Robert Lee, LA, United States
Robert Lee, LA, United States

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Chen S.,Louisiana State University | Wang F.,Louisiana State University | Beaulieu J.C.,Southern Regional Research Center | Stein R.E.,Southern Regional Research Center | Ge B.,Louisiana State University
Applied and Environmental Microbiology | Year: 2011

Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMALAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 108 CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 103 to 6.1 × 104 CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viable Salmonella cell numbers was high (R2 = 0.949 to 0.993), with a quantification range (102 to 105 CFU/reaction in pure culture and 104to 107 CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce. © 2011, American Society for Microbiology.


Calvo A.M.,Northern Illinois University | Cary J.W.,Southern Regional Research Center
Frontiers in Microbiology | Year: 2015

Fungal secondary metabolism and morphological development have been shown to be intimately associated at the genetic level. Much of the literature has focused on the co-regulation of secondary metabolite production (e.g., sterigmatocystin and aflatoxin in Aspergillus nidulans and Aspergillus flavus, respectively) with conidiation or formation of sexual fruiting bodies. However, many of these genetic links also control sclerotial production. Sclerotia are resistant structures produced by a number of fungal genera. They also represent the principal source of primary inoculum for some phytopathogenic fungi. In nature, higher plants often concentrate secondary metabolites in reproductive structures as a means of defense against herbivores and insects. By analogy, fungi also sequester a number of secondary metabolites in sclerotia that act as a chemical defense system against fungivorous predators. These include antiinsectant compounds such as tetramic acids, indole diterpenoids, pyridones, and diketopiperazines. This chapter will focus on the molecular mechanisms governing production of secondary metabolites and the role they play in sclerotial development and fungal ecology, with particular emphasis on Aspergillus species. The global regulatory proteins VeA and LaeA, components of the velvet nuclear protein complex, serve as virulence factors and control both development and secondary metabolite production in many Aspergillus species. We will discuss a number of VeA- and LaeA-regulated secondary metabolic gene clusters in A. flavus that are postulated to be involved in sclerotial morphogenesis and chemical defense. The presence of multiple regulatory factors that control secondary metabolism and sclerotial formation suggests that fungi have evolved these complex regulatory mechanisms as a means to rapidly adapt chemical responses to protect sclerotia from predators, competitors and other environmental stressors. © 2015 Calvo and Cary.


He Z.,Southern Regional Research Center | Olk D.C.,U.S. Department of Agriculture | Cade-Menun B.J.,Agriculture and Agri Food Canada
Soil Science Society of America Journal | Year: 2011

Phosphorus has long been known to be present in soil humic fractions, but little is known about specific P forms in humic fractions or their lability. We extracted the mobile humic acid (MHA) and recalcitrant calcium humate (CaHA) fractions from a Nebraska Hord silt loam soil (a fine-silty, mixed, superactive, mesic Cumulic Haplustoll) under continuous corn (Zea mays L.) receiving either inorganic fertilizer or animal manure. Solution 31P nuclear magnetic resonance spectroscopy demonstrated that P in both MHA and CaHA was predominantly present in organic forms, mostly as orthophosphate monoesters. Spiking experiments indicated no phytate present in these humic fractions, but scyllo-inositol P was identified in all samples. Potato phosphatase hydrolyzed some humicbound P. Fungal phytase released more humic-bound P, which may come from scyllo-inositol P. No additional P was released by including nuclease. Ultraviolet (UV) irradiation increased soluble inorganic P in MHA fractions, but total hydrolyzable P in MHA fractions did not increase, suggesting that the portion of P that was UV labile was also enzymatically hydrolyzable. In contrast, UV irradiation increased soluble inorganic P and total hydrolysable P in CaHA fractions, which suggests that UV-labile P in CaHA fractions did not overlap wiThenzymatically hydrolyzable P. Fertilization management did not significantly alter the lability of humic P in these humic fractions. This research has the potential to improve P management by increasing our knowledge of P lability for more efficient crop uptake. © Soil Science Society of America.


