Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Evaluating Acheta domesticus (Orthoptera: Gryllidae) for the reduction of fumonisin B1 levels in livestock feed.

Mycotoxins that contaminate grain can cause the devaluation of agricultural products and create health risks for the consumer. Fumonisins are one such mycotoxin. Produced primarily by Fusarium verticillioides (Hypocreales: Nectriaceae) (Nirenberg, 1976) on corn, fumonisins' economic impact can be significant by causing various diseases in livestock if contaminated corn is not monitored and removed from animal feed. Finding safe alternatives to the destruction and waste of contaminated grain and restoring its economic value is needed for a sustainable future. Safe reintroduction into the farm food web may be possible through a consumable intermediary such as insects. This study demonstrates the suitability of the house cricket, Acheta domesticus L., as an alternative protein source in domestic animal feed by quantifying fumonisin B1 (FB1) levels in their subsequent insect meal and frass. Small colonies of 2nd instar A. domesticus were reared to 5th instar adults on nutrient-optimized corn-based diets treated with 4 levels of FB1 from 0 to 20 ppm. Increasing levels of FB1 had no adverse effects on the survivorship or growth of A. domesticus. Insect meals prepared from A. domesticus had significantly lower levels of FB1, at 3%-5% of their respective diets, while frass did not differ significantly from their diet. The successful rearing to adulthood of A. domesticus on fumonisin-contaminated diet paired with lower levels of FB1 in their processed insect meal supports the idea that more sustainable agricultural practices can be developed through remediation of low-value mycotoxin-contaminated grain with safer, higher-value insects as livestock feed components.

Mellein: Production in culture by Macrophomina phaseolina isolates from soybean plants exhibiting symptoms of charcoal rot and its role in pathology.

Macrophomina phaseolina (Mp) is a fungal pathogen proposed to enter host roots by releasing toxins that induce local necrosis in roots allowing entry of hyphae. Mp is reported to produce several potent phytotoxins, including (-)-botryodiplodin and phaseolinone, but isolates that do not produce these phytotoxins retain virulence. One hypothesis explaining these observations is that some Mp isolates may produce other unidentified phytotoxin(s) responsible for virulence. A previous study of Mp isolates from soybean found 14 previously unreported secondary metabolites using LC-MS/MS, including mellein, which has various reported biological activities. This study was conducted to investigate the frequency and amounts of mellein produced in culture by Mp isolates from soybean plants exhibiting symptoms of charcoal rot and to investigate the role of mellein in any observed phytotoxicity. LC-MS/MS analysis of cell-free culture filtrates (CCFs) from 89 Mp isolates revealed that 28.1% produced mellein (49-2,203 µg/L). In soybean seedlings in hydroponic culture, Mp CCFs diluted to 25% (vol/vol) in hydroponic growth medium induced phytotoxic symptoms with frequencies of 73% chlorosis, 78% necrosis, 7% wilting, and 16% death, and at 50% (vol/vol) induced phytotoxicity with frequencies of 61% chlorosis, 82% necrosis, 9% wilting, and 26% death. Commercially-available mellein (40-100 µg/mL) in hydroponic culture medium induced wilting. However, mellein concentrations in CCFs exhibited only weak, negative, insignificant correlations with phytotoxicity measures in soybean seedlings, suggesting that mellein does not contribute substantially to observed phytotoxic effects. Further investigation is needed to determine if mellein plays any role in root infection.

Soybean Seed Sugars: A Role in the Mechanism of Resistance to Charcoal Rot and Potential Use as Biomarkers in Selection.

