Farm‐scale evaluation of the impact of Cry1Ab Bt maize on canopy nontarget arthropods: a 3‐year study

The cultivation of Cry1Ab‐expressing genetically modified MON810 (Bt maize) has led to public concern in Europe, regarding its impact on nontarget arthropods (NTAs). We have assessed the potential effects of DKC 6451 YG (MON810) maize on canopy NTAs in a farm‐scale study performed in Central Spain during 3 years. The study focused on hemipteran herbivores (leafhoppers and planthoppers) and hymenopteran parasitic wasps (mymarids) collected by yellow sticky traps, which accounted for 72% of the total number of insects studied. The dynamics and abundance of these groups varied among years, but no significant differences were found between Bt and non‐Bt maize, indicating that Bt maize had no negative effect on these taxa. Nonetheless, the Cry1Ab toxin was detected in 2 different arthropods collected from Bt maize foliage, the cicadellids Zyginidia scutellaris and Empoasca spp. A retrospective power analysis on the arthropod abundance data for our field trials has determined that Z. scutellaris and the family Mymaridae have high capacity to detect differences between the Bt maize and its isogenic counterpart. The use of these canopy NTAs as surrogates for assessing environmental impacts of Bt maize is discussed.     

Turning Maize Cobs into a Valuable Feedstock

With rising energy demand and limited resources, the need for alternative energy production is increasing. Maize cobs have an advantageous composition for the production of biofuels such as cellulosic ethanol but are currently left unused on the fields after harvest. Furthermore, cobs contain a low concentration of nitrogen (<1%). Therefore, cob harvest will not deplete soil fertility. Consequently, maize cobs are a cheap and promising source for sustainable energy production. Yet with primary focus on grain yield, no or little effort has been spent to increase cob biomass yield in addition to grain yield. Both cob and grain yield are complex inherited traits affected by the environment. Breeding of dual-purpose maize varieties with simultaneously increased cob and grain yield requires a deeper understanding of factors influencing both cob and grain development. In this article, the available knowledge on the genetics of cob formation and current and future applications of maize cob utilization are discussed to evaluate the prospects for development of dual-purpose maize varieties.     

Chemical composition,digestibility and feeding value of maize cobs

The digestibility of treated maize cobs was studied using Romney Marsh wether sheep in a 2 × 3 factorial design; maize cobs were ground through a 10- or 6-mm screen and three chemical treatments were applied to each grinding: distilled water (control); sodium hydroxide (4.5 g per 100 g cob dry matter); and Magadi soda (9.0 g per 100 g cob dry matter). Cobs were treated for 24 h using one litre of solution per kg of maize cobs.The digestion coefficient of crude protein was lowered (P< 0.01) by finer grinding of the maize cobs. Dry matter digestibility coefficients were 44.7, 54.2 and 61.6% for maize cobs treated with water, NaOH and Magadi soda, respectively. Digestibility of energy, cell walls and cellulose was increased more by Magadi soda than by NaOH. Digestibility of acid detergent fibre (ADF) was depressed (P< 0.05) by NaOH but improved (P< 0.05) by Magadi soda. Chemical treatment lowered (P< 0.01) the digestibility of crude protein from 28.0 to 17.0% (NaOH) or to 18.7% (Magadi soda). Grinding and chemical treatment of maize cobs interacted (P< 0.01) in their effects on digestibility of crude protein.Friesian cattle, grazed on a predominantly Nandi Setaria (Setaria sphacelata) and Silver Leaf Desmodium (Desmodium uncinatum) pasture, and supplemented with water-, NaOH- and Magadi soda-treated maize cobs, gained 0.41, 0.55 and 0.51 kg per day, respectively. Daily maize cob intake was higher on NaOH-treated than on water- and Magadi soda-treated cobs. Cattle on NaOH- and Magadi soda-treated cobs required more than 1 month to adapt to the cobs and show superior gain.     

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.     

Rapid quantification methods for genetically modified maize contents using genomic DNAs pretreated by sonication and restriction endonuclease digestion for a capillary-type real-time PCR system with a plasmid reference standard

For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established. To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective. The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.     

A Phytotoxin,Betaenone C,and Its Related Metabolites of Phoma betae Fr

For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established. To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective. The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.     

