Aspergillus flavus and Aspergillus parasiticus produce carcinogenic aflatoxins during crop infection, with extensive variations in production among isolates, ranging from atoxigenic to highly toxigenic. Here, we report draft genome sequences of one A. parasiticus isolate and nine A. flavus isolates from field environments for use in comparative, functional, and phylogenetic studies.
ABSTRACT
Aspergillus flavus and Aspergillus parasiticus produce carcinogenic aflatoxins during crop infection, with extensive variations in production among isolates, ranging from atoxigenic to highly toxigenic. Here, we report draft genome sequences of one A. parasiticus isolate and nine A. flavus isolates from field environments for use in comparative, functional, and phylogenetic studies.
ANNOUNCEMENT
Aspergillus flavus and Aspergillus parasiticus are fungi that exist as saprophytes in soil environments and can, under favorable environmental conditions such as drought and heat stress, colonize compromised plant tissues. These adverse environmental conditions stimulate production by the fungi of carcinogenic mycotoxins called aflatoxins (1, 2). Aflatoxin contamination of important crop species such as maize and peanut poses a significant threat to global food safety and security, particularly in developing countries (1). Isolates of these fungi vary in their ability to produce aflatoxins, and the mechanisms contributing to exacerbated aflatoxin production under environmental stresses are hypothesized to be related to oxidative stress tolerance and host plant composition (3, 4). To investigate these phenomena, we sequenced the genomes of one A. parasiticus isolate and nine A. flavus isolates, with varying levels of aflatoxin production and oxidative stress tolerance, from field environments for use in comparative analyses (5, 6).
The A. flavus isolates A1, A9, AF36 (NRRL18543), Afla-Guard (NRRL21882-3), Tox4, VCG1, and VCG4 were obtained from the Louisiana State University AgCenter, Department of Plant Pathology and Crop Physiology (Baton Rouge, LA, USA). The A. flavus isolates K49 (NRRL30797) and K54A were obtained from the USDA Agricultural Research Service (ARS) Biological Control of Pests Research Unit (Stoneville, MS, USA). The A. parasiticus isolate NRRL2999 was obtained from the USDA ARS Northern Regional Research Center (Peoria, IL, USA). The isolates used in this study were isolated from cotton, maize, and peanut plants or fields within the continental United States except for NRRL2999, which originated from Uganda. The isolates were cultured on yeast extract with supplements (YES) liquid medium (2% [wt/vol] yeast extract, 1% [wt/vol] sucrose) for 5 days at 30°C in the dark. Mycelial mats from each culture were collected, ground in liquid nitrogen with a chilled mortar and pestle, and used for DNA isolation with cetyltrimethylammonium bromide (CTAB). Following DNA extraction with chloroform-isoamyl alcohol (24:1), the phases were separated by centrifugation. The DNA was then precipitated using isopropanol and pelleted by centrifugation before being suspended in TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA [pH 8.0]). Isolated DNA was then submitted to the Novogene Corp. (Sacramento, CA, USA) for quality checking, sequencing, and initial data filtering. Libraries (350-bp insert size) were generated using a NEB Ultra II DNA library preparation kit (New England Biolabs, Ipswich, MA, USA) and then were used for short-read paired-end (150 bp) sequencing on a HiSeq 4000 platform (Illumina, San Diego, CA, USA). Generated optical sequencing data were transformed into raw sequencing reads using Casava (v 1.8; Illumina) base calling. Adapters were then trimmed, and low-quality sequencing reads (with a Q score of <20) were filtered and removed by discarding paired-end reads if one read contained >10 bases aligned to adapters (<10% mismatches allowed), >10% N bases, or >50% low-quality bases (with a Phred quality score of <5).
Clean reads were then used for de novo contig assembly using SPAdes (v 3.13.1) with default k values (7). Contigs of <500 bp were removed from each assembly. Contigs were scaffolded using RaGOO (v 1.1) with the Aspergillus flavus AF13 genome as a reference (GenBank accession numbers CP059858 to CP059865) (8). Assembly statistics for each isolate genome are shown in Table 1. For the A. parasiticus NRRL2999 isolate, previous studies showed conserved gene contents, similar chromosome structures, and a high degree of genetic relatedness with respect to A. flavus, allowing A. flavus genomes to be useful references for assembly (8, 9). These new assemblies will be useful for comparative genomic analyses to investigate the root causes of variations in aflatoxin production capability and stress tolerance among these fungi.
