Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

New insights into an RNAi approach for plant defence against piercing‐sucking and stem‐borer insect pests

Insect double‐stranded (ds)RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. Arabidopsis Mob1A is required for organ growth and reproduction. When Arabidopsis roots were soaked in dsMob1A, the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty‐four hours after rice roots were immersed in fluorescent‐labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of Ago and Dicer in rice and planthoppers were induced by dsEYFP. When rice roots were soaked in dsActin, their growth was also significantly suppressed. When planthoppers or Asian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing‐sucking and stem‐borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.     

Spider Venom Toxin Protects Plants from Insect Attack

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the ω-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture. The authors Sher Afzal Khan and Zahid Mukhtar contributed equally to this work.     

Effect of jasmonic acid and salicylic acid induced resistance in groundnut on Helicoverpa armigera

Induced resistance in plants affects insect growth and development as a result of the up‐regulation of defence‐related secondary metabolites or enzyme‐binding proteins. In the present study, the effects of jasmonic acid (JA) and salicylic acid (SA) induced resistance in groundnut on Helicoverpa armigera (Hübner) are examined. Larval survival, larval weights and the activities of digestive enzymes (total serine protease and trypsin) and of detoxifying enzymes [glutathione S‐transferase (GST) and esterase (EST)] are studied in insects fed on four groundnut genotypes with moderate levels of resistance to H. armigera (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697) and a susceptible genotype (JL 24). The plants are pre‐ and/or simultaneously treated with JA and SA, and then infested with H. armigera, which are allowed to feed for 6 days. Significantly lower serine protease and trypsin activities are observed in H. armigera fed on plants treated with JA. Greater GST activity is recorded in insects fed on JA and SA treated plants, whereas EST activity is low in H. armigera larvae fed on plants treated with JA and SA. Serine proteases, trypsin and GST activities and larval weights (r = 0.74–0.95) and larval survival (r = 0.77–0.93) are positively correlated, whereas EST activity and larval weight (r = ?0.55) and larval survival (r = ?0.65) are negatively correlated. The results suggest that midgut digestive and detoxifying enzymes can be used as indicators of the adverse effects of constitutive and/or induced resistance in crop plants on the insect pests and the role of JA and SA in insect pest management.     

Oviposition and feeding avoidance in Helicoverpa armigera (Hübner) against transgenic Bt cotton

Helicoverpa armigera (Hübner), the major target pest of transgenic Bacillus thuringiensis (Bt) cotton, remains susceptible to Bt cotton in China at present. Behavioural avoidance by ovipositing females might lead to reduced exposure to Bt cotton and minimize selection for physiological resistance. We examined the behavioural responses of H. armigera to Bt and non‐Bt cottons to determine whether behavioural avoidance to Bt cotton may be present. In oviposition choice tests, the number of eggs on non‐Bt cotton plants was significantly higher than on Bt cotton plants. Similarly, in no‐choice tests, Bt cotton plants attracted significantly fewer eggs compared with non‐Bt cotton plants. H. armigera neonates showed higher dispersal and lower establishment on Bt cotton than on non‐Bt cotton. First instars were found to feed consistently on non‐Bt cotton leaves, creating large feeding holes, but only produced tiny feeding holes on Bt cotton leaves. The H. armigera population used in this study showed avoidance of oviposition and feeding on Bt cotton. Our results provide important insights into one possible mechanism underlying the durability of Bt cotton resistance and may be useful for improving strategies to sustain the effectiveness of Bt crops.     

Development of a novel‐type transgenic cotton plant for control of cotton bollworm

The transgenic Bt cotton plant has been widely planted throughout the world for the control of cotton budworm Helicoverpa armigera (Hubner). However, a shift towards insect tolerance of Bt cotton is now apparent. In this study, the gene encoding neuropeptide F (NPF) was cloned from cotton budworm H. armigera, an important agricultural pest. The npf gene produces two splicing mRNA variants—npf1 and npf2 (with a 120‐bp segment inserted into the npf1 sequence). These are predicted to form the mature NPF1 and NPF2 peptides, and they were found to regulate feeding behaviour. Knock down of larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight, and dsNPF also caused a decrease of glycogen and an increase of trehalose. Moreover, we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expressed dsNPF. Results showed that H. armigera larvae fed on these transgenic plants or leaves had lower food consumption, body size and body weight compared to controls. These results indicate that NPF is important in the control of feeding of H. armigera and valuable for production of potential transgenic cotton.     

Predator Presence Moves <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Larvae to Distraction

Displacement of herbivorous insects by the presence of predators on whole plants has rarely been studied. By semi-continuous observations of an externally feeding insect herbivore and a predator, we show how the mere presence of the predator, Geocoris lubra Kirkaldy (Hemiptera: Geocoridae), on a plant can have a strong influence on the movement and behaviors of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. The presence of predators, as opposed to mortality by predators, influenced the proportion of larvae feeding, resting and implementing avoidance activities. In addition, the proportion of time individual larvae allocated to feeding, resting and dropping off plants was affected when predators were present with and without contact between the two. Predators do more than just reduce numbers of herbivores; they influence feeding, displacement and subsequently the distribution of plant damage.     

