[Show abstract][Hide abstract] ABSTRACT: Plant β-1,3-glucanases are members of the pathogenesis-related protein 2 (PR-2) family, which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses. One of the differentially expressed proteins (spot 842) identified in a recent proteomic comparison between five pairs of closely-related maize (Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study. Here, the corresponding cDNA was cloned from maize and designated as ZmGns. ZmGns encodes a protein of 338 amino acids containing a potential signal peptide. The expression of ZmGns was detectible in all tissues studied with the highest level in silks. ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus. ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid. In vivo, ZmGns showed a significant inhibitory activity against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis. Its high level expression in the silk tissue and its induced expression by phytohormone treatment, as well as by bacterial and fungal infections suggest it plays a complex role in maize growth, development and defense.
[Show abstract][Hide abstract] ABSTRACT: The persistent occurrence of aflatoxins in food and feed remains a problem for producers of commodities subject to colonization with toxigenic molds. Aflatoxins are secondary metabolites of fungi of the Aspergillus spp. associated with deleterious health effects. Because current screening methods for these toxins are lengthy, destructive, and costly, there is a continuous search for a more rapid, noninvasive, and cost-effective technology. The present study utilized a fluorescence excitation–emission matrix (EEM) of aflatoxin as well as two additional secondary metabolites (kojic acid and the bright greenish-yellow fluorescence (BGYF) compound) of Aspergillus flavus measured with a fluorescence spectrophotometer. The results were compared to image data acquired with a fluorescence hyperspectral sensor in order to evaluate the potential of image-based technology for detecting aflatoxin in grain. The excitation–emission matrix of aflatoxin B1 standard produced overlapping peaks in 340–400 nm of excitation range emitting in the blue range at around 450 nm. The spectral signature extracted from the hyperspectral image was also in the blue range, emitting blue fluorescence. Because the results from both systems were comparable, where all fluorescence peaks were in the blue range, the present study validates the feasibility of image-based technology for nondestructive detection of aflatoxin in corn. Additional peaks were revealed in the aflatoxin EEM in the 260-nm excitation range that were not present in the kojic acid and BGYF compound mixture. This new information allows for the separation of the aflatoxin signature from the potentially confounding overlap of other secondary metabolites occurring in the blue and blue-green spectral ranges.
[Show abstract][Hide abstract] ABSTRACT: A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain (AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn (Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. There appeared to be a population difference between the co-inoculated AF70-GFP+AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto 73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol strain supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.
Frontiers in Microbiology 03/2014; 5:122. DOI:10.3389/fmicb.2014.00122 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The food industry is always on the lookout for sensing technologies for rapid and nondestructive inspection of food products. Hyperspectral imaging technology integrates both imaging and spectroscopy into unique imaging sensors. Its application for food safety and quality inspection has made significant progress in recent years. Specifically, hyperspectral imaging has shown its potential for surface contamination detection in many food related applications. Most existing hyperspectral imaging systems use pushbroom scanning which is generally used for flat surface inspection. In some applications it is desirable to be able to acquire hyperspectral images on circular objects such as corn ears, apples, and cucumbers. Past research describes inspection systems that examine all surfaces of individual objects. Most of these systems did not employ hyperspectral imaging. These systems typically utilized a roller to rotate an object, such as an apple. During apple rotation, the camera took multiple images in order to cover the complete surface of the apple. The acquired image data lacked the spectral component present in a hyperspectral image. This paper discusses the development of a hyperspectral imaging system for a 3-D surface scan of biological samples. The new instrument is based on a pushbroom hyperspectral line scanner using a rotational stage to turn the sample. The system is suitable for whole surface hyperspectral imaging of circular objects. In addition to its value to the food industry, the system could be useful for other applications involving 3-D surface inspection.
