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Published in final edited form as: Mycologia. 2019 Jul 26;111(5):719–729. doi: 10.1080/00275514.2019.1631137

Diversity of Thermophilic and Thermotolerant Fungi in Corn Grain

Katrina Sandona a, Terri L Billingsley Tobias a, Miriam I Hutchinson b, Donald O Natvig b, Andrea Porras-Alfaro a,b,*
PMCID: PMC6801065  NIHMSID: NIHMS1533296  PMID: 31348716

Abstract

Corn bins in the Midwestern United States can reach temperatures up to 52 C. High temperatures combined with sufficient moisture and humidity in bins provide the perfect environment to promote the growth of thermophilic and thermotolerant fungi. In this article, we characterize for the first time thermophilic and thermotolerant fungi in corn grain bins using culture-based methods and pyrosequencing techniques. Corn samples were collected from local farms in western Illinois. Samples were plated and incubated at 50 C using a variety of approaches. Of several hundred kernels examined, more than 90% showed colonization. Species identified using culture methods included Thermomyces lanuginosus, Thermomyces dupontii, Aspergillus fumigatus, Thermoascus crustaceus, and Rhizomucor pusillus. Pyrosequencing was also performed directly on corn grain using fungal specific primers to determine if thermophilic fungi could be detected using this technique. Sequences were dominated by pathogenic fungi, and thermophiles were represented by less than 2% of the sequences despite being isolated from 90% of the grain samples using culturing techniques. The high abundance of previously undocumented viable fungi in corn could have negative implications for grain quality and pose a potential risk for workers and consumers of corn-derived products in the food industry. Members of the Sordariales were absent among thermophile isolates and were not represented in nuc rDNA ITS sequences. This is in striking contrast with results obtained with other substrates such as litter, dung and soils, where mesophilic and thermophilic members of the Sordariaceae and Chaetomiaceae are common. This absence appear to reflect an important difference between the ecology of Sordariales and other orders within the Ascomycota in terms of the ability to compete in microhabitats rich in sugars and living tissues.

Keywords: Aspergillus, Thermomyces, thermophiles, organic toxic dust syndrome

INTRODUCTION

Thermophilic and thermotolerant fungi were first described by Lindt (1886), Tiklinskaya (1899) and Miehe (1907) in the late nineteenth and early twentieth centuries. The comprehensive study of the physiology and systematics of these organisms, however, did not begin until the second World War (Emerson 1968). Although multiple definitions have been employed for fungal thermophiles, one working definition is that thermophilic fungi are those that grow better at 45 C than at 25 C (Powell et al. 2012). Although this definition is somewhat arbitrary, it can be used to distinguish between thermophilic and thermotolerant fungi, which have the ability to grow at 45 C or above but thermotolerant fungi grow better at lower temperatures (Cooney and Emerson 1964). The optimal growth temperature for most thermophilic fungi is between 40 C and 55 C (Maheshwari et al. 2000). There appears to be an upper limit at 60 C, which has also been proposed as an upper limit for the growth of Eukaryotes (Tansey and Brook 1972).

Of the more than 200,000 known fungal species only approximately 30 true thermophiles have been described (Maheshwari et al. 2000). Thermophiles belong to the orders Sordariales, Eurotiales, Onygenales, and Mucorales (Morgenstern et al. 2012). The most common thermophilic and thermotolerant genera include Thermomyces, Aspergillus, Thermoascus, Rhizomucor, Chaetomium and Mycothermus (Maheshwari et al. 2000; Natvig et al. 2015). In both Eurotiales and Sordariales, thermophilic fungi are not monophyletic and are interspersed with mesophiles (Morgenstern et al. 2012).

The early studies of thermophilic fungi focused on compost (Miehe 1907, Emerson 1968), but it is now clear that they inhabit diverse ecosystems and can be isolated from a wide variety of substrates. They have been found in arid ecosystems (Powell et al. 2012), waterlogged grasslands, aquatic sediments, and the dung of birds and mammals (Singh et al. 2003). The ubiquitous nature of thermophilic fungi may be the result of both spore dispersal and their ability to degrade a wide array of organic materials (Salar and Aneja 2007). The broad distribution of thermophiles also likely reflects the abundance of microhabitats that are rich in carbon sources and experience periods of temperature and moisture conditions favorable for their growth. Such microhabitats occur in all temperate, arid, and tropical ecosystems.

