Minicolumn detection methods for aflatoxin in yellow corn: collaborative study

The CPC modified method, the Holaday modified method, and the combination of the 2 procedures have been compared. The CPC modified method involves more cleanup steps but has a more sensitive column. The Holaday modified procedure has fewer cleanup steps, but the column is more difficult to interpret. The combination CPC-Holaday, which has proven to be the most satisfactory, combines the speed and simplicity of the Holaday extraction and the sensitivity of the Velasco minicolumn used in the CPC method. Levels of 10 ng/g were detected by 89% of the collaborating laboratories using the combination, Holaday-Velasco, method. The combination method has been adopted as official first action.     

Simple method for simultaneous detection of aflatoxin and zearalenone in corn.

A method is described for the simultaneous detection of alfatoxin and zearalenone in corn at 5 and 200 ppb, respectively. No evaporation of solvent is required and the procedure is simple enough to be considered for use at marketing locations. The presence of absence of these myocotoxins can be determined in 10-20 min/sample. The procedure involves an initial blender extraction with methanol, partitioning of fat and pigments into 1-1,2-trichlorotrifluoroethane (Freon-113) from an aqueous ammonium sulfate layer, followed by extraction of aflatoxin from the aqueous layer with chlorobenzene. The chlorobenzene extract can be spotted directly onto a thin layer chromatographic plate which requires only 4 min development. Concentrations of aflatoxin and zearalenone can be estimated by visual comparison of sample spots with standards.     

Modification of the rapid screening method for aflatoxin in corn for quantitative use

A study was made to determine if the official AOAC method for screening of aflatoxin in corn could be modified for use as a quantitative method. Several different corn products were analyzed using the modified method, with an average savings of over 1 h/sample vs the CB method. Average recoveries for aflatoxin B1 were 94% for the low level spiked samples and 108% for the high level. Samples of corn and corn products containing naturally incurred aflatoxin were also analyzed with the modified method, and the results compared favorably with those obtained by the CB method.     

Nitrogen fertilizer influence on aflatoxin contamination of corn in Louisiana

Studies were conducted in 1997 and 1998 on a Gigger silt loam at the Macon Ridge Research Station at Winnsboro, LA, to determine the influence of nitrogen (N) rate, timing, and starter nitrogen fertilizer on aflatoxin contamination in corn. Fertilizer N (0, 50, 100, 150, 200, and 250 lb of N/acre), two timings (at planting and six-leaf stage), and starter N fertilizer (a control and 10 lb of N/acre applied in furrow) were evaluated. Application of starter, N rates, and the interaction of starter with N timing and N rates significantly affected aflatoxin levels. Rates of 50-250 lb of N/acre were 34-43% lower in aflatoxin contamination than plots receiving no N. The application of 10 lb of N/acre starter reduced the aflatoxin levels by 20% compared to the no-starter control.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

Simulation of aflatoxin testing plans for shelled peanuts in the United States and in the export market

The 1987 United States aflatoxin testing plan for shelled peanuts was designed with a final accept level of 25 parts per billion (ppb) total aflatoxin. Some of the importers of U.S. peanuts use aflatoxin testing plans with accept levels lower than 25 ppb. For example, the accept level of a testing plan used in The Netherlands is 5 ppb B1 or 10 ppb total aflatoxin. Whenever export lots are re-tested for aflatoxin by an importing country, some lots accepted in the United States will be rejected by the importing country's aflatoxin testing plan. Computer models were developed to determine the effects of decreasing the final accept level of the U.S. testing plan on the number of lots accepted and rejected in the United States and the number of exported lots accepted and rejected by The Netherlands testing plan. Decreasing the final accept level of the U.S. testing plan from 25 to 5 ppb increased the number of lots rejected in the United States by 371% while reducing the number of exported lots rejected by 51%. For every additional 8.3 lots rejected in the United States, one less export lot will be rejected.     

Development of a radioimmunoassay procedure for aflatoxin B1 measurement

A radioimmunoassaay (RIA) procedure to measure aflatoxin B(1) (AfB(1)) in agricultural commodities was developed. AfB(1) oxime derivative was synthesized, characterized, and used for preparation of (125)I-labeled AfB(1). Antiaflatoxin B(1) serum was raised in-house using AfB(1)-bovine serum albumin conjugate as immunogen. The assay system was optimized in the range of 0.2-5 ng/mL, using a liquid phase (PEG) as well as a solid phase (coated polystyrene beads) separation system. Inter-assay and intra-assay variations, recovery, and parallelism studies validated the assay. AfB(1) analysis was carried out in nearly 130 samples of different agricultural commodities. The correlation coefficient was determined using commercial ELISA and in-house-developed RIA methods.     

