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

Effect of temperature, rainfall and planting date on aflatoxin and fumonisin contamination in commercial Bt and non-Bt corn hybrids in Arkansas

Corn (maize, Zea mays) is susceptible to contamination with aflatoxins, fumonisins and other mycotoxins, particularly in the southeastern USA. In principle, mycotoxin contamination could be reduced in commercial corn hybrids with shorter growing seasons by planting at dates which minimize plant stress during the critical kernel-filling period. To evaluate this strategy, commercial Bt and non-Bt hybrids were planted in Arkansas in mid-April and early May of 2002, 2004 and 2005. The mid-April planting date resulted in lower aflatoxin contamination in harvested corn each yr and in significantly less frequent contamination above a regulatory action level in 2005 and overall than did the early-May planting date in both Bt and non-Bt corn. The mid-April planting date resulted in significantly lower total fumonisin contamination in harvested corn and in less frequent contamination above a regulatory advisory level than the early May planting date in 2 of 3 yr and overall in both Bt and non-Bt corn. All fumonisin subtypes studied were reduced. Frequent co-occurrence of aflatoxin and fumonisin was observed. Fumonisin levels averaged lower in Bt hybrids than in non-Bt hybrids at all plantings. Reduced aflatoxin and fumonisin contamination with mid-April planting could not be explained by any measure of heat stress during the kernel-filling period.Le maïs (Zea mays) est sensible à la contamination par les aflatoxines, les fumonisines et d'autres mycotoxines, surtout dans le sud-est des États-Unis. En principe, la contamination par les mycotoxines pourrait être diminuée chez les hybrides commerciaux de maïs par des saisons de croissance plus courtes en plantant à des dates qui minimisent le stress sur les plantes au moment de la période critique du remplissage des grains. Pour évaluer cette stratégie, des hybrides commerciaux Bt et non Bt ont été semés en Arkansas de la mi-avril au début de mai 2002, 2004 et 2005. Pour toutes les années, tant pour le maïs Bt que le non Bt, le grain issu des semis de la mi-avril était moins contaminé aux aflatoxines que celui semé au début de mai. De même, la contamination supérieure aux niveaux légalement acceptés a été moindre en 2005 et dans l'ensemble pour les semis de la mi-avril. Avec les semis de la mi-avril, il y avait significativement moins de contamination par les fumonisines dans le grain récolté et moins de contamination supérieure aux niveaux légalement acceptés qu'avec les semis du début de mai pour deux des trois années et dans l'ensemble, tant pour le maïs Bt que celui non Bt. La quantité de tous les sous-types de fumonisines étudiés a été diminuée. La présence simultanée d'aflatoxines et de fumonisines a fréquemment été observée. Pour tous les semis, les quantités de fumonisines des hybrides Bt ont été inférieures en moyenne à celles des hybrides non Bt. Les moindres contaminations par les aflatoxines et les fumonisines avec les semis de la mi-avril n'ont pu être expliquées par aucune des mesures du stress causé par la chaleur lors de la période du remplissage des grains

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 \ub1 1.51) followed by NI8 + bioplastic (90.25 \ub1 1.52). Both treatments were significantly different when compared with GHA + Tween-80 (82.89 \ub1 2.26) and GHA + bioplastic (70.48 \ub1 3.19), and both GHA formulations did not differ from the control (63.61 \ub1 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

Identification of Select Fumonisin Forming Fusarium Species Using PCR Applications of the Polyketide Synthase Gene and its Relationship to Fumonisin Production in vitro

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1- previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/μl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/μl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification

Genetic Variability of Aspergillus flavus

A nontoxigenic Aspergillus flavus strain, K49, is currently being tested as a biological control agent in corn fields in the Mississippi Delta. However, little is known about the overall genetic diversity of A. flavus from year to year in corn fields and specifically in Mississippi. Our objective was to assess the genetic variability of A. flavus isolates from different seasons, inoculum sources, and years, from a no-till corn field. Of the 175 A. flavus isolates examined, 74 and 97 had the typical norB-cypA type I (1.5 kb) and type II (1.0 kb) deletion patterns, respectively. Variability in the sequence of the omtA gene of the majority of the field isolates (n=118) was compared to strain K49. High levels of haplotypic diversity (24 omtA haplotypes; Hd = 0.61 ± 0.04) were found. Among the 24 haplotypes, two were predominant, H1 (n=71), which consists of mostly toxigenic isolates, and H49 (n=18), which consists of mostly atoxigenic isolates including K49. Toxigenic isolates were prevalent (60%) in this natural population. Nonetheless, about 15% of the population likely shared the same ancestral origin with K49. This study provides valuable information on the diversity of A. flavus. This knowledge can be further used to develop additional biological control strains

