Wheat Lots Research Articles

Distribution of deoxynivalenol, zearalenone, and their respective modified analogues in milling fractions of naturally contaminated wheat grains

Mycotoxins are among the most abundant contaminants in food and feed worldwide. Therefore, in the EU maximum levels are established, e.g. for the frequently occurringFusarium toxins deoxynivalenol (DON) and zearalenone (ZEA). Additional to DON and ZEA, modified mycotoxins are present in naturally contaminated grain products contributing significantly to the exposure of humans and animals with mycotoxins. Up to now data on the spatial distribution of many (masked) mycotoxins in the kernels of wheat are missing. The aim of the present study was to investigate the amounts of DON and ZEA as well as their most abundant derivatives DON-3-glucoside (DON-3G), 3- and 15-acetyl-DON, ZEA-14- and 16-glucoside and ZEA-14-sulphate (ZEA-14S) in mill fractions of naturally contaminated wheat batches using HPLC-MS/MS. The investigated distribution pattern in ten milling fractions is comparable among the three investigated different wheat batches. Interestingly, DON and DON-3G were found to be present to similar amounts in all fractions. In bran, the levels were only slightly higher than in the endosperm. By contrast, for ZEA and ZEA-14S a significantly higher amount of toxin is located in the fibre-rich fractions. The relative mass proportion of DON-3G comprises for only between 2.9 and 11.2% of the free DON, while the relative mass proportion of ZEA-14S is estimated to even exceed the amount of free ZEA in certain fractions. Acetylated DON derivatives and ZEA-glucosides were only detected in low amounts. The experimental results show that a significant reduction of the ZEA and ZEA-14S level in wheat flour is feasible by applying milling technology strategies. However, the almost evenly distribution of DON and DON-3G in all fractions does not allow for the technological removal of relevant toxin amounts. Furthermore, the relative share of masked forms was higher for ZEA derivatives than for the DON conjugates in the investigated wheat lots.

Distribution of Fusarium mycotoxins in wheat milling process

Fusarium head blight (FHB) is a fungal disease that affects cereals and is capable of producing mycotoxins, creating health concerns. In southern Brazil, FHB of wheat is caused by the Fusarium graminearum species complex that produces mainly deoxynivalenol (DON) and zearalenone (ZON) mycotoxins. There is a need for research-based information on how different contamination levels affect these mycotoxins' distribution in the milling process. The objective of this study was to analyze the Fusarium mycotoxin distribution within each milled fraction, extracted from wheat lots artificially contaminated with a crescent gradient of mycotoxins. Wheat samples produced in 2013 season in Southern Brazil region were obtained from plots of breeding program. The wheat samples were artificially contaminated with residues of cleaning and pre-cleaning process, including light and shriveled grains, obtained from a Fusarium nursery screening plot. Pilot-scale milled wheat fractions were collected, comprising finished flour and bran. The Fusarium mycotoxin content was determined by chromatography (UHPLC-MS/MS). The results obtained show that DON presented exponential growth relative to the initial levels of mycotoxin in wheat milled fractions (finished flour and bran). The DON concentration was significativelly higher in bran, when compared with milled wheat and finished flour, in the DON levels lower than 1000 μg kg−1. The finished flour presented lower DON levels when compared with milled wheat, but this reduction was inadequate, to meet the current regulation limits for food.

Occurrence of Fusarium langsethiae Strains Isolated from Durum Wheat in Italy

AbstractSurveys on the occurrence of type A trichothecenes in wheat, and particularly for the T‐2 and HT‐2 toxins, and information on the biology and epidemiology of the causative Fusarium species (i.e. F. langsethiae, F. sporotrichioides) are scarce in Italy, as compared to the more common type B trichothecene, deoxynivalenol and its producers. This 4‐year monitoring of phytopathogenic Fusarium species on 183 seed lots of durum wheat shows wide distribution of F. langsethiae in Italy and the potential of several isolates of this fungus to produce high amounts of T‐2 and HT‐2 in wheat. Fusarium langsethiae was observed for approximately 48% of the analysed samples, with a maximum incidence for a single lot of 10.5%. Fusarium sporotrichioides was observed only in 2011, with an average incidence of 2% (range, 0–3%). A collection of F. langsethiae isolates representative of the main cultivation areas in Italy was established. These isolates showed great variability for their toxin production in vitro. Of 28 strains, all except one isolate can produce the T‐2 and HT‐2 toxins. HT‐2 was generally in greater amounts than T‐2, with an average concentration ratio for HT‐2 to T‐2 of 2.1 (range, 0.7–5.4). The artificial inoculation of wheat with three isolates of F. langsethiae produced no Fusarium head blight symptoms under field conditions. However, significantly higher incidence of F. langsethiae was seen on the kernels of inoculated plants, compared to the uninoculated controls.

