 0.98 aw. Under this condition mycelia formed a mat across the disk surface and conidia were visible after 2 d. Maximum ergosterol content and conidial production were measured at 6 d (FIGS. 1A
; 2B, C). Depletion of glucose in the disks occurred at 4 d (data not shown). By comparison, glucose still was present after 8 d in disks of E. rubrum grown under the same conditions. At 0.86 aw, no growth of A. nidulans or A. flavus was visible after 8 d, and only trace amounts of ergosterol and few conidia were measured (FIGS. 1B; 2B, C). At 0.83 aw and 0.78 aw, the two fungi did not grow. The teleomorph of A. nidulans was not observed on the disks under any condition, nor were sclerotia produced by A. flavus.
Effect of water activity on polyol content.— –
Only trace amounts of total polyols were detectable in the conidial inoculum on the disk (data not shown). The polyol content in E. rubrum reached the maximum after 6 d at both 0.86 aw and 0.98 aw. More total polyols were produced at 0.86 aw than at 0.98 aw (FIG. 3A, B). In contrast A. nidulans and A. flavus reached their maximum polyol content at 2 and 4 days, respectively, when grown at 0.98 aw (FIG. 3A). Total polyol content in these two fungi were low when grown at 0.86 aw (FIG. 3B).
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). Glycerol, erythritol and arabitol also were detected. The amount of glycerol always was greatest at 4 d and decreased over time. The experimental results (FIG. 4A) indicate that glycerol in E. rubrum decreased from nearly 50% of the polyol content at 4 d to about 25% at 8 d. In other experiments glycerol decreased to 5% of the total polyol over the same time period. A similar decrease in glycerol was observed in A. nidulans and A. flavus (FIG. 4C, D). The amount and proportion of erythritol and arabitol also increased during growth at this water activity. Of the small amount of polyols detectable in A. nidulans and A. flavus grown at 0.86 aw, nearly 100% was glycerol (data not shown). The polyols in E. rubrum grown at 0.83 aw were also nearly 100% glycerol (data not shown).
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). In contrast the amount of total polyols in the disks incubated at 0.86 aw decreased only slightly over the same period and glycerol remained the predominate polyol. In the disks transferred to 0.78 aw, total polyol content decreased but glycerol remained the major polyol.
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). When expression data were normalized to expression of UBI1, we observed a similar pattern of expression for two of the genes (MST1, PMA1) regardless of water activity. The expression of TAG1 was higher at 0.86 aw and expression of HYD1 and MPD1 was higher at 0.98 aw. The low average Ct values of UBI1 and HYD1 (<20) suggest a high level of expression for these two genes. The experiment was repeated three times with similar results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Abadias et al 2000, Rosso and Robinson 2001, Nesci et al 2004) or to artificial xerotolerance caused by the uptake of sugar from the media (Brown and Simpson 1973, Pitt and Hocking 1977, Beever and Laracy 1986). Even when liquid-permeable cellophane membranes are used, solute uptake by the fungus is still possible (Beever and Laracy 1986). In addition the amount of sugars needed to adjust the aw of liquid media below 0.90 aw is high, which can be toxic if not prepared properly (Hocking and Pitt 1987).
Based on ergosterol and conidial data, the growth rate of E. rubrum on the disks was essentially the same in the 0.98 and 0.86 aw environments. A direct comparison with the literature was not possible, but several research groups have used microscopic measurements to study growth of xerotolerant fungi, including E. rubrum, over a matrix of conditions (pH, temperature, aw). Guynot et al (2002) studied the growth of E. rubrum on "cake analogue medium," which is a baked product made from wheat flour, vegetable oil, sucrose, eggs and baking powder. This group observed that E. rubrum grew the same at both 0.90 and 0.85 aw (pH 6.5, 25 C) with a marked reduction in growth at 0.80 aw. Ayerst (1969) grew E. rubrum on agar strips suspended over different salt solutions in closed vessels. The growth optimum for three E. rubrum strains varied between 0.96 aw and 0.88 aw and 20 C and 30 C. Gock et al (2003) grew E. rubrum in a medium containing high concentrations of syrup (glucose and fructose). Although these authors did not test 0.98 aw, they reported that E. rubrum grew 1.5 times faster at 0.92 aw (pH 6.5, 25 C), which is near the optimum, than at 0.86 aw. When the incubation temperature was increased to 30 C, the fungus exposed to 0.92 aw grew 2.6 times faster. They also observed a twofold greater germination rate when conidia were incubated at 0.92 aw (pH 6.5) than at 0.86 aw. Growth measurements we obtained in this study on disks with E. rubrum were consistent with these microscopic measures as well as the growth model described by Rosso and Robinsion (2001). One difference we observed was variability in the initial growth on the filters incubated at 0.98 aw. The reason for this variation is not known but might result from delayed germination of conidia under these conditions. By comparison growth of A. flavus and A. nidulans on the disks was robust at 0.98 aw but little measurable growth occurred at 0.86 aw. Other researchers have observed germination and growth at lower aw indicating variability among strains and/or variations due to growth conditions. Ayerst (1969) reported that the optimum temperature/water activity for A. flavus was above 0.98 aw and 30–37 C. At 0.86 aw and 28 C, Ayerst recorded a growth rate of 1 mm/d compared to 8 mm/d when conditions were 0.98 aw and 28 C. Germination also was significantly delayed at the low water activity. Nesci et al (2004) also showed that growth of A. flavus was essentially zero at both 0.937 aw and 0.901 aw. The same results were reported for A. nidulans grown in media with water activity adjusted with NaCl (Beever and Laracy 1986).