ISPDI 2013 - Fifth International Symposium on Photoelectronic Detection and Imaging; 08/2013
[Show abstract][Hide abstract] ABSTRACT: In an effort to address the problem of rapid detection of aflatoxin in grain, particularly oilseeds, the current study assessed the spectral differences of aflatoxin production in kernels from a cornfield inoculated with spores from 2 different strains of toxigenic Aspergillus flavus. Aflatoxin production in corn from the same field due to natural infestation was also assessed. A small corn plot in Baton Rouge, La., U.S.A., was used during the 2008-growing season. Two groups of 400 plants were inoculated with 2 different inocula and 1 group of 400 plants was designated as controls. Any contamination detected in the controls was attributed to natural infestation. A subset of each group was imaged with a visible near infra red (VNIR) hyperspectral system under ultra violet (UV) excitation and subsequently analyzed for aflatoxin using affinity column fluorometry. Group differences were statistically analyzed. Results indicate that when all the spectral data across all groups were averaged, any potential differences between groups (treated and untreated) were obscured. However, spectral analysis based on contaminated “hot” pixel classification showed a distinct spectral shift/separation between contaminated and clean ears with fluorescence peaks at 501 and 478 nm, respectively. All inoculated and naturally infected control ears had fluorescence peaks at 501 nm that differed from uninfected corn ears. Results from this study may be useful in evaluating rapid, noninvasive instrumentation and/or methodology for aflatoxin detection in grain.
The present study evaluated the possible application of fluorescence hyperspectral imaging methodology for the rapid and noninvasive detection of aflatoxin in postharvest corn during various stages of processing. Specific information from this study may be applicable when developing rapid, noninvasive instrumentation and/or methodology for whole ear inspection as well as shucked kernels.
[Show abstract][Hide abstract] ABSTRACT: Aflatoxin contamination caused by Aspergillus flavus infection of corn is a significant and chronic threat to corn being used as food or feed. Contamination of crops at levels of 20 ng g(-1) or higher (as regulated by the USFDA) by this toxin and potent carcinogen makes the crop unsalable, resulting in a significant economic burden on the producer. This review focuses on elimination of this contamination in corn which is a major US crop and the basis of many products. Corn is also "nature's example" of a crop containing heritable resistance to aflatoxin contamination, thereby serving as a model for achieving resistance to aflatoxin contamination in other crops as well. This crop is the largest production grain crop worldwide, providing food for billions of people and livestock and critical feedstock for production of biofuels. In 2011, the economic value of the US corn crop was US$76 billion, with US growers producing an estimated 12 billion bushels, more than one-third of the world's supply. Thus, the economics and significance of corn as a food crop and the threat to food safety due to aflatoxin contamination of this major food crop have prompted the many research efforts in many parts of the world to identify resistance in corn to aflatoxin contamination. Plant breeding and varietal selection has been used as a tool to develop varieties resistance to disease. This methodology has been employed in defining a few corn lines that show resistance to A. flavus invasion; however, no commercial lines have been marketed. With the new tools of proteomics and genomics, identification of resistance mechanisms, and rapid resistance marker selection methodologies, there is an increasing possibility of finding significant resistance in corn, and in understanding the mechanism of this resistance.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 07/2013; 30(8). DOI:10.1080/19440049.2013.812808 · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Naturally occurring Aspergillus flavus strains can be either toxigenic or atoxigenic, indicating their ability to produce aflatoxin or not. The objective was to assess, with the use of a hyperspectral sensor, the difference in fluorescence emission between maize kernels inoculated with toxigenic and atoxigenic inoculums of A. flavus. Maize ears were inoculated with AF13, a toxigenic strain of A. flavus, and AF38, an atoxigenic strain of A. flavus, at dough stage of development and harvested 8 weeks after inoculation. After harvest, single kernels were divided into three groups prior to imaging: control, adjacent, and glowing. Both sides of the kernel, germ and endosperm, were imaged separately using a fluorescence hyperspectral imaging system. After imaging each single kernel was processed with affinity column fluorimetry to determine aflatoxin level. Results from discriminant analysis of the imaging data found that the classification accuracies of the three visually designated groups were not promising. The separation of maize kernels based on different fungal inoculums yielded better results. The best results were achieved with the germ side of the maize kernels. The kernels were later grouped into 'contaminated' and 'healthy' with 20 ppb and 100 ppb thresholds. The contaminated kernels all had longer peak wavelength than did the healthy ones. Results from the discriminant analysis classification indicated overall higher classification accuracy for the 100 ppb threshold on the germ side (94.4%). The germ side was also more useful at discriminating healthy from contaminated kernels for the 20 ppb threshold.