Application of multicriteria analysis for assessment of wheat quality in trade and processing

The quality related value of grains, especially wheat is defined with a number of quality parameters. Numerous parameters that are often mutually inconsistent result in the fact that decision making related to selection of wheat lots based on their quality is a complex multidimensional problem. The aim of the research was to test the possibility of application of multicriteria analysis for ranking of wheat lots in respect to their overall quality and its variation. A modified version of the preference ranking organisation method for enrichment evaluation, MODIPRIM, was used for multicriteria analysis of wheat intended for sales and processing purposes. The results of determining the quality parameters of wheat lots in six consequent crop years were used to form the input database. Multicriteria analysis was conducted with developed optimisation criteria and assessment of the significance coefficients resulted in a reduction of the complexity of the decision making problem involving numerous quality parame...

Quality parameters of wheat grain and flour as influenced by treatments with natural zeolite and diatomaceous earth formulations, grain infestation status and endosperm vitreousness

This study investigated the changes in quality parameters of wheat (mealy and vitreous) non-infested and infested with Sitophilus oryzae (L.) caused by treatments with inert dusts: natural zeolite, two diatomaceous earths from Serbia, and a commercial product enhanced DE Protect-It®.Inert dust treatments, regardless of infestation status, significantly reduced wheat test weight. This was more marked in mealy (8.3%) rather than in vitreous wheat grain (2.7%). Content of silicon dioxide (SiO2) in flour from infested lots was significantly higher than in non-infested ones. Treatments with all formulations of inert dusts significantly affected flour water absorption in the non-infested wheat lots. Non-infested high vitreous wheat treated with Protect-It® showed the highest moisture absorption (61.9 g.100 g−1) in comparison with the control (59.7 g.100 g−1). Significant improvement in dough rheology was observed in the infested soft and hard wheat, particularly through rise of dough energy. Small-deformation tests implied certain dough weakening effects of inert dusts in vitreous wheat but this was not observed in standard large-deformation rheological tests.Application of inert dusts shows an ability to improve some aspects of the technological quality of wheat.

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor®, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.

Comparison of seven methods for stored cereal losses to insects for their application in rural conditions

There are several methods for assessing harvest losses during storage with varying degrees of accuracy, reliability and practical application, however, field conditions may limit their use among subsistence farmers and traders in developing nations. We compared seven standard grain loss methods during natural and controlled infestation by Sitophilus oryzae L. in wheat and barley to select the best procedures for use in farm storage. Methods tested were: 1) visual inspection of infested lots, 2) uncorrected weight loss, 3) modified standard volume/weight ratio, 4) grain count and weight, 5) percentage of damaged grains converted to weight loss, 6) one thousand grain mass, and 7) one thousand grain mass including dust. Previously disinfested cereal lots (wheat, barley, 500 g fresh w) were exposed to 20 newly emerged adult S. oryzae for 90 d at 28 °C and 70% r.h. Naturally- infested wheat lots were also monitored under the same conditions. Before and after this period, fresh and dry weight, number, moisture content of uninfested and damaged grains, weight of 1 L of seeds and dust produced by insect activity, fresh and dry weight of 1000 kernels, and number of adult weevils was determined and applied to appropriate equations of methods (1)–(7). Grain mass loss estimations varied by wide margins (9.3 ± 1.3% to 25.8 ± 2.3% in barley, 2.2 ± 0.7% to 12.5 ± 2.5% in wheat) depending on method employed, suggesting the need for careful selection of the most appropriate procedure under field conditions and farmer/trader interests. While accurate procedures such as method 7 provided dependable results, methods 3 and 5 appear most practical for wheat and barley.