Einfluss von Fusarientoxinen auf die Mast- und Schlachtleistung von Broilern und Puten

In two broiler and two turkey trials the influence of Fusarium toxins in maize on growth and slaughter performance, on residues of toxins in carcass and litters and blood parameters were investigated. In one broiler and turkey trial with naturally contaminated maize the main contaminants were deoxynivalenol (DON), moniliformin (MON and beauvericin (BEA). In one further broiler and turkey trial with inoculated maize (Fusarium subglutinans) the main contaminants were MON and BEA. The level of contamination of mycotoxins in the diets was free, low, medium and high. For every broiler trial 180 and for the turkey trials 60 and 100 one day old chicken were used. The result of these investigations shows that broiler and turkeys are not very sensitive to Fusarium toxins. Growth and slaughter performance and also blood parameters were not negative influenced by higher mycotoxin dosages in the diets.     

Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere

Field studies were done to assess how much of the transgenic, insecticidal protein, Cry1Ab, encoded by a truncated cry1Ab gene from Bacillus thuringiensis (Bt), was released from Bt-maize MON810 into soil and whether bacterial communities inhabiting the rhizosphere of MON810 maize were different from those of the rhizosphere of nontransgenic maize cultivars. Bacterial community structure was investigated by SSCP (single-strand conformation polymorphism) of PCR-amplified 16S rRNA genes from community DNA. Using an improved extraction and detection protocol based on a commercially available ELISA, it was possible to detect Cry1Ab protein extracted from soils to a threshold concentration of 0.07 ng/g soil. From 100 ng of purified Cry1Ab protein added per gram of soil, only an average of 37% was extractable. At both field sites investigated, the amount of Cry1Ab protein in bulk soil of MON810 field plots was always lower than in the rhizosphere, the latter ranging from 0.1 to 10 ng/g soil. Immunoreactive Cry1Ab protein was also detected at 0.21 ng/g bulk soil 7 months after harvesting, i.e. in April of the following year. At this time, however, higher values were found in residues of leaves (21 ng/g) and of roots (183 ng/g), the latter corresponding to 12% of the Cry1Ab protein present in intact roots. A sampling 2 months later indicated further degradation of the protein. Despite the detection of Cry1Ab protein in the rhizosphere of MON810 maize, the bacterial community structure was less affected by the Cry1Ab protein than by other environmental factors, i.e. the age of the plants or field heterogeneities. The persistence of Cry1Ab protein emphasizes the importance of considering post-harvest effects on nontarget organisms.     

Amino acid metabolism in maize earshoots. Implications for assimilate preconditioning and nitrogen signaling

Nitrogen (N) is an essential requirement for kernel growth in maize (Zea mays); however, little is known about how N assimilates are metabolized in young earshoots during seed development. The objective of this study was to assess amino acid metabolism in cob and spikelet tissues during the critical 2 weeks following silking. Two maize hybrids were grown in the field for 2 years at two levels of supplemental N fertilizer (0 and 168 kg N/ha). The effects of the reproductive sink on cob N metabolism were examined by comparing pollinated to unpollinated earshoots. Earshoots were sampled at 2, 8, 14, and 18 d after silking; dissected into cob, spikelet, and/or pedicel and kernel fractions; then analyzed for amino acid profiles and key enzyme activities associated with amino acid metabolism. Major amino acids in the cob were glutamine (Gln), aspartic acid (Asp), asparagine (Asn), glutamate, and alanine. Gln concentrations dropped dramatically from 2 to 14 d after silking in both pollinated and unpollinated cobs, whereas all other measured amino acids accumulated over time in unpollinated spikelets and cobs, especially Asn. N supply had a variable effect on individual amino acid levels in young cobs and spikelets, with Asn being the most notably enhanced. We found that the cob performs significant enzymatic interconversions among Gln, alanine, Asp, and Asn during early reproductive development, which may precondition the N assimilate supply for sustained kernel growth. The measured amino acid profiles and enzymatic activities suggest that the Asn to Gln ratio in cobs may be part of a signal transduction pathway involving aspartate aminotransferase, Gln synthetase, and Asn synthetase to indicate plant N status for kernel development.     

