Atoxigenic Strains Of Aspergillus Flavus Research Articles

Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Biodiversity and Distribution of <i>Aspergillus</i> and Their Toxins in Maize from Western and Eastern Regions of South Africa

Aspergillus species and aflatoxins production are more prevalent during times of high heat and drought. In South Africa, there is frequent occurrence of drought as a result of climate change. The aim of this study was to investigate the biodiversity and distribution of Aspergillus species with their corresponding toxins in maize from main maize producing regions of South Africa; [Western Regions (WR) and Eastern Regions (ER)]. One hundred and twenty-three (64 from WR and 59 from ER) maize samples from the two agro-climatic regions in South Africa were analyzed using cultural, molecular and analytical methods. Across agro-climatic regions, Aspergillus species contaminated about 62% of the maize samples, while Aspergillus flavus was the most prevalent (47.15%) followed by Aspergillus fumigatus (4.69%) while the least was Aspergillus parasiticus (0.81%). The Western Regions showed a higher distribution of varieties of Aspergillus species compared to the Eastern Regions. Aflatoxins contaminated only 27.64% of the maize samples with a mean total aflatoxin concentration of 2.40 μg/kg which is below the South Africa’s set standard for total aflatoxin in food (5 μg/kg). About 10.57% of the samples produce aflatoxins above the 5 μg/kg permissible limit for total aflatoxin in foods. The ratio of toxigenic to atoxigenic strains of Aspergillus flavus was generally low in all the regions of South Africa. This study could aid policy makers to make informed decisions in developing remediation strategies for Aspergillus mycotoxins.

Aflatoxins: Producing-Molds, Structure, Health Issues and Incidence in Southeast Asian and Sub-Saharan African Countries.

This review aims to update the main aspects of aflatoxin production, occurrence and incidence in selected countries, and associated aflatoxicosis outbreaks. Means to reduce aflatoxin incidence in crops were also presented, with an emphasis on the environmentally-friendly technology using atoxigenic strains of Aspergillus flavus. Aflatoxins are unavoidable widespread natural contaminants of foods and feeds with serious impacts on health, agricultural and livestock productivity, and food safety. They are secondary metabolites produced by Aspergillus species distributed on three main sections of the genus (section Flavi, section Ochraceorosei, and section Nidulantes). Poor economic status of a country exacerbates the risk and the extent of crop contamination due to faulty storage conditions that are usually suitable for mold growth and mycotoxin production: temperature of 22 to 29 °C and water activity of 0.90 to 0.99. This situation paralleled the prevalence of high liver cancer and the occasional acute aflatoxicosis episodes that have been associated with these regions. Risk assessment studies revealed that Southeast Asian (SEA) and Sub-Saharan African (SSA) countries remain at high risk and that, apart from the regulatory standards revision to be more restrictive, other actions to prevent or decontaminate crops are to be taken for adequate public health protection. Indeed, a review of publications on the incidence of aflatoxins in selected foods and feeds from countries whose crops are classically known for their highest contamination with aflatoxins, reveals that despite the intensive efforts made to reduce such an incidence, there has been no clear tendency, with the possible exception of South Africa, towards sustained improvements. Nonetheless, a global risk assessment of the new situation regarding crop contamination with aflatoxins by international organizations with the required expertise is suggested to appraise where we stand presently.

The comparative virulence of an atoxigenic strain of Aspergillus flavus (Eurotiales: Trichocomaceae) and the commercial ICIPE 69 Metarhizium anisopliae (Hypocreales: Clavicipitaceae) to the bean leaf beetle Ootheca mutabilis (Coleoptera: Chrysomelidae)

