Long-Term Empirical and Observational Evidence of Practical Helicoverpa zea Resistance to Cotton With Pyramided Bt Toxins.

Evidence of practical resistance of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) to Bt cotton in the United States is debatable, supported with occasional reports of boll damage in the field. Our objective was to provide both empirical and long-term observational evidence of practical resistance by linking both in-season and end-of-season measurements of H. zea damage to pyramided Bt cotton bolls and to provide Cry1Ac diet-based bioassay data in support of these damage estimates. In-season boll damage from H. zea was highly correlated to end-of-season damaged bolls. Across North Carolina, Bt cotton fields with end-of-season bolls damaged by H. zea increased during 2016 compared to previous years. Elevated damage was coupled with an increase in field sprays targeting H. zea during 2016, but not related to an increase in H. zea abundance. Bioassay data indicated that there was a range of Cry1Ac susceptibility across the southeastern United States. Given the range of susceptibility to Cry1Ac across the southeastern United States, it is probable that resistant populations are common. Since H. zea is resistant to cotton expressing pyramided Cry toxins, the adoption of new cotton varieties expressing Vip3Aa will be rapid. Efforts should be made to delay resistance of H. zea to the Vip3Aa toxin to avoid foliar insecticide use.

A Novel, Economical Way to Assess Virulence in Field Populations of Hessian Fly (Diptera: Cecidomyiidae) Utilizing Wheat Resistance Gene H13 as a Model.

Mayetiola destructor (Say) is a serious pest of wheat, Triticum aestivum L., in North America, North Africa, and Central Asia. Singly deployed resistance genes in wheat cultivars have provided effective management of Hessian fly populations for >50 yr. Thirty-five H genes have been documented. Defense mediated by the H gene constitutes strong selection on the Hessian fly population, killing 100% of larvae. A mutation in a matching Hessian fly avirulence gene confers virulence to the H gene, leading to survival on the resistant plant. As the frequency of virulence rises in the population, the H gene loses its effectiveness for pest management. Knowing the frequency of virulence in the population is not only important for monitoring but also for decisions about which H gene should be deployed in regional wheat breeding programs. Here, we present a novel assay for detecting virulence in the field. Hessian fly males were collected in Alabama, Georgia, North Carolina, and South Carolina using sticky traps baited with Hessian fly sex pheromone. Utilizing two PCR reactions, diagnostic molecular markers for the six alleles controlling avirulence and virulence to H13 can be scored based on band size. Throughout the southeast, all three avirulence and three virulence alleles can be identified. In South Carolina, the PCR assay was sensitive enough to detect the spread of virulence into two counties previously documented as 100% susceptible to H13. The new assay also indicates that the previous methods overestimated virulence in the field owing to scoring of the plant instead of the insect.

Effectiveness of Genes for Hessian Fly (Diptera: Cecidomyiidae) Resistance in the Southeastern United States.

The Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), is the most important insect pest of wheat (Triticum aestivum L. subsp. aestivum) in the southeastern United States, and the deployment of genetically resistant wheat is the most effective control. However, the use of resistant wheat results in the selection of pest genotypes that can overcome formerly resistant wheat. We have evaluated the effectiveness of 16 resistance genes for protection of wheat from Hessian fly infestation in the southeastern United States. Results documented that while 10 of the genes evaluated could provide protection of wheat, the most highly effective genes were H12, H18, H24, H25, H26, and H33. However, H12 and H18 have been reported to be only partially effective in field evaluations, and H24, H25, and H26 may be associated with undesirable effects on agronomic traits when introgressed into elite wheat lines. Thus, the most promising new gene for Hessian fly resistance appears to be H33. These results indicate that identified highly effective resistance in wheat to the Hessian fly is a limited resource and emphasize the need to identify novel sources of resistance. Also, we recommend that the deployment of resistance in gene pyramids and the development of novel strategies for engineered resistance be considered.

Species composition of aphid vectors (Hemiptera: Aphididae) of barley yellow dwarf virus and cereal yellow dwarf virus in Alabama and western Florida.