Some thermophilic and thermotolerant fungi are a cause of concern due to their potential effects on human and animal health. For example, Aspergillus fumigatus, a ubiquitous species often considered to be thermotolerant rather than thermophilic, is the primary cause of aspergillosis, a common and often fatal human mycosis (Kousha et al. 2011). Aspergillus flavus, another thermotolerant species, produces aflatoxins that are highly carcinogenic, causing diseases in both humans and livestock (Abbas et al. 2011). Rhizomucor pusillus, a thermophile, is known to cause pulmonary disease, rhinofacial disease, and mycotic endocarditis in immunocompromised patients (Ribes et al. 2000).

In the Midwest, thermophilic and thermotolerant fungi could represent a heretofore unrecognized risk for farmers and consumers. For example, during unloading, the levels of airborne spores in corn silos can reach 105 to 109 CFUs/m3 (Dutkiewicz et al.1989), and the majority of the fungi involved have not been characterized. Health problems associated with grain dust and airborne spores are common, but it is unclear what proportion of dust exposure includes reactions to fungal spores. Organic toxic dust syndrome is a condition associated with the exposure to farm dust during harvesting and grain handling. This condition causes flu like symptoms and can last up to a week with potential complications such as hypersensitive pneumonitis, chronic bronchitis, and asthma (Kirkhorn and Garry 2000).

According to the United States Department of Labor, grain bins are known to reach and maintain high temperatures and emit toxic gases resulting from spoiling grain (https://www.osha.gov/SLTC/grainhandling/). In conjunction with the abundance of organic matter, grain bin conditions can create ideal conditions for the reproduction and growth of thermophilic and thermotolerant fungi.

Because the state of Illinois is a leading producer of corn, with 74,300 farms and over 50 co-op grain storage facilities distributed across 11 million ha, bringing 19 billion dollars to the state annually (https://www.agr.state.il.us/facts-about-illinois-agriculture/), it is important to evaluate the occurrence and any potential risks posed to farmers and the grain supply by thermophilic and thermotolerant fungi. The main objective of this study was to characterize thermophilic fungi in corn storage facilities using a combination of traditional culturing techniques and pyrosequencing and compare them with taxa described in other studies. The use of both techniques will allow us to determine how reliable these methods are for the detection of these poorly studied fungi and will provide the first steps to characterize potential fungi that may be impacting grain quality and farmers health.

MATERIALS AND METHODS

Sample collection. —

Corn grain samples were collected in West Central Illinois, USA. Six samples were collected from available grain at a local farm (182 grams in total) in summer of 2011 and four samples from a commercial co-op were collected (871 grams) in the winter of 2012. The grain was at 15% moisture when sampled in both places. The samples were transported to the laboratory in paper bags and were stored at room temperature prior to processing within a week.

Isolation of thermophilic and thermotolerant fungi from corn grain. —

Fungi were isolated with Emerson yeast starch agar (EYSA; Difco Laboratories, Michigan) with both 50 μg/mL streptomycin and 50 μg/mL tetracycline to prevent bacterial contamination using three methods (approx. 50 seeds per method). In the first method, grain was plated directly on the medium (three corn grains per plate). Plates were incubated at 50 C and monitored for growth every two days for a period of three weeks. Unique fungal morphotypes were transferred to fresh plates of the same medium and were incubated at 50 C to obtain pure cultures.

As a second method of isolation, three hundred corn grains were ground in a mini food processor, and 0.5 g of ground corn was spread on each of 10 EYSA plates with antibiotics and incubated at 50 C. For the third method of isolation, we surfaced sterilized corn grains with 70% ethanol for 5 min, 0.5% bleach solution (5.25% NaOCl) for 5 min, followed by 3×washing with autoclaved deionized water for 3 min per wash. Three seeds were plated per plate on EYSA plates with antibiotics and incubated at 50 C.

All pure isolates were stored in 1.5 mL microcentrifuge tubes with 1 mL sterile mineral oil at 25 C; mycelium samples were also collected and stored at −20 C for DNA extraction.