Use of a biochar-based formulation for coating corn seeds

The series of experiments summarized here were conducted with the objective to evaluate the benefits of using biochar for coating corn seeds. Seeds coated with a slurry containing bio-based ingredients and biochar were tested for germination and vigor, and for their potential to being infected by the fungus Aspergillus flavus, using a novel single seed incubator specifically designed for these purposes. Biochar-treated seeds were also planted for two years in experimental fields in the Mississippi Delta to evaluate their effect on corn yield and aflatoxin contamination of kernels. Experiments were conducted with two types of commercial biochar; one was obtained from hardwood residues and the other from coconut shells. Application of both types of biochar for coating the seeds did not affect seed germination and vigor. However, treated seeds showed increased wettability and a more rapid water uptake. This resulted in a 8.5% shortening of germination time. Microbiological analysis using plate culturing and qPCR methods showed that biochar was not conducive to the growth of A. flavus. This was also confirmed by analyzing soil samples that were collected from experimental fields located in the Mississippi Delta. Most importantly, although aflatoxin contamination was different in the two experimental years, aflatoxin contamination of corn kernels was not affected by biochar-based formulations

Expression Analysis of Stress-Related Genes in Kernels of Different Maize (Zea mays L.) Inbred Lines with Different Resistance to Aflatoxin Contamination

This research examined the expression patterns of 94 stress-related genes in seven maize inbred lines with differential expressions of resistance to aflatoxin contamination. The objective was to develop a set of genes/probes associated with resistance to A. flavus and/or aflatoxin contamination. Ninety four genes were selected from previous gene expression studies with abiotic stress to test the differential expression in maize lines, A638, B73, Lo964, Lo1016, Mo17, Mp313E, and Tex6, using real-time RT-PCR. Based on the relative-expression levels, the seven maize inbred lines clustered into two different groups. One group included B73, Lo1016 and Mo17, which had higher levels of aflatoxin contamination and lower levels of overall gene expression. The second group which included Tex6, Mp313E, Lo964 and A638 had lower levels of aflatoxin contamination and higher overall levels of gene expressions. A total of six “cross-talking” genes were identified between the two groups, which are highly expressed in the resistant Group 2 but down-regulated in susceptible Group 1. When further subjected to drought stress, Tex6 expressed more genes up-regulated and B73 has fewer genes up-regulated. The transcript patterns and interactions measured in these experiments indicate that the resistant mechanism is an interconnected process involving many gene products and transcriptional regulators, as well as various host interactions with environmental factors, particularly, drought and high temperature

The impact of Ramadan intermittent fasting on anthropometric measurements and body composition: Evidence from LORANS study and a meta-analysis

BackgroundAlthough the effect of Ramadan intermittent fasting (RIF) on anthropometry and body composition has been questioned, none of the previous studies tried to explain the reported changes in these parameters. Also, systematic reviews that investigated the topic were limited to healthy individuals or a specific disease group.MethodsThe London Ramadan Study (LORANS) is an observational study on health effects of RIF. We measured weight, waist circumference (WC), hip circumference (HC), body mass index (BMI), waist-to-hip ratio (WHR), basal metabolic rate (BMR), fat percentage (FP), free-fat mass (FFM), extremities predicted muscle mass, total body water (TBW), trunk FM, trunk FFM and trunk predicted muscle mass before and immediately after Ramadan. Using mixed-effects regression models, we investigated the effect of RIF with adjustment for potential confounders. We also conducted a meta-analysis of the results of LORANS with other studies that investigated the effect of RIF on anthropometry and body composition. The review protocol is registered with PROSPERO registry (CRD42020186532).ResultsWe recruited 146 participants (Mean ± SD age = 43.3 ± 15 years). Immediately after Ramadan, compared with before Ramadan, the mean difference was−1.6 kg (P<0.01) in weight,−1.95cm (P<0.01) in WC,−2.86cm (P <0.01) in HC, −0.60 kg/m2 (P < 0.01) in BMI and −1.24 kg (P < 0.01) in FM. In the systematic review and meta-analysis, after screening 2,150 titles and abstracts, 66 studies comprising 7,611 participants were included. In the general population, RIF was followed by a reduction of 1.12 Kg in body weight (−1.89– −0.36, I2 = 0), 0.74 kg/m2 reduction in BMI (−0.96– −0.53, I2 = 0), 1.54cm reduction in WC (−2.37– −0.71, I2 = 0) and 1.76cm reduction in HC (−2.69– −0.83, I2 = 0). The effect of fasting on anthropometric and body composition parameters starts to manifest in the second week of Ramadan and starts to diminish 3 weeks after Ramadan.ConclusionRIF is associated with a reduction in body weight, BMI, WC, HC, FM, FP and TBW. Most of these reductions are partially attributed to reduced FM and TBW. The reductions in these parameters appear to reverse after Ramadan