Hadden J.A.,University of Georgia | French A.D.,Southern Regional Research Center | Woods R.J.,University of Georgia | Woods R.J.,National University of Ireland
Biopolymers | Year: 2013

Molecular dynamics (MD) simulations of cellulose microfibrils are pertinent to the paper, textile, and biofuels industries for their unique capacity to characterize dynamic behavior and atomic-level interactions with solvent molecules and cellulase enzymes. While high-resolution crystallographic data have established a solid basis for computational analysis of cellulose, previous work has demonstrated a tendency for modeled microfibrils to diverge from the linear experimental structure and adopt a twisted conformation. Here, we investigate the dependence of this twisting behavior on computational approximations and establish the theoretical basis for its occurrence. We examine the role of solvent, the effect of nonbonded force field parameters [partial charges and van der Waals (vdW) contributions], and the use of explicitly modeled oxygen lone pairs in both the solute and solvent. Findings suggest that microfibril twisting is favored by vdW interactions, and counteracted by both intrachain hydrogen bonds and solvent effects at the microfibril surface. © 2013 Wiley Periodicals, Inc.


Nishiyama Y.,Joseph Fourier University | Johnson G.P.,Southern Regional Research Center | French A.D.,Southern Regional Research Center
Cellulose | Year: 2012

Powder and fiber diffraction patterns were calculated for model cellulose crystallites with chains 20 glucose units long. Model sizes ranged from four chains to 169 chains, based on cellulose Iβ coordinates. They were subjected to various combinations of energy minimization and molecular dynamics (MD) in water. Disorder induced by MD and one or two layers of water had small effects on the relative intensities, except that together they reduced the low-angle scattering that was otherwise severe enough to shift the 1 1̄ 0 peak. Other shifts in the calculated peaks occurred because the empirical force field used for MD and minimization caused the models to have small discrepancies with the experimental intermolecular distances. Twisting and other disorder induced by minimization or MD increased the breadth of peaks by about 0. 2-0. 3° 2-θ. Patterns were compared with experimental results. In particular, the calculated fiber patterns revealed a potential for a larger number of experimental diffraction spots to be found for cellulose from some higher plants when crystallites are well-oriented. Either that, or further understanding of those structures is needed. One major use for patterns calculated from models is testing of various proposals for microfibril organization. © 2012 Springer Science+Business Media B.V. (outside the USA).


Ehrlich K.C.,Southern Regional Research Center
Frontiers in Microbiology | Year: 2014

Aspergillus flavus is a diverse assemblage of strains that include aflatoxin-producing and non-toxigenic strains with cosmopolitan distribution. The most promising strategy currently being used to reduce preharvest contamination of crops with aflatoxin is to introduce non-aflatoxin (biocontrol) A. flavus into the crop environment. Whether or not introduction of biocontrol strains into agricultural fields is enough to reduce aflatoxin contamination to levels required for acceptance of the contaminated food as fit for consumption is still unknown. There is no question that biocontrol strains are able to reduce the size of the populations of aflatoxin-producing strains but the available data suggests that at most only a four- to five-fold reduction in aflatoxin contamination is achieved. There are many challenges facing this strategy that are both short term and long term. First, the population biology of A. flavus is not well understood due in part to A. flavus's diversity, its ability to form heterokaryotic reproductive forms, and its unknown ability to survive for prolonged periods after application. Second, biocontrol strains must be selected that are suitable for the environment, the type of crop, and the soil into which they will be introduced. Third, there is a need to guard against inadvertent introduction of A. flavus strains that could impose an additional burden on food safety and food quality, and fourth, with global warming and resultant changes in the soil nutrients and concomitant microbiome populations, the biocontrol strategy must be sufficiently flexible to adapt to such changes. Understanding genetic variation within strains of A. flavus is important for developing a robust biocontrol strategy and it is unlikely that a "one size fits all" strategy will work for preharvest aflatoxin reduction. © 2014 Ehrlich.


Moore G.G.,Southern Regional Research Center
World Mycotoxin Journal | Year: 2015

Field inoculation with non-aflatoxigenic Aspergillus flavus is a preferred method for pre-harvest biocontrol of aflatoxin contamination of maize, cottonseed, and groundnut. Rationale for using these A. flavus strains is that they (1) maintain persistent control of aflatoxigenic fungi in the field, and (2) are incapable of out-crossing. Trackable field-released biocontrol strains will be beneficial to study the movement and longevity of non-aflatoxigenic A. flavus strains. Incorporating a naturally-occurring compound such as enhanced green fluorescent protein (eGFP) into a biocontrol strain might allow observation of its behaviour in field settings. The success of long-term field testing of eGFP-expressing A. flavus strains depends on their ability to maintain fluorescence throughout growth. Additionally, to ensure accurate tracking of the fluorescent atoxigenic strain, the likelihood of their out-crossing with individuals from the native population must be determined. In vitro mating experiments paired each of six different eGFP-transformed atoxigenic strains with a highly fertile toxigenic A. flavus isolate. Findings indicate that the eGFP gene, and possibly the aflatoxin cluster, is heritable by the F1 progeny. Not all cultured ascospores were fluorescent, but subsequent growth arising from a single fluorescent ascospore exhibited fluorescence similar to the eGFP parent. Observed mixed-fluorescence among conidia in a single chain suggests heterokaryosis at the moment of conidiogenesis. Mycotoxin assays showed that some fluorescent F1 individuals produce aflatoxin and/or cyclopiazonic acid which would indicate they are recombinant offspring. The findings in this laboratory study lend support to concern that atoxigenic strains are not impervious to genetic recombination and for which, if possible in a natural environment, repeated use could pose a risk of increasing the occurrence of aflatoxigenic individuals in treated fields. © 2014 Wageningen Academic Publishers.


