Flavus Populations Research Articles

Aspergillus flavus resident in Kenya: High genetic diversity in an ancient population primarily shaped by clonal reproduction and mutation-driven evolution.

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.

Effect of Temperature and Relative Humidity on Growth of Aspergillus and Penicillium spp. and Biocontrol Activity of Pseudomonas protegens AS15 against Aflatoxigenic Aspergillus flavus in Stored Rice Grains

In this study, we evaluated the effect of different temperatures (10, 20, 30, and 40 °C) and relative humidities (RHs; 12, 44, 76, and 98%) on populations of predominant grain fungi (Aspergillus candidus, Aspergillus flavus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum) and the biocontrol activity of Pseudomonas protegens AS15 against aflatoxigenic A. flavus KCCM 60330 in stored rice. Populations of all the tested fungi in inoculated rice grains were significantly enhanced by both increased temperature and RH. Multiple linear regression analysis revealed that one unit increase of temperature resulted in greater effects than that of RH on fungal populations. When rice grains were treated with P. protegens AS15 prior to inoculation with A. flavus KCCM 60330, fungal populations and aflatoxin production in the inoculated grains were significantly reduced compared with the grains untreated with strain AS15 regardless of temperature and RH (except 12% RH for fungal population). In addition, bacterial populations in grains were significantly enhanced with increasing temperature and RH, regardless of bacterial treatment. Higher bacterial populations were detected in biocontrol strain-treated grains than in untreated control grains. To our knowledge, this is the first report showing consistent biocontrol activity of P. protegens against A. flavus population and aflatoxin production in stored rice grains under various environmental conditions of temperature and RH.

Characterization of Argentinian Endemic Aspergillus flavus Isolates and Their Potential Use as Biocontrol Agents for Mycotoxins in Maize.

Maize (Zea mays L.) is a highly valuable crop in Argentina, frequently contaminated with the mycotoxins produced by Aspergillus flavus. Biocontrol products formulated with atoxigenic (nontoxic) strains of this fungal species are well known as an effective method to reduce this contamination. In the present study, 83 A. flavus isolates from two maize regions of Argentina were characterized and evaluated for their ability to produce or lack of producing mycotoxins in order to select atoxigenic strains to be used as potential biocontrol agents (BCA). All of the isolates were tested for aflatoxin and cyclopiazonic acid (CPA) production in maize kernels and a liquid culture medium. Genetic diversity of the nonaflatoxigenic isolates was evaluated by analysis of vegetative compatibility groups (VCG) and confirmation of deletions in the aflatoxin biosynthesis cluster. Eight atoxigenic isolates were compared for their ability to reduce aflatoxin and CPA contamination in maize kernels in coinoculation tests. The A. flavus population was composed of 32% aflatoxin and CPA producers and 52% CPA producers, and 16% was determined as atoxigenic. All of the aflatoxin producer isolates also produced CPA. Aflatoxin and CPA production was significantly higher in maize kernels than in liquid medium. The 57 nonaflatoxigenic strains formed six VCG, with AM1 and AM5 being the dominant groups, with a frequency of 58 and 35%, respectively. In coinoculation experiments, all of the atoxigenic strains reduced aflatoxin from 54 to 83% and CPA from 60 to 97%. Members of group AM1 showed a greater aflatoxin reduction than members of AM5 (72 versus 66%) but no differences were detected in CPA production. Here, we described for the first time atoxigenic isolates of A. flavus that show promise to be used as BCA in maize crops in Argentina. This innovating biological control approach should be considered, developed further, and used by the maize industry to preserve the quality properties and food safety of maize kernels in Argentina.

Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya.

Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial β-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

The Role of Drought Stress on Aflatoxin Contamination in Groundnuts (Arachis hypogea L.) and Aspergillus flavus Population in the Soil

