Effects of Soil Rhizobia Abundance on Interactions between a Vector, Pathogen, and Legume Plant Host.

Soil rhizobia promote nitrogen fixation in legume hosts, maximizing their tolerance to different biotic stressors, plant biomass, crop growth, and yield. While the presence of soil rhizobia is considered beneficial for plants, few studies have assessed whether variation in rhizobia abundance affects the tolerance of legumes to stressors. To address this, we assessed the effects of variable soil rhizobia inoculum concentrations on interactions between a legume host (Pisum sativum), a vector insect (Acyrthosiphon pisum), and a virus (Pea enation mosaic virus, PEMV). We showed that increased rhizobia abundance reduces the inhibitory effects of PEMV on the nodule formation and root growth in 2-week-old plants. However, these trends were reversed in 4-week-old plants. Rhizobia abundance did not affect shoot growth or virus prevalence in 2- or 4-week-old plants. Our results show that rhizobia abundance may indirectly affect legume tolerance to a virus, but effects varied based on plant age. To assess the mechanisms that mediated interactions between rhizobia, plants, aphids, and PEMV, we measured the relative expression of gene transcripts related to plant defense signaling. Rhizobia concentrations did not strongly affect the expression of defense genes associated with phytohormone signaling. Our study shows that an abundance of soil rhizobia may impact a plant's ability to tolerate stressors such as vector-borne pathogens, as well as aid in developing sustainable pest and pathogen management systems for legume crops. More broadly, understanding how variable rhizobia concentrations can optimize legume-rhizobia symbiosis may enhance the productivity of legume crops.

The Intricate Role of Ecdysis Triggering Hormone Signaling in Insect Development and Reproductive Regulation.

Insect growth is interrupted by molts, during which the insect develops a new exoskeleton. The exoskeleton confers protection and undergoes shedding between each developmental stage through an evolutionarily conserved and ordered sequence of behaviors, collectively referred to as ecdysis. Ecdysis is triggered by Ecdysis triggering hormone (ETH) synthesized and secreted from peripheral Inka cells on the tracheal surface and plays a vital role in the orchestration of ecdysis in insects and possibly in other arthropod species. ETH synthesized by Inka cells then binds to ETH receptor (ETHR) present on the peptidergic neurons in the central nervous system (CNS) to facilitate synthesis of various other neuropeptides involved in ecdysis. The mechanism of ETH function on ecdysis has been well investigated in holometabolous insects such as moths Manduca sexta and Bombyx mori, fruit fly Drosophila melanogaster, the yellow fever mosquito Aedes aegypti and beetle Tribolium castaneum etc. In contrast, very little information is available about the role of ETH in sequential and gradual growth and developmental changes associated with ecdysis in hemimetabolous insects. Recent studies have identified ETH precursors and characterized functional and biochemical features of ETH and ETHR in a hemimetabolous insect, desert locust, Schistocerca gregaria. Recently, the role of ETH in Juvenile hormone (JH) mediated courtship short-term memory (STM) retention and long-term courtship memory regulation and retention have also been investigated in adult male Drosophila. Our review provides a novel synthesis of ETH signaling cascades and responses in various insects triggering diverse functions in adults and juvenile insects including their development and reproductive regulation and might allow researchers to develop sustainable pest management strategies by identifying novel compounds and targets.

Drought stress affects interactions between potato plants, psyllid vectors, and a bacterial pathogen.

Transmission of insect-borne pathogens is mediated by interactions between insects and plants across variable environments. Water stress, for example, affects the physiology, defense, chemistry, and nutritional balance of plants in ways that alter their tolerance to herbivores and pathogens. However, few studies have explored interactions between water stress and insect-borne pathogens as well as the molecular mechanisms mediating these interactions. Here, we address these knowledge gaps by assessing effects of plant water stress on the transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum (CLs), by the vector Bactericera cockerelli Sulc (potato psyllid). We hypothesized that plant water stress would promote pathogen transmission by inducing plant gene transcripts and phytohormones involved in defense. Our results showed water stress was associated with decreased CLs titer with two psyllid haplotypes. Our analysis of plant gene transcripts suggested water stress affected phytohormone pathways in ways that altered plant tolerance to the CLs pathogen. Our study shows that abiotic stressors like drought may mediate the spread of plant pathogens by altering plant signaling pathways in ways that affect pathogen transmission.