International mycotoxin check sample program: Part II. Report on laboratory performance for determination of aflatoxin M1 in milk

A sample of aflatoxin M1-contaminated lyophilized cow's milk was analyzed by 80 laboratories in 30 countries. Sufficient data were obtained to permit a statistical comparison of the performance of laboratories using AOAC methods I and II and those using high performance liquid chromatography for quantitation. A significant difference was noted between means for laboratories using AOAC method I as opposed to those using HPLC methods. Overall reproducibility (between- plus within-laboratory precision) was best for laboratories using HPLC methods and poorest for those using AOAC method II.     

Reaction to a paper by Whitaker and Dickens on aflatoxin testing plans for shelled peanuts in the U.S. and the export market

The present paper examines a technical paper of Whitaker and Dickens on aflatoxin testing plans that discusses (without a literature reference) a testing plan used in The Netherlands. However, this testing plan has never been in operation. We present the current situation in The Netherlands with respect to legislation and sampling plans on aflatoxin, which has fairly important consequences for the results of the simulation study of Whitaker and Dickens. It is shown that the percentage of rejected U.S.-exported lots in The Netherlands would increase from 16% to 27% based on the actual testing plan in The Netherlands. The need for international harmonization of testing, and the role of Codex Allmentarius is also emphasized.     

Identification of chemical components of corn kernel pericarp wax associated with resistance to Aspergillus flavus infection and aflatoxin production

Kernel pericarp wax of the corn breeding population GT-MAS:gk has been associated with resistance to Aspergillus flavus infection and aflatoxin production. GT-MAS:gk wax, previously compared to waxes of three susceptible genotypes, was presently compared to wax of a different, and more numerous, group of susceptible lines. Wax separation by TLC confirmed previous findings, demonstrating a unique GT-MAS:gk band and a unique "susceptible" band. Only GT-MAS:gk wax inhibited the growth of A. flavus; however, no association was established, as before, between kernel wax abundance and resistance. Gas chromatography-mass spectroscopy (GC-MS) analysis of kernel whole wax showed a higher percentage of phenol-like compounds in wax from GT-MAS:gk than in waxes from the susceptible lines. The GT-MAS:gk unique band contained phenol-like compounds and ethyl-hexadecanoate; butyl-hexadecanoate was preeminent in most of the "susceptible bands". Alkylresorcinol (phenolic compounds) content was dramatically higher in GT-MAS:gk wax than in the wax of susceptible lines. An alkylresorcinol, 5-methylresorcinol, also inhibited in vitro growth of A. flavus. These and other phenolic compounds may contribute to kernel wax inhibition of A. flavus infection/aflatoxin production. Further investigation is needed to confirm a role for them in GT-MAS:gk resistance.     

A rapid aflatoxin B1 ELISA: development and validation with reduced matrix effects for peanuts, corn, pistachio, and Soybeans

Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B(1) (AFB(1))-specific polyclonal antibody (produced from AFB(1)-oxime conjugated to bovine serum albumin (BSA)) and AFB(1)- and AFB(2)-enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB(1)-specific, detecting only AFB(1) in a mixture of four aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), but showed significant cross-reaction with AFG(1) (57-61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC(50) values of 12 +/- 1.5 and 9 +/- 1.5 microg/kg in sample (with 1 in 5 dilution of sample extract) for AFB(1) and AFB(2)-enzyme conjugates, respectively. These assays have narrower detection ranges (7.1-55.5 microg/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC(50) values of 21.6 +/- 2.7 and 12 microg/kg in sample for AFB(1)- and AFB(2)-enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2-99.9 microg/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB(1)-enzyme conjugate indicated relatively good recoveries of AFB(1) spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB(1) at current legal maximum residue limits of 10 microg/kg in food such as corn, peanuts, pistachio, and soybeans.     

Evaluation of some soil and plant analysis procedures as predictors of the need for sulfur for corn production

Abstract

The objective of this study was to evaluate the usefulness of measures of mineralized sulfur (S), soil sulfate‐sulfur (SO₄‐S), concentration of S in plant tissue, and the N: S ratio in plant tissue as predictors of the need for S in a fertilizer program for corn (Zea mays L.). Data to evaluate the use of plant analysis for S as a predictor were obtained from ten sites where various rates of N and S were applied to corn. Regression analysis was used to relate the S concentration in the ear leaf tissue as well as the N: S ratio in the same tissue to relative yield when the rate of applied N was held constant at a rate of 168 kg/ha. These measures of S in plant tissue were not significantly related to relative yield at sites where there was no response to fertilizer S as well as sites where added S increased yield.

Data from the same sites were used to assess the ability of soil tests to predict the need for fertilizer S. A measurement of extractable SO₄‐S in the surface soil (0–15 cm) was not reliable for predicting the need for S for corn grown on soils with a silt loam texture.