TABLE 1.
Aspergillus flavus and Aspergillus parasiticus isolate genomes
| Species | Isolate | Aflatoxin productiona | Amt of filtered data (Mbp) | Score of >Q30 (%) | GC content (%) | Estimated coverage (×) | No. of contigs | Contig N50 (bp) | No. of scaffolds | Scaffold N50 (Mbp) | Assembled length (Mbp) | SRA accession no.b | GenBank accession no.c |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A. flavus | A1 | − | 4,679.8 | 92.0 | 47.3 | 71 | 1,123 | 436,753 | 8 | 5.043 | 37.254 | SRR11486427 | CP051059 (Chr 1), CP051060 (Chr 2), CP051061 (Chr 3), CP051062 (Chr 4), CP051063 (Chr 5), CP051064 (Chr 6), CP051065 (Chr 7), CP051066 (Chr 8) |
| A9 | + + + | 5,631.6 | 92.5 | 47.2 | 83 | 1,581 | 357,152 | 8 | 5.377 | 37.366 | SRR11486426 | CP051035 (Chr 1), CP051036 (Chr 2), CP051037 (Chr 3), CP051038 (Chr 4), CP051039 (Chr 5), CP051040 (Chr 6), CP051041 (Chr 7), CP051042 (Chr 8) | |
| AF36 | − − − | 10,151.0 | 92.2 | 47.5 | 168 | 609 | 355,215 | 8 | 4.959 | 37.642 | SRR11486425 | CP051019 (Chr 1), CP051020 (Chr 2), CP051021 (Chr 3), CP051022 (Chr 4), CP051023 (Chr 5), CP051024 (Chr 6), CP051025 (Chr 7), CP051026 (Chr 8) | |
| Afla-Guard | − − − | 11,128.2 | 91.8 | 47.5 | 171 | 680 | 518,829 | 8 | 5.091 | 37.392 | SRR11486424 | CP051067 (Chr 1), CP051068 (Chr 2), CP051069 (Chr 3), CP051070 (Chr 4), CP051071 (Chr 5), CP051072 (Chr 6), CP051073 (Chr 7), CP051074 (Chr 8) | |
| K49 | − − − | 5,514.9 | 92.5 | 47.5 | 68 | 1,593 | 424,252 | 8 | 5.280 | 37.271 | SRR11486423 | CP051075 (Chr 1), CP051076 (Chr 2), CP051077 (Chr 3), CP051078 (Chr 4), CP051079 (Chr 5), CP051080 (Chr 6), CP051081 (Chr 7), CP051082 (Chr 8) | |
| K54A | − | 6,145.6 | 92.2 | 47.6 | 40 | 805 | 380,569 | 8 | 5.206 | 37.622 | SRR11486422 | CP051083 (Chr 1), CP051084 (Chr 2), CP051085 (Chr 3), CP051086 (Chr 4), CP051087 (Chr 5), CP051088 (Chr 6), CP051089 (Chr 7), CP051090 (Chr 8) | |
| Tox4 | + + + | 5,669.2 | 88.0 | 47.3 | 87 | 895 | 336,276 | 8 | 4.842 | 37.354 | SRR11486421 | CP051043 (Chr 1), CP051044 (Chr 2), CP051045 (Chr 3), CP051046 (Chr 4), CP051047 (Chr 5), CP051048 (Chr 6), CP051049 (Chr 7), CP051050 (Chr 8) | |
| VCG1 | − | 5,501.2 | 94.1 | 48.1 | 141 | 589 | 463,008 | 8 | 5.024 | 36.933 | SRR11486420 | CP051091 (Chr 1), CP051092 (Chr 2), CP051093 (Chr 3), CP051094 (Chr 4), CP051095 (Chr 5), CP051096 (Chr 6), CP051097 (Chr 7), CP051098 (Chr 8) | |
| VCG4 | + + + | 5,939.2 | 94.1 | 48.1 | 152 | 658 | 401,225 | 8 | 5.050 | 37.048 | SRR11486419 | CP051051 (Chr 1), CP051052 (Chr 2), CP051053 (Chr 3), CP051054 (Chr 4), CP051055 (Chr 5), CP051056 (Chr 6), CP051057 (Chr 7), CP051058 (Chr 8) | |
| A. parasiticus | NRRL2999 | + (BG) | 5,699.0 | 92.2 | 47.0 | 45 | 2,321 | 434,276 | 8 | 5.243 | 37.049 | SRR11486418 | CP051027 (Chr 1), CP051028 (Chr 2), CP051029 (Chr 3), CP051030 (Chr 4), CP051031 (Chr 5), CP051032 (Chr 6), CP051033 (Chr 7), CP051034 (Chr 8) |
Aflatoxin production in each isolate: −, atoxigenic; − − −, atoxigenic biological control isolate; + + +, highly toxigenic; + (BG), produces B and G aflatoxins.