Aflatoxins are naturally occurring toxic chemical substances that are produced by fungal species called Aspergillus flavus. The toxic substances are secondary metabolites, which contaminate groundnut while growing in the field and also post-harvest. Drought stress is one of the factors that contribute to increased aflatoxin levels in groundnut during field production. This study was conducted in a screen house at ICRISAT-Chitedze Agricultural Research Station, Malawi to investigate the effects of drought on aflatoxin contamination and A.flavus population in the soil. Four drought stress levels; prolonged (4 weeks), min (3 weeks), mild (2 weeks) and no drought were imposed on five groundnut varieties at pod filling stage. Soil samples were collected from each plot four times; at planting, beginning of drought, end of drought and at harvest. Aflatoxin levels in groundnut grain samples were estimated by use of neogenstrips read with mobile assay tablet reader. Population densities of A.flavus in soil samples collected from the plots were estimated using serial dilutions plated on the selective media, modified dichloran Rose Bengal (MDRB) and quantify A.flavus within 3 days after incubation at 37°C.The results showed that there were significant differences in aflatoxin contamination between drought stress levels (p = 0.011). High aflatoxin contamination was observed under prolonged drought (22.0 ppb) compared to and no drought treatment (1.5 ppb). None of the varieties used in the study showed either resistance or susceptibility to aflatoxin contamination under drought or adequate soil moisture. The results also showed that there were significant differences in A.flavus population at drought period and harvesting time and the mean population of A.flavus in prolonged drought at end of stress and harvesting were 8511 and 6044 cfu/g of soil respectively. It was concluded that drought contribute to aflatoxin contamination in groundnut, and also increased the A.flavus population in soil and also at harvesting.

Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds

To identify predominant isolates for potential use as biocontrol agents, Aspergillus flavus isolates collected from soils of almond, pistachio and fig orchard in the Central Valley of California were tested for their membership to 16 atoxigenic vegetative compatibility groups (VCGs), including YV36, the VCG to which AF36, an atoxigenic isolate commercialized in the United States as biopesticide, belongs. A surprisingly large proportion of isolates belonged to YV36 (13.3%, 7.2% and 6.6% of the total almond, pistachio and fig populations, respectively), while the percentage of isolates belonging to the other 15 VCGs ranged from 0% to 2.3%. In order to gain a better insight into the structure and diversity of atoxigenic A. flavus populations and to further identify predominant isolates, seventeen SSR markers were then used to genetically characterize AF36, the 15 type-isolates of the VCGs and 342 atoxigenic isolates of the almond population. There was considerable genetic diversity among isolates with a lack of differentiation among micro-geographical regions or years. Since isolates sharing identical SSR profiles from distinct orchards were rare, we separated them into groups of at least 3 closely-related isolates from distinct orchards that shared identical alleles for at least 15 out of the 17 loci. This led to the identification of 15 groups comprising up to 24 closely-related isolates. The group which contained the largest number of isolates were members of YV36 while five groups were also found to be members of our studied atoxigenic VCGs. These results suggest that these 15 groups, and AF36 in particular, are well adapted to various environmental conditions in California and to tree crops and, as such, are good candidates for use as biocontrol agents.

Frequent Shifts in Aspergillus flavus Populations Associated with Maize Production in Sonora, Mexico.

Aspergillus flavus frequently contaminates maize, a critical staple for billions of people, with aflatoxins. Diversity among A. flavus L morphotype populations associated with maize in Sonora, Mexico was assessed and, in total, 869 isolates from 83 fields were placed into 136 vegetative compatibility groups (VCGs) using nitrate-nonutilizing mutants. VCG diversity indices did not differ in four agroecosystems (AES) but diversity significantly differed among years. Frequencies of certain VCGs changed manyfold over single years in both multiple fields and multiple AES. Certain VCGs were highly frequent (>1%) in 2006 but frequencies declined repeatedly in each of the two subsequent years. Other VCGs that had low frequencies in 2006 increased in 2007 and subsequently declined. None of the VCGs were consistently associated with any AES. Fourteen VCGs were considered dominant in at least a single year. However, frequencies often varied significantly among years. Only 9% of VCGs were detected all 3 years whereas 66% were detected in only 1 year. Results suggest that the most realistic measurements of both genetic diversity and the frequency of A. flavus VCGs are obtained by sampling multiple locations in multiple years. Single-season sampling in many locations should not be substituted for sampling over multiple years.