Charcoal rot, caused by Macrophomina phaseolina, is a major soybean disease resulting in significant yield loss and poor seed quality. Currently, no resistant soybean cultivar is available in the market and resistance mechanisms to charcoal rot are unknown, although the disease is believed to infect plants from infected soil through the roots by unknown toxin-mediated mechanisms. The objective of this research was to investigate the association between seed sugars (sucrose, raffinose, stachyose, glucose, and fructose) and their role as biomarkers in the soybean defense mechanism in the moderately resistant (MR) and susceptible (S) genotypes to charcoal rot. Seven MR and six S genotypes were grown under irrigated (IR) and non-irrigated (NIR) conditions. A two-year field experiment was conducted in 2012 and 2013 at Jackson, TN, USA. The main findings in this research were that MR genotypes generally had the ability to maintain higher seed levels of sucrose, glucose, and fructose than did S genotypes. Conversely, susceptible genotypes showed a higher level of stachyose and lower levels of sucrose, glucose, and fructose. This was observed in 6 out of 7 MR genotypes and in 4 out of 6 S genotypes in 2012; and in 5 out of 7 MR genotypes and in 5 out of 6 S genotypes in 2013. The response of S genotypes with higher levels of stachyose and lower sucrose, glucose, and fructose, compared with those of MR genotypes, may indicate the possible role of these sugars in a defense mechanism against charcoal rot. It also indicates that nutrient pathways in MR genotypes allowed for a higher influx of nutritious sugars (sucrose, glucose, and fructose) than did S genotypes, suggesting these sugars as potential biomarkers for selecting MR soybean plants after harvest. This research provides new knowledge on seed sugars and helps in understanding the impact of charcoal rot on seed sugars in moderately resistant and susceptible genotypes.

Pigment Produced by Glycine-Stimulated Macrophomina Phaseolina Is a (-)-Botryodiplodin Reaction Product and the Basis for an In-Culture Assay for (-)-Botryodiplodin Production.

An isolate of Macrophomina phaseolina from muskmelons (Cucumis melo)was reported by Dunlap and Bruton to produce red pigment(s) in melons and in culture in the presence of added glycine, alanine, leucine, or asparagine in the medium, but not with some other amino acids and nitrogen-containing compounds. We explored the generality and mechanism of this pigment production response using pathogenic M. phaseolina isolates from soybean plants expressing symptoms of charcoal rot disease. A survey of 42 M. phaseolina isolates growing on Czapek-Dox agar medium supplemented with glycine confirmed pigment production by 71% of isolates at the optimal glycine concentration (10 g/L). Studies in this laboratory have demonstrated that some pathogenic isolates of M. phaseolina produce the mycotoxin (-)-botryodiplodin, which has been reported to react with amino acids, proteins, and other amines to produce red pigments. Time course studies showed a significant positive correlation between pigment and (-)-botryodiplodin production by selected M. phaseolina isolates with maximum production at seven to eight days. Pigments produced in agar culture medium supplemented with glycine, beta-alanine, or other amines exhibited similar UV-vis adsorption spectra as did pigments produced by (±)-botryodiplodin reacting in the same agar medium. In a separate study of 39 M. phaseolina isolates, red pigment production (OD520) on 10 g/L glycine-supplemented Czapek-Dox agar medium correlated significantly with (-)-botryodiplodin production (LC/MS analysis of culture filtrates) in parallel cultures on un-supplemented medium. These results support pigment production on glycine-supplemented agar medium as a simple and inexpensive in-culture method for detecting (-)-botryodiplodin production by M. phaseolina isolates.

Field studies on the deterioration of microplastic films from ultra-thin compostable bags in soil.

In recent years, some countries have replaced single-use plastic bags with bags manufactured from compostable plastic film that can be used for collecting food wastes and composted together with the waste. Because industrial compost contains undeteriorated fragments of these bags, application to field soil is a potential source of small-sized residues from these bags. This study was undertaken to examine deterioration of these compostable film microplastics (CFMPs) in field soil at three different localities in Italy. Deterioration of CFMPs did not exceed 5.7% surface area reduction during the 12-month experimental period in two sites located in Northern Italy. More deterioration was observed in the Southern site, with 7.2% surface area reduction. Deterioration was significantly increased when fields were amended with industrial compost (up to 9.6%), but not with home compost. Up to 92.9% of the recovered CFMPs were associated with the soil fungus Aspergillus flavus, with 20.1%-71.2% aflatoxin-producing isolates. Application of industrial compost resulted in a significant increase in the percentage of CFMPs associated with A. flavus. This observation provides an argument for government regulation of accumulation of CFMPs and elevation of hazardous fungi levels in agricultural soils that receive industrial compost.

