Managing resistance evolution to transgenic Bt maize in corn borers in Spain.

Since 1998, genetically engineered Bt maize varieties expressing the insecticidal Cry1Ab protein (i.e. event MON 810) have been grown in the European Union (EU), mainly in Spain. These varieties confer resistance against the European and Mediterranean corn borer (ECB and MCB), which are the major lepidopteran maize pests in the EU, particularly in Mediterranean areas. However, widespread, repeated and exclusive use of Bt maize is anticipated to increase the risk of Cry1Ab resistance to evolve in corn borer populations. To delay resistance evolution, typically, refuges of non-Bt maize are planted near or adjacent to, or within Bt maize fields. Moreover, changes in Cry1Ab susceptibility in field populations of corn borers and unexpected damage to maize MON 810, due to corn borers, are monitored on an annual basis. After two decades of Bt maize cultivation in Spain, neither resistant corn borer populations nor farmer complaints on unexpected field damage have been reported. However, whether the resistance monitoring strategy followed in Spain, currently based on discriminating concentration bioassays, is sufficiently sensitive to timely detect early warning signs of resistance in the field remains a point of contention. Moreover, the Cry1Ab resistance allele frequency to Bt maize, which has recently been estimated in MCB populations from north-eastern Spain, might exceed that recommended for successful resistance management. To ensure Bt maize durability in Spain, it is key that adequate resistance management approaches, including monitoring of resistance and farmer compliance with refuge requirements, continue to be implemented and are incorporated in integrated pest management schemes.

Transgenic cotton expressing Cry1B protein has no adverse effect on predatory insect Propylea Japonica.

The lady beetle Propylea japonica is a dominant natural predator of insect pests in farmland ecosystems and an important non-target indicator insect for the environmental safety assessment of GM crops. The commercial cultivation of GM crops may cause P. japonica to frequently be exposed to the Bt protein environment. In this study, the biological characteristics, enzyme activity, and expression levels of detoxification and metabolism in P. japonica were studied after Cry1B protein treatment. No significant differences were observed in developmental duration, emergence rate, or body weight at different ages after feeding larvae 0.5 mg/mL of Cry1B protein compared with the control. Furthermore, there were no significant differences in the activities of glutathione S-transferase (GST), catalase (CAT), and peroxidase (POD) after feeding 0.25 mg/mL and 0.5 mg/mL Cry1B protein. However, when the concentration of Cry1B protein increased to 1.0 mg/mL, the activities of the GST, CAT, and POD increased significantly. Compared with the control group, there were no significant differences in the expression levels of most detoxification metabolism related genes; only a few genes had changed expression levels at the individual concentrations (CYP345B1, CYP4Q2, CYP9F2, GST, and microsomal GST). Overall, these results suggest that Cry1B protein has little or no effect on the biological characteristics of P. japonica. Genes related to enzyme activity and detoxification are differentially expressed at high concentration stimulation. Therefore, this research suggests that the potential risks of Cry1B for the predator P. japonica are negligible.

Bacillus thuringiensis protein Vip3Aa does not harm the predator Propylea japonica: A toxicological, histopathological, biochemical and molecular analysis.

The ladybeetle Propylea japonica is a widely distributed natural enemy in many agricultural systems. P. japonica is often used as a test organism for safety assessments of transgenic Bacillus thuringiensis crops. Plant varieties expressing the Vip3Aa insecticidal protein are not currently commercially available in China. In this study, protease inhibitor E-64 was used as a positive control to examine the responses of P. japonica larvae to a high concentration of Vip3Aa proteins. Larvae that were fed E-64 had increased mortality and prolonged developmental period, but these parameters were unaffected when larvae were fed Vip3Aa. The epithelial cells of midguts were intact and closely connected with the basal membrane when larvae were fed Vip3Aa, but the epithelial cells degenerated in the E-64 treatment. The activities of antioxidative enzymes and expression levels of detoxification-related genes in P. japonica larvae were not altered after exposure to Vip3Aa; however, these biochemical and molecular parameters were significantly changed in the E-64 treatment. The results demonstrate that Vip3Aa protein is not harmful to the predator P. japonica.

Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna.

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on different functional traits.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

De novo characterization of venom apparatus transcriptome of Pardosa pseudoannulata and analysis of its gene expression in response to Bt protein.