The recent isolation of an atoxigenic strain of Aspergillus flavus (Link) (Eurotiales: Trichocomaceae), which was virulent to all stages of Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae) has generated interest to investigate virulence of the entomopathogenic fungus against other crop pests, and how it compares with established commercial biopesticides. In this study, we tested the pathogenicity and virulence of the atoxigenic A. flavus strain to Ootheca mutabilis (Sahlberg) (Coleoptera: Chrysomelidae) in the laboratory compared to the commercial Metarhizium anisopliae (Metschnikoff Sorokin) (Hypocreales: Clavicipitaceae; OD®, ICIPE 69). Serial dilutions of both entomopathogenic fungi (1 × 106–1 × 109 conidia/ml) were evaluated against adult O. mutabilis. Distilled water and cypermethrin 5% EC were included as controls. The treatments were replicated four times and the bioassay was repeated once. Both fungi at 1 × 109 conidia/ml killed 100% of tested O. mutabilis within seven days in the 1st and 2nd trials. The concentration of both fungi that killed 50% of O. mutabilis was the same in both trials (5.3 × 106 conidia/ml). The time taken by A. flavus and ICIPE 69 to kill 50% of O. mutabilis was not significantly different in the 1st trial (3.4 ± 0.1 days and 2.9 ± 0.4 days, respectively). However, ICIPE 69 took a significantly shorter time (2.1 ± 0.4 days) than A. flavus (3.6 ± 0.2 days) to kill 50% of O. mutabilis in the 2nd trial. We concluded that A. flavus is comparably as pathogenic and virulent (in terms of cumulative mortality and LC50) against O. mutabilis as ICIPE 69, and therefore both fungi may be viable biopesticides for commercialization against this pest.

Monitoring Aspergillus flavus Genotypes in a Multi-Genotype Aflatoxin Biocontrol Product With Quantitative Pyrosequencing.

Aflatoxins pose significant food security and public health risks, decrease productivity and profitability of animal industries, and hamper trade. To minimize aflatoxin contamination in several crops, a biocontrol technology based on atoxigenic strains of Aspergillus flavus is commercially used in the United States and some African countries. Significant efforts are underway to popularize the use of biocontrol in Africa by various means including incentives. The purpose of this study was to develop quantitative pyrosequencing assays for rapid, simultaneous quantification of proportions of four A. flavus biocontrol genotypes within complex populations of A. flavus associated with maize crops in Nigeria to facilitate payment of farmer incentives for Aflasafe (a biocontrol product) use. Protocols were developed to confirm use of Aflasafe by small scale farmers in Nigeria. Nested PCR amplifications followed by sequence by synthesis pyrosequencing assays were required to quantify frequencies of the active ingredients and, in so doing, confirm successful use of biocontrol by participating farmers. The entire verification process could be completed in 3–4 days proving a savings over other monitoring methods in both time and costs and providing data in a time frame that could work with the commercial agriculture scheme. Quantitative pyrosequencing assays represent a reliable tool for rapid detection, quantification, and monitoring of multiple A. flavus genotypes within complex fungal communities, satisfying the requirements of the regulatory community and crop end-users that wish to determine which purchased crops were treated with the biocontrol product. Techniques developed in the current study can be modified for monitoring other crop-associated fungi.

Performance of Broilers Fed with Maize Colonized by Either Toxigenic or Atoxigenic Strains of Aspergillus flavus with and without an Aflatoxin-Sequestering Agent.

In warm agricultural areas across the globe, maize, groundnut, and other crops become frequently contaminated with aflatoxins produced primarily by the fungus Aspergillus flavus. Crop contamination with those highly toxic and carcinogenic compounds impacts both human and animal health, as well as the income of farmers and trade. In Nigeria, poultry productivity is hindered by high prevalence of aflatoxins in feeds. A practical solution to decrease crop aflatoxin content is to use aflatoxin biocontrol products based on non-toxin-producing strains of A. flavus. The biocontrol product Aflasafe® was registered in 2014 for use in maize and groundnut grown in Nigeria. Its use allows the production of aflatoxin-safe maize and groundnut. A portion of the maize treated with Aflasafe in Nigeria is being used to manufacture feeds used by the poultry industry, and productivity is improving. One of the conditions to register Aflasafe with the national regulator was to demonstrate both the safety of Aflasafe-treated maize to avian species and the impact of Aflasafe as a public good. Results presented here demonstrate that the use of maize colonized by an atoxigenic strain of Aflasafe resulted in superior (p < 0.05) broiler performance in all evaluated parameters in comparison to broilers fed with toxigenic maize. Use of an aflatoxin-sequestering agent (ASA) was not sufficient to counteract the harmful effects of aflatoxins. Both the safety and public good value of Aflasafe were demonstrated during our study. In Nigeria, the availability of aflatoxin-safe crops as a result of using Aflasafe allows poultry producers to improve their productivity, their income, and the health of consumers of poultry products.