Effects of Charcoal Rot on Soybean Seed Composition in Soybean Genotypes That Differ in Charcoal Rot Resistance under Irrigated and Non-Irrigated Conditions.

Charcoal rot is a major disease of soybean (Glycine max) caused by Macrophomina phaseolina and results in significant loss in yield and seed quality. The effects of charcoal rot on seed composition (seed protein, oil, and fatty acids), a component of seed quality, is not well understood. Therefore, the objective of this research was to investigate the impact of charcoal rot on seed protein, oil, and fatty acids in different soybean genotypes differing in their charcoal rot susceptibility under irrigated and non-irrigated conditions. Two field experiments were conducted in 2012 and 2013 in Jackson, TN, USA. Thirteen genotypes differing in charcoal rot resistance (moderately resistant and susceptible) were evaluated. Under non-irrigated conditions, moderately resistant genotypes showed either no change or increased protein and oleic acid but had lower linolenic acid. Under non-irrigated conditions, most of the susceptible genotypes showed lower protein and linolenic acid but higher oleic acid. Most of the moderately resistant genotypes had higher protein than susceptible genotypes under irrigated and non-irrigated conditions but lower oil than susceptible genotypes. The different responses among genotypes for protein, oil, oleic acid, and linolenic acid observed in each year may be due to both genotype tolerance to drought and environmental conditions, especially heat differences in each year (2012 was warmer than 2013). This research showed that the increases in protein and oleic acid and the decrease in linolenic acid may be a possible physiological mechanism underlying the plant's responses to the charcoal rot infection. This research further helps scientists understand the impact of irrigated and non-irrigated conditions on seed nutrition changes, using resistant and susceptible genotypes.

First Report of the Production of Mycotoxins and Other Secondary Metabolites by Macrophomina phaseolina (Tassi) Goid. Isolates from Soybeans (Glycine max L.) Symptomatic with Charcoal Rot Disease.

Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57-79.9 µg/L), moniliformin (61.8% of cultures at 0.011-12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71-1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012-0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants.

Draft Genome Sequences of One Aspergillus parasiticus Isolate and Nine Aspergillus flavus Isolates with Varying Stress Tolerance and Aflatoxin Production.

Aspergillus flavus and Aspergillus parasiticus produce carcinogenic aflatoxins during crop infection, with extensive variations in production among isolates, ranging from atoxigenic to highly toxigenic. Here, we report draft genome sequences of one A. parasiticus isolate and nine A. flavus isolates from field environments for use in comparative, functional, and phylogenetic studies.

Effects of Harvest-Aids on Seed Nutrition in Soybean under Midsouth USA Conditions.

Interest in using harvest aids (defoliants or crop desiccants) such as paraquat, carfentrazone-ethyl, glyphosate, and sodium chlorate (NaClO3) have become increasingly important to assure harvest efficiency, producer profit, and to maintain seed quality. However, information on the effects of harvest aids on seed nutrition (composition) (protein, oil, fatty acids, sugars, and amino acids) in soybean is very limited. The objective of this research was to investigate the influence of harvest aids on seed protein, oil, fatty acids, sugars, and amino acids in soybean. Our hypothesis was that harvest aid may influence seed nutrition, especially at R6 as at R6 the seeds may still undergo biochemical changes. Field experiments were conducted in 2012 and 2013 under Midsouth USA environmental conditions in which harvest aids were applied at R6 (seed-fill) and R7 (yellow pods) growth stages. Harvest aids applied included an untreated control, 0.28 kg ai ha-1 of paraquat, 0.28 kg ai ha-1 of paraquat, and 1.015 kg ai ha-1 of carfentrazone-ethyl (AIM); 6.72 kg ai ha-1 sodium chlorate, 1.015 kg ai ha-1 carfentrazone-ethyl; and 2.0 kg ae ha-1 glyphosate. Results showed that the application of harvest aids at either R6 or R7 resulted in the alteration of some seed composition such as protein, oil, oleic acid, fructose, and little effects on amino acids. In addition, harvest aids affected seed composition constituents differently depending on year and growth stage. This research demonstrated the possible alteration of some nutrients by harvest aids. This research helps growers and scientists to advance the understanding and management of harvest aids and investigate possible effects of harvest aids on seed nutrition.

