Molecular Characterization of Aspergillus flavus in Imported Maize in Kenya | InformativeBD Informative bd | Sharing Knowledge Informativebd

Joseph Oduor Odongo, Paul O. Angi’enda, Bramwel Wanjala, Catherine Taracha, and David M. Onyango, from the different institute of the Kenya. wrote a research article about, Molecular Characterization of Aspergillus flavus in Imported Maize in Kenya. entitled, Molecular characterisation of Aspergillus flavus on imported maize through gazetted and ungazetted points of Entries in Kenya. This research paper published by the International journal of Microbiology and Mycology (IJMM). an open access scholarly research journal on Microbiology. under the affiliation of the International Network For Natural Sciences | NNSpub. an open access multidisciplinary research journal publisher.

Abstract

Maize is a vital staple crop in Kenya, serving as a primary source of food and feed. Contamination of maize (Zea mays) by Aspergillus flavus and the subsequent production of aflatoxins pose significant threats to food safety and human health. The risk of A. flavus contamination on imported maize at both gazetted and un-gazetted points of entry has not been extensively studied. The primary objective of this study was to examine the genotypic, phenotypic, and aflatoxigenic traits of A. flavus biovars derived from imported maize at Gazetted and Un-gazetted Points of Entries in Kenya. Furthermore, the study sought to establish the phylogenetic relationships among the identified A. flavus strains. A total of 600 imported maize samples were tested for aflatoxin contamination using the Total aflatoxin ELISA test. Out of 600 samples, 4.17% tested positive and were further subjected to morphological and molecular studies. The morphological analysis revealed the presence of 13 biovars of A. flavus. Micro-morphologically, variations were observed in spore color, size, structure, conidiophore structure, and vesicle shape. The specific primers Calmodulin (CaM), the ITS1-5.8S-ITS2 region of the ribosomal DNA was successfully amplified in 10 out of the 13 biovars that were presumed to be A. flavus, confirming their positive identification as A. flavus. A single band of approximately 700 bp, which corresponds to the expected size of the ITS region in Aspergillus flavus, was observed in 10 out of the 13 biovars. This indicates the presence of A. flavus DNA in those biovars. The amplification of the ITS region provides a specific molecular marker for the identification of A. flavus. These findings highlight the significance of aflQ (ordA) and aflD (nor-1) genes as reliable markers for evaluating the aflatoxigenic potential of A. flavus biovars. Regarding aflatoxigenicity, DV-AM method was used, and qualitative analysis was conducted. Out of the 13 biovars of A. flavus biovars tested, 23.08% exhibited aflatoxigenicity, while the remaining 10 biovars did not show any aflatoxigenicity. These findings indicate the presence of both aflatoxigenic and non-aflatoxigenic strains of A. flavus among the imported maize samples. The phylogenetic analysis revealed that Taxon 31 (AY495945.1 Aspergillus flavus biovar 92016f aflR-aflJ intergenic region partial sequence) and Taxon 32 (NR 111041.1 Aspergillus flavus ATCC 16883 ITS region from TYPE material). This genotypic and phenotypic characterization provides valuable information for understanding the diversity and potential toxigenicity of A. flavus strains on imported maize. This study contributes to the understanding of the genotypic and phenotypic characteristics of A. flavus on imported maize in Kenya.

Introduction

Maize plays a central role in the food security and livelihoods of Kenyan populations. It serves as a staple food crop for a significant portion of the population, contributing to both dietary needs and income generation. Moreover, maize is an essential component of livestock feed, supporting the growth of the domestic livestock industry. In sub-Saharan Africa as a whole, maize is ranked third in importance among cereal crops, following rice and wheat (Shiferaw et al., 2011). The cultivation and trade of maize have a considerable impact on regional economies and food systems. Maize (Zea mays) is often contaminated by Aspergillus fungal species during pre- and post-harvest practices, storage, and transportation. Studies by Horn (2007) showed that Aspergillus species are commonly found in the soil, which acts as a source of primary inoculum for infecting developing maize kernels during the growing season. Aspergillus flavus is distributed globally with a high frequency of occurrence in warm climates which favor the growth of the fungus (Cotty et al., 1994).

Understanding the population structure and genetic diversity of A. flavus is crucial for diversification of effective management strategies. Different strains of A. flavus may have varying levels of aflatoxin production and pathogenicity, which can influence the severity of contamination in maize (Abbas et al., 2013). Additionally, certain strains may exhibit resistance or susceptibility to control measures, such as biological control agents or fungicides. Therefore, identifying specific strains or groups within the A. flavus population can aid in the selection of appropriate control strategies to minimize aflatoxin contamination. Moreover, the genetic diversity of A. flavus may also have implications for host-pathogen interactions and disease development. Different strains may exhibit variations in their ability to infect maize kernels, colonize host tissues, and compete with other microorganisms in the maize ecosystem (Atehnkeng et al., 2014). Understanding these interactions can help in the development of resistant maize varieties and cultural practices that can limit fungal growth and subsequent aflatoxin production. The population structure and genetic diversity of A. flavus strains isolated from maize play a significant role in aflatoxin contamination and disease development. The existence of multiple strains within the A. flavus population highlights the need for comprehensive investigations to characterize their phenotypic and genotypic traits. Such studies will provide insights into the factors influencing aflatoxin production, the design of effective control strategies, and the development of resistant maize varieties to minimize the health and economic risks associated with aflatoxin contamination. Aspergillus species, including Aspergillus flavus, are of great concern due to their ability to produce aflatoxins, potent carcinogens and toxins that contaminate various agricultural commodities, including maize. The accurate identification and characterization of Aspergillus species is crucial for assessing their potential to produce aflatoxins and understanding their impact on food safety.