BACKGROUND: Pardosa pseudoannulata is a prevailing spider species, and has been regarded as an important bio-control agent of insect pests in farmland of China. However, the available genomic and transcriptomic databases of P. pseudoannulata and their venom are limited, which severely hampers functional genomic analysis of P. pseudoannulata. Recently high-throughput sequencing technology has been proved to be an efficient tool for profiling the transcriptome of relevant non-target organisms exposed to Bacillus thuringiensis (Bt) protein through food webs. RESULTS: In this study, the transcriptome of the venom apparatus was analyzed. A total of 113,358 non-redundant unigenes were yielded, among which 34,041 unigenes with complete or various length encoding regions were assigned biological function annotations and annotated with gene ontology and karyotic orthologous group terms. In addition, 3726 unigenes involved in response to stimulus and 720 unigenes associated with immune-response pathways were identified. Furthermore, we investigated transcriptomic changes in the venom apparatus using tag-based DGE technique. A total of 1724 differentially expressed genes (DEGs) were detected, while 75 and 372 DEGs were functionally annotated with KEGG pathways and GO terms, respectively. qPCR analyses were performed to verify the DEGs directly or indirectly related to immune and stress responses, including genes encoding heat shock protein, toll-like receptor, GST and NADH dehydrogenase. CONCLUSION: This is the first study conducted to specifically investigate the venom apparatus of P. pseudoannulata in response to Bt protein exposure through tritrophic chain. A substantial fraction of transcript sequences was generated by high-throughput sequencing of the venom apparatus of P. pseudoannulata. Then a comparative transcriptome analysis showing a large number of candidate genes involved in immune response were identified by the tag-based DGE technology. This transcriptome dataset will provide a comprehensive sequence resource for furture molecular genetic research of the venom apparatus of P. pseudoannulata.

Assessing the potential for interaction between the insecticidal activity of two genetically engineered cotton events combined by conventional breeding: An example with COT102 × MON 15985.

Bollgard(®) III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex(®) cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity.

Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops.

In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).

Aquatic degradation of Cry1Ab protein and decomposition dynamics of transgenic corn leaves under controlled conditions.

The increasing cultivation of genetically modified corn plants (Zea mays) during the last decades is suggested as a potential risk to the environment. One of these genetically modified variety expressed the insecticidal Cry1Ab protein originating from Bacillus thuringiensis (Bt), resulting in resistance against Ostrinia nubilalis, the European corn borer. Transgenic litter material is extensively studied regarding the decomposition in soils. However, only a few field studies analyzed the fate of the Cry1Ab protein and the impact of green and senescent leaf litter from corn on the decomposition rate and related ecosystem functions in aquatic environments. Consequently, a microbial litter decomposition experiment was conducted under controlled semi-natural conditions in batch culture using two maize varieties: one variety with Cry1Ab and another one with the appertaining Iso-line as control treatment. The results showed no significant differences between the treatment with Cry1Ab and the Iso-line regarding loss of total mass in dry weight of 43% for Iso-line and 45% for Bt-corn litter, lignin content increased to 137.5% (Iso-line) and 115.7% (Bt-corn), and phenol loss decreased by 53.6% (Iso-line), 62.2% (Bt-corn) during three weeks of the experiment. At the end of the experiment Cry1Ab protein was still detected with 6% of the initial concentration. A slightly but significant lower cellulose content was found for the Cry1Ab treatment compared to the Iso-line litter at the end of the experiment. The significant higher total protein (25%) and nitrogen (25%) content in Bt corn, most likely due to the additionally expression of the transgenic protein, may increase the microbial cellulose degradation and decrease microbial lignin degradation. In conclusion a relevant year by year input of protein and therefore nitrogen rich Bt corn litter into aquatic environments may affect the balanced nutrient turnover in aquatic ecosystems.

Impact of Transgenic Maize Ruifeng125 on Diversity and Dynamics of Bacterial Community in Rhizosphere Soil.