DNA extraction, PCR and phylogenetics. —

DNA from isolates was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Wisconsin) following the manufacturer’s protocol. The nuc rDNA ITS1-5.8S‐ITS2 (ITS barcode) was amplified using the ITS fungal specific primers: ITS1F (5´-CTTGGTCATTTAGAGGAAGTAA-3´) and ITS4 (5´- TCCTCCGCTTATTGATATGC-3´) (White et al. 1990; Gardes and Bruns 1993). Final PCR concentrations included μ1X GoTaq Green PCR Master Mix (Promega, Wisconsin), 0.2 μM of both ITS1F and ITS4 primers , 0.1% bovine serum albumin (Sigma-Aldrich, Missouri), and < 250 ng of DNA for each sample. PCR was performed under the following conditions: 95 C for 5 min, then 30 cycles at 94 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 45 s, followed by a final extension of 72 C for 7 min. DNA was quantified with gel electrophoresis (1% agarose in Tris acetate EDTA) and a low DNA mass ladder (Invitrogen, California) (Porras-Alfaro et al. 2008).

The PCR products were cleaned using 2 μl Exo-SAP-IT ® (Affymetrix, Ohio) with 3 μL of each PCR product. Sequencing was performed using the BigDye® v1.1 Cycle Sequencing Kit (Applied Biosystems, California). Samples were sequenced in the Department of Biology at the University of New Mexico. Contigs of forward and reverse sequences were edited using Sequencher 4.9 (Gene Codes Corporation, Michigan). The closest database match of each fungal isolate was determined using the Basic Local Alignment Search Tool (BLASTN) in GenBank and UNITE databases (Kõljalg et al. 2013). Alignments were created in MUSCLE and trimmed and edited in MEGA 6 (Tamura et al. 2013). Phylogenetic trees were inferred with the Maximum Likelihood method in MEGA6. We tested several nucleotide substitution models (with “gamma distributed” rates), including Tamura-Nei (results presented here), General Time Reversible, and Jukes-Cantor. All models produced trees with identical or nearly-identical topologies for a given Eurotiales or Mucorales alignment. Bootstrap analysis was conducted for 1000 replicates. Sequences were deposited in GenBank under numbers KT365186-KT365231. Alignments were deposited at TreeBASE (Submission ID 23897).

Next generation sequencing. —

We employed pyrosequencing (454) to determine the extent to which sequences from thermophilic and thermotolerant fungi could be detected in samples obtained directly from corn silos without additional incubation at high temperatures in the laboratory. In addition, this analysis allowed us to determine whether sequences from members of the Sordariales could be detected in samples from which they were absent in culture studies. DNA was extracted from the each individual corn sample described above following the protocol of a MoBio plant extraction kit (MoBio Laboratories, California). Corn was ground in liquid nitrogen prior to the addition of extraction buffer. Samples were processed and subjected to amplicon pyrosequencing by MR DNA laboratories (Molecular Research LP, Texas, www.mrdnalab.com) following the procedures described by Dowd et al. (2008) and Dean et al. (2013). PCR was performed to each sample with the ITS fungal specific primers: ITS1F and ITS4 (see above) with a single step 30-cycle PCR using HotStarTaq polymerase and the Plus Master Mix Kit (Qiagen, California) under the following conditions: 94 C for 60 s, 53 C for 40 s, 72 C for 60 s, and a final elongation of 72 C for 5 min. After PCR, all amplicon products were combined in equal concentrations and purified using Agencourt Ampure beads (Agencourt, Bioscience Corporation, Massachusetts). The samples were sequenced using a Roche 454 FLX Titanium instrument and reagents following the manufacturer’s procedures.

Sequences in a FASTQ file derived from the original 454 Standard Flowgram File (SFF) file were processed using USEARCH v8.1 (Edgar 2013) to remove barcode and primer sequences and to exclude sequences with any barcode nucleotide mismatches or >2 primer nucleotide mismatches. The output sequences were trimmed to 250 nucleotides, filtered with a maximum expected error of 1.0 (such that a maximum of 1 nucleotide per sequence is expected to be incorrectly assigned), and singletons were discarded.