The breeding biology, nest success, habitat and behavior of the endangered Saffron-cowled Blackbird, Xanthopsar flavus (Aves: Icteridae), at an Important Bird Area (IBA) in Rio Grande do Sul, Brazil

ABSTRACT The Saffron-cowled Blackbird, Xanthopsar flavus (Gmelin, 1788), is a globally vulnerable icterid endemic to grasslands and open areas, and a priority species for research and conservation programs. This contribution provides information on the population size, habitat, behavior, breeding biology and nest success of X. flavus in two conservation units (CUs) in Viamao, state of Rio Grande do Sul, Brazil: the Environmental Protection Area Banhado Grande, and the Wildlife Refuge Banhado dos Pachecos, classified as an “Important Bird Area”. Searches for X. flavus were carried out mainly in open areas, the type of habitat favored by the species. Outside the breeding season individual behavior was recorded by the ad libitum method; during the breeding season, selected X. flavus pairs were observed following the sequence sampling method. The research areas were visited once a month, totaling approximately 530 hours of observations (September 2014 to June 2016) over 84 days, which included two breeding seasons. The species was observed across all months (not necessarily within the same year) and several X. flavus flocks were encountered, some with more than one hundred individuals (range = 2-137). Additionally, the behavior and feeding aspects, habitat use and breeding information on X. flavus were recorded. Two breeding colonies were found, and eleven nests were monitored. The estimated nesting success was 10% in Colony 1, but zero in Colony 2, where all eggs and nestlings were predated. Saffron-cowled Blackbirds were recorded in mixed flocks, mostly with Pseudoleistes guirahuro (Vieillot, 1819), P. virescens (Vieillot, 1819) and Xolmis dominicanus (Vieillot, 1823), the last also a globally endangered species. The collected information highlights the importance of CUs for the maintenance of X. flavus populations in the region. Maintenance of proper areas for feeding and breeding is necessary and urgent. Information from current research is being employed in the management plan of the Wildlife Refuge Banhado dos Pachecos in which X. flavus is one of the conservation target-species.

Fungal screening and aflatoxin production by Aspergillus section Flavi isolated from pre-harvest maize ears grown in two Argentine regions

Aflatoxin contamination in maize kernels grown in central Argentina has increased since 2008. Pre-harvest maize ear samples were collected during two growing seasons to determine prevalent fungal genera as well as aflatoxin production capacity by Aspergillus sect. Flavi in two maize regions. A direct planting procedure was performed for fungal screening. Aspergillus section Flavi was identified via a morphological analysis and some colonies were isolated. Strains were subjected to molecular analysis and aflatoxin production was studied. Fusarium was the fungus genus with the highest severity, followed by Penicillium and Aspergillus in both regions. Aspergillus sect. flavi were detected in 27 (73%) samples. PCR amplifications were observed in 94% of the isolates. Aspergillus sect. flavi severity varied significantly between years. A great proportion of toxigenic strains were observed in a native Aspergillus sect. flavi population (95%) producing B- and G-type aflatoxins. Maize ears grown in regions I and IV in Argentina are prone to degradation by fungi. Toxigenic strains are predominant in native Aspergillus sect. Flavi populations. Aflatoxin contamination of maize kernels is not restricted to post-harvest; rather, it is a serious issue that begins with colonization by Aspergillus sect. Flavi in ears.

Flexible thin-layer plasma inactivation of bacteria and mold survival in beef jerky packaging and its effects on the meat's physicochemical properties

The aims of the present study were to examine the use of a flexible thin-layer plasma system in inactivating bacteria and mold on beef jerky in a commercial package and to evaluate the physicochemical changes of the jerky. After plasma treatment for 10min, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Aspergillus flavus populations on the beef jerky were reduced by approximately 2 to 3Log CFU/g. No significant changes in metmyoglobin content, shear force, and myofibrillar fragmentation index were found in the plasma-treated beef jerky. On the other hand, the peroxide content and L⁎ value were decreased whereas the a⁎ and ΔE value were increased in the plasma-treated sample. Sensory evaluation indicated negative effects of plasma treatment on flavor, off-odor, and overall acceptability of the beef jerky. In conclusion, the flexible thin-layer plasma system could be employed as a means for decontamination of beef jerky, with slight changes to the physicochemical quality of the product.

Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.