[Show abstract][Hide abstract] ABSTRACT: Aflatoxin is a mycotoxin produced mainly by Aspergillus flavus (A.flavus) and Aspergillus parasitiucus fungi that grow naturally in corn. Very serious health problems such as liver damage and lung cancer can result from exposure to high toxin levels in grain. Consequently, many countries have established strict guidelines for permissible levels in consumables. Conventional chemical-based analytical methods used to screen for aflatoxin such as thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) are time consuming, expensive, and require the destruction of samples as well as proper training for data interpretation. Thus, it has been a continuing effort within the research community to find a way to rapidly and non-destructively detect and possibly quantify aflatoxin contamination in corn. One of the more recent developments in this area is the use of spectral technology. Specifically, fluorescence hyperspectral imaging offers a potential rapid, and non-invasive method for contamination detection in corn infected with toxigenic A.flavus spores. The current hyperspectral image system is designed for scanning flat surfaces, which is suitable for imaging single or a group of corn kernels. In the case of a whole corn cob, it is preferred to be able to scan the circumference of the corn ear, appropriate for whole ear inspection. This paper discusses the development of a hyperspectral imaging system for whole corn ear imaging. The new instrument is based on a hyperspectral line scanner using a rotational stage to turn the corn ear.
[Show abstract][Hide abstract] ABSTRACT: Support Vector Machine (SVM) was used in the Genetic Algorithms (GA) process to select and classify a subset of hyperspectral image bands. The method was applied to fluorescence hyperspectral data for the detection of aflatoxin contamination in Aspergillus flavus infected single corn kernels. In the band selection process, the training sample classification accuracy was used as fitness function. Two aflatoxin thresholds, 20 ppb and 100 ppb, were used to divide the single corn kernels into clean and contaminated samples. The validation accuracy was 87.7% for the 20 ppb threshold and 90.5% for the 100 ppb threshold. The results were generated from the GA selected 36 bands and 11 bands, respectively. Compared to the full wavelength classification, the subset of image bands had slightly better or similar performance. A reduced image space could save time both in spectral data acquisition and analysis, which is crucial in the development of rapid and none invasive methods for contamination detection.
2012 4th Workshop on Hyperspectral Image and Signal Processing: Evolution in Remote Sensing (WHISPERS); 06/2012
[Show abstract][Hide abstract] ABSTRACT: Naturally occurring Aspergillus flavus strains can be either toxigenic
or atoxigenic, indicating their ability to produce aflatoxin or not,
under specific conditions. Corn contaminated with toxigenic strains of
A. flavus can result in great losses to the agricultural industry and
pose threats to public health. Past research showed that fluorescence
hyperspectral imaging could be a potential tool for rapid and
non-invasive detection of aflatoxin contaminated corn. The objective of
the current study was to assess, with the use of a hyperspectral sensor,
the difference in fluorescence emission between corn kernels inoculated
with toxigenic and atoxigenic inoculums of A. flavus. Corn ears were
inoculated with AF13, a toxigenic strain of A. flavus, and AF38, an
atoxigenic strain of A. flavus, at dough stage of development and
harvested 8 weeks after inoculation. After harvest, single corn kernels
were divided into three groups prior to imaging: control, adjacent, and
glowing. Both sides of the kernel, germplasm and endosperm, were imaged
separately using a fluorescence hyperspectral imaging system. It was
found that the classification accuracies of the three manually
designated groups were not promising. However, the separation of corn
kernels based on different fungal inoculums yielded better results. The
best result was achieved with the germplasm side of the corn kernels.
Results are expected to enhance the potential of fluorescence
hyperspectral imaging for the detection of aflatoxin contaminated corn.
Proceedings of SPIE - The International Society for Optical Engineering 05/2012; 8369:8-. DOI:10.1117/12.919580 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.
Biologia Plantarum 03/2012; 56(1). DOI:10.1007/s10535-012-0027-0 · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genomic study of filamentous fungi has made significant advances in recent years, and the genomes of several species in the genus Aspergillus have been sequenced, including Aspergillus flavus. This ubiquitous mold is present as a saprobe in a wide range of agricultural and natural habits, and can function as an opportunistic animal and plant pathogen. A. flavus produces many secondary metabolites including aflatoxins, aflatrem and cyclopiazonic acid. In this chapter, our main focus is on the current status of the genomics of A. flavus as well as on the potential applications of genomics-based approaches to understanding mycotoxin production and fungal pathogenicity. It is hoped that the results of A. flavus genomics and functional genomics studies will empower researchers to find effective controlling strategies to eliminate mycotoxin contamination and to yield a safer and more abundant food and feed supply.
