Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan
Abstract
:1. Introduction
2. Results
2.1. Sesame Seed Samples and Isolation of Fungi Belonging to Aspergillus Section Flavi
2.2. Examination of the Aflatoxigenic Potential of 260 Aspergillus Section Flavi Isolates
2.3. Whole Genome Sequencing and Comparative Analyses of 12 Pakistani Isolates
2.3.1. Phylogenetic Analysis
2.3.2. Nucleotide Variation Analysis
2.3.3. Prediction of High Impact Mutations
2.3.4. Copy Number Variation in the Aflatoxin Cluster Locus
3. Discussions
4. Conclusions
5. Materials and Methods
5.1. Study Area and Sesame Seeds Samples Collection
5.2. Isolation and Morphological Identification of Aspergillus Section Flavi
5.3. Aflatoxigenic and Non-Aflatoxigenic Potential of Aspergillus Section Flavi
5.3.1. Fungal Culture Preparations and Aflatoxin Extraction
5.3.2. HPLC Analysis of Aflatoxins
5.4. Molecular Characterization
5.4.1. DNA Extraction
5.4.2. Genome Sequencing and Quality Control
5.4.3. Variant Calling and Annotation
5.4.4. Phylogenetic Analysis
5.4.5. Genomic Analysis of Aflatoxin Loci
5.5. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Agro-Ecological Zones | Collection Site | Seed Condition | Fr (%) | RD (%) |
---|---|---|---|---|
Rain fed | Rawalpindi | Fresh | 100 | 6.18 |
Stored | 100 | 36.36 | ||
Attock | Fresh | 80 | 7.69 | |
Stored | 100 | 9.82 | ||
Chakwal | Fresh | 60 | 5.10 | |
Stored | 100 | 20 | ||
Irrigated | Hafiz Abad | Fresh | 69.2 | 1.92 |
Stored | 100 | 3.87 | ||
Gujranwala | Fresh | 100 | 5.09 | |
Stored | 100 | 7.85 | ||
Gujrat | Fresh | 40 | 0.89 | |
Stored | 100 | 14.87 | ||
Sargodha | Fresh | 100 | 10.27 | |
Stored | 100 | 18.42 | ||
Bahawalpur | Fresh | 50 | 0.99 | |
Stored | 66.6 | 6.83 |
Parameters | Seed Conditions | Rainfed | Irrigated | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Rawalpindi | Attock | Chakwal | Total | Hafizabad | Gujranwala | Gujrat | Sargodha | Bahawalpur | Total | |||
Aflatoxigenic isolates n(%) | Fresh | 8 (72.73) ** | 6 (54.55) * | 6 (50.00) * | 20(58.82) * | 16 (80.00) *** | 8 (72.73) ** | 3 (20) * | 3 (50) ** | 2 (50) ** | 32 (57.14) ** | |
Store | 20 (80.00) *** | 22 (73.33) ** | 30 (81.08) *** | 72(78.26) ** | 20 (80.00) *** | 12 (80) *** | 16 (80) *** | 9 (90) *** | 7 (87.5) *** | 64 (82.05) *** | ||
No. of isolates showing AFB1, AFB2, AFG1 and AFG2 | AFB1 | Fresh | 8 | 4 | 3 | 15 | 16 | 8 | 3 | 3 | 2 | 32 |
Store | 15 | 18 | 27 | 60 | 19 | 11 | 14 | 8 | 7 | 59 | ||
AFB2 | Fresh | 5 | 4 | 3 | 12 | 3 | 3 | 0 | 2 | 0 | 8 | |
Store | 8 | 7 | 14 | 29 | 7 | 7 | 7 | 4 | 2 | 27 | ||
AFG1 | Fresh | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Store | 0 | 2 | 2 | 4 | 2 | 3 | 0 | 2 | 0 | 7 | ||
AFG2 | Fresh | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Store | 0 | 2 | 2 | 4 | 0 | 0 | 0 | 2 | 0 | 2 | ||
Non-aflatoxigenic isolates n (%) | Fresh | 3 (27.27) * | 5 (45.45) * | 6 (50.00) * | 14(41.10) * | 4 (20.00) * | 3 (27.27) * | 12 (80) *** | 3 (50) ** | 2 (50) ** | 24 (42.85) * | |
Store | 5 (20.00) * | 8 (26.67) * | 7 (18.92) | 20(21.74) * | 5 (20.00) * | 3 (20.00) * | 4 (20) * | 1 (10)c | 1 (12.5) | 14 (17.95) |