Efficacy of water-dispersible formulations of biological control strains of Aspergillus flavus for aflatoxin management in corn

ABSTRACTField experiments were conducted in 2011 and 2012 to evaluate the efficacy of water-dispersible granule (WDG) formulations of biocontrol strains of Aspergillus flavus in controlling aflatoxin contamination of corn. In 2011, when aflatoxin was present at very high levels, there was no WDG treatment that could provide significant protection against aflatoxin contamination. The following year a new WDG formulation was tested that resulted in 100% reduction in aflatoxin in one field experiment and ≥ 49% reduction in all five WDG treatments with biocontrol strain 21882. Large sampling error, however, limited the resolution of various treatment effects. Corn samples were also subjected to microbial analysis to understand better the mechanisms of successful biocontrol. In the samples examined here, the size of the A. flavus population on the grain was associated with the amount of aflatoxin, but the toxigenic status of that population was a poor predictor of aflatoxin concentration.

Sexual Reproduction in Aspergillus flavus Sclerotia: Acquisition of Novel Alleles from Soil Populations and Uniparental Mitochondrial Inheritance.

Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. flavus is heterothallic and laboratory crosses between strains of the opposite mating type produce progeny showing genetic recombination. Sclerotia formed in crops are dispersed onto the soil surface at harvest and are predominantly produced by single strains of one mating type. Less commonly, sclerotia may be fertilized during co-infection of crops with sexually compatible strains. In this study, laboratory and field experiments were performed to examine sexual reproduction in single-strain and fertilized sclerotia following exposure of sclerotia to natural fungal populations in soil. Female and male roles and mitochondrial inheritance in A. flavus were also examined through reciprocal crosses between sclerotia and conidia. Single-strain sclerotia produced ascospores on soil and progeny showed biparental inheritance that included novel alleles originating from fertilization by native soil strains. Sclerotia fertilized in the laboratory and applied to soil before ascocarp formation also produced ascospores with evidence of recombination in progeny, but only known parental alleles were detected. In reciprocal crosses, sclerotia and conidia from both strains functioned as female and male, respectively, indicating A. flavus is hermaphroditic, although the degree of fertility depended upon the parental sources of sclerotia and conidia. All progeny showed maternal inheritance of mitochondria from the sclerotia. Compared to A. flavus populations in crops, soil populations would provide a higher likelihood of exposure of sclerotia to sexually compatible strains and a more diverse source of genetic material for outcrossing.

Efficacy of 2, 6-di (t-butyl)-p-cresol (BHT) and the entomopathogenic fungus Purpureocillium lilacinum, to control Tribolium confusum and to reduce aflatoxin B1 in stored maize

The objective of this study was to evaluate the effectiveness of the treatment with the entomopathogenic fungus Purpureocillium lilacinum and the antioxidant BHT alone or combined in sublethal doses and at different water activity (aW) levels, as insecticides and/or fungicide in sterile maize. Effect on accumulation of aflatoxin B1 (AFB1) was also evaluated. In addition, we studied the resilience of P. lilacinum in maize grains and the effect of the treatments on Aspergillus flavus populations and on Tribolium confusum, an insect pest vector of aflatoxigenic fungi. The combined treatment showed approximately 80% of insecticidal efficacy at all of the tested aW levels. A low incidence and prevention of contamination with A. flavus was observed in the live insects under all of the tested conditions. The A. flavus populations increased significantly at the end of the incubation period in all of the treatments. However, P. lilacinum populations showed no significant differences, when compared to the control treatment, in the presence of the toxigenic fungus. Maximum levels of AFB1 reduction (of around 90%) were observed after treating maize with the combination of P. lilacinum + BHT. Thus the obtained results prove that the treatment of combination was the most effective and show that it is a promising strategy for an integrated management of pests in stored maize.

AtoxigenicAspergillus flavus biological control of aflatoxin contamination: what is the mechanism?

The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic strain to the seed target has been used to describe the mechanism of biological control of aflatoxin contamination. However, recent evidence suggests that a form of intraspecific aflatoxin inhibition requiring growth of the competing strains together during the infection process in such a way that hyphae physically interact or touch is the trigger for preventing induction of aflatoxin synthesis. This direct touch-based inhibition of aflatoxin synthesis is posited to be the mechanistic basis of biological control in this system. Evidence for this idea comes from the published observations that co-culture of toxigenic and atoxigenic strains in a suspended disc system, in which the hyphae physically interact, prevents aflatoxin production. However, growth of the same strains in the same medium in the two compartments of a filter insert plate well system, separating the atoxigenic and toxigenic strains with a 0.4 μm or 3.0 μm filter, allows aflatoxin production approaching that of the toxigenic strain alone. When the strains are mixed and placed in both the insert and the well compartments, the intraspecific aflatoxin inhibition occurs as it did in the suspended disc culture system. This further suggests that neither nutrient competition nor soluble signal molecules, which should pass through the filter, are involved in intraspecific aflatoxin inhibition. When the two strains are separated by a 12 μm filter that would allow some passage of conidia or hyphae between the compartments the aflatoxin synthesis is approximately half that of the toxigenic strain alone. This phenomenon could be termed ‘competitive inclusion’ or ‘competitive phenotype conversion’. Work of others that relates to understanding the phenomenon is discussed, as well as anAspergillus flavus population biology study from the Louisiana maize agro-ecosystem which has biological control implications.