Characterization of maize allergens - MON810 vs. its non-transgenic counterpart

One of the main concerns about genetically modified foods and their potential impacts on human health is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. We intended to evaluate, and to compare, using quantitative real time RT-PCR technique, the expression of 5 already known maize allergens (Zea m14, Zea m25, Zea m27kD, 50kD Zein and trypsin inhibitor) in MON 810 vs. its non-transgenic counterpart, throughout seed development (10, 16 and 23days after pollination). We have shown that none of the tested allergen genes presented differential expression, with statistic significance, along all tested seed development stages, in MON810 vs. its conventional counterpart. We have also used bidimensional gel electrophoresis followed by Western blotting with plasma from two maize allergic subjects to characterize their immunologic responses against MON 810 vs. its non-transgenic control. Immunoreactive spots were characterized by MS. We have identified fourteen new IgE-binding proteins present in both transgenic and non-transgenic maize.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.     

Maize seed selection by East African smallholder farmers and resistance to Maize streak virus*

Interviews identified that most small‐scale maize farmers in central Uganda and in the Southern Highlands of Tanzania plant home‐saved seed of landraces or seed derived from various open‐pollinated and hybrid varieties. Some farmers also bought a portion of their seed, either certified seed, locally traded seed or even maize sold for consumption. Selection for home‐saved seed was generally among harvested cobs. Big cobs with many, regularly arranged, large, white, flint kernels were preferred. A maize cob may bear several hundred seeds, so a farmer needs to save <1% of cobs for seed. A form of resistance in which plants show only moderate symptoms and suffer only a small reduction in yield when infected has been incorporated in some released varieties. Because not all plants in most crops are infected and because plants uninfected with Maize streak virus (MSV) tend to produce bigger cobs than infected resistant plants, the few cobs selected by a farmer for seed may all be from the uninfected ‘escapes’, with no preferential selection of resistant types. On‐station simulation of the farmers’ selection process in a crop of the MSV‐resistant maize variety, Longe 1, confirmed this. An alternative very strong form of MSV resistance was identified.     

Volatile and gaseous metabolites released by germinating seeds of lentil and maize cultivars with different susceptibilities to fusariosis and smut

The effect of volatile and gaseous metabolites released by germinating seeds of lentil cultivars more and less susceptible to fusariosis on the germination of spores ofMucor racemosus, Trichoderma viride, Verticillium dahliae andBotrytis cinerea was found to depend rather on the fungal genus than on the lentil cultivar. However, spores ofFusarium oxysporum reacted more sensitively during germination to the presence of exudates of both cultivars, when the more susceptible lentil displayed a stimulation, the less susceptible one an inhibition of spore germination. The greatest difference in the effect of exudates was observed in the more and less susceptible maize cultivars with respect to the germination of chlamydospores ofUstilago maydis, especially during the first hours of seed germination. Analysis of the exudates of germinating seeds showed the release of a greater amount of ethanol and methanol with acetaldehyde by the more susceptible cultivars of lentil and particularly maize.     

Genetic and Morphometric Analysis of Cob Architecture and Biomass-Related Traits in the Intermated B73 × Mo17 Recombinant Inbred Lines of Maize

Expected future cellulosic ethanol production increases the demand for biomass in the US Corn Belt. With low nutritious value, low nitrogen content, and compact biomass, maize cobs can provide a significant amount of cellulosic materials. The value of maize cobs depends on cob architecture, chemical composition, and their relation to grain yield as primary trait. Eight traits including cob volume, fractional diameters, length, weight, tissue density, and grain yield have been analyzed in this quantitative trait locus (QTL) mapping experiment to evaluate their inheritance and inter-relations. One hundred eighty-four recombinant inbred lines of the intermated B73?×?Mo17 (IBM) Syn 4 population were evaluated from an experiment carried out at three locations and analyzed using genotypic information of 1,339 public SNP markers. QTL detection was performed using (1) comparison-wise thresholds with reselection of cofactors (α?=?0.001) and (2) empirical logarithm of odds score thresholds (P?=?0.05). Several QTL with small genetic effects (R ²?=?2.9–13.4 %) were found, suggesting a complex quantitative inheritance of all traits. Increased cob tissue density was found to add value to the residual without a commensurate negative impact on grain yield and therefore enables for simultaneous selection for cob biomass and grain yield.     