Yellow dwarf is a major disease problem of wheat, Triticum aestivum L., in Alabama and is estimated to cause yield loss of 21-42 bu/acre. The disease is caused by a complex of viruses comprising several virus species, including Barley yellow dwarf virus-PAV and Cereal yellow dwarf virus-RPV. Several other strains have not yet been classified into a specific species. The viruses are transmitted exclusively by aphids (Hemiptera:Aphididae). Between the 2005 and 2008 winter wheat seasons, aphids were surveyed in the beginning of each planting season in several wheat plots in Alabama and western Florida Collected aphids were identified and bioassayed for their yellow dwarf virus infectivity. This survey program was designed to identify the aphid species that serve as fall vectors of yellow dwarf virus into winter wheat plantings. From 2005 to 2008, bird cherry-oat aphid, Rhopalosiphum padi (L.); rice root aphid, Rhopalosiphum rufiabdominale (Sasaki); and greenbug, Schizaphis graminum (Rondani), were found consistently between October and December. The species of aphids and their timing of appearance in wheat plots were consistent with flight data collected in North Alabama between 1996 and 1999. Both R. padi and R. rufiabdominale were found to carry and transmit Barley yellow dwarf virus-PAV and Cereal yellow dwarf virus-RPV. The number of collected aphids and proportion of viruliferous aphids were low. Although this study has shown that both aphids are involved with introduction of yellow dwarf virus to winter wheat in Alabama and western Florida, no conclusions can be made as to which species may be the most important vector of yellow dwarf virus in the region.

Virulence in Hessian fly (Diptera: Cecidomyiidae) field collections from the southeastern United States to 21 resistance genes in wheat.

Genetic resistance in wheat, Triticum aestivum L., is the most efficacious method for control of Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae). However, because of the appearance of new genotypes (biotypes) in response to deployment of resistance, field collections of Hessian fly need to be evaluated on a regular basis to provide breeders and producers information on the efficacy of resistance (R) genes with respect to the genotype composition of Hessian fly in regional areas. We report here on the efficacy of 21 R genes in wheat to field collections of Hessian fly from the southeastern United States. Results documented that of the 21 R genes evaluated only five would provide effective protection of wheat from Hessian fly in the southeastern United States. These genes were H12, H18, H24, H25, and H26. Although not all of the 33 identified R genes were evaluated in the current study, these results indicate that identified genetic resistance to protect wheat from Hessian attack in the southeastern United States is a limited resource. Historically, R genes for Hessian fly resistance in wheat have been deployed as single gene releases. Although this strategy has been successful in the past, we recommend that in the future deployment of combinations of highly effective previously undeployed genes, such as H24 and H26, be considered. Our study also highlights the need to identify new and effective sources of resistance in wheat to Hessian fly if genetic resistance is to continue as a viable option for protection of wheat in the southeastern United States.

Using haplotypes to monitor the migration of fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Texas and Florida.

Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), infestations in most of North America north of Mexico arise from annual migrations of populations that overwinter in southern Texas and Florida. A comparison of the cytochrome oxidase I haplotype profiles within the fall armyworm corn-strain, the subgroup that preferentially infests corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), identified significant differences in the proportions of certain haplotypes between the Texas and Florida populations. These proportional differences were preserved as the populations migrated, providing a molecular metric by which the source of a migrant population could be identified. The migratory pattern derived from this method for several southeastern states was shown to be consistent with predictions based on analysis of historical agricultural and fall armyworm infestation data. These results demonstrate the utility of haplotype proportions to monitor fall armyworm migration, and they also introduce a potential method to predict the severity of cotton crop infestations in the short term.

Trapping Phyllophaga spp. (Coleoptera: Scarabaeidae: Melolonthinae) in the United States and Canada using sex attractants.

The sex pheromone of the scarab beetle, Phyllophaga anxia, is a blend of the methyl esters of two amino acids, L-valine and L-isoleucine. A field trapping study was conducted, deploying different blends of the two compounds at 59 locations in the United States and Canada. More than 57,000 males of 61 Phyllophaga species (Coleoptera: Scarabaeidae: Melolonthinae) were captured and identified. Three major findings included: (1) widespread use of the two compounds [of the 147 Phyllophaga (sensu stricto) species found in the United States and Canada, males of nearly 40% were captured]; (2) in most species intraspecific male response to the pheromone blends was stable between years and over geography; and (3) an unusual pheromone polymorphism was described from P. anxia. Populations at some locations were captured with L-valine methyl ester alone, whereas populations at other locations were captured with L-isoleucine methyl ester alone. At additional locations, the L-valine methyl ester-responding populations and the L-isoleucine methyl ester-responding populations were both present, producing a bimodal capture curve. In southeastern Massachusetts and in Rhode Island, in the United States, P. anxia males were captured with blends of L-valine methyl ester and L-isoleucine methyl ester.

TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis.

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.

Assessment of experimental Bt events against fall armyworm and corn earworm in field corn.

Performance of experimental Bacillus thuringiensis (Bt) MON events alone and pyramided with MON810 were evaluated over 3 yr in Georgia and Alabama. Ability of events to prevent whorl defoliation by the fall armyworm, Spodoptera frugiperda (J. E. Smith), and natural ear feeding damage by the corn earworm, Helicoverpa zea (Boddie) was assessed. In each year, near-isogenic hybrids with novel single transformation events and crosses pyramided with the MON810 event were compared with the standard single MON810 event and nontransformed susceptible control. Events were tested for resistance to whorl damage by manual infestations of fall armyworm and ear damage by natural infestations of corn earworm. All Bt events tested reduced fall armyworm whorl damage ratings per plant compared with the susceptible hybrid. All Bt treatments also had considerably less ear infestation and damage by corn earworm compared with the nontransgenic isoline. The MON841, MION849, and MON851 events reduced ear damage by H. zea but were not as effective as other novel events and were not advanced for further testing after the 1999 season. Pyramiding events compared with single events did not improve control of fall armyworm whorl damage, but they generally did prevent more ear damage by corn earworm. The MON84006 event singly and pyramided with MON810 had superior control of whorl-stage damage by S. frugiperda and ear damage by H. zea compared with MON810. Deployment of new events and genes could provide additional tools for managing the potential for insect resistance to Bt toxins. Furthermore, improved control of whorl and ear infestations by H. zea and S. frugiperda would increase the flexibility of planting corn, Zea mays L., and permit double cropping of corn in areas where these pests perennially reach damaging levels.

Damage loss assessment and control of the cereal leaf beetle (Coleoptera: Chrysomelidae) in winter wheat.

Cereal leaf beetle, Oulema melanopus (L.), invaded northern Alabama and Georgia more than a decade ago and since has become an economic pest of winter wheat and other cereal crops in the southeastern United States. A series of trials was conducted beginning in 1995 to determine optimal rate and timing of applications of selected foliar insecticides for managing cereal leaf beetle in soft red winter wheat. These trials, cage studies with larvae, and a manual defoliation experiment were used to provide information on cereal leafbeetle yield loss relationships and to develop economic decision rules for cereal leaf beetle in soft red winter wheat. Malathion, methomyl, carbaryl, and spinosad effectively controlled larval infestations when treatments were applied after most eggs had hatched. Encapsulated endotoxin of Bacillus thuringiensis, methyl parathion, and disulfoton applied at the lowest labeled rates were not effective treatments. Organophosphate insecticides generally were not effective when applied before most eggs had hatched. The most effective treatments were the low rates of lambda cyhalothrin when applied early while adults were still laying eggs and before or near 50% egg hatch. These early applications applied at or before spike emergence virtually eliminated cereal leaf beetle injury. The manual defoliation study demonstrated that defoliation before spike emergence has greater impact on grain yield and yield components than defoliation after spike emergence. Furthermore, flag leaf defoliation causes more damage than injury to lower leaves. Grain test weight and kernel weight were not affected by larval injury in most trials. Regression of larval numbers and yield losses calculated a yield loss of 12.65% or 459 kg/ha per larva per stem, which at current application costs suggested an economic threshold of 0.4 larvae per stem during the spike emergence to anthesis stages.

Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone.

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.

Incidence of Viruses Infecting Winter Wheat in Alabama.

The most important viral diseases of wheat are caused by Barley yellow dwarf virus (BYDV, strains PAV and MAV) and Cereal yellow dwarf virus (CYDV, strain RPV). Starting in 2000, winter wheat crops growing in northern, central, and southern Alabama were evaluated for the occurrence of BYDV-PAV and CYDV-RPV. In addition to these viruses, samples were tested for the presence of Soilborne wheat mosaic virus (SBWMV), Wheat spindle streak mosaic virus (WSSMV), and Wheat streak mosaic virus (WSMV). BYDV-PAV and CYDV-RPV were found throughout the state, alone or as co-infections, in 14.6% of the samples collected in 2000 and 12.2% of samples in 2001. PAV was found at a lower incidence than RPV (4.3 and 9.9%, respectively) in 2000; however, in 2001, PAV was detected in 8.2% and RPV in 1.9% of the samples. There was less rainfall than the 30-year average during the 1999-2000 growing season, and this may have contributed to differences in the relative incidence of PAV and RPV between the 2 years. SBWMV, WSSMV, and WSMV also were detected in Alabama in 2, 7.8, and 5.4% of the samples collected in 2000, respectively, and in 9.6, 34.3, and 18.5% of the samples collected in 2001, respectively. This is the first report of WSMV in Alabama winter wheat.

