**6. PCR based identification of mycotoxigenic** *Fusarium* **spp**

### **6.1 Primers for PCR amplification**

Primers specific for fumonisins producing *Fusarium* spp. (*FUM1* Forward-CCATCAC AGTGGGACACAGT, *FUM1* Reverse-CGTATC GTCAGCATGATGTAGC) were used previously [72]. PCR were performed in mixture 15 μl volume containing 1 μl of DNA sample, 7.5 μl of master mix, 1 μl *FUM1* forward primer, 1 μl *FUM1* reverse primer, 4.5 μl nuclease free water. PCR was performed using T100 Thermocycler (BioRad, Hercules, USA). The PCR condition for *FUM1* regions include 94° C for 4 min for initial denaturation, followed by 35 cycles of denaturation at 94° C for 1 min, primer annealing at 58° C for 1 min, primer extension at 72° C for 1 min. The final extension was set at 72° C for 10 min. 4 μl of the PCR product was electrophoresed on 1.5 % agarose gel, stained with ethidium bromide, illuminated and documented using Gel documentation system (Dynamica, GelView Master).

## **7. Statistical analyses**

The collected data were analyzed statistically by using Minitab software version 17 (www.minitab.com). The mean of all the treatments were compared by critical difference value at 5 % level of significance.

### **8. Results**

### **8.1 Determination of total Aflatoxins contamination in stored maize grain samples collected from some selected growing areas of Bangladesh**

The study was performed at the Laboratory of Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh. Composite stored maize grain samples were collected from 15 maize growing areas of Bangladesh including Panchagarh, Thakurgaon, Dinajpur, Nilphamari, Rangpur, Lalmonirhat, Gaibandha, Bogura, Natore, Kushtia, Jashore, Chuadanga, Kishoreganj, Manikganj and Cumilla.

In terms of total aflatoxins concentration in μg/kg, the highest and lowest amount of aflatoxins concentration was recorded in Chuadanga (101.57 μg/kg) and Dinajpur (1.08 μg/kg) which exposed no significant relationship to each other. The moderate amount of afalatoxin level was detected in Gaibandha (68.73 μg/kg), Kushtia (31.48 μg/ kg), Kishoreganj (30.86 μg/kg), Rangpur (20.56 μg/kg) and Cumilla (11.91 μg/kg) revealing more aflatoxins contamination than the regulatory limit (10 μg/kg) in which only aflatoxins concentration from Kushtia and Kishoreganj revealed statistically significant data, besides, rest of the location exhibited below level of aflatoxins contamination of regulatory limit showing more or less statistically significant data.

Total aflatoxins associated with maize grains were detected in 2020, with the supreme value was detected in Chuadanga (30.5 %) followed by Kushtia (29.5 %), Nilphamari (22.5 %), Panchagarh (19.25 %) and the minimal aflatoxins was detected in Manikganj (3.2 %), rest of the samples from other districts revealed lower to moderate level of aflatoxins, moreover, data from Chuadanga and Kushtia, Cumilla, Jashore and Natore, Thakurgaon and Rangpur, Lalmonirhat and Kishoreganj regions revealed ststistically similar data while data from other regions exhibited statistically dissimilar data.

In case of infection rate, toxgenic maize samples were obtained from Panchagarh, Thakurgoan, Gaibandha, Chuadanga, Kishoreganj exhibiting 100 % infection by

*A. flavus* and no atoxigenic samples were found in those area. Moderate amount of toxigenic *A. flavus* was detected in Jashore followed by Cumilla, Natore, Lalmonirhat, Nilphamari which were 78 %, 75 %, 66 %, 50 % respectively and atoxigenic fungi was detected 22 %, 25 %, 34 %, 50 %, 50 % were detected respectively. Rest of the locations (Dinajpur, Rangpur, Bogura, Kushtia, Manikganj) exhibited higher amount of atoxigenic *A. flavus* compared to toxigenic *A. flavus* (**Table 1**).

The outmost percent aflatoxins concentration over standard limit was found in Chuadanga (915.7 %) followed by Gaibandha (587.3 %), Kushtia (214.8 %), Kishoreganj (208.85 %), Rangpur (105.6 %), Cumilla (19.5 %) revealing that the aflatoxins contamination from those area were beyond the regulatory limit set by EU for aflatoxins (10 ppb), conversely, aflatoxin concentration from other nine locations were below the regulatory limit of aflatoxins (**Table 1**).