Static incubation techniques were used to evaluate the amount of S mineralized from soil collected from seven sites. The amount of SO₄‐S measured after four and twelve weeks of incubation was curvilinearly related (p <.05) to yield increase from a S fertilizer. Net mineralized S was less than 2.1 and 3.7 ppm SO₄‐S after four and twelve weeks of incubation, respectively, for soils taken from sites where response to fertilizer S was obtained. Data collected in this study indicate that a measure of mineralized S could improve the ability to predict S needs for corn production on soils with a silt loam texture and a low organic matter content.     

Rapid screening method for aflatoxin M1 in milk

A rapid screening method for detecting aflatoxin M1 in milk has been developed, based on minicolumn chromatography and requiring 8-10 min for each test. The minicolumn is packed with dry Florisil (100-200 mesh) on the bottom, anhydrous Na2SO4 as the next layer, topped with neutral alumina (70-200 mesh) to which 8% water (wet basis) has been added. A blue fluorescent band at the Florisil-Na2SO4 interface indicates the presence of aflatoxin M1. The limit of detection is estimated to be about 0.2 microgram/kb. Because several items are disposable, both the time to maintain glassware and the cost per determination are reduced.     

Collaborative study of a method for chemical confirmation of the identity of aflatoxin.

The chemical method for confirmation of the identity of aflatoxin by derivative formation directly on the TLC plate was studied collaboratively by 8 participants. The results show that aflatoxin B-1 was confirmed in 17 of 17 sample extracts representing 15 mu-g aflatoxin B-1/kg peanut butter, in 13 of 16 extracts representing 5 mu-g/kg, and in none of the 7 aflatoxin-free extracts. Collaborators commented that the method was easily performed and gave good results. The method has been adopted as official first action.     

Presidedress soil nitrogen test for corn in Virginia

Abstract

The presidedress soil nitrate test (PSNT) and the presidedress tissue nitrogen test (PTNT) have been developed to assess residual soil nitrogen (N) sufficiency for corn (Zea mays L.) in the humid eastern U.S. We conducted field studies at 47 sites during 1990 and 1991 to evaluate the use of the PSNT and PTNT for corn in Coastal Plain, Piedmont, and Appalachian Ridge and Valley regions of Virginia. Seven rates of fertilizer N (0, 45, 90, 135, 180, 225, and 270 kg/ha) were applied at corn height of 0.40 to 0.50 m and replicated four times in a randomized complete block design. Whole corn plants and soil to a depth of 0.30 m were sampled when corn height was 0.15 to 0.30 m to estimate available soil N prior to the application of fertilizer N treatments. Corn grain yield response to fertilizer N was used to assess residual soil N availability. Nitrogen concentration of whole corn plants at 0.15 to 0.30 m height was not an accurate indicator of plant‐available soil N. Corn yields were maximized without sidedress N at the 19 sites where soil NO₃‐N was at least 18 mg‐kg^‐1 and at the 17 sites where soil (NO₃+NH₄)‐N was at least 22 mg‐kg^‐1. The PSNT predicted corn N sufficiency regardless of soil physiographic region or surface texture; however, the critical values for NO₃‐N and (NO₃+NH₄)‐N were 3 to 5 mg‐kg^‐1 lower than those established in Pennsylvania and Maryland, where cooler soil temperatures may permit greater residence time of inorganic N.     

Medium-term effects of corn biochar addition on soil biota activities and functions in a temperate soil cropped to corn

Biochar addition to soil has been generally associated with crop yield increases observed in some soils, and increased nutrient availability is one of the mechanisms proposed. Any impact of biochar on soil organisms can potentially translate to changes in nutrient availability and crop productivity, possibly explaining some of the beneficial and detrimental yield effects reported in literature. Therefore, the main aim of this study was to assess the medium-term impact of biochar addition on microbial and faunal activities in a temperate soil cropped to corn and the consequences for their main functions, litter decomposition and mineralization. Biochar was added to a corn field at rates of 0, 3, 12, 30 tons ha⁻¹ three years prior to this study, in comparison to an annual application of 1 t ha⁻¹.Biochar application increased microbial abundance, which nearly doubled at the highest addition rate, while mesofauna activity, and litter decomposition facilitated by mesofauna were not increased significantly but were positively influenced by biochar addition when these responses were modeled, and in the last case directly and positively associated to the higher microbial abundance. In addition, in short-term laboratory experiments after the addition of litter, biochar presence increased NO₂ + NO₃ mineralization, and decreased that of SO₄ and Cl. However, those nutrient effects were not shown to be of concern at the field scale, where only some significant increases in SOC, pH, Cl and PO₄ were observed.Therefore, no negative impacts in the soil biota activities and functions assessed were observed for the tested alkaline biochar after three years of the application, although this trend needs to be verified for other soil and biochar types.     