An attempt to develop surface plasmon resonance based immunosensor for Karnal bunt ( Tilletia indica) diagnosis based on the experience of nano-gold based lateral flow immuno-dipstick test

Karnal bunt (KB) of wheat incited by Tilletia indica is an economically important quarantined fungal pathogen. Similarity in teliospore configuration makes it difficult to differentiate KB teliospores from the teliospores of other bunt fungi using conventional approaches. In order to determine the correct identity of KB teliospore— an infectious entity, it is essential to develop specific diagnostic probes and high quality of immunological reagents against infectious entities. The immuno-dipstick assay developed in our lab is quite sensitive to detect the antigens of even five teliospores. For on-site testing of KB, a nano-gold based lateral flow immuno-dipstick assay (LFID) was also developed in our lab using anti-teliospore antibodies. For development of LFID, colloidal gold was prepared and conjugated with anti-teliospore antibodies which were further characterized. However, species specific detection is yet to be achieved by generation of monoclonal antibodies against diagnostic antigens (34 and 66 kD teliospore's proteins). Based on the experience of nano-gold based LFID, we are proposing to develop surface plasmon resonance (SPR) based label-free detection system. The same may be employed not only for determination of the quality of immunological reagents in terms of sensitivity, specificity and precision but also development of suitable design of immunosensor for differential diagnosis of KB ( T. indica). The approach involves the use of a mouse monoclonal antibody against diagnostic antigens and a suitable design of SPR sensor by the preparation of immuno-affinity layer over self assembled monolayer surfaces. The signal to noise ratio can further be enhanced using nano-gold particles. This will be first attempt for real-time monitoring of KB teliospores in wheat lots by SPR sensor and in a fully automated manner for establishing seed certification and plant quarantine standards.

Bulk Functionality Diversification by Unsupervised Single‐Kernel Near‐Infrared (SKNIR) Sorting of Wheat

ABSTRACTWe explored the effects of fractioning heterogeneous bulk wheat by fast unsupervised single‐kernel near‐infrared (SKNIR) sorting according to an internal complex NIR functionality trait using a fast prototype kernel sorter designed for postharvest bulk sorting. Sorting into three functionality fractions was performed on low quality lots from an organic field experiment from two growth years and two locations. Sorted lots were mixtures originally diversified by three different preceding catch crops. The resulting 12 fractions, as well as the 12 original wheat lots were characterized by 20 standard quality variables of grains and flours. The data was analyzed by principal component analysis (PCA) and analysis of variance (ANOVA). Within each year and location/cultivar, the SKNIR fractionation had significant positive effect on bulk grain density, protein, wet gluten content, Zeleny sedimentation volume, farinograph water absorption, farinograph softening, falling number, gelatinization temperature, and hardness index. Using the NIR fingerprint directly for sorting without calibration to a univariate reference showed that the resulting fractions were based on the major variance in the entire physicochemical quality trait within each lot as expressed by NIR. This novel unsupervised approach may become a powerful tool for sorting according to complex functionality traits, thus increasing overall quality, applicability, and value of the sorted crop.

Rapid Assessment of Insect Fragments in Flour Milled from Wheat Infested with Known Densities of Immature and Adult Sitophilus oryzae (Coleoptera: Curculionidae)

Milling wheat, Triticum aestivum L., infested with low densities of internal feeding insects can result in flour containing insect fragments. The Food and Drug Administration (FDA) enforces a standard or defect action level stating that a maximum of 75 insect fragments per 50 g of flour is allowed. However, the relationship between level of infestation and number of resulting fragments is not well documented, and a more rapid method for enumerating insect fragments is needed. We characterized the number of insect fragments produced from milling small lots of wheat spiked with known densities and life stages of Sitophilus oryzae (L.) (Coleoptera: Curculionidae). Insect fragments were enumerated with near-infrared spectroscopy (NIRS), a quick nondestructive procedure, and with the industry standard flotation method. Results showed that an individual small larva, large larva, pupa, or adult produced 0.4, 0.7, 1.5, and 27.0 fragments, respectively. NIRS-predicted counts of ≤51 (from small larvae), ≤53 (from large larvae), ≤43 (from pupae), or 0 (from adults) indicated that there were <75 actual fragments in that sample, because the upper bound of associated 95% inverse prediction confidence intervals was less than the standard; NIRS-predicted counts of ≥98, ≥117, ≥108, or ≥225 fragments (same life stages as above) signaled that these flour samples contained >75 actual fragments. These data suggest that NIRS could be adopted for rapid assessment of insect fragments resulting from relatively low levels of infestation with immature life states, but that it was not accurate enough for enumerating insect fragments, relevant to FDA standards, resulting from adults.

