Geographical Distribution and Molecular Characterization of aToxigenic and a toxigenic Isolates of Aspergillus Flavus and Aspergillus Parasiticus in kenyax

Abstract

Grainsof important food and export crops in Africa, such as maize, cassava and peanuts, are susceptible and prone to contamination with different toxin producing moulds. Aflatoxinsare mycotoxins associated with liver damage and cancer in humans and animals. Thesetoxic substances are produced by fungi (such as Aspergillus flavus and Aspergillus parasiticus)in food and feed commodities that are exposed to poor conditions during crop cultivationor during storage and processing of the harvest. The presence of aflatoxins especiallyin maize and peanuts in Kenya is of great concern. Reports of aflatoxin poisoning inmaizeand peanuts in Kenya are an indication of the high level of exposure of people whoselives depend on these crops as their staple foods. Recent developments in the applicationofatoxigenic strains of these fungi as biological control agents against toxigenic strainscould provide a solution to the problem. In Kenya the distribution and characterizationof toxigenic' and atoxigenic Aspergillus strains is not known. The main objectiveof this project 'was to isolate, identify and characterize atoxigenic and toxigenic strainsof A. jlavus and A. i/arasiticus in Kenya. Fungal isolates from soils under maize and peanutcultivation from various locations in Kenya were examined to determine the distributionsof toxigenic Aspegillus species and to identify endemic atoxigenic strains. In thisstudy,soils samples were collected randomly from Kenyan fields located in seven agroecologicalzones (AEZ) (CL3, CL4, L3, L4, LM3, LM4 and LM5) and characterized by use ofmorphological,physiological and molecular techniques. In this study, Aspergillus section Flaviwasdetected in all the 57 soil samples. In total, 220 isolates belonging to A. flavus and A.parasiticus were obtained. Aspergillus section Flavi L-strain was the most commonly isolatedmember (54%), followed by Aspergillus section Flavi S-starins (35%). Aspergillus jlavus was the most predominant (63.2%), followed by A. parasitic us (27.7%), A. tamari (5.5%)and A. nomius (2.7%). The mean Colony Forming Units (CFU) of the Aspergillus coloniesper gram of soil was extremely variable among the districts, ranging from 3.0xl03 to1.72x106(p<0.05). The mean temperatures across the collection sites also varied (from 24 toWC) according to the respectiveAgroecological zones AEZ. The pH varied from 5.5 - 6.8, whichis within the optimal pH requirement for the members of section Flavi. Each of theregionshad atoxigenic strains of potential value which can be employed as biological controlagents in the management of afl~toxicoses. For molecular characterization in the presentstudy, internal transcribed spacer (ITS 4 and ITS 5 primers) targeting the 5.8S regionof the rDNA and AB28rrW81 primers targeting 18S ribosomal DNA region of Aspergillusspp were used. The two primers generated a specific target band ::::600bp that wassubsequently sequenced. The sequences obtained were edited by Bioedit and other analysisperformed using Mega 5. The ITS-PCR and AB 28ffW 81-PCR products were digestedusing HinjI endonuclease and generated polymorphic bands that ranged from= 100 to350bp.Aspergillus section Flavi sequences for the ITSl- 5.8S-ITS2 rDNA genes were comparedagainst all other sequences available online with the basic local alignment search tool algoritlim (BLAST, National Center for Biotechnology Information). Sequence homologysearches within the Aspergillus section Flavi revealed that the majority of the isolates(36.8%) were Aspergillus flavus , followed by A. terreus ( 15.8%), A. insuetus (15.8%), A. parasiticus (10.5%), A. oryzae (5.2%), A. ochraceus (5.2%), A. variecolor (5.2%)and A. calidoustus (5.2%). These results strongly support the need for using molecularmarkers as an auxiliary tool in differentiating fungal species.