Insect Resistance Using Native Genes in Tomato and Pepper: An Entomologist’s Perspective on Successes & Challenges N. K. Krishna Kumar Deputy Director General (Hort.Sci.) ICAR, New Delhi

Types of Insect Resistance Non-preference (antixenosis) - Not A GUEST e.g. Trichomes GlandularNon-glandular Density, length, chemical profile Photos:

Types of Insect Resistance…. Antibiosis : True form of resistance - Adverse on the growth & development of the insect - e.g. gossypol, tannins, heliocides, silica etc. - Evolution of Secondary metabolites Tolerance: - ability of the plant to yield more as compared to the susceptible one even after infestation

Contrasting perceptions - Entomologist Vs Plant Breeder - More success in breeding for disease resistance - ETL for scoring pest vis-à-vis vector - Mechanical Vs. Vector inoculation - Vector or Virus or both resistance - Immunity Vs. Resistance - Laboratory susceptible and Field tolerant

Contrasting perceptions - Good success limited to Gall midges? - Resistance vs. Horticultural traits - GM Bt altered the whole scenario - Gene silencing, inter-specific hybrids etc. - Are GM crops good for NE? - Public Sector vs. Private sector - Resistance management - Sucking pests a major problem

Important sucking pests of tomato & pepper Importance – Direct pest or vector Tomato - Frankliniella shultzei?, T. palmi? - tospo Bemisia tabaci – TLCV, TYLCV Pepper - Scirtothrips dorsalis? – tospo -Red spider mites, Aphids etc - Gall midges

Field screening of chilli Accessions for Resistance to S. dorsalis Resistant Susceptible

Thrips

Thrips & Tospoviruses If a Crop is infected by Tospovirus, then it can be safely assumed that a species of thrips is involved Without thrips, Tospoviruses can not transfer from one plant to another: But, without thrips Tospoviruses survive from one season to another only in infected plants -Laurence A. Mound

Validation of species-specific marker for T. tabaci and T. palmi 0.5 kb T. tabaci T. palmi Marker for Scirtothrips dorsalis to be developed

Gall midge

Capsicum gall midge Gall midge Damage

Gall midge and seed formation

Brinjal Gall midge, Asphondylia capparis Damaged Undamaged Damaged

Brinjal Gall midge, Asphondylia capparis Damaged Undamaged Damaged

Brinjal Gall midge, Asphondylia capparis damaged fruits Undamaged Damaged

Variability in Chilli C. baccatum C. pubescens C. frutescens C. annuum C. chinense

Tomato wild relatives

Wild species show reproductive barriers

Potential of S. chilense Unlocking the phenome of S. chilense Solanum chilense Begomovirus resistance Tospovirus resistance Water use efficiency Salinity tolerance Robust roots for efficient water uptake Flavonoids

Interspecific hybridization: Solanum macrocarpon X Solanum melongena L. Desirable qualities for use in breeding programs (brinjal) 1.Shoot and fruit borer resistance 2.Nematode resistance 3.Leaf hopper resistance 4.Bacterial wilt resistance 5.Drought tolerance 6.Edible fruits

Newer approaches in Resistance Breeding DNA Barcoding & Molecular phylogeny RNA interference (insect, pathogen) Metabolomics Insect genomics

WC-M35 T1-4-M17 T1-4-M29b T1-7-B2 T1-7-B9b T1-7-B15a Chimeric RNAi Construct for GBNV, CVMV, CMV, CaCV Resistance Manamohan, Krishna Reddy & Asokan, IIHR (2014)

T1-5-C30b T1-7-B9b T1-7-B2 M35- (WILD CONTROL) BIOASSAY RESULTS: A) GBNV and CMV infected transgenic and wild type tomato plants after 90 days infection. B ) Phenotypic variations in the leaf indicating resistant to GBNV and CMV viruses. A B Manamohan, Krishna Reddy & Asokan, IIHR (2014)