He Z.,Southern Regional Research Center | Chapital D.C.,Southern Regional Research Center
Journal of Visualized Experiments | Year: 2015

Recently, the interest in plant seed meal-based products as wood adhesives has steadily increased, as these plant raw materials are considered renewable and environment-friendly. These natural products may serve as alternatives to petroleum-based adhesives to ease environmental and sustainability concerns. This work demonstrates the preparation and testing of the plant seed-based wood adhesives using cottonseed and soy meal as raw materials. In addition to untreated meals, water washed meals and protein isolates are prepared and tested. Adhesive slurries are prepared by mixing a freeze-dried meal product with deionized water (3:25 w/w) for 2 hr. Each adhesive preparation is applied to one end of 2 wood veneer strips using a brush. The tacky adhesive coated areas of the wood veneer strips are lapped and glued by hot-pressing. Adhesive strength is reported as the shear strength of the bonded wood specimen at break. Water resistance of the adhesives is measured by the change in shear strength of the bonded wood specimens at break after water soaking. This protocol allows one to assess plant seed-based agricultural products as suitable candidates for substitution of synthetic-based wood adhesives. Adjustments to the adhesive formulation with or without additives and bonding conditions could optimize their adhesive properties for various practical applications. © 2015 Journal of Visualized Experiments.


Ehrlich K.C.,Southern Regional Research Center | Mack B.M.,Southern Regional Research Center
Toxins | Year: 2014

Fifty six secondary metabolite biosynthesis gene clusters are predicted to be in the Aspergillus flavus genome. In spite of this, the biosyntheses of only seven metabolites, including the aflatoxins, kojic acid, cyclopiazonic acid and aflatrem, have been assigned to a particular gene cluster. We used RNA-seq to compare expression of secondary metabolite genes in gene clusters for the closely related fungi A. parasiticus, A. oryzae, and A. flavus S and L sclerotial morphotypes. The data help to refine the identification of probable functional gene clusters within these species. Our results suggest that A. flavus, a prevalent contaminant of maize, cottonseed, peanuts and tree nuts, is capable of producing metabolites which, besides aflatoxin, could be an underappreciated contributor to its toxicity. © 2014 by the authors; licensee MDPI, Basel, Switzerland.


Shih F.F.,Southern Regional Research Center
JAOCS, Journal of the American Oil Chemists' Society | Year: 2012

Because of the large amount of rice produced annually, milled rice co-products, such as rice flour, rice bran, rice bran oil, wax, and rice hulls are plentiful and readily available. These co-products are valuable sources of food ingredients, but they are currently under-utilized. Rice bran and flour contain different levels of protein. When treated with carbohydrate-hydrolyzing enzymes, high-protein products can be produced, which are nutritious and used widely in health and baby food formulations. Recently, subcritical water processing has also been studied, in which water is put under high pressure to maintain its liquid state and used as an environmental-friendly media for the recovery of rice bran proteins. Rice flour contains more than 90% rice starch (RS), and functional properties of RS are normally modified to suit the needs for food applications. For example, the addition of small amounts of phosphorylated RS esters or pregelatinized rice flour to frying batters made of rice flour was found to impart superior sensory characteristics and lower the amount of oil-uptake. Reductions in oil uptake of up to 50% were observed with the rice based formulations compared with batters made from traditional wheat ingredients. Treatment of RS with octenylsuccinic anhydride produces modified starch products that are useful as an emulsifying agent for the encapsulation of lipophilic compounds. Similarly, other rice-based products, such as microcrystalline celluloses from rice hulls, edible films formed with rice wax, various components with antioxidant properties, and germinated brown rice flours have also been developed. © 2011 AOCS (outside the USA).

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