With the development of commercialized planting of genetically modified crops, their ecological security risks remain a key topic of public concern. Insect-resistant genetically modified maize, Ruifeng125, which expresses a fusion Bt protein (Cry1Ab-Cry2Aj), has obtained the application safety certificate issued by the Chinese government. To determine the effects of Ruifeng125 on the diversity and dynamics of bacterial communities, the accumulation and degradation pattern of the fusion Bt protein in the rhizosphere soil of transgenic maize were detected. Results showed that the contents of Bt protein varied significantly at different developmental stages, but after straw was returned to the field, over 97% of Bt proteins were degraded quickly at the early stages (≤10 d) and then they were degraded at a relatively slow rate. In addition, the variations in bacterial community diversity in the rhizosphere soil were detected by 16S ribosomal RNA (Rrna) high-throughput sequencing technology. A total of 44 phyla, 435 families, and 842 genera were obtained by 16S rRNA sequencing, among which Proteobacteria, Actinobacia, Acidobacter Acidobacterium, and Chloroflexi were the dominant taxa. At the same developmental stage, no significant differences in soil bacterial diversity were detected between Ruifeng125 and its non-transgenic control variety. Further analysis revealed that developmental stage, rather than the transgenic event, made the greatest contribution to the changes in soil microbial diversity. This research provides important information for evaluating the impacts of Bt crops on the soil microbiome and establishes a theoretical foundation for their environmental safety assessment.

Transgenic early japonica rice: Integration and expression characterization of stem borer resistance Bt gene.

BACKGROUND: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines. METHODS: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages. RESULTS: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues. CONCLUSIONS: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops.

Response of the Propylea japonica Microbiota to Treatment with Cry1B Protein.

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a dominant natural enemy of insect pests in farmland ecosystems. It also serves as an important non-target insect for environmental safety evaluations of transgenic crops. Widespread planting of transgenic crops may result in direct or indirect exposure of P. japonica to recombinant Bacillus thuringiensis (Bt) protein, which may in turn affect the biological performance of this natural enemy by affecting the P. japonica microflora. However, the effects of Bt proteins (such as Cry1B) on the P. japonica microbiota are currently unclear. Here, we used a high-throughput sequencing method to investigate differences in the P. japonica microbiota resulting from treatment with Cry1B compared to a sucrose control. The results demonstrated that the P. japonica microbiome was dominated by Firmicutes at the phylum level and by Staphylococcus at the genus level. Within-sample (α) diversity indices demonstrated a high degree of consistency between the microbial communities of P. japonica treated with the sucrose control and those treated with 0.25 or 0.5 mg/mL Cry1B. Furthermore, there were no significant differences in the abundance of any taxa after treatment with 0.25 mg/mL Cry1B for 24 or 48 h, and treatment with 0.5 mg/mL Cry1B for 24 or 48 h led to changes only in Staphylococcus, a member of the phylum Firmicutes. Treatment with a high Cry1B concentration (1.0 mg/mL) for 24 or 48 h caused significant changes in the abundance of specific taxa (e.g., Gemmatimonades, Patescibacteria, Thauera, and Microbacterium). However, compared with the control, most taxa remained unchanged. The statistically significant differences may have been due to the stimulatory effects of treatment with a high concentration of Cry1B. Overall, the results showed that Cry1B protein could alter endophytic bacterial community abundance, but not composition, in P. japonica. The effects of Bt proteins on endophytes and other parameters in non-target insects require further study. This study provides data support for the safety evaluation of transgenic plants.

Impact of Insect-Resistant Transgenic Maize 2A-7 on Diversity and Dynamics of Bacterial Communities in Rhizosphere Soil.

Artificial modification of Bacillus thuringiensis (Bt) proteins can effectively improve their resistance to target pests, but the effect of such modification on the diversity of rhizosphere microorganisms remains unclear. Transgenic maize 2A-7 contains two artificially modified Bt proteins, mCry1Ab and mCry2Ab. These proteins can enter soil and pose a potential threat to soil microbial diversity. To assess their impacts on rhizosphere bacteria communities, the contents of the two Bt proteins and changes in bacterial community diversity in the rhizosphere soils of transgenic maize 2A-7 and its control variety were analyzed at different growth stages in 2020. The results showed that the two Bt proteins were detected at low levels in the rhizosphere soils of 2A-7 plants. No significant differences in soil bacterial diversity were detected between 2A-7 and its control variety at any of the growth stages. Bioinformatics analysis indicated that the growth stage, rather than the cultivar, was the main factor causing changes in bacterial communities. This research provides valuable data for understanding the impact of Bt crops on the soil microbiome, and establishes a theoretical basis for evaluation of their safety.

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera.

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight; pupation rate; adult longevity; fecundity; relative growth rate, RGR; efficiency of conversion of digested food, ECD; and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.

Synergistic interaction of Cry1Ah and Vip3Aa19 proteins combination with midgut ATP-binding cassette subfamily C receptors of Conogethes punctiferalis (Guenée) (Lepidoptera: Crambidae).