Biological Control Products for Aflatoxin Prevention in Italy: Commercial Field Evaluation of Atoxigenic Aspergillus flavus Active Ingredients.

Since 2003, non-compliant aflatoxin concentrations have been detected in maize produced in Italy. The most successful worldwide experiments in aflatoxin prevention resulted from distribution of atoxigenic strains of Aspergillus flavus to displace aflatoxin-producers during crop development. The displacement results in lower aflatoxin concentrations in harvested grain. The current study evaluated in field performances of two atoxigenic strains of A. flavus endemic to Italy in artificially inoculated maize ears and in naturally contaminated maize. Co-inoculation of atoxigenic strains with aflatoxin producers resulted in highly significant reductions in aflatoxin concentrations (>90%) in both years only with atoxigenic strain A2085. The average percent reduction in aflatoxin B1 concentration in naturally contaminated maize fields was 92.3%, without significant differences in fumonisins between treated and control maize. The vegetative compatibility group of A2085 was the most frequently recovered A. flavus in both treated and control plots (average 61.9% and 53.5% of the A. flavus, respectively). A2085 was therefore selected as an active ingredient for biocontrol products and deposited under provisions of the Budapest Treaty in the Belgian Co-Ordinated Collections of Micro-Organisms (BCCM/MUCL) collection (accession MUCL54911). Further work on development of A2085 as a tool for preventing aflatoxin contamination in maize produced in Italy is ongoing with the commercial product named AF-X1™.

Infection with toxigenic and atoxigenic strains of Aspergillus flavus induces different transcriptional signatures in maize kernels

ABSTRACTThe application of atoxigenic Aspergillus flavus strains in maize fields has been shown to be an effective strategy for controlling contamination of aflatoxins, potent carcinogens produced by the fungus. This study monitored the expression levels of 18 defense genes against toxigenic and atoxigenic A. flavus strains in developing maize kernels over a time course of 96 h after inoculation. A stronger upregulation of genes encoding pathogenesis-related proteins, oxidative stress-related proteins, transcriptional factors and lipoxygenases were observed in response to the atoxigenic strain. On the other side, this strain showed a significant enhanced growth in the later stages of infection, measured as copy number of the constitutive calmodulin gene. These results suggest that overexpression of maize-defense-associated genes observed in response to the atoxigenic strain could contribute to an aflatoxin reduction. The identification of genes significantly affecting the resistance to A. flavus or aflatoxin accumulation would accelerate the development of resistant cultivars.

Aflatoxin M1 in milk and milk products: a short review

Aflatoxin M1 (AFM1) is associated with carcinogenicity, genotoxicity, mutagenicity, and teratogenicity and as a result, represents a human health problem worldwide. This review will detail the toxicity, analytical methodology, occurrence, and prevention and control of AFM1 in milk and milk products. The probable daily intakes (PDI) per bodyweight (bw) worldwide ranged from 0.002 to 0.26 ng/kg bw/day for AFM1. Nevertheless, the high occurrence of AFM1 demonstrated in this review establishes the need for monitoring to reduce the risk of toxicity to humans. The recommended extraction method of AFM1 from milk is liquid-liquid with acetonitrile because of the acceptable recoveries (85-97%), compatibility with the environment, and cleanest extracts. The recommended analytical technique for the determination of AFM1 in milk is the high performance-liquid chromatography-fluorescence detector (HPLC-FLD), achieving a 0.001 µg/kg detection limit. The HPLC-FLD is the most common internationally recognised official method for the analysis of AFM1 in milk. The suggested extraction and analytical method for cheese is dichloromethane (81-108% recoveries) and ELISA, respectively. This review reports the projected worldwide occurrence of AFM1 in milk of 2010-2015. Of the 7,841 samples, 5,873 (75%) were positive for AFM1, 26% (2,042) exceeded the maximum residue levels (MRL) of 0.05 µg/kg defined by the European Union and 1.53% (120) exceeded the MRL of 0.5 µg/kg defined by the US Food and Drug Administration. The most effective way of preventing AFM1 occurrences is to reduce contamination of AFB1 in animal feed using biological control with atoxigenic strains ofAspergillus flavus, proper storage of crops, and the addition of binders to AFB1-contaminated feed. Controllable measures include the addition of binders and use of biological transforming agents such as lactic acid bacteria applied directly to milk. Though the one accepted method for the control of AFM1 in milk and milk products is the enforcement of governmental MRL.

