**10. PCR based identification and confirmation of fumonisins producing**  *Fusarium* **species obtained from maize grain samples**

F01\_Pan, F02\_Tha, F03\_Din, F04\_Nil, F05\_Ran, F06\_Lal, F07\_Gai, F08\_Bog, F09\_Nat, F010\_Kus, F011\_ Jes, F012\_Chu, F013\_Kis, F014\_Man and F015\_Cum 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 *Fusarium.* PCR products were then sequenced. (**Figure 2A**). Out of fifteen maize growing areas, *F. oxysporum* was found in Panchagarh (*F. oxysporum* strain EP19), Thakurgaon (*F. oxysporum* strain En3), Dinajpur (*F. oxysporum* strain EP19), Nilphamari (*F. oxysporum* strain EP19), Rangpur (*F. oxysporum* strain En3), Natore

### **Figure 5.**

*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 FUM1 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, F01\_Pan: Panchagarh, 2, F02\_Tha: Thakurgoan, 3, F03\_Din: Dinajpur, 4, F04\_Nil: Nilphamari, 5, F05\_Ran: Rangpur, 6, F06\_Lal: Lalmonirhat, 7, F07\_Gai: Gaibandha, 8, F08\_Bog: Bogura, 9, F09\_Nat: Natore, 10, F010\_Kus: Kushtia, 11, F011\_Jes: Jashore, 12, F012\_Chu: Chuadanga, 13, F013\_Kis: Kishoreganj, 14, F014\_Man: Manikganj and 15, F015\_Cum:Cumilla.*

(*F. oxysporum* isolate FH10 18S), Kushtia (*F. oxysporum* strain EP19), Jashore (*F. oxysporum* strain En3), Chuadanga (*F. oxysporum* isolate H200714-017) Manikganj (*F. oxysporum* strain EP19), Cumilla (*F. oxysporum* strain En3) and *F. proliferatum* was found in Lalmonirhat (*F. proliferatum* strain TH11-3), Gaibandha (*F. proliferatum* strain TH11-3), Bogura (*F. proliferatum* strain TH11-3) and Kishoreganj (*F. proliferatum* strain TH11-3).

Fungal isolates F06\_Lal, F07\_Gai, F08\_Bog and F013\_Kis obtained from maize grain samples were collected from Lalmonirhat, Gaibandha, Bogura and Kishoreganj showed the highest homology with *F. proliferatum* strain TH11-3 (**Table 4**). The fungal isolates obtained from maize grain samples collected from


*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 Fusarium species based on the homology percentage with their closest relatives available in the NCBI database. F01\_Pan: Panchagarh, F02\_Tha: Thakurgoan, F03\_Din: Dinajpur, 4, F04\_Nil: Nilphamari, F05\_Ran: Rangpur, F06\_Lal: Lalmonirhat, F07\_Gai: Gaibandha, F08\_Bog: Bogura, F09\_Nat: Natore, F010\_Kus: Kushtia, F011\_Jes: Jashore, F012\_Chu: Chuadanga, F013\_Kis: Kishoreganj, F014\_Man: Manikganj and F015\_Cum: Cumilla.*

### **Table 4.**

*List of Fusarium isolates identified by homology search of sequences of ITS region by BLAST program obtained from maize grain samples collected from fifteen growing areas of Bangladesh.*

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

Panchagarh (F01\_Pan), Thakurgaon (F02\_Tha), Dinajpur (F03\_Din), Nilphamari (F04\_Nil), Rangpur (F05\_Ran), Lalmonirhat (F06\_Lal), Gaibandha (F07\_Gai), Bogura (F08\_Bog), Natore (F09\_Nat), Kustia (F010\_Kus), Jessore (F011\_ Jes), Chuadanga (F012\_Chu), Kishoreganj (F013\_Kis), Manikganj (F014\_Man) and Cumilla (F015\_Cum) showed significant homology with different strains of *F. oxysporum* (**Table 4**).

When the isolates of *Fusarium* species were analyzed by PCR for fumonisins producing ability using *FUM1* gene based primers from fifteen maize growing areas. The result showed the amplified DNA fragment was 183 bp confirmed that the *Fusarium* had the ability to produce fumonisin that encode *FUM1* gene (**Figure 5B**). Only two *Fusarium* species showed a positive result with *FUM1* gene set of primers. The result was contrary as *F. proliferatum* and *F. oxysporum* (**Table 4**) were fumonisin-producers as it was evident from our investigation.