Sequences were clustered into operational taxonomic units (OTUs) with both UPARSE implemented in USEARCH and UCLUST implemented in QIIME (Edgar 2010, Caporaso et al. 2010). Representative OTU sequences were sorted into taxonomic groups using three different methods: 1) comparison against the UNITE database (version 7, 97% threshold, released August 2015; Kõljalg et al. 2013) using taxonomy assignment with BLAST (Altschul 1990) implemented in QIIME, 2) analysis with the RDP Classifier using the Warcup ITS training set (Wang et al. 2007; Deshpande et al. 2016), and 3) direct BLASTN searches against the NCBI nr database (Altschul 1990). Possible amplicon chimeras were identified and eliminated when different regions of a given query sequence matched taxonomically different subject sequences in BLASTN searches. Such presumed chimeras often resulted in discrepancies across the methods used for taxonomic identifications.

In order to assess the relative frequencies of ITS sequences from specific fungal groups, we created a BLAST database for our pyrosequences on a local server and performed searches using BLASTN 2.2.26. This effort included BLASTN searches with ITS sequences from the thermophilic and thermotolerant fungi obtained during the course of this study. In addition, we conducted BLASTN searches with ITS sequences derived from several mesophilic and thermophilic strains from the Sordariales (GenBank accession numbers in parentheses): Chaetomiaceace species Mycothermus thermophilus (JF412007), Chaetomium thermophilum var. coprophilum (KP336795), Thielavia terrestris (CP003011), and Myceliophthora heterothallica (JN659479); and Sordariaceae species Neurospora crassa (AY681193), and Sordaria macrospora (AF246293). The pyrosequences were deposited in Sequence Read Archive (SRA)-NCBI under accession number PRJNA521234.

RESULTS

Thermophilic and thermotolerant fungi were abundant and readily isolated from corn collected from silos at a local grain elevator co-op and a farm in Illinois. Fungal spores and mycelium were visible in surface of the corn samples collected in this study. A total of 62 cultures were isolated and 46 sequences were successfully amplified from pure isolates using ITS primers (TABLE 1). Thermophilic and thermotolerant isolates belonged to two fungal phyla. Ascomycota was the dominant phylum with 85% of the sequences and Mucoromycota was represented by 15% of the sequences. All sequences within Ascomycota and Mucoromycota were represented by the orders Eurotiales and Mucorales, respectively.

TABLE 1.

Fungi isolated from corn in western Illinois, USA and identification using the ITS region.