Co-Transcriptomic Analysis of the Maize-Western Corn Rootworm Interaction.

The Western corn rootworm (WCR; Diabrotica virgifera virgifera) is an economically important belowground pest of maize. Belowground feeding by WCR is damaging because it weakens the roots system, diminishes nutrient uptake, and creates entry points for fungal and bacterial pathogens and increases lodging, all of which can significantly suppress maize yields. Previously, it was demonstrated that belowground herbivory can trigger plant defense responses in the roots and the shoots, thereby impacting intraplant communication. Although several aspects of maize-WCR interactions have been reported, co-transcriptomic remodeling in the plant and insect are yet to be explored. We used a maize genotype, Mp708, that is resistant to a large guild of herbivore pests to study the underlying plant defense signaling network between below and aboveground tissues. We also evaluated WCR compensatory transcriptome responses. Using RNA-seq, we profiled the transcriptome of roots and leaves that interacted with WCR infestation up to 5 days post infestation (dpi). Our results suggest that Mp708 shoots and roots had elevated constitutive and WCR-feeding induced expression of genes related to jasmonic acid and ethylene pathways, respectively, before and after WCR feeding for 1 and 5 days. Similarly, extended feeding by WCR for 5 days in Mp708 roots suppressed many genes involved in the benzoxazinoid pathway, which is a major group of indole-derived secondary metabolites that provides resistance to several insect pests in maize. Furthermore, extended feeding by WCR on Mp708 roots revealed several genes that were downregulated in WCR, which include genes related to proteolysis, neuropeptide signaling pathway, defense response, drug catabolic process, and hormone metabolic process. These findings indicate a dynamic transcriptomic dialog between WCR and WCR-infested maize plants.

Documenting pollinators, floral hosts, and plant-pollinator interactions in U.S. Pacific Northwest agroecosystems.

The abundance and diversity of pollinator populations are in global decline. Managed pollinator species, like honey bees, and wild species are key ecosystem service providers in both natural and managed agroecosystems. However, relatively few studies have exhaustively characterized pollinator populations in diverse agroecosystems over multiple years, while also thoroughly documenting plant-pollinator interactions. Yet, such studies are needed to fulfill the national pollinator protection plans that have been released by the United States and other nations. Our research is among the first studies to respond to these directives by systematically documenting bee and plant biodiversity, bee-plant interactions, and bee-mediated pollen movement in farming systems of the Pacific Northwest, USA. Our data provides insight into the processes mediating pollinator and plant community assembly, persistence, and resilience across landscapes with variable crop and landscape diversity and agroecosystem management practices. These data will also contribute to the development of a United States pollinator database, supporting the United States' plan to promote pollinators. With few publicly available data sets that systematically take account of agroecosystem practices, plant populations, and pollinators, our research will provide future users the means to conduct synesthetic studies of pollinators and ecosystem function in a period of rapid and global pollinator declines. There are no copyright or proprietary restrictions for research or teaching purposes. Usage of the data set must be cited.

Responses of pea plants to multiple antagonists are mediated by order of attack and phytohormone crosstalk.

Plants are often attacked by multiple antagonists and traits of the attacking organisms and their order of arrival onto hosts may affect plant defences. However, few studies have assessed how multiple antagonists, and varying attack order, affect plant defence or nutrition. To address this, we assessed defensive and nutritional responses of Pisum sativum plants after attack by a vector herbivore (Acrythosiphon pisum), a nonvector herbivore (Sitona lineatus), and a pathogen (Pea enation mosaic virus, PEMV). We show viruliferous A. pisum induced several antipathogen plant defence signals, but these defences were inhibited by S. lineatus feeding on peas infected with PEMV. In contrast, S. lineatus feeding induced antiherbivore defence signals, and these plant defences were enhanced by PEMV. Sitona lineatus also increased abundance of plant amino acids, but only when they attacked after viruliferous A. pisum. Our results suggest that diverse communities of biotic antagonists alter defence and nutritional traits of plants through complex pathways that depend on the identity of attackers and their order of arrival onto hosts. Moreover, we show interactions among a group of biotic stressors can vary along a spectrum from antagonism to enhancement/synergism based on the identity and order of attackers, and these interactions are mediated by a multitude of phytohormone pathways.