Rapid Assessment of Insect Fragments in Flour Milled from Wheat Infested with Known Densities of Immature and Adult &lt;I&gt;Sitophilus oryzae&lt;/I&gt; (Coleoptera: Curculionidae)

Milling wheat, Triticum aestivum L., infested with low densities of internal feeding insects can result in flour containing insect fragments. The Food and Drug Administration (FDA) enforces a standard or defect action level stating that a maximum of 75 insect fragments per 50 g of flour is allowed. However, the relationship between level of infestation and number of resulting fragments is not well documented, and a more rapid method for enumerating insect fragments is needed. We characterized the number of insect fragments produced from milling small lots of wheat spiked with known densities and life stages of Sitophilus oryzae (L.) (Coleoptera: Curculionidae). Insect fragments were enumerated with near-infrared spectroscopy (NIRS), a quick nondestructive procedure, and with the industry standard flotation method. Results showed that an individual small larva, large larva, pupa, or adult produced 0.4, 0.7, 1.5, and 27.0 fragments, respectively. NIRS-predicted counts of < or =51 (from small larvae), < or =53 (from large larvae), < or =43 (from pupae), or 0 (from adults) indicated that there were <75 actual fragments in that sample, because the upper bound of associated 95% inverse prediction confidence intervals was less than the standard; NIRS-predicted counts of > or =98, > or =117, 108, or > or =225 fragments (same life stages as above) signaled that these flour samples contained >75 actual fragments. These data suggest that NIRS could be adopted for rapid assessment of insect fragments resulting from relatively low levels of infestation with immature life states, but that it was not accurate enough for enumerating insect fragments, relevant to FDA standards, resulting from adults.

Biological control of snow mould (Microdochium nivale) in winter cereals by Pseudomonas brassicacearum, MA250

Seed lots of winter wheat and rye, naturally infested with Microdochium nivale and Fusarium spp., were treated with an isolate of Pseudomonas, which was recovered from roots of Brassica napus. Seeds were treated with bacterial fermentate and dried before sowing or they were directly sprayed in the furrow-opener at the moment of sowing. Besides field experiments, parallel climate chamber bioassays were performed to assess the effect of bacterial treatment on snow mould caused by seed-borne M. nivale and Fusarium spp. The biocontrol effect was assessed by plant density counts and by measuring yield. Significant biocontrol activity, measured by plant density counts, was detected both in field and climate chamber experiments sown with wheat. Biocontrol effect after spray application at sowing was less pronounced, although a slight increase in plant density was observed. The cell concentration required to obtain adequate biocontrol effect was 109 CFU per ml for the dose used. The bacterial isolate was identified by 16S rDNA sequencing and biochemical tests as a Pseudomonas brassicacearum strain.

Protein heterogeneity in wheat lots using single-seed NIT — A Theory of Sampling (TOS) breakdown of all sampling and analytical errors

An in-depth heterogeneity analysis of wheat lots from varying field experiments with respect to protein concentration was conducted in order to quantify and compare both sampling and analytical errors as defined by the Theory of Sampling (TOS). Thirty wheat samples of forty-two seeds were extracted from three different wheat lots. Half of these were extracted using a non-optimal spoon (grab sampling) and the other half were extracted using a riffle splitter. Ten additional samples of forty-two seeds were extracted using a riffle splitter from ten different wheat lots. The protein content of every single-seed was determined by Near-Infrared Transmission (NIT) spectroscopy based on a multivariate calibration to Kjeldahl with Partial Least Squares Regression (PLS-R). The effect of orientation and number of replicate measurements of the individual seeds in the NIT beam was investigated for minimizing the analytical error as well the resulting time requirements. Remarkably, the best prediction model with respect to Root Mean Squared Error of Cross Validation (RMSECV) was obtained by only recording three replicate NIT spectra of the seeds in only one specific orientation.The variance of the Global Estimation Error (GEE) of both the riffle splitter and the spoon sampling processes was estimated as well as its components, the Fundamental Sampling Error (FSE), the Grouping and Segregation Error (GSE), the Incorrect Sampling Errors (ISE) and the Total Analytical Error (TAE). The bias induced by non-probabilistic spoon extraction was also estimated. The GEE variance of the spoon extractions was seventy percent higher than that of the riffle split samples. The sampling variances of FSE, GSE and ISE were all of the same order of magnitude, each approximately ten times higher than the TAE variance. The squared bias of the spoon sampling was approximately twice the magnitude of the sampling variances and thus contributed significantly to the representativity score. Spoon sampling representativity was three times the size of that for the riffle splitter. Order of magnitude estimates of the Constitutional Heterogeneity (CHL) as well as the Distributional Heterogeneity (DHL) for a forty-two seed riffle split sample was derived. In this investigation the fundamental concepts of TOS have been investigated and estimates of all sampling and analytical errors have been presented for a specific zero-dimensional composite material. The ability of TOS for quantifying and evaluating the various error contributions to the overall estimation of the protein concentration was confirmed.