At the onset of this study our hypothesis was that fluctuating levels of polyols control the internal water activity in E. rubrum. In line with published literature (Adler et al 1982, Hocking and Norton 1983, Beever and Laracy 1986, Hallsworth and Magan 1995) total polyol content was higher in E. rubrum when grown at 0.86 aw environments than at 0.98 aw, even though growth rates were the same. In terms of composition, mannitol predominated at 0.98 aw, often comprising 80 % of the total polyol. Glycerol, erythritol and arabitol also were detected under these conditions. During growth of the fungus, the amount of glycerol decreased relative to the other polyols, suggesting that at 0.98 aw, glycerol is highest during the germination period. Similar results have been reported for other fungi, including A. nidulans and A. flavus (Hallsworth and Magan 1995, Clark et al 2003, de Vries et al 2003, Ruijter et al 2003, Nesci et al 2004). In contrast glycerol comprised more than 90% of the polyols in E. rubrum grown under 0.86 aw conditions. This indicates that, like other fungi, E. rubrum synthesizes glycerol for osmotic maintenance under low water activities.
When a disk from one water activity is transferred to a vial at higher or lower water activity, time (estimated to be at least 6 h) is necessary for moisture equilibrium to be established on the disk. The response of E. rubrum to changes in water activity revealed that, when transferred to a hypoosomotic environment, polyols in the fungus changed from predominately glycerol at 0.86 aw to predominately mannitol at 0.98 aw within 24 h. The lack of significant amounts of glycerol after 24 h suggests that E. rubrum does not respond as Saccharomyces cerevisiae with the rapid efflux of glycerol via an aquaglyceroporin (Luyten et al 1995). The glycerol is presumably metabolized, shunted to the glucogenesis pathway, and then enters the mannitol cycle (Ruijter et al 2003). The hyperosmotic switch from 0.86 aw to 0.78 aw did not induce an increase in total polyol, and glycerol remained the predominate polyol.
As a method to analyze gene expression, ample RNA for qPCR analysis was isolated from the disk. The generally low Ct values observed in this study were generated with a 1:500 dilution of the cDNA produced from a fourth of the RNA extracted from individual disks. These results suggest that the filter disk technique would be sensitive enough to evaluate gene expression across the entire course of the experiment. Even though measurements were taken late in the growth phase on the disks, significant differences were observed in gene expression of three genes. The nearly twofold higher expression of MPD1 under the 0.98 aw conditions is consistent with its proposed role in mannitol biosynthesis (Ruijter et al 2003). The higher expression of TAG1 under conditions of low water activity suggests that a triacylglycerol lipase is active in the biosynthesis of glycerol. This enzyme has been associated with increased turgor in the appresoria of Magnaporthe grisea during pathogenesis, which is modified by a rapid accumulation of glycerol (Thines et al 2000). We also measured higher expression of HYD1, which encodes a putative hydrophobin, under conditions of higher water activity. Hydrophobins are cell wall proteins that provide surface tension to aerial hyphae (Wösten 2001). Because hydrophobins are also an important component of the cell walls of conidia (Wösten 2001), it is not possible to determine whether the differences observed in expression of HYD1 are in response to water activity or the status of conidia production when samples were collected.
In conclusion this study demonstrates that the disk method is a useful tool to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The method mimics the conditions the fungus experiences in a grain bin environment where changes in moisture are not abrupt. We also have shown differential expression of three E. rubrum genes in response to two moisture environments as well as differential accumulation of polyols.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: woloshuk{at}purdue.edu
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LITERATURE CITED |
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