Responses to predation risk cues and alarm pheromones affect plant virus transmission by an aphid vector.

Herbivores assess predation risk in their environment by identifying visual, chemical, and tactile predator cues. Detection of predator cues can induce risk-avoidance behaviors in herbivores that affect feeding, dispersal, and host selection in ways that minimize mortality and reproductive costs. For herbivores that transmit plant pathogens, including many aphids, changes in herbivore behavior in response to predator cues may also affect pathogen spread. However, few studies have assessed how aphid behavioral responses to different types of predator cues affect pathogen transmission. Here, we conducted greenhouse experiments to assess whether responses of pea aphids (Acyrthosiphon pisum) to predation risk and alarm pheromone (E-ß-Farnesene), an aphid alarm signal released in response to predation risk, affected transmission of Pea enation mosaic virus (PEMV). We exposed A. pisum individuals to risk cues, and quantified viral titer in aphids and pea (Pisum sativum) host plants across several time periods. We also assessed how A. pisum responses to risk cues affected aphid nutrition, reproduction, and host selection. We show that exposure to predator cues and alarm pheromone significantly reduced PEMV acquisition and inoculation. Although vectors avoided hosts with predator cues, predator cues did not alter vector reproduction or reduce nutrient acquisition. Overall, these results suggest that non-consumptive effects of predators may indirectly decrease the spread of plant pathogens by altering vector behavior in ways that reduce vector competence and pathogen transmission efficiency.

Orchard Management and Landscape Context Mediate the Pear Floral Microbiome.

Crop-associated microbiota are a key factor affecting host health and productivity. Most crops are grown within heterogeneous landscapes, and interactions between management practices and landscape context often affect plant and animal biodiversity in agroecosystems. However, whether these same factors typically affect crop-associated microbiota is less clear. Here, we assessed whether orchard management strategies and landscape context affected bacterial and fungal communities in pear (Pyrus communis) flowers. We found that bacteria and fungi responded differently to management schemes. Organically certified orchards had higher fungal diversity in flowers than conventional or bio-based integrated pest management (IPM) orchards, but organic orchards had the lowest bacterial diversity. Orchard management scheme also best predicted the distribution of several important bacterial and fungal genera that either cause or suppress disease; organic and bio-based IPM best explained the distributions of bacterial and fungal genera, respectively. Moreover, patterns of bacterial and fungal diversity were affected by interactions between management, landscape context, and climate. When examining the similarity of bacterial and fungal communities across sites, both abundance- and taxon-related turnovers were mediated primarily by orchard management scheme and landscape context and, specifically, the amount of land in cultivation. Our study reveals local- and landscape-level drivers of floral microbiome structure in a major fruit crop, providing insights that can inform microbiome management to promote host health and high-yielding quality fruit. IMPORTANCE Proper crop management during bloom is essential for producing disease-free tree fruit. Tree fruits are often grown in heterogeneous landscapes; however, few studies have assessed whether landscape context and crop management affect the floral microbiome, which plays a critical role in shaping plant health and disease tolerance. Such work is key for identification of tactics and/or contexts where beneficial microbes proliferate and pathogenic microbes are limited. Here, we characterize the floral microbiome of pear crops in Washington State, where major production occurs in intermountain valleys and basins with variable elevation and microclimates. Our results show that both local-level (crop management) and landscape-level (habitat types and climate) factors affect floral microbiota but in disparate ways for each kingdom. More broadly, these findings can potentially inform microbiome management in orchards for promotion of host health and high-quality yields.

Role of viral suppressors governing asymmetric synergism between tomato-infecting begomoviruses.

Mixed viral infections are common in fields and frequently exacerbate disease severity via synergistic interactions among individual viral genomic components leading to major crop loss. Two predominant species of tomato-infecting begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl Gujarat virus (ToLCGuV), are known to cause severe leaf curl disease of tomato in India. Previously, we have demonstrated asymmetric synergism between these two distinct begomovirus species during mixed infection in solanaceous hosts. In the present study, we have identified the underlying proteins that positively regulate asymmetric synergism and their effect on plant defense machinery. During co-infection, the AC2 and AV2 of ToLCGuV enhanced ToLCNDV DNA accumulation in Nicotiana benthamiana as well as in their natural host, tomato. Furthermore, we found that AC2 and AV2 of ToLCNDV and AV2 of ToLCGuV play a critical role in suppression of post transcriptional gene silencing (PTGS) machinery. Taken together, AC2 and AV2 encoded proteins of ToLCGuV are the crucial viral factors promoting asymmetric synergism with ToLCNDV. KEY POINTS: • Begomoviral suppressors play vital roles in viral synergism. • AC2 and AV2 of ToLCGuV asymmetrically enhance ToLCNDV accumulation. • AC2 and AV2 of ToLCNDV and ToLCGuV AV2 are major PTGS suppressors.

