Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.
ABSTRACT
Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.
ANNOUNCEMENT
Aspergillus flavus is an opportunistic pathogen that infects plants, animals, and humans and produces aflatoxin, a natural carcinogen (1–4). Aflatoxin consumed in grains or milk is acutely toxic, stunts children’s growth, and induces liver cancer (1, 5–8). Aspergillus flavus has the potential to produce several toxic secondary metabolites beyond aflatoxins B1 and B2, including cyclopiazonic acid, kojic acid, β-nitropropionic acid, aspertoxin, aflatrem, and aspergillic acid (1–4). The fungus may also cause allergic reactions in humans and aspergillosis diseases, notably in immunocompromised people’s lungs (5–8). Under favorable hot and dry field conditions, A. flavus can cause corn ear rot, which is frequently associated with aflatoxin contamination inside diseased corn kernels (9–12). Aflatoxin contamination in corn threatens consumer food safety and grower economic stability, causing an estimated $52 million to over $1 billion of economic loss per year in the United States (12). In Louisiana, a severe corn aflatoxin outbreak in 1998 resulted in almost total crop loss, and periodic aflatoxin outbreaks still occur (https://www.farmprogress.com/aflatoxin-levels-low-louisiana-corn). Aspergillus flavus populations are very diverse and may consist of many different vegetative compatibility groups (VCGs) (2, 3, 9, 13–15). Members within a VCG share identical het locus alleles and belong to quasiclonal lineages exhibiting similar quantities of aflatoxin production (some VCGs do not produce any aflatoxins), sclerotial sizes, and mating types (2, 3, 9, 13, 15). Aspergillus flavus was isolated on differential/selective medium from 50 soil samples (255 isolates) and 25 surface-sterilized corn kernels (612 isolates) from 85 individual ears collected at maturity from 11 cornfields in Louisiana (15). The isolates belonged to at least 16 VCGs (15). A selection of 20 isolates encompassing all 16 VCGs were deposited in the USDA-SRRC’s culture collection.
To prepare for sequencing, 75-ml potato dextrose broth conidial cultures were shaken overnight at 125 rpm and 30°C. Mycelial pellets were collected, flash frozen with liquid nitrogen, and freeze-dried, and DNA was extracted with a Quick-DNA fungal/bacterial DNA miniprep kit (Zymo Research, Irvine, CA). Genomic DNA (gDNA) quality and purity were assessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE), and quality control was performed using a Qubit 2.0 fluorometer (Life Technologies, Burlington, Canada). Sequencing libraries for each gDNA extract were prepared using a Nextera XT DNA sample prep kit (Illumina, San Diego, CA) in 2 × 150-bp paired-end format and pooled in a single NextSeq 500/550 midoutput kit v2 (n = 8) for sequencing with an Illumina NextSeq sequencer. Low-quality reads were trimmed to a quality threshold of Q > 30 using Trimmomatic (16) with a NexteraPE adapter file. Trimmed reads were subjected to de novo assembly using the SPAdes assembler v3.12.0 (17), and assembly quality assessment utilized QUAST (18). Default settings were used for all software programs unless otherwise noted.
Data availability.
Genome sequences of these 20 Aspergillus flavus isolates were deposited in DDBJ/ENA/GenBank under BioProject number PRJNA482816. The genome sequencing and assembly statistics (contig number, read number, genome size, N50 value, G+C content, and SRA and whole-genome sequence [WGS] accession numbers) for each isolate are shown in Table 1. This announcement reports the first versions of these genome sequences.
TABLE 1.
