Seed Myco-priming improves crop yield and herbivory induced defenses in maize by coordinating antioxidants and Jasmonic acid pathway.

BACKGROUND: Seed Myco-priming based on consortium of entomopathogenic fungi is very effective seed treatment against Ostrinia furnacalis herbivory. Maize regulates defense responses against herbivory by the production of defense-related enzymatic and non-enzymatic antioxidants, phytohormones, and their corresponding genes. Jasmonic acid (JA) plays a key role in plant-entomopathogenic fungi-herbivore interaction. RESULTS: To understand how a consortium of the entomopathogenic fungi Beauveria bassiana and Trichoderma asperellum induce changes in the response of maize to herbivory and increase the crop yield, 2-year field experiment, antioxidant enzymes, leaf transcriptome, and phytohormone were performed. Fungal inoculation enhanced the production of antioxidant enzymes and JA signaling pathway more than the normal herbivory. The comparison between single inoculated, consortium inoculated, and non-inoculated plants resulted in distinct transcriptome profiles representing a considerable difference in expression of antioxidant- and JA- responsive genes identified through Weighted gene co-expression network analysis (WGCNA) and expression analysis, respectively. Seed priming with a consortium of B. bassiana and T. asperellum significantly enhanced the expression of genes involved in antioxidants production and JA biosynthesis cascade, with the highest expression recorded at 24-h post O. furnacalis larval infestation. They reduced the larval nutritional indices and survival up to 87% and enhancing crop yield and gross return up to 82-96% over the year 2018 and 2019. CONCLUSION: From our results we suggest that a consortium of B. bassiana and T. asperellum can be used synergistically against O. furnacalis in maize under field condition and can mediate antioxidants- and JA- associated maize defense response by boosting up the expression of their responsive genes, thereby enhancing crop yield.

Myco-Synergism Boosts Herbivory-Induced Maize Defense by Triggering Antioxidants and Phytohormone Signaling.

BACKGROUND: Biocontrol strategies are the best possible and eco-friendly solution to develop resistance against O furnacalis and improve the maize yield. However, the knowledge about underlying molecular mechanisms, metabolic shifts, and hormonal signaling is limited. METHODS: Here, we used an axenic and a consortium of entomopathogenic Beauveria bassiana OFDH1-5 and a pathogen-antagonistic Trichoderma asperellum GDFS1009 in maize and observed that consortium applications resulted in higher chlorophyll contents and antioxidants activities [superoxide dismutase (SOD), peroxidase (POD), proline, protease, and polyphenol oxidase (PPO)] with a decrease in O. furnacalis survival. We performed a comprehensive transcriptome and an untargeted metabolome profiling for the first time at a vegetative stage in fungal inoculated maize leaves at 0-, 12-, 24-, 48-, and 72-h post insect infestation. RESULTS: The consortium of B. bassiana and T. asperellum leads to 80-95% of O. furnacalis mortality. A total of 13,156 differentially expressed genes were used for weighted gene coexpression network analysis. We identified the six significant modules containing thirteen candidate genes [protein kinase (GRMZM2G025459), acyl-CoA dehydrogenase (GRMZM5G864319), thioredoxin gene (GRMZM2G091481), glutathione S-transferase (GRMZM2G116273), patatin-like phospholipase gene (GRMZM2G154523), cytochrome P450 (GRMZM2G139874), protease inhibitor (GRMZM2G004466), (AC233926.1_FG002), chitinase (GRMZM2G453805), defensin (GRMZM2G392863), peroxidase (GRMZM2G144153), GDSL- like lipase (AC212068.4_FG005), and Beta-glucosidase (GRMZM2G031660)], which are not previously reported that are highly correlated with Jasmonic acid - Ethylene (JA-ET) signaling pathway and antioxidants. We detected a total of 130 negative and 491 positive metabolomic features using a ultrahigh-performance liquid chromatography ion trap time-of-flight mass spectrometry (UHPLC-QTOF-MS). Intramodular significance and real time-quantitative polymerase chain reaction (RT-qPCR) expressions showed that these genes are the true candidate genes. Consortium treated maize had higher jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) levels. CONCLUSION: Our results provide insights into the genetics, biochemicals, and metabolic diversity and are useful for future biocontrol strategies against ACB attacks.

Bacillus thuringiensis and Chlorantraniliprole Trigger the Expression of Detoxification-Related Genes in the Larval Midgut of Plutella xylostella.