Parameters | Seed Conditions | Rainfed | Irrigated | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Rawalpindi | Attock | Chakwal | Total | Hafizabad | Gujranwala | Gujrat | Sargodha | Bahawalpur | Total | |||
Average Aflatoxin (µgkg−1) | AFB1 | Fresh | 128.31 ** | 18.25 | 50.23 * | 65.14 * | 20.84 | 55.98 * | 23.55 | 121.13 ** | 55.29 * | 41.67 * |
Stored | 117.82 * | 128.23 ** | 78.06 * | 105.23 ** | 196.14 ** | 93.50 * | 302.99 *** | 20.91 | 37.64 | 165.08 ** | ||
AFB2 | Fresh | 10.90 | 8.34 | 6.98 | 8.69 | 5.61 | 3.55 | 0.00 | 5.71 | 0.00 | 3.31 | |
Stored | 11.42 | 4.44 | 12.13 | 9.43 | 5.00 | 13.60 | 6.65 | 16.85 | 8.00 | 8.90 | ||
AFG1 | Fresh | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Stored | 0.00 | 2.07 | 1.54 | 1.29 | 0.8196 | 1.41 | 0.00 | 4.64 | 0.00 | 1.13 | ||
AFG2 | Fresh | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | |
Stored | 0.00 | 1.05 | 0.99 | 0.74 | 0.00 | 0.00 | 0.00 | 3.92 | 0.00 | 0.50 | ||
Range of Aflatoxin (µgkg−1) | AFB1 | Fresh | 1.48–361.20 | 6.20–75.33 | 8.90–363.26 | 1.48–363.2 | 6.50–34.22 | 5.39–135.01 | 4.89–180.70 | 11.33–405.50 | 20.70–200 | 4.89–405.57 |
Stored | 2.78–357.60 | 49.97–350 | 3.74–171.10 | 2.78–357.6 | 21.84–616.40 | 2.72–367.94 | 9.43–2503.10 | 15.34–48.95 | 8.25–75.5 | 2.72–2503.1 | ||
AFB2 | Fresh | 15.00–33.00 | 8.91–28.60 | 2.00–50.78 | 2.00–50.78 | 10.27–56.13 | 3.52–20.27 | – | 5.25–29.03 | – | 3.52–56.13 | |
Stored | 20.00–50.10 | 10.0–29.57 | 5.33–64.40 | 29.50–64.4 | 15.16–60.25 | 15.27–40.60 | 20.22–40.40 | 30.32–60.20 | 5.67–58.34 | 5.67–60.25 | ||
AFG1 | Fresh | – | – | – | – | – | – | – | – | – | – | |
Stored | – | 5.40–56.58 | 10.60–46.40 | 5.40–56.58 | 2.26–18.23 | 2.28–10.90 | – | 8.74–37.60 | – | 2.2–37.64 | ||
AFG2 | Fresh | – | – | – | – | – | – | – | – | – | – | |
Stored | – | 2.23–29.29 | 10.23–26.70 | 2.23–29.29 | – | – | – | 14.11–25.10 | – | 14.11–25.10 |
Isolates Number | Morphological Identification | Aflatoxin Production | Aflatoxin Level (µgkg−1) | Molecular Identification | NCBI Accession Number |
---|---|---|---|---|---|
AFP1 | Aspergillus flavus | High aflatoxin production | 2503.71 | Aspergillus flavus | SRX9628150 |
AFP2 | Aspergillus flavus | High aflatoxin production | 1599.62 | Aspergillus flavus | SRX9630624 |
AFP3 | Aspergillus flavus | High aflatoxin production | 616.48 | Aspergillus flavus | SRX9630884 |
AFP4 | Aspergillus flavus | High aflatoxin production | 405.57 | Aspergillus flavus | SRX9631392 |
AFP5 | Aspergillus flavus | High aflatoxin production | 399.26 | Aspergillus flavus | SRX9631389 |
AFP6 | Aspergillus flavus | High aflatoxin production | 367.94 | Aspergillus flavus | SRX9631420 |
AFP7 | Aspergillus flavus | Medium aflatoxin production | 110.54 | Aspergillus flavus | SRX9631445 |
AFP8 | Aspergillus flavus | Medium aflatoxin production | 119.45 | Aspergillus flavus | SRX9631446 |
AFP9 | Aspergillus flavus | Medium aflatoxin production | 139.23 | Aspergillus flavus | SRX9631391 |
AFP10 | Aspergillus flavus | Very low aflatoxin production | 4.15 | Aspergillus flavus | SRX9631390 |
AFP11 | Aspergillus flavus | Very low aflatoxin production | 16.80 | Aspergillus flavus | SRX9631387 |
AFP12 | Aspergillus flavus | Very low aflatoxin production | 26.72 | Aspergillus flavus | SRX9631388 |