[Show abstract][Hide abstract] ABSTRACT: To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in
developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been
hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for
breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant
lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75–94% genetic similarity
within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel
embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide
gel electrophoresis. Differentially expressed (≥1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified
as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in
maize that confer resistance to A. flavus infection and/or aflatoxin production.
KeywordsTwo-dimensional gel electrophoresis–Corn–Protein profile comparison–Aflatoxin resistance–Antifungal protein–Stress-related protein
[Show abstract][Hide abstract] ABSTRACT: A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC. Antiproliferative effects were measured in human breast, ovarian, and prostate cancer cell lines, with all three chiral forms exhibiting growth inhibition (GI) in the low to mid μM range for all cells. The natural enantiomer, and in some cases the racemate, gave significantly greater GI than the unnatural stereoisomer for estrogen receptor positive (ER(+)) versus ER(-) breast/ovarian cell lines as well as for androgen receptor positive (AR(+)) versus AR(-) prostate cancer cells. Surprisingly, differences between ER(+) and ER(-) cell lines were not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be more complicated than strictly ER interactions.
[Show abstract][Hide abstract] ABSTRACT: Aflatoxin is produced by the fungus Aspergillus flavus when the fungus invades developing corn kernels. Because of its potent toxicity, the levels of aflatoxin are regulated by the Food and Drug Administration (FDA) in the US, allowing 20 ppb (parts per billion) limits in food, and feed intended for interstate commerce. Currently, aflatoxin detection and quantification methods are based on analytical tests. These tests require the destruction of samples, can be costly and time consuming, and often rely on less than desirable sampling techniques. Thus, the ability to detect aflatoxin in a rapid, non-invasive way is crucial to the corn industry in particular. This paper described how narrow-band fluorescence indices were developed for aflatoxin contamination detection based on single corn kernel samples. The indices were based on two bands extracted from full wavelength fluorescence hyperspectral imagery. The two band results were later applied to two large sample experiments with 25 g and 1 kg of corn per sample. The detection accuracies were 85% and 95% when 100 ppb threshold was used. Since the data acquisition period is significantly lower for several image bands than for full wavelength hyperspectral data, this study would be helpful in the development of real-time detection instrumentation for the corn industry.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus flavus is the most infamous species among the over 185 known species within the genus Aspergillus. It is not only one of the most abundant and widely distributed soil-borne molds that can be found anywhere on earth but,also
produces aflatoxins, among the most carcinogenic natural products ever discovered (Jelinek et al., 1989). A. flavus is a saprobe capable of surviving on many organic nutrient sources like plant debris, tree leaves, decaying wood, animal
fodder, cotton, compost piles, dead insects and animal carcasses, stored grains, and even human and animal patients (Klich,
1998). Its optimal range for growth is between 25 – 37°C, but it can grow in a wide range of temperatures from 12 to 48°C.
The ability of the fimgus to grow at relatively high temperatures of the fungus contributes to its pathogenicity in humans
and other warm blooded animals. For most of its life-cycle, the fungus exists in the form of mycelia or asexual spores known
as conidia. Stress from adverse conditions such as lack of adequate nutrients or water, causes the mycelia to form resistant
structures called sclerotia. The fungus over-winters either as spores, as scierotia or as mycelia in debris. Under favorable
conditions scierotia germinate directly to produce new colonies or conidiophores with conidia (Bennett et al., 1986; Cotty, 1988; Chang et al., 2002)
[Show abstract][Hide abstract] ABSTRACT: Selective principal component regression analysis (SPCR) uses a subset of the original image bands for principal component transformation and regression. For optimal band selection before the transformation, this paper used genetic algorithms (GA). In this case, the GA process used the regression correlation as its fitness function. This algorithm was used for analyzing fluorescence hyperspectral images of aflatoxin contaminated corn kernels. The results showed that SPCR could produce results similar to the standard PCR approach. However, the data dimension was much less for the SPCR process. The SPCR correlation coefficient was 0.8 when 33 of the original 74 bands were used for the SPCR transformation. The results demonstrated that SPCR could be used as a combined dimension reduction and data analysis tool for high dimensionality data processing.
Hyperspectral Image and Signal Processing: Evolution in Remote Sensing (WHISPERS), 2011 3rd Workshop on; 01/2011