Acoustic identification and measurement of activity patterns of white grubs in soil.

Activity patterns of Phyllophaga crinita (Burmeister), Phyllophaga congrua (LeConte), Phyllophaga crassissima (Blanchard), and Cyclocephala lurida (Bland) grubs were monitored with acoustic sensors in small pots of bluegrass, Poa arachnifera Torr, at varying and constant temperatures over multiple-day periods. Experienced listeners readily distinguished three types of sound with distinct differences in frequency and temporal patterns, intensities, and durations. Of approximately 3,000 sounds detected from P. crinita larvae, 7% were identifiable as snaps, with large amplitudes and short durations typically associated with root breakage or clipping activity. Approximately 60% were identifiable as rustles, suggestive of surfaces sliding or rubbing past each other during general movement activity. Another 2% of sounds contained patterns of repeated pulses suggestive of surfaces scraping across a pointed ridge. The remaining 31% had spectral or temporal patterns that fell outside the ranges of easily recognizable sound types. Because the behavioral significance of the different sound types has not yet been fully established, the classified and unclassified sounds were pooled together in analyses of the effects of species, temperature, weight, and time of day. Grubs of all four species produced detectable sounds at rates that increased with temperature [0.45 sounds/((min)(degrees C))] and larval weight [6.3 sounds/((min)(g))]. Mean sound rates were independent of species and time of day. At temperatures <9 degrees C, mean sound rates fell below the typical levels of background noise observed under field conditions. This reduced activity at low temperatures is likely to reduce the effectiveness of acoustic monitoring in the field in cold weather. The consistency of results obtained in these tests over multiple-day periods suggests that acoustic systems have potential as tools for nondestructive monitoring of the efficacy of insect management treatments as well as for biological and ecological studies.

Acoustic estimation of infestations and population densities of white grubs (Coleoptera: Scarabaeidae) in turfgrass.

Incidental sounds produced by Phyllophaga crinita (Burmeister) and Cyclocephala lurida (Bland) (Coleoptera: Scarabaeidae) white grubs were monitored with single- and multiple-sensor acoustic detection systems in turf fields and golf course fairways in Texas. The maximum detection range of an individual acoustic sensor was measured in a greenhouse as approximately the area enclosed in a 26.5-cm-diameter perimeter (552 cm2). A single-sensor acoustic system was used to rate the likelihood of white grub infestation at monitored sites, and a four-sensor array was used to count the numbers of white grubs at sites where infestations were identified. White grub population densities were acoustically estimated by dividing the estimated numbers of white grubs by the area of the detection range. For comparisons with acoustic monitoring methods, infestations were assessed also by examining 10-cm-diameter soil cores collected with a standard golf cup-cutter. Both acoustic and cup-cutter assessments of infestation and estimates of white grub population densities were verified by excavation and sifting of the soil around the sensors after each site was monitored. The single-sensor acoustic method was more successful in assessing infestations at a recording site than was the cup-cutter method, possibly because the detection range was larger than the area of the soil core. White grubs were recovered from >90% of monitored sites rated at medium or high likelihood of infestation. Infestations were successfully identified at 23 of the 24 sites where white grubs were recovered at densities >50/m2, the threshold for economic damage. The four-sensor array yielded the most accurate estimates of the numbers of white grubs in the detection range, enabling reliable, nondestructive estimation of white grub population densities. However, tests with the array took longer and were more difficult to perform than tests with the single sensor.

Enhanced Recovery of Listeria from Dairy-Plant Processing Environments through Combined Use of Repair Enrichment and Selective Enrichment/Detection Procedures.