Persistence and Efficacy of Three Diatomaceous Earth Formulations Against &lt;I&gt;Sitophilus oryzae&lt;/I&gt; (Coleoptera: Curculionidae) on Wheat and Barley

The insecticidal and residual efficacy of three diatomaceous earth (DE) formulations, Insecto, PyriSec, and SilicoSec, against Sitophilus oryzae (L.) on barley and wheat was assessed. For this purpose, 4-kg lots of barley and wheat were treated with the above-mentioned DE formulations, in three dose rates (0.75, 1, and 1.5 g/kg grain) and stored at 26 degrees C. Samples of these lots were taken at the day of storage, and every 45 d, until the completion of a 450-d period of storage. Bioassays were conducted by exposing S. oryzae adults to these samples, at 26 degrees C and 57% RH. In these bioassays, the DE efficacy was evaluated by recording adult mortality after 24 h, 48 h, 7 d, and 14 d of exposure on the treated grains. After the 14-d count, all adults were removed, and the samples were left at the same conditions for an additional 45 d, to evaluate the capacity for progeny production in the treated grains. Adult mortality after 14 d of exposure was exponentially decreased with time. During the first 270 d of storage, mortality was > 90%, and progeny production was < 1 adult per sample, whereas after 270 d a gradual decrease in adult mortality occurred, with a resulting increase in progeny production. Generally, the three DE formulations tested were equally effective against S. onyzae adults. During the first 270 d of storage, the DE formulations were equally effective on both grains tested, but from 315 d of storage and on, S. oryzae mortality was higher on barley than on wheat. At this interval, progeny production was gradually increased, especially on grains treated with the lowest DE dose rate. However, even this rate caused a satisfactory level of mortality (> 90% after 14 d of exposure) during the first 270 d of storage.

Distribution of Deoxynivalenol in Wheat, Wheat Flour, Bran, and Gluten, and Variability Associated with the Test Procedure

Analytical data obtained on deoxynivalenol (DON) concentration in naturally contaminated wheat during processing in an industrial mill were statistically analyzed, and the distribution functions of DON concentration in lots of wheat, bran, wheat flour, and gluten were estimated. The analytical method had acceptable precision (HORRAT 0.25-0.32) for each test sample. The total variance combined sampling, sample preparation, and analytical variances were 0.188, 0.033, 0.42, and 0.0014 ppm2 for wheat, 1.93; flour, 0.99; bran, 4.68; and gluten, 0.29, respectively. The distribution function of DON contamination presented an asymmetric tail for high values of concentration in wheat grains and wheat flour; in bran it seemed to be bimodal with 2 separated peaks of different concentrations; in gluten the normal distribution function gave a reasonably good fit to empirical data. The function eta(c) = -In(-Inp), where p (c) is the cumulative distribution function was linear with c in the so-called extreme-value type I distribution and could be fitted by a cubic polynomial in c in the distributions determined for all the products. This variability and distributional information contributes to the design of better sampling plans in order to reduce the total variability and to estimate errors in the evaluation of DON concentration in lots of wheat and wheat products.

Broadband Microwave Wheat Permittivity Measurements in Free Space

Use of broadband horn/lens antennas with a collimated beam in a free-space measurement set-up with a network analyzer operating between 3 and 13 GHz and applying time-domain gating improved the accuracy of permittivity (dielectric properties) measurements on grain. Measurements were taken in the transmission mode on samples from five different classes of wheat over ranges of moisture content, temperature, and bulk density. Dielectric properties were divided by bulk density of the grain for comparison of different wheat lots. No differences in permittivity data were noted among the different wheat classes, so data were combined for presentation. The new data agree well with those reported previously. For further research, to provide dielectric properties over a very broad frequency range for a given grain sample, use of time-domain spectroscopy is suggested.