Gene sequencing has emerged as a powerful tool for the accurate identification and classification of Aspergillus species. In recent years, numerous studies have utilized gene sequencing data to characterize Aspergillus biovars from different sources. By comparing the genetic sequences of specific genes, such as the internal transcribed spacer (ITS) region, researchers can determine the species and genetic diversity within a population. In addition to genetic characterization, a polyphasic approach is commonly employed to identify and characterize Aspergillus biovars. This approach combines morphological and molecular analyses to provide a comprehensive understanding of the biovars. Morphological characteristics, such as colony color, texture, spore color, size and structure, conidiophore structure and vesicle shape are observed and recorded. These characteristics help in differentiating between various Aspergillus species and subgroups. Furthermore, molecular techniques, including polymerase chain reaction (PCR) amplification and sequencing of specific genetic markers, allow for a more precise identification of aflatoxigenic and nonaflatoxigenic A. flavus biovars. These methods target genes associated with aflatoxin production, such as the aflatoxin biosynthesis cluster genes, to determine the potential of a biovar to produce aflatoxins. The combination of gene sequencing and polyphasic approaches provides a comprehensive understanding of the genetic diversity, population structure, and aflatoxinproducing potential of Aspergillus species, particularly A. flavus. This information is essential for risk assessment, development of effective control strategies, and ensuring the safety and quality of imported maize and other agricultural commodities.

This study contributed to the understanding of the population dynamics and potential risks associated with A. flavus in imported maize. Given the prominence of maize in Kenya, research efforts focusing on this crop are crucial. The genotypic and phenotypic characterization of A. flavus on imported maize assumes particular significance in the Kenyan context. A thorough understanding of the genetic diversity and potential for mycotoxin production in A. flavus populations is essential for developing effective control strategies and mitigating the health risks associated with mycotoxin contamination. Gazetted and un-gazetted points of entry play a crucial role in facilitating the importation of maize. However, the risk of A. flavus contamination in imported maize has not been thoroughly investigated, warranting a comprehensive genotypic and phenotypic characterization of this fungus. Understanding the genotypic and phenotypic characteristics of A. flavus on imported maize is essential for several reasons. Firstly, it allows for the identification of specific genetic traits and phenotypic features associated with higher aflatoxin production, thus enabling the development of targeted control strategies. Secondly, it provides insights into the diversity of A. flavus biovars present in imported maize and their potential for aflatoxin contamination. This knowledge can contribute to risk assessment and management strategies aimed at preventing or minimizing aflatoxin contamination in the domestic maize supply chain.

Genotypic characterization involves studying the genetic makeup of A. flavus biovars to determine their relatedness, genetic diversity, and potential for toxin production. Several molecular techniques have been used for genotyping A. flavus, including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and multilocus sequence typing (MLST) (Abdallah et al., 2018). These methods have provided valuable insights into the genetic diversity and population structure of A. flavus, highlighting the presence of distinct genotypes in different geographic regions (Klich et al., 2015). Phenotypic characterization involves studying the observable traits and behaviors of A. flavus, such as growth patterns, conidiation, and mycotoxin production. Phenotypic characterization is essential for understanding the pathogenicity and virulence of A. flavus strains on imported maize. Researchers have observed variations in colony morphology, growth rate, and sporulation among different A. flavus biovars (Calvo et al., 2016). Furthermore, studies have demonstrated the production of mycotoxins, particularly aflatoxins, by certain A. flavus strains (Chang et al., 2019). Phenotypic characterization provides valuable information for risk assessment and identifying high-risk A. flavus biovars in imported maize. The genotypic and phenotypic characterization of A. flavus on imported maize plays a crucial role in assessing the potential health risks associated with mycotoxin contamination. By combining genotypic and phenotypic data, researchers can identify highly toxigenic A. flavus strains and evaluate their prevalence in imported maize.

This information is essential for implementing targeted control measures, such as crop management strategies, post-harvest interventions, and storage practices, to minimize mycotoxin contamination and ensure food safety (Li et al., 2020). Investigating A. flavus on imported maize specifically at gazetted and ungazetted points of entry in Kenya is crucial. Gazetted points of entry are official border checkpoints designated for the importation of agricultural products, while un-gazetted points of entry refer to informal channels through which goods, including maize, are smuggled into the country. Analyzing both types of entry points can provide a comprehensive understanding of the risks associated with A. flavus contamination in imported maize, as well as the efficacy of control measures implemented at official checkpoints. In this study, we aim to conduct a detailed genotypic and phenotypic characterization of A. flavus on imported maize at both gazetted and un-gazetted points of entry in Kenya. We will analyze the genetic diversity, aflatoxin production capability, and other phenotypic traits of A. flavus biovars obtained from imported maize samples. By doing so, we hope to gain insights into the potential sources and pathways of A. flavus contamination in imported maize and develop targeted strategies to ensure the safety and quality of imported maize in Kenya.

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