The efficacy of using a repair step to increase sensitivity of recovery of injured Listeria from environmental sources in dairy processing plants was investigated. The USDA-FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth medium University of Vermont (UVM) for primary enrichment was the standard method chosen for comparison. UVM broth was used in conjunction with rapid methods (Organon Teknika and Gene-Trak™), following manufacturer's guidelines. Listeria Repair Broth (LRB) was used as the repair enrichment medium in modified protocols of the standard and rapid procedures. LRB employs a nonselective period (2-5 hours) for repair of injured Listeria prior to selective-agent addition. Of 80 environmental sites positive by any method, UVM and LRB showed similar recovery rates (87.5% and 88.8%, respectively). Thus LRB provided little advantage over current procedures for use in contaminated sites. UVM was superior when used in conjunction with either rapid method. The USDA and modified USDA (mUSDA) procedures gave identical recovery rates (93%), but 10 additional positive sites were attributed to the use of two enrichment broths. The culture method combined with either rapid method from each broth increased the sensitivity to 97.5-98.8% when data from UVM and LRB was combined. False negative rates in the USDA method (7.1%) were attributed to the lack of color change in Fraser secondary broth. Fraser broth also yielded many false positive results (overall 66.2%) making this broth of limited value as a screening tool for highly contaminated samples. In order to optimize methodology for detection of Listeria , suppression of background flora and the recovery of potentially injured Listeria in the processing environment must be addressed. Overgrowth occurring during the nonselective enrichment period was suspected of causing suboptimal sensitivity in LRB; however, the combination of UVM and LRB showed promising recovery rates. Ceftazidime was evaluated against 68 background isolates that survived throughout the various enrichment and detection methods. Inhibition of 57 of the contaminants indicates a potential role for ceftazidime in the LRB selective-agent regime for sites with high microbial background.

Environmental Surveillance of Dairy Processing Plants for the Presence of Yersinia Species.

Thirty dairy processing plants were surveyed to determine if the psychrotrophic nature of Yersinia influenced its environmental niche within the plants. Three hundred forty-seven samples obtained from 30 dairy processing plants were tested for the presence of Yersinia spp. Ten (33.3%) plants had one or more positive sites. A total of 20 of the 347 (5.8%) sites tested were positive for one or more species of Yersinia . Yersinia enterocolitica was present at 85% (17/20) of the positive sites. It was identified by itself on 15 occasions and twice with other species of Yersinia . Other species identified included Yersinia ruckeri in 3 of the 20 positive samples (15%); Yersinia kristensenii and Yersinia frederiksenii both in 1 of the 20 (5%) positive samples. Ten of the 20 (50%) positive samples were obtained from coolers and entrances to freezers. Yersinia -positive cooler/freezer sites were obtained from 9 of the 10 positive plants. The remaining 10 Yersinia -positive sites were from raw milk receiving areas (4/10) and drains around machinery (6/10), including an ice maker, a case washer, two holding tanks, one filler machine and a packaging area. These results reinforce both the psychrotrophic nature of Yersinia and the evidence that raw milk can be a primary source of the organism. Our findings show that sanitation schemes need to be developed for coolers and freezers to prohibit the colonization and proliferation of Yersinia within these areas.

Increased Incidence of Listeria Species in Dairy Processing Plants Having Adjacent Farm Facilities.

The processing environments of 30 dairy facilities were surveyed for the presence of Listeria species. Two different primary enrichment media - University of Vermont Modified Listeria Enrichment Broth and Listeria Repair Broth - were employed to increase the probability of identifying positive samples. Samples were also tested using both an enzyme-linked immunosorbent-based (ELISA-based) assay and a gene probe assay. A total of 346 sponge samples were evaluated for the presence of Listeria . Listeria spp. were identified via one or more of the assays 122 (35.3%) times. Fifty-five of the positive samples (37.2%) contained Listeria monocytogenes and 93 (62.8%) contained Listeria innocua . Of the 30 plants tested, 9 had a dairy farm contiguous to the processing facilities. Our results show that these plants are more likely to be contaminated (9/9) than those plants without on site dairy farms (17/21). Analysis of the Listeria spp. results indicated that contamination was significantly higher (α = 0.1) at those plants with an on-site dairy farm (x = 50.1 %) than those plants without an on-site dairy farm (x = 33.5%). Plants producing dairy ingredients, frozen milk products or fluid milk were all shown to have significantly higher incidence rates than expected. Conversely, plants producing cultured dairy foods, or a combination of cultured dairy foods and fluid milk were found to have significantly lower incidence rates than expected. There was no statistically significant difference in contamination by area within the plants.