Persistence in soil of microplastic films from ultra-thin compostable plastic bags and implications on soil Aspergillus flavus population.

An increasing number of states and municipalities are choosing to reduce plastic litter by replacing plastic items, particularly single-use ones, with same-use products manufactured from compostable plastics. This study investigated the formation and persistence of compostable film microplastic particles (CFMPs) from ultra-thin compostable carrier bags in soil under laboratory conditions, and the potential impact of CFMPs on Aspergillus flavus populations in the soil. During a 12-month incubation period, compostable film samples in soils with small, medium or large populations of indigenous A. flavus, underwent 5.9, 9.8, and 17.1% reduction in total surface area, respectively. Despite the low levels of deterioration, the number of CFMPs released increased steadily over the incubation period, particularly fragments with size < 0.05 mm. Up to 88.4% of the released fragments had associated A. flavus and up to 68% of isolates from CFMPs produced aflatoxins. A. flavus levels associated with CFMPs increased rapidly during the initial part of the 12-month incubation period, whereas the percent aflatoxigenicity continued to increase even after A. flavus density leveled off later. During 12 months incubation, A. flavus DNA amounts recovered from CFMPs increased in soils with all levels of indigenous A. flavus, with the largest increases (119.1%) occurring in soil containing the lowest indigenous A. flavus. These results suggest that burying compostable film in soil, or application of compost containing CFMPs, may reduce soil quality and increase risk of adverse impacts from elevated aflatoxigenic A. flavus populations in soil.

The Influence of Agricultural Practices, the Environment, and Cultivar Differences on Soybean Seed Protein, Oil, Sugars, and Amino Acids.

Information on the effects of agricultural practices such as seeding rate (S), row spacing (RS), herbicide apical treatment (T), and nitrogen application (N) on soybean seed nutrition (protein, oil, fatty acids, sugars, and amino acids) is limited. Although seed composition (nutrition) constituents are genetically controlled, agricultural practices and environmental conditions significantly influence the amount and quality of seed nutrition. Therefore, the objective of this research was to understand the responses of these seed composition constituents to these practices, the environment, and cultivar differences. Two-field experiments were conducted, in 2015 and 2016, in Milan, TN, USA. The experiments were irrigated with four replications and included: two soybean cultivars, two seeding rates, three different row spacings, two N rates, and Cobra herbicide apical treatment. The results showed significant effects of S, RS, N, and T on some seed composition constituents, including protein; oleic, linolenic, and stearic acids; sugars; and some amino acids. The current research demonstrated that single or twin row with a seeding rate of 40,000 seeds ha-1 resulted in higher protein, oleic, some sugars, and some amino acids. However, a high seeding rate of 56,000 seeds ha-1 resulted in lower protein, oleic acid, some sugars, and some amino acids due to plant competition for soil nutrients. Herbicide apical application of Cobra1X resulted in higher linolenic acid and some amino acids. Application of nitrogen resulted in higher protein, linolenic, and some amino acids. This research is beneficial to the scientific communities, including breeders and physiologists through advancing knowledge on the interactions between cultivars and environment for seed nutritional quality selection, and to soybean producers through consideration of best agricultural management to maintain high seed nutritional qualities.

Phytotoxic Responses of Soybean (Glycine max L.) to Botryodiplodin, a Toxin Produced by the Charcoal Rot Disease Fungus, Macrophomina phaseolina.