Aspergillus flavus strains, aflatoxin production, and genomic statistics data
| Genome identifier | Strain/VCG | Aflatoxigenicity | Aflatoxin B1 production (ppb) ± SEb | No. of contigs | No. of reads | Genome size (bp) | N50 (bp) | Coverage (×) | % G+C | SRA accession no. | GenBank accession no. |
|---|---|---|---|---|---|---|---|---|---|---|---|
| MOD1-573 | RRS4 | Aflatoxigenic | 11,900 ± 836 | 670 | 34,952,640 | 37,023,989 | 132,464 | 42 | 48.3 | SRR11596619 | JABVYR000000000 |
| MOD1-575 | RRS7 | Aflatoxigenic | 20 ± 6 | 624 | 31,401,164 | 36,757,764 | 142,633 | 39 | 48.3 | SRR11596618 | JABVYS000000000 |
| MOD1-576 | RRS5 | Aflatoxigenic | 24,006 ± 3,918 | 688 | 34,833,608 | 37,068,150 | 126,389 | 42 | 48.2 | SRR11596607 | JABVYT000000000 |
| MOD1-578 | RRS9 | Aflatoxigenic | 3,872 ± 1,026 | 652 | 40,610,742 | 37,193,948 | 152,925 | 48 | 48.3 | SRR11596606 | JABVYU000000000 |
| MOD1-580 | RRS1 | Aflatoxigenic | < 0 | 683 | 35,257,442 | 36,938,434 | 121,985 | 42 | 48.3 | SRR11596605 | JABVYV000000000 |
| MOD1-581 | RRS10 | Nonaflatoxigenic | 0 ± 0 | 723 | 39,417,907 | 36,954,782 | 112,933 | 39 | 48.3 | SRR11596604 | JABVYW000000000 |
| MOD1-584 | RRS11 | Aflatoxigenic | 1,714 ± 120 | 595 | 29,738,152 | 36,865,287 | 144,290 | 41 | 48.3 | SRR11596603 | JABVYX000000000 |
| MOD1-586 | RRS3 | Aflatoxigenic | 16,162 ± 150 | 756 | 35,670,908 | 37,089,482 | 143,643 | 42 | 48.3 | SRR11596602 | JABVYY000000000 |
| MOD1-587 | RRS12 | Aflatoxigenic | 42,548 ± 9,686 | 701 | 30,328,260 | 36,985,390 | 136,341 | 40 | 48.3 | SRR11596601 | JABVYZ000000000 |
| MOD1-590 | RRS2 | Aflatoxigenic | 14,032 ± 4,858 | 635 | 32,785,962 | 36,946,864 | 138,751 | 45 | 48.3 | SRR11596600 | JABVZA000000000 |
| MOD1-591 | RRS13 | Nonaflatoxigenic | 0 ± 0 | 626 | 35,283,328 | 36,626,824 | 141,548 | 49 | 48.3 | SRR11596617 | JABVZB000000000 |
| MOD1-595 | RRS14 | Nonaflatoxigenic | 0 ± 0 | 623 | 32,152,852 | 36,915,067 | 152,851 | 45 | 48.2 | SRR11596616 | JABVZC000000000 |
| MOD1-599 | RRS8 | Aflatoxigenic | 27,998 ± 9,260 | 723 | 38,755,062 | 37,006,081 | 152,745 | 42 | 48.2 | SRR11596615 | JABVZD000000000 |
| MOD1-601 | RRS5 | Aflatoxigenic | 19,114 ± 332 | 867 | 40,051,400 | 37,973,590 | 170,945 | 57 | 48.2 | SRR11596614 | JABVZE000000000 |
| MOD1-605 | RRS6 | Nonaflatoxigenic | 0 ± 0 | 627 | 36,745,078 | 36,775,823 | 155,768 | 52 | 48.2 | SRR11596613 | JABVZF000000000 |
| MOD1-607 | RRS15 | Aflatoxigenic | 3,968 ± 432 | 1,167 | 21,467,712 | 38,231,283 | 85,770 | 30 | 48.2 | SRR11596612 | JABVZG000000000 |
| MOD1-618 | RRSSOLOa | Aflatoxigenic | 12,460 ± 2,650 | 546 | 36,203,968 | 36,963,370 | 169,107 | 51 | 48.3 | SRR11596611 | JABVZH000000000 |
| MOD1-619 | RRS1 | Aflatoxigenic | 35 | 908 | 39,434,866 | 36,935,936 | 129,357 | 110 | 48.3 | SRR11596610 | JABVZI000000000 |
| MOD1-620 | RRS1 | Aflatoxigenic | 3 | 1,226 | 42,922,302 | 37,016,679 | 143,589 | 108 | 48.2 | SRR11596609 | JABVZJ000000000 |
| MOD1-621 | RRS1 | Aflatoxigenic | 3 | 1,660 | 37,110,996 | 36,895,533 | 138,343 | 105 | 48.3 | SRR11596608 | JABVZK000000000 |
RRSSOLO refers to an isolate that did not complement any others and therefore was in a singleton VCG.
Aflatoxin B1 (AFB1) was quantified with ultraperformance liquid chromatography from each of three independent 4-day-old cultures grown in glucose-salts medium. AFB2 was detected in lower quantities for each extract with AFB1. MOD1-619, MOD1-620, and MOD1-621 were previously quantified from single cultures grown on rice (15).
ACKNOWLEDGMENTS
This project is supported by the U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment.
We thank Kanniah Rajasekaran of USDA-ARS (New Orleans, LA) for his continued support of aflatoxin research.
The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the FDA, or the U.S. Government. References to commercial materials, equipment, or processes do not in any way constitute endorsement.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Genome sequences of these 20 Aspergillus flavus isolates were deposited in DDBJ/ENA/GenBank under BioProject number PRJNA482816. The genome sequencing and assembly statistics (contig number, read number, genome size, N50 value, G+C content, and SRA and whole-genome sequence [WGS] accession numbers) for each isolate are shown in Table 1. This announcement reports the first versions of these genome sequences.
TABLE 1.