Seq ID Organism Isolation method Similarity % in Genbank ITS GenBank Number
TAFCg16 Aspergillus fumigatus ground corn 100 KT365186
TAFCg4 Thermomyces lanuginosus ground corn 100 KT365187
TAFCg5 Thermomyces lanuginosus ground corn 100 KT365188
TAFCg7 Thermomyces dupontii (Talaromyces thermophilus)a ground corn 100 KT365189
TAFCg8 Rhizomucor pusillus ground corn 99 KT365190
TAFCn11 Rhizomucor pusillus non-sterilized corn 100 KT365191
TAFCn17 Thermomyces lanuginosus non-sterilized corn 96 KT365192
TAFCn19 Thermomyces lanuginosus non-sterilized corn 100 KT365193
TAFCn2 Rhizomucor pusillus non-sterilized corn 100 KT365194
TAFCN20 Rhizomucor pusillus non-sterilized corn 99 KT365195
TAFCn22 Thermomyces lanuginosus non-sterilized corn 100 KT365196
TAFCn23 Thermomyces lanuginosus non-sterilized corn 100 KT365197
TAFCn24 Thermomyces dupontii non-sterilized corn 100 KT365198
TAFCn26 Thermomyces lanuginosus non-sterilized corn 100 KT365199
TAFCn27 Thermomyces lanuginosus non-sterilized corn 100 KT365200
TAFCn32 Thermomyces lanuginosus non-sterilized corn 99 KT365201
TAFCn33 Thermomyces lanuginosus non-sterilized corn 100 KT365202
TAFCn35 Rhizomucor pusillus non-sterilized corn 98 KT365203
TAFCn36 Thermomyces lanuginosus non-sterilized corn 99 KT365204
TAFCn38 Rhizomucor pusillus non-sterilized corn 99 KT365205
TAFCn48 Thermomyces dupontii non-sterilized corn 100 KT365206
TAFCn5 Thermomyces lanuginosus non-sterilized corn 98 KT365207
TAFCn8 Thermomyces lanuginosus non-sterilized corn 99 KT365208
TAFCn9 Thermomyces lanuginosus non-sterilized corn 100 KT365209
TAFCs1 Thermomyces dupontii sterilized corn 100 KT365210
TCSB113 Thermomyces lanuginosus non-sterilized corn 100 KT365211
TCSB141 Thermomyces lanuginosus non-sterilized corn 100 KT365212
TCSB19 Thermomyces lanuginosus non-sterilized corn 98 KT365213
TCSB2351 Thermomyces lanuginosus non-sterilized corn 100 KT365214
TCSB240 Aspergillus fumigatus non-sterilized corn 100 KT365215
TCSB32 Thermomyces lanuginosus non-sterilized corn 99 KT365216
TCSB34 Thermomyces lanuginosus non-sterilized corn 100 KT365217
TCSB341 Thermomyces lanuginosus non-sterilized corn 100 KT365218
TCSB342 Aspergillus fumigatus non-sterilized corn 99 KT365219
TCSB354 Thermomyces lanuginosus non-sterilized corn 100 KT365220
TCSB36 Thermoascus crustaceus non-sterilized corn 99 KT365221
TCSB5 Thermomyces lanuginosus non-sterilized corn 100 KT365222
TGCB4 Thermomyces lanuginosus non-sterilized corn 100 KT365223
TGCB5 Thermomyces lanuginosus non-sterilized corn 99 KT365224
TGCB6 Thermomyces lanuginosus non-sterilized corn 100 KT365225
TGCB8 Thermomyces lanuginosus non-sterilized corn 99 KT365226
TGCB9 Thermomyces lanuginosus non-sterilized corn 100 KT365227
TGCH10 Thermomyces lanuginosus non-sterilized corn 100 KT365228
TGCH11 Thermomyces lanuginosus non-sterilized corn 100 KT365229
TGCH9 Thermomyces lanuginosus non-sterilized corn 100 KT365230
TGHC6 Thermomyces lanuginosus non-sterilized corn 99 KT365231
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a

The use of Thermomyces dupontii follows the recent combination of Houbraken and Samson (Houbraken et al. 2014)

In the order Eurotiales, Thermomyces was the most abundant genus with 32 isolates of T. lanuginosus (70% of sequences) and four isolates of T. dupontii (9%), followed by Aspergillus fumigatus with three isolates (7%), and Thermoascus crustaceus with one isolate (2%) (TABLE 1). In addition to species in the Eurotiales, we obtained 6 isolates of a species in the Mucorales, Rhizomucor pusillus (12%)(TABLE 1). All fungal taxa were found in the non-sterilized whole corn. In contrast, only Thermomyces dupontii was isolated from surface sterilized seeds (approx. 2% of the seeds). All taxa except species of Thermoascus were found in the ground corn grain showing that sample processing can affect detection of rare taxa. Phylogenetic analysis of the fungi within the order Eurotiales was performed using Genbank sequences as well as fungi isolated from corn grain (TABLE 1, FIG. 1). The thermophilic and thermotolerant isolates from this study in the order Eurotiales clustered with three genera (Thermoascus, Thermomyces, and Aspergillus). The tree topology (FIG. 1) was consistent with previously published phylogenetic studies of thermophiles (Morgenstern et al. 2012; Houbraken et al. 2014), and the major clades exhibited good bootstrap support. We note that until recently Thermomyces dupontii was commonly referred to as Talaromyces thermophilus. The new binomial (Houbraken et al. 2014) resolves a previous problem of polyphyly in Talaromyces.

Figure 1.

Figure 1.

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Phylogenetic analysis of thermophiles in the order Eurotiales. Maximum likelihood tree based on ITS. Bootstrap values (1000 replicates) are shown for branches with > 70% support. Sequences from strains of Coccidioides immitis were used as the outgroup. Sequences from this study are shown in bold. Sequences designated with “ThNM” numbers are from a previous study of thermophiles obtained from an arid grassland in New Mexico (Powell et al. 2012). Genbank numbers for sequences used for comparison are shown in parentheses.

