## 4 Discussion  
### Presence of Aflatoxin-Producing Genes in Wild Aspergillus sp. Isolates  
Aflatoxin contamination is of great concern in several agricultural countries owing to its high toxicity to poultry, which potentially consume AFB-contaminated feeds via AFB-containing raw materials or grains. Recently, [PERSON] et al. [30] isolated 15 presumptive _A. flavus_ strains from various feed and grain samples in Bangladesh and determined their AFB-producing capacities by measuring AFB-producing gene expression. In this study, only one isolate, designated isolate no. 41, possessed seven major AFB-producing genes, namely _afIR_, _afIS_, _afID_ (known as _nor-1_), _afIM_ (known as _ver-1_), _afIO_ (known as _omtB_), _afIP_ (known as _omtA_), and _afIQ_ (_ordA_), while the other 14 isolates did not express at least one major AFB-producing gene [30]. Interestingly, even if they did not express certain genes related to AFB production in isolated _A. flavus_, only one isolate (designated isolate no. 26) did not produce AFB\({}_{1}\) but yielded a minute amount of AFB\({}_{2}\) (up to 12.3 \(\mu\)g/g agar). Isolate no. 26 did not express _afIM_, which encodes the 28 kDa reduced nicotinamide adenine dinucleotide phosphate-dependent ketoneductase protein to bio-transform versicolorin A to demethylisterigmatocystin; thus, this gene potentially plays a crucial role in AFB production in _Aspergillus_. On the other hand, among the 15 isolates, 11 produced both AFB\({}_{1}\) and AFB\({}_{2}\) in a 3:2 ratio and in quantities exceeding 6.3 and 4.5 \(\mu\)g/g agar, respectively [30]. However, in addition to the _afIM_ gene, other AFB-producing genes may also serve an essential role in AFB biosynthesis as a key component. As AFB biosynthesis recruits various enzyme systems to produce four main AFBs, the first gene group involved in AFB biosynthesis comprises genes encoding two fatty acid synthases (FASs) and a polyketide synthase (_PksA_) [31]. These initial components play an important role in natural product formation, as they mediate a hexanoyl starter subunit to form corresponding polyketides, such as norsolorinic acid; however, they are also involved in various natural product biosynthesis systems in fungi apart from AFB biosynthesis [32]. Therefore, down-regulating FAS and PKS genes in the fungal community is considerably challenging owing to the production of diverse natural products, and the presence or status of other AFB-producing genes may be important to non-AFB-producing _Aspergillus_ strains. In our study, the AFB-producing capacities of three _A. flavus_ strains, namely ATCC 9643, ATCC 22546, and ATCC 96045, with or without co-cultivation, were compared in the presence of non-AFB\({}_{1}\)-producing strains that suppress AFB\({}_{1}\)-producing strains, and two of them, ATCC 9643 and ATCC 96045, did not produce AFB\({}_{1}\). Compared with ATCC 22546, ATCC 9643 did not express _afIK_ and _afIL_, which encode versicolorin B synthase and desaturase, respectively. Recently, 4-hydroxy-7-methyl-3-phenylcoumarin was found to inhibit AFB1 production in ATCC 22546 by down-regulating _afIK_ gene expression [15]. Moreover, 1,8-cineole also suppressed _afIL_ gene expression in ATCC 22546, and 1,8-cineole-treated ATCC 22546 exhibited dramatically reduced AFB\({}_{1}\) production [33]. Based on these results, _Aspergillus_ may not produce AFB1 or potentially display suppressed AFB production with the down-regulation or absence of the _afIK_ and _afIL_ genes. Taken together, _afIK_, _afIL_, and _afIM_ gene expression serves an important role in AFB production in _Aspergillus_. Similarly, the ATCC 96045 strain neither expresses _afIE_, _afIK_, _afIL_, or _afIO_ nor produces AFBs. It is already being used for the biocontrol of AFB-producing _A. flavus_ strains in the US under the name AF36, and this biocontrol has proven successful in agricultural fields.