Study of Aflatoxigenicity and Biocontrol of Aspergillus flavus and Impact of Imizoquins on its Physiology

Abstract

Aspergillus flavus is a world-wide threat to human, animal and plant health due to the production of aflatoxins in many food and feed products. In first phase of the study, the incidence of aflatoxigenic strains of A. flavus was evaluated in cattle feed, water and milk from three districts of Punjab province of Pakistan (Rawalpindi, Gujrat and Okara) due to high incidence of aflatoxins in dairy products of those areas. In total, 39 of the hundred samples collected were contaminated by A. flavus. Specifically, the incidence of A. flavus was 54.28 %, 53.3 % and 11.42 % in feed (n=35), water (n=30) and milk (n=35) samples, respectively. These strains were further investigated for their aflatoxigenic nature using cultural (fluorescence under UV-light and NH4OH vapor induced color change test) and molecular (PCR) methods. The UV method indicated aflatoxigenic potential in 62 % of strains, and the ammonia vapor test showed 54 % of samples to be positive for aflatoxin production. The UV test is more sensitive than the ammonia test; however, comparable results from both methods strengthened our confidence in the findings. PCR detection of aflatoxin producing cluster of A. flavus was done by employing primers for four structural genes i.e. nor-1, ver-1, omt-A, aflR and two primers were used to distinguish A. flavus from A. parasiticus. The omt-A and aflR genes were regarded as potential markers for aflatoxins production because these genes were amplified in all those strains that were regarded as aflatoxigenic based on cultural methods for aflatoxin detection. It was demonstrated that, in addition to feed, water also acts as a potential threat for aflatoxigenic A. flavus entry into the food chain of the studied region. Moreover, the combined use of cultural and molecular methods, used in this study, can provide a cheaper and faster way to detect aflatoxigenic A. flavus in food and feed samples in developing countries. Fungal secondary metabolites have long been investigated for their pathogenic and therapeutic roles. Numerous biosynthetic gene clusters (BGCs) have been studied in order to decipher the roles of these genetic clusters. A previous study found that Ralstonia solanacearum, a soil-borne bacterium produces a lipopeptide, ralsolamycin, that lowers the expression of a BCG, named as imq, in A. flavus. The transcription factor of this gene cluster, imqK, regulates the synthesis of tripeptide-derived alkaloids, imizoquins. In second phase of this study, imizoquins were found to promote spore germination in A. flavus and few related fungi by acting as endogenous antioxidants to provide protection against germination inhibitory effects of reactive oxygen species (ROS). In addition to their role in fungal development, imizoquins also counteract the delaying effects of ralsolamycin extracts on germination and inhibit the growth of R. solanacearum in a bacteriostatic manner. Thus, this study highlighted the role of secondary metabolites in bacterial-fungal interactions. In third phase of the study, the use of generally regarded as safe (GRAS) microorganisms for the biocontrol strategy against aflatoxigenic A. flavus was investigated. Thirteen Geotrichum candidum strains were used in the study, of which ten strains were locally isolated from dairy products and three strains were procured from University of the Caen, France. These strains were individually assessed for anti-A. flavus activity as well as in combination with four lactic acid bacteria (LAB) strains which included three Lactococcus garvieae strains QAULG01, QAULG02, QAULG03 and a Lactococcus lactis strain QAULG04. The antagonistic potential of G. candidum strains and their combinations with the lactic acid bacteria were assessed by well diffusion assays. G. candidum strain (QAUGC01) and the combination of QAUGC01+QAULG01 gave the best antagonism and yielded the inhibition zones of 19 mm and 30 mm, respectively. The organic extracts obtained from the cell free supernatants of the two samples i.e. QAUGC01 and QAUGC01+QAULG01 yielded the most promising results, and the organic extract from QAUGC01 provided 20 mm inhibitory zone against A. flavus mycelial growth. One of the active metabolites of the cell free supernatants of the two samples was identified to be a derivative of butanoic acid. In conclusion, it is demonstrated through these studies that microbial interaction studies provided useful information regarding the small biomolecules that microorganisms (e.g. LAB from cattle gut, G. candidum from dairy products and R. solanacearum from rhizosphere) use to antagonize and halt the proliferation of nearby residing microbial competitor (e.g. A. flavus in this study).