We acknowledge that a compelling argument has been made that A. fumigatus is more properly referred as Neosartorya fumigata (Pitt and Taylor 2014; Taylor et al. 2016). Here, however, to avoid confusion due to the medical importance of this fungus we follow the convention of Genbank taxonomy reflected in submission entries.

Mucoromycota were represented by a single monophyletic clade of Rhizomucor pusillus with 99% bootstrap support (FIG. 2), which is consistent with the report by Hoffmann et al. (2013). The Rhizomucor pusillus isolates were similar to cultures isolated from wine starters and pathogenic strains described in Walther et al. (2013).

Figure 2.

Figure 2.

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Phylogenetic analysis of thermophiles in the order Mucorales. Maximum likelihood tree with the highest log likelihood based on ITS. Bootstrap values (1000 replicates) are shown for branches with > 70% support. Fungal species Phycomyces blakesleeanus was used as the outgroup. Sequences from this study are shown in bold. Genbank numbers for sequences used for comparison are shown in parentheses.

Next-generation sequencing analysis.–

A total of 42,047 sequences were recovered using pyrosequencing. In all, 15,954 sequences were recovered from three samples from 2011, and 26,093 sequences were recovered from four samples from 2012. UPARSE clustering at 97% similarity produced 63 putative OTUs, whereas de novo clustering with UCLUST at 97% similarity produced 178 putative OTUs. Results from taxonomic analyses suggested that the UCLUST analysis overestimated diversity at the level of OTUs. When OTUs were combined at the genus level based on taxonomic analyses, however, the outcomes were nearly identical for the two clustering procedures. Likewise, at the genus level, taxonomic results obtained with the UNITE database, the Warcup RDP classifier and BLAST were in substantial agreement. The numerical results presented here reflect UPARSE clustering combined with BLAST (TABLE 2).

Table 2.

Most common genera detected by 454 Next Generation Sequencing in 2011 and 2012.

Genus Ave. Counts1 Year 2011 SE Ave. Counts Year 2012 SE
Fusarium/Gibberella 1 0.71 2832.3 455
Aspergillus2 2283 2280 501.5 116.5
Eurotium 1853 1131 460.3 63.7
Stenocarpella 0 - 1787 380
Ascosphaera 592 689 41.8 5.5
Alternaria 1.33 1.6 80.8 17.1
Penicillium 11.3 6.6 56.8 10.8
Trichoderma 0 - 51 14.1
Thermoascus2 41.7 20 1.8 1
Epicoccum 0 - 29.3 12.6
Cladosporium 0.33 0.41 27 6.4
Candida 0.67 0.82 22 7.6
Acremonium 0 - 15 9
Cystofilobasidium 0 - 10.8 4.6
Cryptococcus/Filobasidium 0 - 10.5 8.4
Guehomyces 0 - 6.8 4.8
Hyphopichia 0 - 6.3 2.6
Pichia 0 - 5.8 6.6
Wallemia 1 1.2 2 1.6
Galactomyces 0 - 2 2.3
Sporobolomyces 0 - 2 2.3
Diaporthe 0 - 1.5 1
Blastobotrys 1.7 2 0
Hannaella 0 - 1.25 1.44
Pseudogymnoascus 0 - 1.3 1.4
Davidiella 0 - 1 0.8
Khuskia 0 - 1 1.2
Trichosporon 0 - 1 1.2
Debaryomyces 0 - 0.75 0.55
Geotrichum 0 - 0.75 0.55
Oidiodendron 0 - 0.75 0.87
Rhodotorula 0 - 0.8 0.9
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1

Average number of reads for all OTUs assigned to a given genus after sequences were processed and given taxonomic assignments as described in Materials and Methods.

2

Thermoascus was the only genus of known thermophilic fungi represented among the most common genera. As discussed in the Results, although there were no top BLASTN hits to the thermotolerant species Aspergillus fumigatus, we cannot rule out the possibility that sequences from A. fumigatus were entirely absent from the 454 data.

The majority of the taxa in 2011 was represented by a few genera, mainly saprobes and entomopathogens including Aspergillus, Eurotium, and Ascosphaera (TABLE 2). The sequences obtained from the 2012 samples included a substantially larger number from potential plant pathogens. The most common genera recovered included Fusarium/Gibberella, Stenocarpella, Aspergillus, and Eurotium (TABLE 2).

