**8.3 Identification of** *A. flavus* **from the stored maize grain samples collected from some selected growing areas of Bangladesh**

Morphological identification of *A.flavus* was detected by using petridish and culture plate method as well as observing microscopic figures under compound and stereo microscope (**Figure 2A(a)–(d)**). Thirty five fungal isolates were identified using primers specific to ITS 1 and ITS 4 regions. PCR assays of *A. flavus* DNA with ITS 1 and ITS 4 primers amplified a single fragment of about 600 bp which revealed that all the isolates obtained were fungi. Sequence analysis of ITS region by BLAST program revealed that all the isolates obtained from maize were belong to *A. flavus* (**Figure 3A**).

### **8.4 PCR based identification and confirmation of aflatoxin producing**  *Aspergillus flavus* **species obtained from maize grain samples**

AF02\_Ran, AF01\_Lal, AF01\_Bog, AF02\_Bog, AF03\_ Jas, AF04\_ Jas, AF01\_Chu, AF03\_Kis, AF04\_Kis, AF01\_Man were identified by PCR amplification of ITS region using ITS1 and ITS4 primers and the results of PCR showed an amplification size 600 bp confirmed the fungal isolates (**Figure 3A**) and their several strains were found in Rangpur (*A. flavus* strain 64-A1), Lalmonirhat (*A. flavus* strain SGE22), Bogura (*A. flavus* strain SGE22 and *A. flavus* strain bpo4), Jashore (*A. flavus* and *A. flavus* isolate AA221), Chuadanga (*A. flavus* strain JN-YG-3-5), Kishoreganj (*A. flavus* strain 64-A1 and *A. flavus* strain ND26), Manikganj (*A. flavus* strain SU-16).

PCR products were then sequenced and analysis of sequence data of amplified ITS region using BLAST program revealed that fungal isolates AF01\_Man, AF03\_ Jas, AF02\_Ran obtained from maize grain samples collected from Manikganj, Jashore, Rangpur revealed the highest homology of 99.33 %, 99.17 %, 95.74 % with the *A. flavus strain* SU-16, *A. flavus, A. flavus strain* 64-A1. Other sevel isolates obtained from Lalmonirhat (AF01\_Lal), Bogura (AF01\_Bog), Bogura (AF02\_Bog), Jashore (AF04\_ Jes), Chuadanga (AF01\_Chu), Kishoreganj (AF03\_Kis), Kishoreganj (AF04\_Kis) showed significant homology with different strains of *A. flavus* (**Table 2**).

**Figure 1.**

*Linear correlations between toxigenic* A. flavus *infected maize grains and total aflatoxins concentration.*

### **Figure 2.**

*(A) Composite photographs of* Aspergillus *spp*. *in different sections. (a) Apparent growth of* Aspergillus *spp. on the maize grain surface, (b) enlarged view of individual maize grain showing the growth of* Aspergillus *spp., morphology of suspected* Aspergillus *spp. (c) Yellowish green colonies of* A. flavus *on PDA, (d) vesicle with less conidial ornamentation with conidiphores of* A. flavus. *(B) Composite photographs of* Fusarium *spp. in different sections. (a) Apparent growth of* Fusarium *spp. on the maize grain surface, (b) enlarged view of individual maize grain showing the growth of* Fusarium *spp., morphology of suspected* Fusarium *spp., (c) pinkish white growth of* F. proliferatum *on PDA, (d) microconidia of* F. proliferatum *without septum under microscope with 40× magnification, (e) whitish growth of* F. oxysporum *on PDA and (f) Micro and macroconidia (with septum) of* F. oxyporum *without septum. Culture photographs were taken at 7 days after inoculation and microscopic photographs were taken with 40× magnification using compound light microscope equipped with a digital camera.*

### **Figure 3.**

*(A) PCR amplification of ITS region from the genomic DNA of the fungal isolates using ITS-1 and ITS-4 primers and (B) PCR amplification of nor, omt, apa-2 gene from the genomic DNA of the fungal isolates obtained from obtained from fifteen maize growing areas of Bangladesh M: 1 kb plus DNA ladder, 1, AF02\_Ran: Rangpur, 2, AF01\_Lal: Lalmonirhat, 3, AF01\_Bog: Bogura, 4, AF02\_Bog: Bogura, 5, AF03\_Jas: Jassore, 6, AF04\_Jas: Jashore, 7, AF01\_Chu: Chuadanga, 8, AF03\_Kis: Kishoreganj, 9, AF04\_Kis:Kishoreganj, 10, AF01\_Man: Manikgan.*


### *Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat… DOI: http://dx.doi.org/10.5772/intechopen.101647*



### *Maize Genetic Resources - Breeding Strategies and Recent Advances*

*Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat… DOI: http://dx.doi.org/10.5772/intechopen.101647*

When the isolates of *Aspergillus* Spp. were analyzed by PCR for aflatoxin producing ability using *nor*, *omtA*, *apa-2* genes based primers from fifteen maize growing areas. The result showed the amplified DNA fragment was 400 bp, 1024 bp, 1032 bp confirmed that the *A. flavus* isolates had the ability to produce aflatoxin that encode *nor*, *omtA*, *apa-2* genes (**Figure 3B**). Only six species showed a positive result with *nor*, *omtA*, *apa-2* genes set of primers. The result indicated *A. flavus* strains were aflatoxins producers as those were an evident from our investigation (**Figure 3B**).

PCR products were sequenced using ITS-1 primer and sequence data were analyzed by homology search using BLAST Nucleotide program. Isolates were identified as different *A. flavus* based on the homology percentage with their closest relatives available in the NCBI database.