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.

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.

Health economic impacts and cost-effectiveness of aflatoxin-reduction strategies in Africa: case studies in biocontrol and post-harvest interventions

Advances in health economics have proven useful in evaluating the cost-effectiveness of interventions, where the benefit usually takes the form of improved health outcomes rather than market outcomes. The paper performs health-based cost-effectiveness analyses of two potential aflatoxin control strategies in Africa: (1) pre-harvest biocontrol, using atoxigenic strains of Aspergillus flavus competitively to exclude toxigenic strains from colonizing maize in Nigeria; and (2) post-harvest interventions in a package to reduce aflatoxin accumulation in groundnuts in Guinea. It is described how health benefits gained from each intervention, in terms of fewer aflatoxin-induced hepatocellular carcinoma cases, can be compared with the costs of implementing the interventions. It is found that both interventions would be extremely cost-effective if applied widely in African agriculture. That is, the monetized value of lives saved and quality of life gained by reducing aflatoxin-induced hepatocellular carcinoma far exceeds the cost of either biocontrol or the post-harvest intervention package to achieve those health benefits. The estimated cost-effectiveness ratio (CER; gross domestic product multiplied by disability-adjusted life years saved per unit cost) for biocontrol in Nigerian maize ranged from 5.10 to 24.8; while the estimated CER for the post-harvest intervention package in Guinean groundnuts ranged from 0.21 to 2.08. Any intervention with a CER > 1 is considered by the World Health Organization (WHO) to be ‘very cost-effective’, while an intervention with a CER > 0.33 is considered ‘cost-effective’. Aside from cost-effectiveness, public health interventions must be readily accepted by the public, and must have financial and infrastructural support to be feasible in the parts of the world where they are most needed.

Classification of toxigenic and atoxigenic strains of Aspergillus flavus with hyperspectral imaging

Aspergillus flavus ( A. flavus) produces secondary metabolites, aflatoxins, that are harmful to both humans and animals. Because of stringent federal regulation requirements as well as the limitations of available detection methods, there is an urgent need for rapid, non-invasive and effective techniques such as hyperspectral imaging, for the detection of the toxigenic strains of A. flavus. Hyperspectral images of toxigenic and atoxigenic strains of A. flavus were classified. Principal component analysis (PCA) was applied for data decorrelation and dimensionality reduction. A Genetic Algorithm (GA) was implemented for the selection of principal components (PCs) based on Bhattacharya Distance (B-Distance). A Support Vector Machine (SVM) was successfully applied for the classification. Under halogen light sources, in average 83% of the toxigenic fungus pixels and 74% of the atoxigenic fungus pixels were correctly classified; while under UV light sources, 67% of the toxigenic fungus pixels and 85% of the atoxigenic fungus pixels were correctly classified. The pair-wise classification accuracies between toxigenic AF13 and each atoxigenic fungus species (AF38, AF283 and AF2038) were 80%, 91% and 95% under halogen light sources, and 75%, 97% and 99% under UV lights, respectively.

Use of Pyrosequencing to Quantify Incidence of a SpecificAspergillus flavusStrain Within Complex Fungal Communities Associated with Commercial Cotton Crops