Bacillus thuringiensis Cry and Vip proteins are highly effective at controlling agricultural pests and could be used in pyramided transgenic crops. However, the molecular mechanism underlying the Cry1Ah and Vip3Aa19 synergistic interaction has never been investigated at the molecular level in Yellow peach moth (YPM) Conogethes punctiferalis. Binding affinity and synergism of Cry1Ah and Vip3Aa19 proteins with ABC transporter subfamily C receptors ABCC1, ABCC2 and ABCC3 proteins from the midgut of YPM larva by using surface plasmon resonance (SPR) and pull-down assays. Both assays revealed that Cry1Ah could interact with ABCC1, ABCC2, and ABCC3, whereas Vip3Aa19 only interacts with ABCC1 and ABCC3, but not with ABCC2. Hence, when compared to the Vip3Aa19 protein, Cry1Ah had a higher binding affinity for ABCC1, ABCC2, and ABCC3. Furthermore, competitive binding assay between Cry1Ah and Vip3Aa19 protein with ABC transporter subfamily C receptors resulted in the final eluted protein samples displaying vibrant blue bands of Cry1Ah and very faint bands of Vip3Aa19. Suggesting that Cry and Vip proteins could deliver a synergistic effect after cleaving the midgut proteases. Therefore, this finding indicated that the Cry1Ah and Vip3Aa19 do not compete for interacting with midgut receptors and thus provide strong synergism against YPM.

Bt protein hasten entomopathogenic fungi-induced death of nontarget pest whitefly by suppressing protective symbionts.

The risk assessment of Bacillus thuringiensis (Bt) crops on nontarget pests has received much attention. Despite the knowledge of various beneficial bacterial symbionts in pests, whether Bt proteins affect these symbionts and subsequently alter the pest's ecology remains largely unknown. The whitefly Bemisia tabaci is one of the most serious nontarget pests in Bt cotton. Here, we explored the Bt Cry1Ac protein-induced changes in whitefly symbiont abundance and the subsequent effects on whitefly response against a naturally prevalent entomopathogenic fungus Cordyceps javanica. The obligate symbiont 'Candidatus Portiera aleyrodidarum' (hereafter P. aleyrodidarum) as well as facultative symbionts 'Candidatus Hamiltonella defensa' (hereafter H. defensa), 'Candidatus Cardinium hertigii' (hereafter C. hertigii) and 'Candidatus Rickettsia bellii' (hereafter R. bellii) dominate the microbial community of whiteflies. The Bt exposure had no effects on H. defensa infected (H) and H. defensa-C. hertigii doubly infected (HC) whiteflies, but decreased the total copy number of symbionts as well as the R. bellii proportion in H. defensa-C. hertigii- R. bellii triply infected whiteflies (HCR). C. javanica caused whitefly adults 100 % mortality within 8 days. Without Bt protein exposure, HCR whiteflies survived significantly longer than H and HC whiteflies sprayed by C. javanica, suggesting that R. bellii confers protection. However, in Bt-exposed groups, C. javanica generated synchronous death of H, HC and HCR whiteflies. Specifically, in H and HC whiteflies, Bt protein-exposure showed no significant difference in progress of death caused by C. javanica. But in HCR whiteflies, Bt exposure hastened death induced by C. javanica, suppressing the R. bellii-conferred protection. This is the first report revealing that Bt protein altered symbiont community conferred adverse effects on nontarget pests, providing a new perspective for Bt risk assessment and biocontrol strategies of nontarget pests.

One-Pot Synthesis of HRP&SA/ZIF-8 Nanocomposite and Its Application in the Detection of Insecticidal Crystalline Protein Cry1Ab.

This study reported the functionality integration of zeolitic imidazolate framework-8 (ZIF-8) with horseradish peroxidase (HRP) and streptavidin (SA) for the synthesis of a HRP&SA/ZIF-8 nanocomposite through one-pot coprecipitation. The synthesized HRP&SA/ZIF-8 nanocomposite was then employed as the ideal signal tag for application in the enzyme-linked immunosorbent assay (ELISA) and exhibited excellent sensitivity, selectivity and accuracy in the detection of insecticidal crystalline (Cry) protein Cry1Ab as a transgenic biomarker with a detection limit of 4.8 pg/mL. This proposed method provides a new way for the detection of transgenic biomarkers in food and may inspire further integration of a variety of biomolecules into ZIF-8 for applications ranging from biosensing, biomedicine, and catalysis to energy.