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR.

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5kb from 20kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A.flavus not captured with other methods. Aflatoxins, potent naturally-occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A.flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A.flavus populations.

Global health issues of aflatoxins in food and agriculture: challenges and opportunities.

Global health issues of aflatoxins in food and agriculture: challenges and opportunities.

Studies on Aspergillus Flavus Link. Isolated From Maize in Iran

Abstract The Aspergillus flavus population structure from maize kernels was examined. During 2011, samples were collected from two main grain maize production areas in Iran (Fars and Ardebil provinces), shortly before harvest. One-hundred nine A. flavus isolates were recovered on Dichloran Rose Bengal Chloramphenicole (DRBC) agar and Aspergillus flavus/parasiticus medium (AFPA) and grouped into morphotypes and Vegetative Compatibility Groups (VCGs) based on morphological (e.g. sclerotia production), physiological (e.g. aflatoxin-producing ability) and genetic criteria (e.g. heterokaryosis). In general, morphotype and VCG composition were highly dissimilar in both provinces. In total, 43.8% and 44.3% of A. flavus isolates from Ardebil and Fars, respectively, produced sclerotia. Sclerotia producers were identified as A. flavus L and S strain morphotypes in Ardebil (66.7% and 33.3%, respectively) and Fars (29.6% and 70.4%, respectively). Furthermore, 71 isolates (65.1%) were able to produce aflatoxin (Ardebil 40.8%, Fars 59.2%). The aflatoxin values were categorized into four different classes (< 10, 10-100, 100-1,000 and > 1,000 ppb). In total, 51 aflatoxin producing isolates of A. flavus (Ardebil n = 22, Fars n = 29) were assigned into 26 VCGs by complementation of nit auxotrophs on nitrate medium. None of the A. flavus isolates from Ardebil complemented with any isolates from Fars. Genetic diversity of A. flavus isolates was 59.1% and 41.8% for Ardebil and Fars, respectively. The different geographical adaptation and genetic make-up of A. flavus isolates may be due to different climatic conditions, soil types and crop sequences in both maize production areas.

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters.

Field efficacy of a mixture of atoxigenic Aspergillus flavus Link:Fr vegetative compatibility groups in preventing aflatoxin contamination in maize (Zea mays L.)

Competitive exclusion of aflatoxin producers by endemic atoxigenic strains of Aspergillus flavus is a proven tool for aflatoxin management being adapted for use in Africa. Field efficacy of an experimental formulation consisting of four native atoxigenic strains (La3303, La3304, La3279 and Ka16127) was evaluated on maize in 2007 and 2008 in four agroecological zones in Nigeria. The four atoxigenic strains were individually formulated on sterile sorghum grain and subsequently mixed in equal proportions. The blended product was applied on soil (40kg/ha), 2–3weeks before flowering. Grains from treated and untreated fields were analyzed for aflatoxins at harvest and after storage. Proportions of the A. flavus population composed of each of the four applied strains in soil before treatment and in harvested grains were determined using vegetative compatibility analyses. Application of the strain mixture resulted in reduced aflatoxin content and significantly (P<0.05) increased the combined frequencies of the vegetative compatibility groups (VCGs) of the applied strains recovered from the soil and grain. Aflatoxin reductions of 67–95% were associated with a 74–80% combined incidence of the VCGs of the four atoxigenic strains on the treated crops. The applied atoxigenic strains remained with the crop into storage and reduced postharvest increases in contamination. The results suggest that the evaluated multi-strain formulated product has potential to contribute to reduced aflatoxin contamination in Nigeria. This is the first report of a field evaluation of an endemic strain mixture effective at reducing aflatoxin contamination during crop development.