Sequence Read Archive (SRA) accession numbers for raw data.
GenBank accession numbers for scaffolded chromosomes (Chr).
Data availability.
The genome sequences for these A. flavus and A. parasiticus isolates have been deposited in the NCBI GenBank under BioProject PRJNA607981. Accession numbers for each isolate genome are listed in Table 1.
ACKNOWLEDGMENTS
We thank Billy Wilson and Sheron Simpson for technical assistance in the laboratory. We also thank Kenneth Damann and Hamed Abbas for providing the isolates sequenced in this work.
This work is partially supported by the USDA ARS, USDA National Institute for Food and Agriculture (NIFA) Agriculture and Food Research Initiative Proposal 2017-07176, the Georgia Agricultural Commodity Commission for Corn, the National Corn Growers Association Aflatoxin Mitigation Center of Excellence, the Georgia Peanut Commission, the National Peanut Board, and the Peanut Research Foundation.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.
REFERENCES
- 1.Amaike S, Keller NP. 2011. Aspergillus flavus. Annu Rev Phytopathol 49:107–133. doi: 10.1146/annurev-phyto-072910-095221. [DOI] [PubMed] [Google Scholar]
- 2.Fountain JC, Scully BT, Ni X, Kemerait RC, Lee RD, Chen ZY, Guo B. 2014. Environmental influences on maize-Aspergillus flavus interactions and aflatoxin production. Front Microbiol 5:40. doi: 10.3389/fmicb.2014.00040. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Roze LV, Hong SY, Linz JE. 2013. Aflatoxin biosynthesis: current frontiers. Annu Rev Food Sci Technol 4:293–311. doi: 10.1146/annurev-food-083012-123702. [DOI] [PubMed] [Google Scholar]
- 4.Yang L, Fountain JC, Ji P, Ni X, Chen S, Lee RD, Kemerait RC, Guo B. 2018. Deciphering drought‐induced metabolic responses and regulation in developing maize kernels. Plant Biotechnol J 16:1616–1628. doi: 10.1111/pbi.12899. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fountain JC, Scully BT, Chen ZY, Gold SE, Glenn AE, Abbas HK, Lee RD, Kemerait RC, Guo B. 2015. Effects of hydrogen peroxide on different toxigenic and atoxigenic isolates of Aspergillus flavus. Toxins (Basel) 7:2985–2999. doi: 10.3390/toxins7082985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Fountain JC, Bajaj P, Pandey MK, Nayak SN, Yang L, Kumar V, Jayale AS, Chitikineni A, Zhuang W, Scully BT, Lee RD, Kemerait RC, Varshney RK, Guo B. 2016. Oxidative stress and carbon metabolism influence Aspergillus flavus transcriptome composition and secondary metabolite production. Sci Rep 6:38747. doi: 10.1038/srep38747. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Fountain JC, Clevenger JP, Nadon B, Youngblood RC, Korani W, Chang P-K, Starr D, Wang H, Isett B, Johnston HR, Wiggins R, Agarwal G, Chu Y, Kemerait RC, Pandey MK, Bhatnagar D, Ozias-Akins P, Varshney RK, Scheffler BE, Vaughn JN, Guo B. 18 August 2020. Two new Aspergillus flavus reference genomes reveal a large insertion potentially contributing to isolate stress tolerance and aflatoxin production. G3 (Bethesda) doi: 10.1534/g3.120.401405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Linz JE, Wee J, Roze LV. 2014. Aspergillus parasiticus SU-1 genome sequence, predicted chromosome structure, and comparative gene expression under aflatoxin-inducing conditions: evidence that differential expression contributes to species phenotype. Eukaryot Cell 13:1113–1123. doi: 10.1128/EC.00108-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The genome sequences for these A. flavus and A. parasiticus isolates have been deposited in the NCBI GenBank under BioProject PRJNA607981. Accession numbers for each isolate genome are listed in Table 1.