Effects of Exogenous Salicylic Acid Application to Aboveground Part on the Defense Responses in Bt (Bacillus thuringiensis) and Non-Bt Corn (Zea mays L.) Seedlings.

Bt (Bacillus thuringiensis) corn is one of the top three large-scale commercialized anti-insect transgenic crops around the world. In the present study, we tested the Bt protein content, defense chemicals contents, and defense enzyme activities in both the leaves and roots of Bt corn varieties 5422Bt1 and 5422CBCL, as well as their conventional corn 5422 seedlings, with two fully expanded leaves which had been treated with 2.5 mM exogenous salicylic acid (SA) to the aboveground part for 24 h. The result showed that the SA treatment to the aboveground part could significantly increase the polyphenol oxidase activity of conventional corn 5422, the Bt protein content, and peroxidase activities of Bt corn 5422Bt1, as well as the polyphenol oxidase and peroxidase activity of Bt corn 5422CBCL in the leaves. In the roots, the polyphenol oxidase and peroxidase activity of conventional corn 5422, the polyphenol oxidase and superoxide dismutase activities of Bt corn 5422Bt1, the DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content, and four defense enzymes activities of Bt corn 5422CBCL were systematically increased. These findings suggest that the direct effect of SA application to aboveground part on the leaf defense responses in Bt corn 5422CBCL is stronger than that in non-Bt corn. Meanwhile, the systemic effect of SA on the root defense responses in Bt corn 5422CBCL is stronger than that in conventional corn 5422 and Bt corn 5422Bt1. It can be concluded that the Bt gene introduction and endogenous chemical defense responses of corns act synergistically during the SA-induced defense processes to the aboveground part. Different transformation events affected the root defense response when the SA treatment was applied to the aboveground part.

Effect of Urea Spray on Boll Shell Insecticidal Protein Content in Bt Cotton.

Reproductive organs of Bacillus thuringiensis transgenic cotton, which contribute to cotton final yield, have low insect resistant efficacy, so it is important to improve their insect resistance. This study was conducted to find out the impact of different urea spray doses on the expression of Cry1A protein in boll shell of Bt cotton (Sikang 1 and Sikang 3), and nitrogen metabolism in this process was also studied to uncover the physiological mechanism. The experiment with six urea doses was organized during peak boll stage in 2017 and 2018. The results showed that urea spray could significantly increase boll shell insecticidal protein contents in both cultivars, with the highest Bt protein content observed at 28-32 kg ha-1 urea dose. In addition, urea spray increased the contents of soluble protein and free amino acid and the activities of GS, GOGAT, GOT, and GPT, but decreased the activities of peptidase and protease in boll shell. Correlation analysis showed that the amount of boll shell Bt protein was positively correlated with levels of soluble protein and amino acid, and activities of GS, GOGAT, GOT, and GPT, but negatively correlated with peptidase and protease activities. Thus, this study demonstrated that higher protein synthesis ability and lower proteolysis ability were related to increased Bt protein content in urea-sprayed boll shell.

Performance of Seed Treatments Applied on Bt and Non-Bt Maize Against Fall Armyworm (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is the main pest of maize in Brazil, attacking plants from emergence to reproductive stages. Here, we conducted studies to evaluate the efficacy of two seed treatments (chlorantraniliprole alone and imidacloprid combined with thiodicarb) on Bt and non-Bt maize in laboratory bioassays with distinct FAW strains that are susceptible, selected for resistance to Bt-maize single (Cry1F) or pyramided (Cry1A.105 + Cry2Ab2) events and F1 hybrids of the selected and susceptible strains (heterozygotes), and in the field against a natural infestation. In the laboratory, leaf-discs from seed treated Bt-maize plants at 7 d after emergence (DAE) increased the mortality of FAW resistant, heterozygote, and susceptible strains up to 24.8%, when compared with the respective maize grown without a seed treatment. In the field against natural infestations of FAW, Bt maize with a seed treatment had ~30% less FAW damage than non-Bt maize with the same seed treatment at 7 and 14 DAE. No differences in FAW damage was observed between Bt and non-Bt maize grown with and without a seed treatment at 21 DAE. Maize seeds treated with chlorantraniliprole alone or imidacloprid and thiodicarb combined presented limited protection against early infestations of FAW strains under laboratory and field studies.