Increasing knowledge of the deleterious health and economic impacts of aflatoxin in crop commodities has stimulated global interest in aflatoxin mitigation. Current evidence of the incidence of Aspergillus flavus isolates belonging to vegetative compatibility groups (VCGs) lacking the ability to produce aflatoxins (i.e., atoxigenic) in Ghana may lead to the development of an aflatoxin biocontrol strategy to mitigate crop aflatoxin content. In this study, 12 genetically diverse atoxigenic African A. flavus VCGs (AAVs) were identified from fungal communities associated with maize and groundnut grown in Ghana. Representative isolates of the 12 AAVs were assessed for their ability to inhibit aflatoxin contamination by an aflatoxin-producing isolate in laboratory assays. Then, the 12 isolates were evaluated for their potential as biocontrol agents for aflatoxin mitigation when included in three experimental products (each containing four atoxigenic isolates). The three experimental products were evaluated in 50 maize and 50 groundnut farmers’ fields across three agroecological zones (AEZs) in Ghana during the 2014 cropping season. In laboratory assays, the atoxigenic isolates reduced aflatoxin biosynthesis by 87–98% compared to grains inoculated with the aflatoxin-producing isolate alone. In field trials, the applied isolates moved to the crops and had higher (P < 0.05) frequencies than other A. flavus genotypes. In addition, although at lower frequencies, most atoxigenic genotypes were repeatedly found in untreated crops. Aflatoxin levels in treated crops were lower by 70–100% in groundnut and by 50–100% in maize (P < 0.05) than in untreated crops. Results from the current study indicate that combined use of appropriate, well-adapted isolates of atoxigenic AAVs as active ingredients of biocontrol products effectively displace aflatoxin producers and in so doing limit aflatoxin contamination. A member each of eight atoxigenic AAVs with superior competitive potential and wide adaptation across AEZs were selected for further field efficacy trials in Ghana. A major criterion for selection was the atoxigenic isolate’s ability to colonize soils and grains after release in crop field soils. Use of isolates belonging to atoxigenic AAVs in biocontrol management strategies has the potential to improve food safety, productivity, and income opportunities for smallholder farmers in Ghana.

The utility of the cytochrome oxidase I gene barcode region for diagnosis of the Mediterranean fruit fly, Ceratitis capitata (Weidemann), is evaluated using African fruit fly collections. The method fails to discern C. capitata from its close relative Ceratitis caetrata Munro, based on genetic distances, parsimony networks, or nucleotide diagnostic characters observed in the DNA barcode sequences. When treated as a single taxon, it is possible to discern the C. capitata + C. caetrata lineage from other Ceratitis species. Levels of intraspecific diversity vary within the genus Ceratitis and multiple copies of the mitochondrial gene are reported for Ceratitis cosyra (Walker). The DNA barcoding method based on genetic distance is compared with a molecular identification method using restriction fragment length polymorphism. The DNA barcode and restriction fragment-length polymorphism methods provide similar identification results, but the DNA sequence information is more suitable for quantitative analysis of the information.

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Aspergillus flavus has long been considered to be an asexual species. Although a sexual stage was recently reported for this species from in vitro studies, the amount of recombination ongoing in natural populations and the genetic distance across which meiosis occurs is largely unknown. In the current study, genetic diversity, reproduction and evolution of natural A. flavus populations endemic to Kenya were examined. A total of 2744 isolates recovered from 629 maize-field soils across southern Kenya in two consecutive seasons were characterized at 17 SSR loci, revealing high genetic diversity (9-72 alleles/locus and 2140 haplotypes). Clonal reproduction and persistence of clonal lineages predominated, with many identical haplotypes occurring in multiple soil samples and both seasons. Genetic analyses predicted three distinct lineages with linkage disequilibrium and evolutionary relationships among haplotypes within each lineage suggesting mutation-driven evolution followed by clonal reproduction. Low genetic differentiation among adjacent communities reflected frequent short distance dispersal.

BackgroundHuanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited α- proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed.ResultsA panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide.ConclusionsOur results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.

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There has been considerable interest in understanding biological, ecological, historical, and evolutionary processes that contribute to the diversification of species and populations among tephritid fruit flies. Only a limited number of studies have examined the genetic diversity and population biology of species belonging to the genus Anastrepha considering fine-scale differentiations associated to locality as well as hosts over an entire fruiting season. To expand our understanding of population structure and genetic diversity in one of the critical Anastrepha fruit flies populations in a highly diverse tropical environment we analyzed Anastrepha obliqua (Macquart) (Diptera: Tephritidae) in the Mexican state of Veracruz from five host fruit species and 52 geographic collections using sequence data from mtDNA and microsatellite markers from nuclear DNA. Indeed, we examined the population structure of this pest in a micro-geographic region and report on relationships and historical processes for individuals collected within a small portion of the geographic range of its distribution. Analyses of 1055 bp mtDNA sequences from CO1and ND1genes across 400 individuals detected 34 haplotypes. Haplotype and nucleotide diversity was low, with 53% of the individuals exhibiting a single haplotype (OBV1). Host association and fine-scale differentiation at 17 microsatellite markers across 719 individuals from 32 of the 52 geographic collections reveal fragmented A. obliqua populations. These findings have important implications for the implementation of the Sterile Insect Technique (SIT) and other pest management programs used to control this pestiferous fruit fly.

Human populations in Kenya are repeatedly exposed to dangerous aflatoxin levels through consumption of contaminated crops. Biocontrol with atoxigenic Aspergillus flavus is an effective method for preventing aflatoxin in crops. Although four atoxigenic A. flavus isolates (C6E, E63I, R7H and R7K) recovered from maize produced in Kenya are registered as active ingredients for a biocontrol product (Aflasafe KE01) directed at preventing contamination, natural distributions of these four genotypes prior to initiation of commercial use have not been reported. Distributions of the active ingredients of KE01 based on haplotypes at 17 SSR loci are reported. Incidences of the active ingredients and closely related haplotypes were determined in soil collected from 629 maize fields in consecutive long and short rains seasons of 2012. The four KE01 haplotypes were among the top ten most frequent. Haplotype H-1467 of active ingredient R7K was the most frequent and widespread haplotype in both seasons and was detected in the most soils (3.8%). The four KE01 haplotypes each belonged to large clonal groups containing 27-46 unique haplotypes distributed across multiple areas and in 21% of soils. Each of the KE01 haplotypes belonged to a distinct vegetative compatibility group (VCG), and all A. flavus with haplotypes matching a KE01 active ingredient belonged to the same VCG as the matching active ingredient as did all A. flavus haplotypes differing at only one SSR locus. Persistence of the KE01 active ingredients in Kenyan agroecosystems is demonstrated by detection of identical SSR haplotypes six years after initial isolation. The data provide baselines for assessing long-term influences of biocontrol applications in highly vulnerable production areas of Kenya.