Mitigation options for N2O emission from a corn field in Kalimantan,Indonesia

Abstract

Field experiments were designed to quantify N₂O emissions from corn fields after the application of different types of nitrogen fertilizers. Plots were established in South Kalimantan, Indonesia, and given either urea (200 kg ha^?1), urea (170 kg ha^?1) + dicyandiamide ([DCD] 20 kg ha^?1) or controlled-release fertilizer LP-30 (214 kg ha^?1) prior to the plantation of corn seeds (variety BISI 2). Each fertilizer treatment was equivalent to 90 kg N ha^?1. Plots without chemical N fertilizer were also prepared as a control. The field was designed to have three replicates for each treatment with a randomized block design. Nitrous oxide fluxes were measured at 4, 8, 12, 21, 31, 41, 51, 72 and 92 days after fertilizer application (DAFA). Total N₂O emission was the highest from the urea plots, followed by the LP-30 plots. The emissions from the urea + DCD plots did not differ from those from the control plots. The N₂O emission from the urea + DCD plots was approximately one thirtieth of that from the urea treatment. However, fertilizer type had no effect on grain yield. Thus, the use of urea + DCD is considered to be the best mitigation option among the tested fertilizer applications for N₂O emission from corn fields in Kalimantan, Indonesia.     

Evaluation of penicylinders used in disinfectant testing: bacterial attachment and surface texture

Two possible deficiencies in the AOAC use-dilution method for registration of chemical disinfectants by the Environmental Protection Agency are examined: (1) the physical disparities among brands of penicylinders and (2) the variability of bacterial numbers on penicylinders depending upon test strain and penicylinder surface texture. Textural differences of 2 brands of stainless steel penicylinders, one brand of porcelain, and one brand of glass were assessed by scanning electron microscopy. A considerable variation in smoothness of both inner and outer surfaces of stainless steel and porcelain penicylinders was observed. Glass penicylinders were very smooth. Numbers of bacteria attached to a penicylinder were assessed by vortexing the penicylinders 30 s at No. 4 after using the AOAC method of bacterial inoculation and drying 40 min at 37 degrees C. With this methodology, stainless steel carriers retained the 3 AOAC-recommended bacterial test strains differentially: ca 10(7) for Pseudomonas aeruginosa, 5 X 10(6) for Staphylococcus aureus, and 10(6) for Salmonella choleraesuis; glass retained 10(6)-10(7) organisms of all 3 test strains; porcelain retained about that amount of S. aureus but 10(5)-10(6) P. aeruginosa and 10(3)-10(4) S. choleraesuis. These data suggest that disinfectants are not similarly challenged with the AOAC-recommended test bacteria and that an alternative method should be considered to ensure comparable numbers of bacteria on penicylinders.     

Sources of resistance to aflatoxin production in maize

Drought-tolerant maize genotypes (Huffman, Z08-004, Tuxpan, PH 9, NRC 5348, Chunco, Saint Croix, and Arizona) were compared in the field and laboratory to toxin-resistant GT-MAS:gk and Yellow Creole. SDS-PAGE, scanning electron microscopy of kernel cuticle, amount of kernel wax, Aspergillus flavus kernel colonization, Aspergillus ear rot, insect damage, aflatoxin production, and their relationships were examined. SDS-PAGE showed the presence of a 14 kDa trypsin inhibitor in the kernels of all genotypes except Chunco, which contains a protein of a larger molecular weight. The 14 kDa trypsin inhibitor protein content in these genotypes was higher than in GT-MAS:gk and Yellow Creole. Scanning electron microscopy revealed that Arizona, Huffman, and Chunco genotypes had abundant wax deposits on kernel surfaces and the amount of pericarp wax was equal to or above that from GT-MAS:gk and Yellow Creole. Differences in Aspergillus ear rot ratings, fungal colonization, and insect damage by corn earworm were observed in all drought-tolerant maize genotypes as well as in the controls. Kernel screening assays showed that aflatoxin B(1) levels in inoculated drought-tolerant genotypes differed significantly from those in GT-MAS:gk and Yellow Creole (LSD = 576). Aflatoxin B(1) levels in the inoculated genotypes differed significantly from those of GT-MAS:gk or Yellow Creole (LSD = 1389) when grown under drought stress conditions. Pearson correlation coefficients were significant between ear rot ratings and insect damage (r = 0.75; P = 0.01) and between Aspergillus ear rot and aflatoxin levels (r = 0.54; P = 0.05). On the basis of the parameters studied, there are indications that these genotypes were potential sources of A. flavus resistance.