Toxicity of Fusarium mycotoxins on maize plants

Synergistic effects of Fusarium — toxins mixture (moniliformin, fumonisin B₁, fusaproliferin, zearalenone, zearalenol and deoxynivalenol, each at concentration 3.5 μg mL-1) on maize plants of resistant and susceptible cultivars were studied. After 72-hour treatment the biomass production with both cultivars was approximately 6% lower than in the respective controls. In the resistant cultivar chlorophyll content was increased comparing to control, with higher increase in chlorophyll b. In susceptible cultivar chlorophyll content was slightly decreased, particularly with chlorophyll b.No significant differences between treated and non-treated plants were found in the cell ultrastructure. In susceptible plants the young root cells were more vacuolated and plasmolysis occurred in the cells of outer cortex. In leaves of this cultivar also disorganization of thylakoids in some chloroplasts was observed.     

The Cry1Ab Protein Has Minor Effects on the Arbuscular Mycorrhizal Fungal Communities after Five Seasons of Continuous Bt Maize Cultivation

The cultivation of genetically modified plants (GMP) has raised concerns regarding the plants’ ecological safety. A greenhouse experiment was conducted to assess the impact of five seasons of continuous Bt (Bacillus thuringiensis) maize cultivation on the colonisation and community structure of the non-target organisms arbuscular mycorrhizal fungi (AMF) in the maize roots, bulk soils and rhizospheric soils using the terminal restriction fragment length polymorphism (T-RFLP) analysis of the 28S ribosomal DNA and sequencing methods. AMF colonisation was significantly higher in the two Bt maize lines that express Cry1Ab, 5422Bt1 (event Bt11) and 5422CBCL (MON810) than in the non-Bt isoline 5422. No significant differences were observed in the diversity of the AMF community between the roots, bulk soils and rhizospheric soils of the Bt and non-Bt maize cultivars. The AMF genus Glomus was dominant in most of the samples, as detected by DNA sequencing. A clustering analysis based on the DNA sequence data suggested that the sample types (i.e., the samples from the roots, bulk soils or rhizospheric soils) might have greater influence on the AMF community phylotypes than the maize cultivars. This study indicated that the Cry1Ab protein has minor effects on the AMF communities after five seasons of continuous Bt maize cultivation.     

An evaluation of methods for assessing the impacts of Bt‐maize MON810 cultivation and pyrethroid insecticide use on Auchenorrhyncha (planthoppers and leafhoppers)

1 Auchenorrhyncha (Planthoppers and Leafhoppers) are not only pests of many crops, but they are also nontarget organisms with respect to Bt‐protein expressing genetically modified plants. As herbivorous arthropods, planthoppers and leafhoppers ingest Cry proteins depending on their feeding behaviour. Consequently, they are directly exposed to these entomotoxic proteins and can also serve as a source of Cry protein exposure to predatory arthropods. Therefore, it is reasonable to use Auchenorrhyncha in the risk assessment of genetically modified crops. 2 During a 2‐year field study, we evaluated four different methods in terms of their feasibility to assess the impacts of plant‐incorporated protectants from Bt‐maize and of insecticide use on this group of arthropods. Visual assessment of plants, sweep netting, yellow traps and custom made sticky traps were utilized in field plots of Bt‐maize MON810, untreated near‐isogenic maize and insecticide‐treated near‐isogenic maize and were compared with respect to their capability to reflect the diversity and abundance of Auchenorrhyncha species. 3 Zyginidia scutellaris (Herrich‐Schäffer) (Cicadomorpha: Cicadellidae) represented more than 94% of all captured individuals in both years. The analysis of Z. scutellaris data showed no consistent differences between Bt‐maize MON810 and the untreated near isogenic hybrid, demonstrating no negative impact of MON810 on this species. Insecticide treatment, on the other hand, was not equivalent to the isogenic maize in terms of Z. scutellaris densities. Based on the collected data and on practical considerations, we recommend the combined use of transect‐wise sweep netting and sticky traps for the sampling of Auchenorrhyncha in maize.