Atoxigenic strains of Aspergillus flavus have been used as aflatoxin management tools on over 50,000 hectares of commercial crops since 2000. To assess treatment efficacy, atoxigenic strain incidence is routinely monitored by vegetative compatibility analyses (VCA) that require culturing, generation of auxotrophs, and complementation with tester mutants. Two pyrosequencing assays (PA) that require no culturing were developed for monitoring incidences of atoxigenic strains on ginned cottonseed. The assays, which quantify frequencies of characteristic single nucleotide polymorphisms (SNPs) in the aflR and pksA genes, were validated against standard VCA on cottonseed collected from commercial gins in South Texas, Arizona, and Southern California where the atoxigenic strain AF36 is used to manage aflatoxin contamination. Cottonseed washings were subjected to both VCA and PA. PA was performed directly on DNA isolated from particulates pelleted from the wash water by centrifugation. Addition of CaCl(2) and diatomaceous earth prior to pelleting increased the amount of DNA isolated. Accuracy and reproducibility of the PA were contrasted with those for the VCA that has been used for over a decade. Correlation coefficients between VCA and PA indicated good correspondence between the results from the two assays (r = 0.91 for aflR assay and r = 0.80 for pksA assay). PAs were highly variable for samples with low incidences of A. flavus due to variability in the initial polymerase chain reaction step. This held for both DNA isolated from cottonseed washes and for mixtures of purified DNA. For samples yielding low quantities of A. flavus DNA, averaging of results from 4 to 5 replicates was required to achieve acceptable correlations with VCA. Pyrosequencing has the potential to become a powerful tool for monitoring atoxigenic strains within complex A. flavus communities without limitations imposed by traditional culturing methods.

Effects of methyl jasmonate on phytoalexin production and aflatoxin control in the developing cotton boll

Artificially wounded 22–27-day old developing cotton bolls were initially inoculated with, (1) a cell-free, hot water-soluble mycelial extract (CFME) of an atoxigenic strain of Aspergillus flavus or with, (2) chitosan lactate (CHL) or with, (3) CFME or CHL and then exposed to gaseous methyl jasmonate (MJ) or, (4) exposed to MJ alone. Five days after these treatments, the induction of the sesquiterpenoid naphthol phytoalexins, 2,7-dihydroxycadalene (DHC) and 2-hydroxy-7-methoxy cadalene (HMC), lacinilene C, lacinilene C7-methyl ether, and the coumarin phytoalexin-scopoletin was determined on the excised carpel discs surrounding the inoculated surfaces of the developing cotton bolls. The results indicated a two- or three-fold increase in the production of the phytoalexins when gaseous MJ was added in combination to the CFME or the CHL elicitors. In a separate experiment, 22–27-day old developing cotton bolls were pretreated for a five-day period as described above and then a spore suspension of a toxigenic strain of A. flavus was introduced into a second artificial wound which was produced adjacent to the first wound. On boll maturity, the cottonseeds located within the locules underlying the areas that were pretreated with both elicitors and MJ then later infected with toxigenic A. flavus exhibited a 75–95% aflatoxin B1 inhibition. These results suggest a host defense mechanism which may be triggered by both elicitors and MJ.

The Effect of Sterilization, pH, Filler and Spore Inoculum Concentration on the Preparation of Alginate Pellets

Alginate encapsulation of an atoxigenic strain of Aspergillus flavus was studied in order to optimize encapsulation of fungal inocula with alginic acid. Sterilization by autoclaving is known to depolymerize sodium alginate. Buffered solutions (pH = 7-8) reduced this effect. Autoclaving the alginate solution with a filler/nutrient further inhibited the depolymerization reaction. Autoclaving under optimal conditions allowed a less expensive alginate (medium viscosity) to be used at a lower concentration (1%) to produce a stable product. The lowest cost pellets resulted from use of 1% medium viscosity sodium alginate with 10% cotton-seed meal. Further savings may be achieved by performing fermentations directly in alginate-nutrient mixtures and thus eliminating the mixing and blending steps. In such formulations, the nutrient composition and length of fermentation must be adjusted to prevent alginate hydrolysis. The ultimate composition of alginate pellets is influenced by the diffusion of nutrients during gelation. Up to 65% of water-soluble nutrients were lost from alginate pellets during gelation. Once pellets are introduced into the environment, organisms other than the formulated agent compete for pelleted nutrients. A minimum concentration of the biocontrol agent must be present to ensure the agent excludes competitors and successfully converts the nutrients to biomass. For A. flavus, 5000 spores g−1 were required.