Effects of climate change and crop planting structure on the abundance of cotton bollworm,Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The interactions between plants and insects play an important role in ecosystems. Climate change and cropping patterns can affect herbivorous pest insect dynamics. Understanding the reasons for population fluctuations can help improve integrated pest management strategies. Here, a 25‐year dataset on climate, cropping planting structure, and the population dynamics of cotton bollworms (Helicoverpa armigera) from Bachu County, south Xinjiang, China, was analyzed to assess the effects of changes in climate and crop planting structure on the population dynamics of H. armigera. The three generations of H. armigera showed increasing trends in population size with climate warming, especially in the third generation. The relative abundances of the first and second generations decreased, but that of the third generation increased. Rising temperature and precipitation produced different impacts on the development of different generations. The population numbers of H. armigera increased with the increase in the non‐Bacillus thuringiensis (Bt) cotton‐planted area. Asynchrony of abrupt changes existed among climate change, crop flowering dates, and the phenology of H. armigera moths. The asynchronous responses in crop flowering dates and phenology of H. armigera to climate warming would expand in the future. The primary factors affecting the first, second, and third generations of moths were T_mean in June, the last appearance date of the second generation of moths, and the duration of the third generation of moths, respectively. To reduce the harm to crops caused by H. armigera, Bt cotton should be widely planted.     

Field evidence for indirect interactions between foliar‐feeding insect and root‐feeding nematode communities on Nicotiana tabacum

Abstract 1. As herbivory often elicits systemic changes in plant traits, indirect interactions via induced plant responses may be a pervasive feature structuring herbivore communities. Although the importance of this phenomenon has been emphasised for herbivorous insects, it is unknown if and how induced responses contribute to the organisation of other major phytoparasitic taxa. 2. Survey and experimental field studies were used to investigate the role of plants in linking the dynamics of foliar‐feeding insects and root‐feeding nematodes on tobacco, Nicotiana tabacum. 3. Plant‐mediated interactions between insects and nematodes could largely be differentiated by insect feeding guild, with positive insect–nematode interactions predominating with leaf‐chewing insects (caterpillars) and negative interactions occurring with sap‐feeding insects (aphids). For example, insect defoliation was positively correlated with the abundance of root‐feeding nematodes, but aphids and nematodes were negatively correlated. Experimental field manipulations of foliar insect and nematode root herbivory also tended to support this outcome. 4. Overall, these results suggest that plants indirectly link the dynamics of divergent consumer taxa in spatially distinct ecosystems. This lends support to the growing perception that plants play a critical role in propagating indirect effects among a diverse assemblage of consumers.     

Silicon‐mediated multiple interactions: Simultaneous induction of rice defense and inhibition of larval performance and insecticide tolerance of Chilo suppressalis by sodium silicate

The rice striped stem borer (SSB, Chilo suppressalis) is one of the most destructive pests of rice plants. Si‐mediated rice defense against various pests has been widely reported, and sodium silicate (SS) has been used as an effective source of silicon for application to plants. However, there is quite limited information about the direct effects of Si application on herbivorous insects. SSB larval performance and their insecticide tolerance were examined after they had been reared either on rice plants cultivated in nutrient solution containing 0.5 and 2.0 mM SS or on artificial diets with 0.1% and 0.5% SS. SS amendment in either rice culture medium or artificial diets significantly suppressed the enzymatic activities of acetylcholinesterase, glutathione S‐transferases, and levels of cytochrome P450 protein in the midgut of C. suppressalis larvae. Larvae fed on diets containing SS showed lower insecticide tolerance. Additionally, RNA‐seq analysis showed that SS‐mediated larval insecticide tolerance was closely associated with fatty acid biosynthesis and pyruvate metabolism pathways. Our results suggest that Si not only enhances plant resistance against insect herbivore, but also impairs the insect's capacity to detoxify the insecticides. This should be considered as another important aspect in Si‐mediated plant–insect interaction and may provide a novel approach of pest management.     

Compatibility of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.     

Inhibitor of apoptosis is an effective target gene for RNAi-mediated control of Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double-stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB-specific genes to identify targets for RNAi-mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat-killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.     

Expression of a synthetic cry1AcF gene in transgenic Pigeon pea confers resistance to Helicoverpa armigera

Pigeon pea is an important legume. Yield losses due to insect pests are enormous in the cultivation of this crop. Expression of cry proteins has led to increased resistance to pests in several crops. We report in this paper, expression of a chimeric cry1AcF (encoding cry1Ac and cry1F domains) gene in transgenic pigeon pea and its resistance towards Helicoverpa armigera. PCR, Southern hybridization, RT‐PCR and Western analysis confirmed stable integration and expression of the cry1AcF gene in pigeon pea transgenics. When screened for efficacy of the transformants for resistance against H. armigera, the transgenics showed not only high mortality of the larva but could also resist the damage caused by the larvae. Analysis for the stable integration, expression and efficacy of the transgenics resulted in the identification of four T3 plants arising from two T1 backgrounds as highly promising. The results demonstrate potentiality of the chimeric cry1AcF gene in developing H. armigera‐resistant pigeon pea.