OPTIMIZATION OF WHEAT BLENDING TO PRODUCE BREADMAKING FLOUR

ABSTRACTLinear programming was utilized to optimize the blending of wheat lots which have different quality characteristic and costs. Using best subsets regression three quality tests (particle size index, dough volume and falling number value) were selected in relation to loaf volume of bread to be produced. The chosen criteria were set up in a linear programming format as a model for the computerized solution. The model's applicability was assessed in a commercial mill. As a result of applying the model it was found possible to produce breadmaking flour with a reasonable quality and at a lower cost.

Seedling Blight Development and Control in Spring Wheat Damaged by Fusarium graminearum Group 2.

Aspects of seedborne infection in Fusarium-damaged and undamaged seed lots of hard red spring wheat were studied. In a 22°C environment, seedling blight developed from plantings of Fusarium-damaged seed that were characterized by both reduction and delay in emergence and postemergence seedling death. After 28 days, stand counts from the Fusarium-damaged seed lot averaged 46%, compared with 98% from the undamaged seed lot. However, the mean dry weight of surviving seedlings from the Fusarium-damaged lot was not statistically different from that of seedlings from the undamaged seed lot. Surface-sterilization reduced recovery of Fusarium graminearum group 2 from the damaged lot by 7% and increased germination by 32%, suggesting that seed are both infected and contaminated with viable propagules of the fungus. In field experiments, seed treatment with maneb- or thiabendazole-containing fungicide combinations significantly reduced seedling blight and improved stands derived from the Fusarium-damaged seed lot. Triazole fungicides, including difenoconazole, fenbuconazole, and tetraconazole, were less effective. Triazole fungicides and carboxin-containing fungicide combinations controlled loose smut in these trials, while maneb seed treatments did not. In four field tests, seedling blight incidence in plantings of Fusarium-damaged seed increased as soil temperatures during emergence increased. The distribution of blighted seedlings was random and not clustered. Seedling blight did not develop in field plantings of the undamaged seed lot.

Micropycnometer Measurement of Single‐Kernel Density of Healthy, Sprouted, and Scab‐Damaged Wheats

ABSTRACTSamples from four market lots of hard red winter and soft red winter wheat containing sprout‐ and scab‐damaged kernels were used to test a prototype single‐kernel density micropycnometer. Fifteen kernels for each damage type and an equal number of healthy kernels were weighed to the nearest 0.01 mg, then measured for volume to the nearest 1.0 μL. Volume measurements for all kernels were performed three consecutive times with the micropycnometer, then kernels were evaluated for weight, size, moisture, and hardness using a Single Kernel Characterization System. The structure of the sampling plan and the goals of the study indicated that a mixed‐model statistical analysis was needed. The fixed effects were wheat class and type of kernel, and the random effects included lot, the interaction of lot with class and kernel type, kernels within each lot, and repeated measures of single‐kernel density. Results indicated that variability of the three measurements per kernel did not depend on type of kernel or class of wheat. The standard deviation for repeated density measurements was 0.0029 g/cm3. Kernel‐to‐kernel variability changed depending on the type of kernel; healthy and sprout‐damaged kernels showed similar variability in density, whereas scab damaged kernels had a variance about four to five times higher. Type of kernel significantly affected mean density; healthy kernels averaged 1.28 g/cm3, sprout‐damaged kernels averaged 1.19 g/cm3, and scab‐damaged kernels averaged 1.08 g/cm3. Wheat class did not exert a significant influence on singlekernel density. Attempts to predict single‐kernel density using kernel weight, size, moisture, and hardness found no relationships of practical importance.

Development of Seed Immunoblot Binding Assay for Detection of Karnal bunt (Tilletia indica) of Wheat

Polyclonal antisera were produced in albino white rabbits against intact teliospores of Karnal bunt (Tilletia indica). The Immunoprobe generated was used for the development of Immunoblot binding assay for detecting Karnal bunt (KB) infections in wheat seed samples. The antiserum reacted strongly with intact teliospores of T. indica, Pantnagar isolate in agglutination reaction. Wheat grains with different grades of infection could be readily detected by Seed Immunoblot Binding Assay (SIBA). Karnal bunt infected wheat seeds when kept for vigour testing on nitrocellulose paper, formed a coloured imprint after the paper was immunoprocessed. The SIBA would not only be a better Indication of teliospores load on seed but also quality of seed in terms of vigour. This method is expected to be useful in routine monitoring of wheat lots for the presence of KB teliospores.