Toxins have been proposed to facilitate fungal root infection by creating regions of readily-penetrated necrotic tissue when applied externally to intact roots. Isolates of the charcoal rot disease fungus, Macrophomina phaseolina, from soybean plants in Mississippi produced a phytotoxic toxin, (-)-botryodiplodin, but no detectable phaseolinone, a toxin previously proposed to play a role in the root infection mechanism. This study was undertaken to determine if (-)-botryodiplodin induces toxic responses of the types that could facilitate root infection. (±)-Botryodiplodin prepared by chemical synthesis caused phytotoxic effects identical to those observed with (-)-botryodiplodin preparations from M. phaseolina culture filtrates, consistent with fungus-induced phytotoxicity being due to (-)-botryodiplodin, not phaseolinone or other unknown impurities. Soybean leaf disc cultures of Saline cultivar were more susceptible to (±)-botryodiplodin phytotoxicity than were cultures of two charcoal rot-resistant genotypes, DS97-84-1 and DT97-4290. (±)-Botryodiplodin caused similar phytotoxicity in actively growing duckweed (Lemna pausicostata) plantlet cultures, but at much lower concentrations. In soybean seedlings growing in hydroponic culture, (±)-botryodiplodin added to culture medium inhibited lateral and tap root growth, and caused loss of root caps and normal root tip cellular structure. Thus, botryodiplodin applied externally to undisturbed soybean roots induced phytotoxic responses of types expected to facilitate fungal root infection.

Toxin Production in Soybean (Glycine max L.) Plants with Charcoal Rot Disease and by Macrophomina phaseolina, the Fungus that Causes the Disease.

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14-6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA.

Field Displacement of Aflatoxigenic Aspergillus flavus Strains Through Repeated Biological Control Applications.

A popular pre-harvest strategy to mitigate aflatoxin contamination of corn involves field application of non-aflatoxigenic strains of Aspergillus flavus. The basis of this biological control may involve multiple factors, but competitive displacement of aflatoxigenic strains by the biocontrol strains is a likely mechanism. Three biocontrol strains (NRRL 21882, 18543, and 30797) were applied annually, over a 4-year period, to the same 3.2-ha commercial corn field in the Mississippi Delta, where we monitored their post-release establishment, spread, and persistence. Within 2 months of the first biocontrol application, the percentage of soil-inhabiting aflatoxigenic A. flavus strains in some plots was reduced from 48 to 9% of the population. The frequency of aflatoxigenic A. flavus strains was also significantly reduced in the rest of field. After 4 years, neighboring plots that had never received a biocontrol treatment, and distanced from our treatment plots by at least 20 meters, had less than 20% aflatoxigenic isolates. This significant halo effect might be attributed to movement of soil through tillage operations, but the aflatoxigenicity shift could be detected in the untreated plots within 2 months of the initial applications, at a time when there was no tillage. The A. flavus populations that colonized the grain were also monitored and found to be less than 15% toxigenic in the fourth year for all treatments. Over all treatments and years, less than 2 ppb of aflatoxin was detected, which could be a consequence of the field-wide shift of the inherent A. flavus population to predominately non-aflatoxigenic strains. This study supports the efficacy of using non-aflatoxigenic A. flavus strains as pre-harvest biocontrol, and shows that most of its effectiveness occurs with the first application.

Degradation of microplastic seed film-coating fragments in soil.

Encapsulating fungicides and/or insecticides in film-coatings applied to agronomic seeds has become a widely accepted method for enhancing seed germination and overall seedling health by protecting against many diseases and early-season insect pests. Despite advancements in seed film-coating technologies, abrasion of the seed coating can occur during handling and mechanical planting operations, resulting in variable amounts of detached fragments entering the soil. The present study investigated the degradation in soil of these plastic-like, small-sized fragments, referred to here as microplastic coating fragments. Degradation of microplastic coating fragments in soil was found to be highly variable. The lowest degradation rate (≤48 days) was observed in fragments detached from seeds coated with a commercial polymer mixture, while fragments from a biodegradable plastic formulation degraded completely within 32 days. When spores of the plant growth-promoting bacterium, Bacillus subtilis, were incorporated into the bioplastic, degradation was even more rapid (≤24 days). The fragment degradation rate was unaffected by incorporating two commonly used neonicotinoid insecticides, imidacloprid or thiacloprid, into either coating formulations, but insecticide dissipation rates in soil were more rapid when added associated with seed coating fragments than when spiked in directly. Half-lives of these two insecticides were reduced by up to 27% in fragments from bioplastic-coated seeds. These results are consistent with variable and not easily predicted soil degradation rates for seed coating fragments, with enhanced dissipation of coating-entrapped pesticides and with a higher degradation rate for biodegradable seed coating incorporating selected microbial strains.