Based on BLAST analyses and sequence alignments, sequences from only one thermophilic species was identified among the pyrosequences, and it was in low abundance relative to non-thermophiles. Thermophile sequences were first tentatively identified by performing BLASTN searches with sequences derived from cultured thermophiles against our pyrosequencing database. Pyrosequences with good BLAST hits were then employed in BLASTN searches against the nr database at GenBank. This procedure identified 176 sequences with top Genbank hits to Thermoacsus crustaceus (identified in all seven samples across both years). The smaller number of sequences reported for Thermoascus in TABLE 2 (132) relative to the number of T. crustaceus sequences identified with BLAST searches (176) reflects the stringent filtering used for OTU identification.

We identified no pyrosequences for the other three species obtained in our culture studies—namely, Thermomyces dupontii, T. lanuginosus, Rhizomucor pusillus and Aspergillus fumigatus. Although sequences from species of Aspergillus were common in the pyrosequencing data (TABLE 2) , when we employed these sequences in GenBank BLAST searches all had top hits to A. niger and species other than A. fumigatus. However, given the high levels of ITS similarity among species of Aspergillus, it may not be possible to rule out completely that sequences of A. fumigatus were present in our data.

In BLASTN searches against our pyrosequencing data, we also failed to identify sequences for any mesophilic or thermophilic members of the Sordariales, using the sequences from members of the Chaetomiaceae and Sordariaceae listed in Materials and Methods. Rather, the sequences from our pyrosequencing data that best matched sequences from the Sordariales were in fact from the Hypocreales and Diaporthales.

DISCUSSION

Thermophilic and thermotolerant fungi constitute a small portion of described fungi, and their detection requires the use of specific temperatures. Therefore, they tend to be overlooked and are understudied in diversity studies (Maheshwari et al. 2000). This study reports for the first time thermophilic and thermotolerant fungi isolated in high rates from grain bins in the United States Midwest. These fungi are difficult to detect using direct sequencing, highlighting potential problems to monitor and study these specialized microbial communities.

Thermophilic and thermotolerant fungi have a wide geographic distribution, for example the isolates of T. dupontii from corn were closely related to taxa found in New Mexico and Finland (Hultman et al. 2010; Powell et al. 2012). The Thermomyces lanuginosus sequences were similar to those of isolates from soil in New Mexico and Japan (Boonlue et al. 2003; Powell et al. 2012). The Thermoascus sequences were similar to those of isolates from New Mexico and Illinois (Peterson 2008; Powell et al. 2012; Higginbotham et al. 2013). The Aspergillus strains were similar to strains isolated from New Mexico and Washington (Rakeman et al. 2005; Powell et al. 2012).

Under suitable conditions, thermophiles can become an important part of microbial biomass. Traditionally, these fungi have been isolated from compost piles and other agricultural refuse (Maheshwari et al. 2000). High temperatures are common in grain storage facilities even during the winter months, and the abundance of organic matter combined with favorable moisture and humidity provide a suitable habitat for thermophiles considering their ability to grow at high temperatures as a competitive strategy (Powell et al. 2012).

We recovered one thermophilic genus from the pyrosequencing data, and different culturing and sterilization techniques resulted in the differential but high recovery of taxa using cultures, demonstrating that thermophilic fungal detection in environmental or culturing surveys may be dependent on isolation methods and the molecular techniques employed. For example, in a previous study, potential thermophilic fungi were detected in very low amounts using direct sequencing of environmental samples (Porras-Alfaro et al. 2011), even though Powell et al. (2012) showed that taxa rarely recovered using molecular methods could be isolated at rates of up to 60% at the same site using culturing techniques. A similar study using pyrosequencing in pressmud (a sugar cane filtrate ) detected only a subset of thermophilic and thermotolerant fungi with respect to ones isolated in culture (de Oliveira et al. 2016). It is not entirely clear whether the low recovery rates in this study reflect the low mass abundance of thermophilic fungi in substrates that have been examined or, instead, represent a failure of current molecular methods to detect thermophiles that are present in environmental samples.