Aspergillus flavus strains, aflatoxin production, and genomic statistics data
| Genome identifier | Strain/VCG | Aflatoxigenicity | Aflatoxin B1 production (ppb) ± SEb | No. of contigs | No. of reads | Genome size (bp) | N50 (bp) | Coverage (×) | % G+C | SRA accession no. | GenBank accession no. |
|---|---|---|---|---|---|---|---|---|---|---|---|
| MOD1-573 | RRS4 | Aflatoxigenic | 11,900 ± 836 | 670 | 34,952,640 | 37,023,989 | 132,464 | 42 | 48.3 | SRR11596619 | JABVYR000000000 |
| MOD1-575 | RRS7 | Aflatoxigenic | 20 ± 6 | 624 | 31,401,164 | 36,757,764 | 142,633 | 39 | 48.3 | SRR11596618 | JABVYS000000000 |
| MOD1-576 | RRS5 | Aflatoxigenic | 24,006 ± 3,918 | 688 | 34,833,608 | 37,068,150 | 126,389 | 42 | 48.2 | SRR11596607 | JABVYT000000000 |
| MOD1-578 | RRS9 | Aflatoxigenic | 3,872 ± 1,026 | 652 | 40,610,742 | 37,193,948 | 152,925 | 48 | 48.3 | SRR11596606 | JABVYU000000000 |
| MOD1-580 | RRS1 | Aflatoxigenic | < 0 | 683 | 35,257,442 | 36,938,434 | 121,985 | 42 | 48.3 | SRR11596605 | JABVYV000000000 |
| MOD1-581 | RRS10 | Nonaflatoxigenic | 0 ± 0 | 723 | 39,417,907 | 36,954,782 | 112,933 | 39 | 48.3 | SRR11596604 | JABVYW000000000 |
| MOD1-584 | RRS11 | Aflatoxigenic | 1,714 ± 120 | 595 | 29,738,152 | 36,865,287 | 144,290 | 41 | 48.3 | SRR11596603 | JABVYX000000000 |
| MOD1-586 | RRS3 | Aflatoxigenic | 16,162 ± 150 | 756 | 35,670,908 | 37,089,482 | 143,643 | 42 | 48.3 | SRR11596602 | JABVYY000000000 |
| MOD1-587 | RRS12 | Aflatoxigenic | 42,548 ± 9,686 | 701 | 30,328,260 | 36,985,390 | 136,341 | 40 | 48.3 | SRR11596601 | JABVYZ000000000 |
| MOD1-590 | RRS2 | Aflatoxigenic | 14,032 ± 4,858 | 635 | 32,785,962 | 36,946,864 | 138,751 | 45 | 48.3 | SRR11596600 | JABVZA000000000 |
| MOD1-591 | RRS13 | Nonaflatoxigenic | 0 ± 0 | 626 | 35,283,328 | 36,626,824 | 141,548 | 49 | 48.3 | SRR11596617 | JABVZB000000000 |
| MOD1-595 | RRS14 | Nonaflatoxigenic | 0 ± 0 | 623 | 32,152,852 | 36,915,067 | 152,851 | 45 | 48.2 | SRR11596616 | JABVZC000000000 |
| MOD1-599 | RRS8 | Aflatoxigenic | 27,998 ± 9,260 | 723 | 38,755,062 | 37,006,081 | 152,745 | 42 | 48.2 | SRR11596615 | JABVZD000000000 |
| MOD1-601 | RRS5 | Aflatoxigenic | 19,114 ± 332 | 867 | 40,051,400 | 37,973,590 | 170,945 | 57 | 48.2 | SRR11596614 | JABVZE000000000 |
| MOD1-605 | RRS6 | Nonaflatoxigenic | 0 ± 0 | 627 | 36,745,078 | 36,775,823 | 155,768 | 52 | 48.2 | SRR11596613 | JABVZF000000000 |
| MOD1-607 | RRS15 | Aflatoxigenic | 3,968 ± 432 | 1,167 | 21,467,712 | 38,231,283 | 85,770 | 30 | 48.2 | SRR11596612 | JABVZG000000000 |
| MOD1-618 | RRSSOLOa | Aflatoxigenic | 12,460 ± 2,650 | 546 | 36,203,968 | 36,963,370 | 169,107 | 51 | 48.3 | SRR11596611 | JABVZH000000000 |
| MOD1-619 | RRS1 | Aflatoxigenic | 35 | 908 | 39,434,866 | 36,935,936 | 129,357 | 110 | 48.3 | SRR11596610 | JABVZI000000000 |
| MOD1-620 | RRS1 | Aflatoxigenic | 3 | 1,226 | 42,922,302 | 37,016,679 | 143,589 | 108 | 48.2 | SRR11596609 | JABVZJ000000000 |
| MOD1-621 | RRS1 | Aflatoxigenic | 3 | 1,660 | 37,110,996 | 36,895,533 | 138,343 | 105 | 48.3 | SRR11596608 | JABVZK000000000 |
RRSSOLO refers to an isolate that did not complement any others and therefore was in a singleton VCG.
Aflatoxin B1 (AFB1) was quantified with ultraperformance liquid chromatography from each of three independent 4-day-old cultures grown in glucose-salts medium. AFB2 was detected in lower quantities for each extract with AFB1. MOD1-619, MOD1-620, and MOD1-621 were previously quantified from single cultures grown on rice (15).