## 4 Discussion
### Growth Competition between ATCC 22546 and ATCC 9643  
The biocontrol of _A. parasitic_ by selected non-conventional yeast strains has recently been suggested. Two wild yeasts, _Aureobashium pullulans_ and _Saitozyma podzolica_, have demonstrated their potential to suppress the mycelian growth of _A. flavus_ and _A. parasitic_ and reduce mycotoxin production [34]. _S. podzolica_ produces chitinase, \(\beta\)-1,3-glucanase, andamylase, and these three enzymes play an important role in controlling fungal growth. In addition, _A. pullulans_ generates protease and cellulase, as well as chitinase, \(\beta\)-1,3-glucanase, and amylase. The above two yeasts were found to reduce AFB production in _A. parasisticus_ by approximately 20% in a bread model [34]. Chitinases mediate hydrolytic reactions that break down glycosidic bonds in chitin and are the chief components of fungal cell walls [35], while \(\beta\)-1,3-glucanase decomposes fungal \(\beta\)-1,3-glucanas (Curdlan and laminarin) [36]. Amylases, proteases, and cellulases are all extracellular enzymes known to control fungal growth by breaking the a-1,4-linkages in starch, degrading the proteins in fungal cell walls, and breaking down celluloses in fungal cell walls, respectively [37; 38; 39].  
_A. pullulans_ has been used to control _A. flavus_ in vitro and on tomato fruits, and it has been found to produce chitinase and \(\beta\)-1,3-glucanase after five days of cultivation, leading to a one-third decrease in lesion diameter on tomato fruits compared with that in the control [28]. Therefore, these extracellular enzymes secreted by microbes potentially contribute to _A. flavus_ and _A. parasiticus_ control for the safety of foods and feeds.  
In a previous study using non-AFB-producing _A. parasiticus_ as a biocontrol agent against AFB contamination, the co-inoculation of non-AFB-producing _A. parasiticus_ strains inhibited AFB production in corn under laboratory conditions [40]. To further examine this finding, another study also employed non-AFB-producing _A. flavus_ and found it to exhibit potential in controlling AFB contamination in cotton fields [41]. Both non-AFB-producing _A. flavus_ and _A. parasiticus_ strains were administered to peanuts fields in three different formulations, and they each yielded a large soil population of non-AFB-producing strains [42]. Similarly, in cotton fields, treatment with non-AFB-producing strains decreased AFB\({}_{1}\) production from 75% to 99% [43; 44].  
When applying non-aflatoxigenic strains to fields, two major parameters, application time and inoculum rate, must be considered. [PERSON] and [PERSON] [45] recommended the simultaneous co-inoculation of non-aflatoxigenic and aflatoxinemia; however, a one-day difference in inoculation timing led to failed AFB reduction. Regarding the inoculation rate of non-AFB-producing strains, AFB contamination in peanuts significantly decreased with the increasing non-aflatoxigenic inoculum rate [46].  
To use the ATCC 9643 strain as a biological agent against AFB contamination, the optimal application timing was employed based on simultaneous co-inoculation [45]. At this inoculation time, ATCC 9643 was co-inoculated with ATCC 22546 in five different ATCC 9643/ATCC 22546 inoculation ratios of 1:1, 1:2, 1:4, 4:1, and 2:1. In these treatments, ATCC 9643 displayed competitive growth against ATCC 22546, significantly reducing AFB\({}_{1}\) and AFB\({}_{2}\) production. However, this competitiveness was potentially attenuated when ATCC 9643 was applied to crop fields, as the co-inoculation of ATCC 9643 with ATCC 22546 on water-soaked rice exhibited different growth patterns between the two strains, with ATCC 22546 readily dominating ATCC 9643.