Laboratory and Field Investigations on Compatibility of Beauveria bassiana (Hypocreales: Clavicipitaceae) Spores With a Sprayable Bioplastic Formulation for Application in the Biocontrol of Tarnished Plant Bug in Cotton.

Two isolates of Beauveria bassiana (Balsamo) Vuillemin, including the commercial strain GHA and the Mississippi Delta native NI8 strain, and two emulsifiers, Tween-80 and a starch-based sprayable bioplastic, were evaluated in the laboratory and field for pathogenicity and infectivity against the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae). The effect on fruit damage based on within-season cotton plant mapping was also examined. The highest mortality 10 d after treatment was found with insects caged on cotton terminals sprayed with NI8 + Tween-80, followed by those exposed to NI8 + bioplastic. Similarly, sporulation was shown to be higher in NI8 + Tween-80 than in other treatments. Plots sprayed with B. bassiana showed at least a twofold decrease in tarnished plant bug adults 3 d after treatment compared with control plots. Little to no variation was observed in tarnished plant bug nymph populations between treated and untreated plots. Within-season plant mapping provided clear evidence of damage to cotton caused by tarnished plant bug. The highest percentage retention of all first position fruiting structures was observed in plots treated with NI8 + Tween-80 (93.41 ± 1.51) followed by NI8 + bioplastic (90.25 ± 1.52). Both treatments were significantly different when compared with GHA + Tween-80 (82.89 ± 2.26) and GHA + bioplastic (70.48 ± 3.19), and both GHA formulations did not differ from the control (63.61 ± 2.96). Overall, these results indicated that B. bassiana application resulted in >50% mortality of tarnished plant bug regardless of the isolates by direct spray or by contact. However, the superior performance of the Mississippi Delta native NI8 strain was observed in all treatment applications and evaluation times.

Genome Sequences of Three Strains of Aspergillus flavus for the Biological Control of Aflatoxin.

Aflatoxin is a carcinogenic contaminant of many commodities that are infected by Aspergillus flavus Nonaflatoxigenic strains of A. flavus have been utilized as biological control agents. Here, we report the genome sequences from three biocontrol strains. This information will be useful in developing markers for postrelease monitoring of these fungi.

Biological Control of Aflatoxin Contamination in U.S. Crops and the Use of Bioplastic Formulations of Aspergillus flavus Biocontrol Strains To Optimize Application Strategies.

Aflatoxin contamination has a major economic impact on crop production in the southern United States. Reduction of aflatoxin contamination in harvested crops has been achieved by applying nonaflatoxigenic biocontrol Aspergillus flavus strains that can out-compete wild aflatoxigenic A. flavus, reducing their numbers at the site of application. Currently, the standard method for applying biocontrol A. flavus strains to soil is using a nutrient-supplying carrier (e.g., pearled barley for Afla-Guard). Granules of Bioplastic (partially acetylated corn starch) have been investigated as an alternative nutritive carrier for biocontrol agents. Bioplastic granules have also been used to prepare a sprayable biocontrol formulation that gives effective reduction of aflatoxin contamination in harvested corn kernels with application of much smaller amounts to leaves later in the growing season. The ultimate goal of biocontrol research is to produce biocontrol systems that can be applied to crops only when long-range weather forecasting indicates they will be needed.

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

Field 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.