Background: Diamondback moth (DBM), Plutella xylostella (L.), has developed resistance to many insecticides. The molecular mechanism of DBM resistance to Bt-G033A combined with chlorantraniliprole (CL) remains undefined. Methods: In this study, field-resistant strains of Plutella xylostella to three pesticides, namely, Bacillus thuringiensis (Bt) toxin (Bt-G033A), CL, and a mixture of Bt + CL, were selected to evaluate the resistance level. Additionally, transcriptomic profiles of a susceptible (SS-DBM), field-resistant (FOH-DBM), Bt-resistant (Bt-DBM), CL-resistant (CL-DBM), and Bt + CL-resistant (BtC-DBM) strains were performed by comparative analysis to identify genes responsible for detoxification. Results: The Bt-G033A was the most toxic chemical to all the DBM strains among the three insecticides. The comparative analysis identified 25,518 differentially expressed genes (DEGs) between pairs/combinations of strains. DEGs were enriched in pathways related to metabolic and catalytic activity and ABC transporter in resistant strains. In total, 17 metabolic resistance-related candidate genes were identified in resistance to Bt-G033A, CL, and Bt + CL by co-expression network analysis. Within candidate genes, the majority was upregulated in key genes including cytochrome P450, glutathione S-transferase (GST), carboxylesterase, and acetylcholinesterase in CL- and BtC-resistant strains. Furthermore, aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, trypsin, and ABC transporter genes were eminent as Bt-resistance-related genes. Expression patterns of key genes by the quantitative real-time PCR (qRT-PCR) proved the credibility of transcriptome data and suggest their association in the detoxification process. Conclusion: To date, this study is the most comprehensive research presenting functional transcriptome analysis of DBM using Bt-G033A and CL combined insecticidal activity.

The impact of different plant extracts on population suppression of Helicoverpa armigera (Hub.) and tomato (Lycopersicon esculentum Mill) yield under field conditions.

Helicoverpa armigera (Hub.) is a destructive pest of the tomato (Lycopersicon esculentum Mill) crop in Pakistan. Although insecticides are the primary management strategy used to control H. armigera, most of them are not effective due to considerable toxic residual effects on the fruits. Nonetheless, H. armigera is rapidly evolving resistance against the available pesticides for its management. This situation calls upon the need of alternative management options against the pest. Different plant extracts have been suggested as a viable, environment-friendly option for plant protection with minimal side effects. Furthermore, the plant extracts could also manage the insect species evolving resistance against pesticides. This study evaluated the efficacy of different plant extracts (i.e., Neem seed, turmeric, garlic and marsh pepper) against H. armigera. Furthermore, the impact of the plant extracts on growth and yield of tomato crop was also tested under field conditions. The results revealed that all plant extracts resulted in higher mortality of H. armigera compared to control. Similarly, the highest plant height was observed for the plants treated with the plant extracts compared to untreated plants. Moreover, the highest tomato yield was observed in plants treated with plant extracts, especially with neem seed (21.013 kg/plot) followed by pepper extract (19.25 kg/plot), and garlic extract 18.4 kg/plot) compared to the untreated plants (8.9 kg/plot). It is concluded that plant extracts can be used as eco-friendly approaches for improving tomato yield and resistance management of H. armigera.

The glutathione S-transferase (PxGST2L) may contribute to the detoxification metabolism of chlorantraniliprole in Plutella xylostella(L.).

The diamondback moth (Plutella xylostella L.), is an economic pest of cruciferous plants worldwide, which causes great economic loss to cruciferous plants production. However, the pest has developed resistance to insecticides. One of such insecticides is chlorantraniliprole. The study of the mechanisms underlying resistance is key for the effective management of resistance. In this study, a comparative proteomics approach was used to isolate and identify various proteins that differed between chlorantraniliprole-susceptible and -resistant strains of P. xylostella. Eleven proteins were significantly different and were successfully identified by MALDI-TOF-MS. Metabolism-related proteins accounted for the highest proportion among the eleven different proteins. The function of the PxGST2L protein was validated by RNAi. Knockdown of PxGST2L reduced the GST activity and increased the toxicity of chlorantraniliprole to the diamondback moth. The resistance ratio of diamondback moth to chlorantraniliprole was reduced from 1029 to 505. The results indicated that PxGST2L is partly responsible for chlorantraniliprole insecticide resistance in DBM. Our finding contributes to the understanding of the mechanism underlying resistance to chlorantraniliprole in the DBM, to develop effective resistance management tactics.

Assessing the Single and Combined Toxicity of Chlorantraniliprole and Bacillus thuringiensis (GO33A) against Four Selected Strains of Plutella xylostella (Lepidoptera: Plutellidae), and a Gene Expression Analysis.