Rebijith et al. (2011) Indian populations of T. palmi is genetically distinct Indian populations

Genome Sequencing of Arthropods 50 billion USD on insect control but exterminates beneficial insects. The mandate of future research is to specifically target only the pests and exclude the non-targets. One way to achieve this end is to understand the genetic composition and thus phenotypic capability of insects. The i5k insect and arthropod genome sequencing initiative is a step in this direction. Returns for investment: With the annotated genomic data we will be able to understand better certain relationships like vector-parasite; host- endosymbionts and certain pest features which make insects highly invasive and adaptable to changing environment. Total # of species targeted for sequencing Hexapoda 702 Chelicerata 64 Crustacea 20 Myriapoda 6 Total # of species targeted for sequencing Hexapoda 702 Chelicerata 64 Crustacea 20 Myriapoda 6

Genome (90 megabases) sequencing of Tetranychus urticae Repertoire of gene families implicated in digestion (polyphagy), detoxification and transport of xenobiotics. Some gene families are more developed and often expanded compared to insects (highly proliferated cysteine peptidase genes; 86 cytochrome oxidase P450 gene families; Carboxyl / cholinesterases gene family; expansion within 32 gene families of glutathion-S-transferase (GST) and a new Mu-class GST (believed to be only in vertebrates). Detoxification gene families acquired through lateral transfer of bacterial and fungal origin are intradiol ring cleavage dioxygenases and cynate lyase. In short, the genome of T. urticae has made impressive gene gains including T. urticae specific expansion within gene-families and lateral gains from bacteria and fungi with regard to detoxification Implying rapid new host adaptation and pesticides Miodrag Grbic et al., Nature (2011)

Novel methods for mass producing insect sex pheromones Pheromone-blend in a model tobacco plants by transgenically expressing a set of genes of a bio-synthetic pathway for alcohols (11-tetradecenol, 11- hexadecenol) and corresponding acetates, components of pheromone blend of two species of ermine moths, Yponomenta evonymella and Y. padella. As a step further, what if these sex pheromones are made to emit the volatiles, at a time when the plant is not flowering or in a trap-crop, it could result in pest suppression when the crop of interest actually comes to bloom. Source: A plant factory for moth pheromone production Nature communications, 25 Feb 2014, DOI: /ncomms4353

Breaking of the Endosymbionts – Insect Link  Essential role in insect adaptation to various food types e.g. ligocellulosic biomass degradation in termites and carbon recyclers  Nutrient production e.g. vitamins, growth promoters  Detoxification  Immune modulation and evidences point out to enhancement in immune competence in insects e.g. protection from pathogens and parasites  Environmental adaptation Several laboratories are involved in studying the metagenomics of the microbial consortia residing the guts of insects which have adapted themselves to diverse environments Future research: Can we target the metabolism of endosymbionts through RNAi or other approaches so as to suppress the insect pest population ?

Comparative Genomic Analysis Comparative metagenome analysis of the gut symbionts of three different insects from different insect orders, having different diets and life histories revealed dramatic diversity of microbial species. The insect species are (i) grasshopper, Acrida cinerea (Orthoptera), (ii) Cutworm, Agrotis ipsilon (Lepidoptera) and (iii) termite, Nasutitermes sp. (Isoptera:Termitidae). Abundance of bacterial phyla based on the predicted gene models in the gut microbiota of grasshopper (GH), cutworm (CW), and termite (TM), respectively Shi W, Xie S, Chen X, Sun S, et al. (2013)

Composition of grasshopper (G) and cutworm (C) gut microbiomes as revealed by 16S analysis. Source: Shi W, Xie S, Chen X, Sun S, et al. (2013) Comparative Genomic Analysis of the Endosymbionts of Herbivorous Insects Reveals Eco- Environmental Adaptations: Biotechnology Applications. PLoS Genet 9(1): e doi: /journal.pgen