SNPs | |||||||
---|---|---|---|---|---|---|---|
Isolate | Annotation | Gene_id | CHR | SITE | REF | ALT | Type |
AFP10 | aflLa/hypB | AFLA_139240 | EQ963478 | 2205372 | C | T | stop gained |
AFP10 | aflU/cypA/P450 monooxygenase | AFLA_139430 | EQ963478 | 2252122 | A | G | splice donor variant & intron variant |
AFP10 | EQ963478 | 2252148 | G | A | stop gained | ||
Indels | |||||||
Isolate | Annotation | Gene_id | CHR | SITE | REF | ALT | Type |
AFP10 | aflY/hypA/hypP | AFLA_139150 | EQ963478 | 2,187,887 | CTGG | TTGA | stop gained |
AFP2 | aflI/avfA/cytochrome P450 monooxygenase | AFLA_139230 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained |
AFP1 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained | ||
AFP10 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained | ||
AFP4 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained | ||
AFP8 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained | ||
AFP11 | EQ963478 | 2,204,748 | CAGC | TAGG | stop gained | ||
AFP2 | aflL/verB/desaturase/P450 monooxygenase | AFLA_139250 | EQ963478 | 2,205,720 | CACTGAGCTGGCCCC | GATTGACCTGGGCGCA | frameshift variant & missense variant |
AFP1 | EQ963478 | 2,205,720 | CACTGAGCTGGCCCC | GATTGACCTGGGCGCA | frameshift variant & missense variant | ||
AFP4 | EQ963478 | 2,205,720 | CACTGAGCTGGCCCC | GATTGACCTGGGCGCA | frameshift variant & missense variant | ||
AFP8 | EQ963478 | 2,205,720 | CACTGAGCTGGCCCC | GATTGACCTGGGCGCA | frameshift variant & missense variant | ||
AFP11 | EQ963478 | 2,205,720 | CACTGAGCTGGCCCC | GATTGACCTGGGCGCA | frameshift variant & missense variant | ||
AFP10 | aflN/verA/monooxygenase | AFLA_139280 | EQ963478 | 2,211,911 | ATACA | GTACC | splice donor variant & splice region variant & synonymous variant & intron variant |
AFP10 | aflT/aflT/transmembrane protein | AFLA_139420 | EQ963478 | 2,250,339 | CTTGATA | TTTAATG | stop gained |
2,250,372 | AAGAGAGAGAGAGAGAGAGAAAGAAAGAAGAAT | AAGAGAGAGAGAGAGAGAGAGAAAGAAAGAAGAAG | frameshift variant & missense variant | ||||
AFP10 | aflU/cypA/P450 monooxygenase | AFLA_139430 | EQ963478 | 2,251,958 | TGA | TAGA | frameshift variant |
2,251,976 | GAATTTCA | GATGGTTCA | frameshift variant & stop gained |
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Ajmal, M.; Alshannaq, A.F.; Moon, H.; Choi, D.; Akram, A.; Nayyar, B.G.; Gibbons, J.G.; Yu, J.-H. Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan. Toxins 2022, 14, 117. https://doi.org/10.3390/toxins14020117
Ajmal M, Alshannaq AF, Moon H, Choi D, Akram A, Nayyar BG, Gibbons JG, Yu J-H. Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan. Toxins. 2022; 14(2):117. https://doi.org/10.3390/toxins14020117
Chicago/Turabian StyleAjmal, Maryam, Ahmad F. Alshannaq, Heungyun Moon, Dasol Choi, Abida Akram, Brian Gagosh Nayyar, John G. Gibbons, and Jae-Hyuk Yu. 2022. "Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan" Toxins 14, no. 2: 117. https://doi.org/10.3390/toxins14020117
APA StyleAjmal, M., Alshannaq, A. F., Moon, H., Choi, D., Akram, A., Nayyar, B. G., Gibbons, J. G., & Yu, J.-H. (2022). Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan. Toxins, 14(2), 117. https://doi.org/10.3390/toxins14020117