Twin-screw extrusion of ‘Pesta’-encapsulated biocontrol agents

'Pesta' granules in which fungal propagules are encapsulated in a wheat gluten matrix were prepared in multi-pound quantities by twin-screw extrusion and fluid bed drying. Dough formulations for extrusion contained wheat flour and kaolin, or wheat flour, kaolin and rice flour, plus water and fungal inoculum. Conidial inoculum of Colletotrichum truncatum, a pathogen of the weed hemp sesbania (Sesbania exaltata), survived laboratory scale dough preparation [100% retention of colony-forming units (c.f.u.)] better than dough preparation for twin-screw extrusion (8-10% c.f.u. retention). The loss in viability was linked to the lower water content of dough used in the twin-screw extruder. Fluid bed drying reduced viability further to 1%. Retention of viability after twin-screw extrusion and fluid bed drying at 35-50 °C was 35% with Alternaria conjuncta/infectoria, a pathogen of swamp dodder (Cuscuta gronovii). Retention was 86-100% with atoxigenic strains of Aspergillus flavus and Aspergillus parasiticus used as biocompetitors to reduce aflatoxin levels in peanuts. In the greenhouse, twin-screw-extruded granules containing C. truncatum (at about 5 x 10 4 c.f.u. g -1 ) caused high levels of infection and mortality in hemp sesbania seedlings.

Formulating Atoxigenic Aspergillus flavus for Field Release

A procedure was developed to encapsulate mycelia of an atoxigenic strain of Aspergillus flavus in alginate pellets for seeding into agricultural fields in order to reduce aflatoxin contamination via competitive exclusion. Kaolin, a clay filler commonly employed in alginate formulations, was detrimental to pellet performance as measured by spore yield. Corn cob grits, a by-product of the corn industry, was found to be an excellent replacement for kaolin. Of nine nutritive adjuvants tested, wheat gluten improved pellet performance the most, although gluten concentrations above 5% were difficult to process. The best formulation tested consisted of 1% sodium alginate, 5% corn cob grits and 5% wheat gluten. On a 'per gram' basis, this alginate formulation yielded more spores than either A. flavus sclerotia or colonized wheat seed. Pesticides were also tested as adjuvants with potential use for protecting pellets under field conditions. Only one (chloramphenicol) of four tested pesticides (the others were dichloran, rose Bengal and cyfluthrin) reduced pellet sporulation. Formulations with or without pesticide adjuvants retained similar spore yield potential during a 2-year storage at 8 C. However, spore production in stored products lagged behind that of fresh products. At 75% relative humidity (RH), pellet storage stability decreased with increasing temperature from 27 to 42 C. Pellet spore yield at 32 C decreased as RH decreased from 100 to 90%. Sporulation occurred at 90% RH but not at 88% RH. Spore yield varied widely in four field tests, and the cumulative spore yield was inversely correlated (r2= -0.798, P 0.01) with rainfall. The results suggest that alginate pellets may be effective formulations for delivery of atoxigenic A. flavus strains to furrow-irrigated cotton in desert environments, where aflatoxin contamination of cottonseed is most severe.

Influence of Field Application of an Atoxigenic Strain ofAspergillus flavuson the Populations ofA. flavusInfecting Cotton Bolls and on the Aflatoxin Content of Cottonseed

An atoxigenic strain of Aspergillus flavus was applied to soils planted with cotton in Yuma, Arizona, to assess the ability of the atoxigenic strain to competitively exclude aflatoxin-producing strains during cotton boll infection and thereby prevent aflatoxin contamination of cottonseed. In both 1989 and 1990, the atoxigenic strain displaced other infecting strains during cotton boll development. Displacement was associated with significant reductions in the quantity of aflatoxins contaminating the crop at maturity. Although frequency of infected locules differed between years (1% versus 25%), in both years displacement occurred without increases in the amount of infection as measured by the quantity of locules with bright-green-yellow-fluorescence (BGYF)

Competitive Exclusion of a Toxigenic Strain ofAspergillus flavusby an Atoxigenic Strain

Several experiments were employed to test the role of competition in the ability of an atoxigenic strain of Aspergillus flavus to inhibit the aflatoxin contamination of developing cotton bolls. In initial tests, nitrate-nonutilizing mutants were used to follow seed infection by toxigenic and atoxigenic strains of A. flavus in coinoculated bolls. Competitive exclusion was found to contribute to the effect of the atoxigenic strain on contamination, but results suggested a second mechanism may also have been in effect. Aflatoxin contamination by the toxigenic strain was similarly inhibited by an atoxigenic strain in vivo and in liquid fermentation, and the atoxigenic strain was equally effective when applied at spore concentrations either equal to those of the toxigenic strain or one-half those of the toxigenic strain [...]