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Molecular Characterization of Aspergillus flavus Strains Isolated from Animal Feeds

Hadjer Saber
Hadjer Saber
, 
Yahia Chebloune
Yahia Chebloune
 e 
Abdallah Moussaoui
Abdallah Moussaoui
   | 21 dic 2022
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Article Category: Original Paper
Pubblicato online: 21 dic 2022
Pagine: 589 - 599
Ricevuto: 09 giu 2022
Accettato: 21 set 2022
DOI: https://doi.org/10.33073/pjm-2022-048
Parole chiave
© 2022 Hadjer Saber et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Phenotypic characterization of A. flavus isolates grown on A. flavus/parasiticus agar (AFPA) medium. Colony color: white-green colony diameter 18 mm, colony reverse color: yellowish orange after seven days of incubation at 25°C.
Phenotypic characterization of A. flavus isolates grown on A. flavus/parasiticus agar (AFPA) medium. Colony color: white-green colony diameter 18 mm, colony reverse color: yellowish orange after seven days of incubation at 25°C.

Fig. 2

PCR amplification of intergenic sequences of the four isolates. IGS-F and IGS-R primers were used for PCR amplification of the intergenic sequences in total DNA isolated from the four fungal isolates as described in Material and Methods. A total of 5 μl of each PCR product was separated in a 1.5% agarose gel by electrophoresis. Lane 1 kb ladder (L) DNA marker, Aspergillus flavus isolates are indicated on the top of each lane. The sizes of selected DNA fragments are indicated on the sides of the panels.
PCR amplification of intergenic sequences of the four isolates. IGS-F and IGS-R primers were used for PCR amplification of the intergenic sequences in total DNA isolated from the four fungal isolates as described in Material and Methods. A total of 5 μl of each PCR product was separated in a 1.5% agarose gel by electrophoresis. Lane 1 kb ladder (L) DNA marker, Aspergillus flavus isolates are indicated on the top of each lane. The sizes of selected DNA fragments are indicated on the sides of the panels.

Fig. 3

PCR and RFLP analyses of fungal amplicons. A total of 5 μl of PCR products of each of the isolates (FZM1, FAK45, FSZ47 and FDY50) was digested with Bgl II restriction endonuclease as described in Materials and Methods. Nondigested (ND) and digested (D) products were loaded and separated by electrophoresis in a 2% agarose gel. A 100 bp DNA ladder (L) was used as molecular weight marker, the positions of 100, 500 and 1,000 bp fragments are indicated. Positions of the 362, 210 and 102 bp fragments generated by BglII digestion are indicated.
PCR and RFLP analyses of fungal amplicons. A total of 5 μl of PCR products of each of the isolates (FZM1, FAK45, FSZ47 and FDY50) was digested with Bgl II restriction endonuclease as described in Materials and Methods. Nondigested (ND) and digested (D) products were loaded and separated by electrophoresis in a 2% agarose gel. A 100 bp DNA ladder (L) was used as molecular weight marker, the positions of 100, 500 and 1,000 bp fragments are indicated. Positions of the 362, 210 and 102 bp fragments generated by BglII digestion are indicated.

Fig. 4

Nucleotide sequence alignment of the four isolates with four reference strains. The names of the (.) four isolates are in green. Nucleotide sequence alignment was performed using Clustal software (–) represent similarity (.), represent deletion. Nucleotide changes are shown in bold font.
Nucleotide sequence alignment of the four isolates with four reference strains. The names of the (.) four isolates are in green. Nucleotide sequence alignment was performed using Clustal software (–) represent similarity (.), represent deletion. Nucleotide changes are shown in bold font.

Fig. 5

Maximum Likelihood phylogenic tree showing the relationships between the examined A. flavus isolates and reference strains, based on aflR/aflS(J) intergenic sequence.
Maximum Likelihood phylogenic tree showing the relationships between the examined A. flavus isolates and reference strains, based on aflR/aflS(J) intergenic sequence.

Nucleotide variations in aflR-aflS(J) intergenic region sequences of Aspergillus flavus isolates.

Nucleotide position Nucleotide variation Isolate
103, 385, 478 C with T (substitution) FZM1: Aspergillus flavus strain FZM1 aflR-aflS(J) intergenic region, partial sequence
161 Insertion of G
380, 477, 508 T with C (substitution)
475 C with G (inversion)
479–481 Insertion of ACA
482 A with G (substitution)
483 G with T (substitution)
544 A with T (inversion)
50 T with C (substitution) FAK45: Aspergillus flavus strain FAK45 aflR-aflS(J) intergenic region, partial sequence
103 C with T (substitution)
3 C with T (substitution) FDY50: Aspergillus flavus strain aflR-aflS(J) intergenic region, partial sequence
5, 28 G with A (substitution)

Genbank accession numbers of the Aspergillus flavus isolates.

Isolate Accession number Strain
FZM1 OL944584.1 A. flavus strain FZM1 aflR-aflJ intergenic region, partial sequence
FAK45 OL944586.1 A. flavus strain FAK45 aflR-aflJ intergenic region, partial sequence
FSZ47 OL944587.1 A. flavus strain FSZ47 aflR-aflJ intergenic region, partial sequence
FDY50 OL944588.1 A. flavus strain FDY50 aflR-aflJ intergenic region, partial sequence

Aspergillus flavus isolates identity based on the BLAST NCBI data.

Strain Identity (%)
FZM1 Aspergillus flavus strain A9 chromosome 3 (97.48%)
FAK45 Aspergillus flavus strain A9 chromosome 3 (98.12%)
FSZ47 Aspergillus flavus strain SU-16 chromosome 3 (99.37%)
FDY50 Aspergillus flavus strain SU-16 chromosome 3 (99.67%)

Genbank accession numbers of the Aspergillus flavus reference strains.

Accession number Strain
CP051037.1 A. flavus strain A9 chromosome 3
CP051085.1 A. flavus strain K54A chromosome 3
CP051045.1 A. flavus strain Tox4 chromosome 3
CP047251.1 A. flavus strain SU-16 chromosome 3
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