Members of the Sordariales were absent among cultured isolates and pyrosequences. This was true not only with respect to thermophiles, but it was also the case that BLAST searches employing sequences from a diversity of species (listed in Materials and Methods) from the Sordariaceae and Chaetomiaceae failed to reveal any sequences from the Sordariales. Thermophilic members of the Chaetomiaceae are common in surveys from diverse substrates, including compost, soil, litter and other plant materials (Cooney and Emerson 1964; Straatsma et al. 1994; Pan et al. 2010; Powell et al. 2012). Moreover, mesophilic members of the Sordariales are common in surveys of fungi from diverse plant materials, including seeds (e.g., Gallery et al. 2007). We hypothesize that the lack of Sordariales in corn is due to the abundance of simple and non-cellulosic carbohydrates in this substrate that favor the growth of other fungi. Although many members of the Sordariales are capable of growing on media with simple sugars under laboratory conditions, most thermophilic and non-thermophilic species in this group are best known to grow on decaying plant material rich in cellulose (Li et al. 2011a; Busk and Lange 2013).

It is also interesting that the samples from the two different silos differed substantially in the dominant sequences obtained by pyrosequencing. The samples obtained in 2011 were dominated by the Eurotiales, whereas those obtained in 2012 were dominated by members of the Hypocreales with strong representation from the corn pathogen Stenocarpella maydis (Diaporthales). These differences could be a result of multiple factors including different environmental conditions during growing season and harvest including sampling time, as well as potential successional processes as living tissues die in response to aging and rising temperatures during storage. This variability in community composition reflects the challenges of studying specialized microbial communities that could impact the quality of grain as either substrate for ethanol production or feedstock consumption. For example, thermophilic fungi can degrade lignocellulosic biomass at higher temperatures (Berka et al. 2011) and produce secondary metabolites (Guo et al. 2012). Thermophilic fungi may also interact with other important fungi in the corn industry, such as species of Aspergillus and Fusarium that are responsible for mycotoxin production. Both Aspergillus and Fusarium were detected in high numbers in our pyrosequencing data (TABLE 2).

High levels of thermophilic and thermotolerant fungal spores in grain storage facilities are likely to play a role in the multiple health problems reported by farmers that handle grain, including different respiratory illnesses. Although there have not been studies directly linking thermophilic fungi to acute respiratory illnesses such as Organic Toxic Dust Syndrome (OTDS), these health conditions are common on agricultural workers exposed to toxic dust that contains fungal spores in addition of other substances. The CDC’s alert in 1994 reported at least 29 workers affected by microorganisms present on grain (CDC 1994). doPico et al. (1982) and Rask-Andersen (1989) estimated that 30 to 40% of agricultural workers exposed to organic dusts will develop OTDS. Unfortunately, little documentation exists regarding the specific fungi that may be partially responsible for OTDS and related conditions. Endotoxins are present in organic dust and are likely involved in the onset of OTDS (Olenchock et al. 1990; Dutkiewicz 1987). Davis et al. (1975) found that almost 70% of thermophilic and thermotolerant fungi, including Thermomyces and Thermoascus, were highly toxic in in vitro assays, representing a direct potential threat to human health. Despite the economic importance of corn and the high numbers of farmers potentially affected, few efforts have been made to characterize these fungi in corn grain (Dutkiewicz et al. 1989; Linaker and Smedley 2002). Our study represents one of the first reports of thermophiles in corn, but additional studies with a larger number of samples and sites are necessary to determine their potential role in common respiratory conditions observed in farmers and the distribution of these fungi in corn silos. The propagules for these organisms are very likely present in the field prior to and during harvest, and remain in the grain during transport and in storage. High temperature, grain quality, and moisture in silos are likely to favor propagation and sporulation of these fungi.

ACKNOWLEDGMENTS

Authors would like to thank the farmers in Western Illinois that donated the samples and Marina Small and Antonio Rosales for their help processing them. This project was partially supported by Western Illinois University Graduate Student Research and Professional Development Award and the Department of Biological Sciences. We acknowledge technical support from the UNM Department of Biology’s Molecular Biology Facility under National Institutes of Health grant P30GM110907. We thank the UNM Center for Advanced Research Computing, supported in part by the National Science Foundation, for providing high performance computing resources used in this work.

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