Concerns about resistance development to conventional insecticides in diamondback moth (DBM) Plutella xylostella (L.), the most destructive pest of Brassica vegetables, have stimulated interest in alternative pest management strategies. The toxicity of Bacillus thuringiensis subsp. aizawai (Bt GO33A) combined with chlorantraniliprole (Chl) has not been documented. Here, we examined single and combined toxicity of chlorantraniliprole and Bt to assess the levels of resistance in four DBM strains. Additionally, enzyme activities were tested in field-original highly resistant (FOH-DBM), Bt-resistant (Bt-DBM), chlorantraniliprole-resistant (CL-DBM), and Bt + chlorantraniliprole-resistant (BtC-DBM) strains. The Bt product had the highest toxicity to all four DBM strains followed by the mixture of insecticides (Bt + Chl) and chlorantraniliprole. Synergism between Bt and chlorantraniliprole was observed; the combination of Bt + (Bt + Chl) (1:1, LC50:LC50) was the most toxic, showing a synergistic effect against all four DBM strains with a poison ratio of 1.35, 1.29, 1.27, and 1.25. Glutathione S-transferase (GST) and carboxyl-esterase (CarE) activities showed positive correlations with chlorantraniliprole resistance, but no correlation was observed with resistance to Bt and Bt + Chl insecticides. Expression of genes coding for PxGST, CarE, AChE, and MFO using qRT-PCR showed that the PxGST and MFO were significantly overexpressed in Bt-DBM. However, AChE and CarE showed no difference in the four DBM strains. Mixtures of Bt with chlorantraniliprole exhibited synergistic effects and may aid the design of new combinations of pesticides to delay resistance in DBM strains substantially.

Analysis of Cry1Ah Toxin-Binding Reliability to Midgut Membrane Proteins of the Asian Corn Borer.

Evolution of insect resistance to Bt toxins challenges the use of Cry toxins to control agricultural pests. In lepidopterans, Cry toxin affinity towards multiple midgut epithelial receptors has become a matter of dispute. Cry1Ah toxin-binding proteins were identified in the larval midgut of susceptible (ACB-BtS) and resistant (ACB-AhR) strains of the Asian corn borer (ACB). A pull-down assay was performed using biotinylated Cry1Ah toxin, and the binding proteins were identified by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study aimed to find the binding consistency of the midgut epithelial protein to the Cry1Ah toxin. The binding proteins from different fractions of SDS-PAGE showed a different pattern. We observed an isoform of prophenoloxidase PPO1b (UniProt Acc No. A0A1Q1MKI0), which was found only in the ACB-AhR fractions. Prophenoloxidase (proPO) is an extraordinary defense molecule activated in insect species during pathogen invasion and the wound healing process. Importantly, this prophenoloxidase might have direct/indirect interaction with the Cry1Ah toxin. Our data also suggest that factors like techniques, enrichment of binding proteins in the sample and the reversible and irreversible nature of the brush border membrane vesicles (BBMVs) to Cry toxins could cause the inconsistency in the protein-protein interactions. Moreover, inside the larva midgut, the influence of the Cry toxins under physiological conditions might be different from the laboratory procedures.

Contribution of phenoloxidase activation mechanism to Bt insecticidal protein resistance in Asian corn borer.

Phenoloxidase (PO) is a crucial enzyme in the Arthropods melanization process, in which synthesized melanin rapidly acts at the site of injury and infection. In this study, we observed significant changes in humoral and cellular responses after exposing susceptible and resistant strains to a sub-lethal concentration of Cry1Ah toxin. Based on STRING v 11.0 computational protein-protein interaction analysis, we selected seven immune genes namely Prophenoloxidase PPO1b, PP03, Serpin-3, Serpin-5, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 reported in Asian corn borer. Quantitative real-time PCR gene expression studies showed Cry1Ah resistant strain had higher expression of PPO1b, PP03, Serpin-3, Beta-1,3-glucan recognition protein, Immulectin-3 and Serine protease SP105 genes in midgut and hemocyte samples. This study also investigated and found that the level of prophenoloxidation (proPO) activity in Cry1Ah resistant strains was significantly higher than susceptible strains. Cry1Ah toxin significantly increased the resistant strain's immune responses, the difference was observed through assays of bacterial agglutination and phagocytosis. Additionally, immune response induced by Cry1Ah toxin influences the microbiome composition associated with the host system. These parameters seem to explain the contribution of PO/PO regulating proteins render the host to resist the Cry1Ah toxin.

Identification of Cry1Ah-binding proteins through pull down and gene expression analysis in Cry1Ah-resistant and susceptible strains of Ostrinia furnacalis.

Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis, Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling Cry1Ah to NHS-activated Sepharose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI, and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO.

Transcriptome and Proteome Alternation With Resistance to Bacillus thuringiensis Cry1Ah Toxin in Ostrinia furnacalis.

Background: Asian corn borer (ACB), Ostrinia furnacalis can develop resistance to transgenic Bacillus thuringiensis (Bt) maize expressing Cry1Ah-toxin. However, the mechanisms that regulate the resistance of ACB to Cry1Ah-toxin are unknown. Objective: In order to understand the molecular basis of the Cry1Ah-toxin resistance in ACB, "omics" analyses were performed to examine the difference between Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains of ACB at both transcriptional and translational levels. Results: A total of 7,007 differentially expressed genes (DEGs) and 182 differentially expressed proteins (DEPs) were identified between ACB-AhR and ACB-BtS and 90 genes had simultaneous transcription and translation profiles. Down-regulated genes associated with Cry1Ah resistance included aminopeptidase N, ABCC3, DIMBOA-induced cytochrome P450, alkaline phosphatase, glutathione S-transferase, cadherin-like protein, and V-ATPase. Whereas, anti-stress genes, such as heat shock protein 70 and carboxylesterase were up-regulated in ACB-AhR, displaying that a higher proportion of genes/proteins related to resistance was down-regulated compared to up-regulated. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 578 and 29 DEGs and DEPs, to 27 and 10 pathways, respectively (P < 0.05). Furthermore, real-time quantitative (qRT-PCR) results based on relative expression levels of randomly selected genes confirmed the "omics" response. Conclusion: Despite the previous studies, this is the first combination of a study using RNA-Seq and iTRAQ approaches on Cry1Ah-toxin binding, which led to the identification of longer length of unigenes in ACB. The DEGs and DEPs results are valuable for further clarifying Cry1Ah-mediated resistance.

Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker).

The oriental armyworm (OAW), Mythimna separata (Walker), is a destructive pest of agricultural crops in Asia and Australia. Commercialized Bt crops have performed very well against their target pests; however, very few studies have been done on the susceptibility of OAW to Bt toxins in either sprays or expressed in Bt crops. In this work, we evaluated the toxicities of Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, Cry2Aa, Cry2Ab, Cry1Ie, Vip3Aa19, Vip3Aa16, and Vip3Ca against OAW neonate larvae, as well as the interaction between Cry and Vip toxins. The results from bioassays revealed that LC50 (lethal concentration for 50% mortality) values ranged from 1.6 to 78.6 µg/g (toxin/diet) for those toxins. Among them, Vip3 proteins, along with Cry1A proteins and Cry2Aa, were the ones with the highest potency, with LC50 values ranging from 1.6 to 7.4 µg/g. Synergism between Cry and Vip toxins was observed, being high in the combination of Vip3Aa16 with Cry1 toxins, with synergetic factors ranging from 2.2 to 9.2. The Vip3Ca toxin did not show any synergistic effect with any of the toxins tested. These results can help in designing new combinations of pyramiding genes in Bt crops, as well as in recombinant bacteria, for the control of OAW as well as for resistance management programs.

Characterization of the Cry1Ah resistance in Asian corn Borer and its cross-resistance to other Bacillus thuringiensis toxins.

Transgenic crops producing insecticidal proteins are effective to manage lepidopteran pests. Development of insect-resistance is the major threat to Bacillus thuringiensis (Bt) crops such as Cry1Ah-Maize. Laboratory selection with Bt-Cry1Ah toxin incorporated in artificial diet, during 48 generations of Asian corn borer (ACB) Ostrinia furnacalis produced 200-fold resistance. This resistant colony ACB-AhR readily consumed and survived on Cry1Ah-expressing Bt-maize. Cross-resistance analysis showed high cross-resistance to Cry1F (464-fold), moderate cross-resistance to Cry1Ab (28.38-fold), Cry1Ac (22.11-fold) and no cross-resistance to Cry1Ie toxin. This ACB-AhR cross-resistant phenotype is different from ACB-Cry1Fa resistant population that showed no cross resistance to Cry1Ah, suggesting that different mechanisms of resistance were selected in these two populations. Bioassays of reciprocal F1 crosses-progeny suggested autosomal inheritance of Cry1Ah resistance with no maternal effects. The dominance of resistance increased as concentration decreased. In Cry1Ah-maize tissues the progeny of reciprocal F1 crosses behaved as functionally recessive. Progenies analysis from backcrosses (F1 × resistant strain) suggested polygenic contribution to Cry1Ah- resistance in ACB-AhR. The use of multiple toxins is an imperative factor for delaying evolution of resistance to Cry1Ah-corn in ACB. However, the fact that ACB-AhR showed cross resistance to Cry1Fa indicates that selection of toxins for pyramided plants should be carefully done.