Insect alarm pheromones in response to predators: Ecological trade-offs and molecular mechanisms.

Insect alarm pheromones are chemical substances that are synthesized and released in response to predators to reduce predation risk. Alarm pheromones can also be perceived by predators, who take advantage of alarm cues to locate prey. While selection favors evolution of alarm pheromone signals that are not easily detectable by predators, predator evolution selects for better prey detection ability. Here, we review the diversity of alarm signals, and consider the behavioral and ecological conditions under which they have evolved. We show that components of alarm pheromones are similar across many insects, although aphids exhibit different behavioral responses to alarm cues compared to social insects. The effects of alarm pheromones on prey behavior depend on factors such as the concentration of pheromones and the density of conspecifics. We also discuss the molecular mechanisms of alarm pheromone perception underlying the evolutionary arms race between predators and prey, and the function of olfactory proteins and receptors in particular. Our review provides a novel synthesis of the diversity and function of insect alarm pheromones, while suggesting avenues that might better allow researchers to exploit population-level responses to alarm signaling for the sustainable management of pests and vector-borne pathogens.

Plant-mediated interactions between a vector and a non-vector herbivore promote the spread of a plant virus.

Herbivores that transmit plant pathogens often share hosts with non-vector herbivores. These co-occurring herbivores can affect vector fitness and behaviour through competition and by altering host plant quality. However, few studies have examined how such interactions may both directly and indirectly influence the spread of a plant pathogen. Here, we conducted field and greenhouse trials to assess whether a defoliating herbivore (Sitona lineatus) mediated the spread of a plant pathogen, Pea enation mosaic virus (PEMV), by affecting the fitness and behaviour of Acrythosiphon pisum, the PEMV vector. We observed higher rates of PEMV spread when infectious A. pisum individuals shared hosts with S. lineatus individuals. Using structural equation models, we showed that herbivory from S. lineatus increased A. pisum fitness, which stimulated vector movement and PEMV spread. Moreover, plant susceptibility to PEMV was indirectly enhanced by S. lineatus, which displaced A. pisum individuals to the most susceptible parts of the plant. Subsequent analyses of plant defence genes revealed considerable differences in plant phytohormones associated with anti-herbivore and anti-pathogen defence when S. lineatus was present. Given that vectors interact with non-vector herbivores in natural and managed ecosystems, characterizing how such interactions affect pathogens would greatly enhance our understanding of disease ecology.

Tri-trophic interactions mediate the spread of a vector-borne plant pathogen.

Many insect herbivores are vectors that transmit plant pathogens as they forage. Within food webs, vectors interact with a range of host plants, other herbivores, and predators. Yet, few studies have examined how tri-trophic interactions involving vectors affect the spread of pathogens. Here we assessed effects of food web structure on aphid vectors and the prevalence of an aphid-borne persistent pathogen (Pea enation mosaic virus, PEMV) in pea plants. We experimentally manipulated ladybird predators, alternative host plants, and non-vector herbivores and assessed responses of pea aphids and PEMV using structural equation models. We show that variation in bottom-up, top-down, and horizontal interactions mediated PEMV prevalence. Predators reduced PEMV prevalence by consuming aphids, but an alternative host plant (vetch) had the opposite effect by promoting aphid movement and abundance. Non-vector herbivores (pea leaf weevil) increased PEMV susceptibility in peas. In turn, weevils offset the positive effects of predators on PEMV, but increased the negative effects of vetch. Our results show that tri-trophic interactions within insect and plant food webs can mediate vector biology with synergistic and opposing effects on pathogens. Continuing to assess how community-wide interactions affect vectors will aid in our understanding of vector-borne pathosystems.

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber).

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), ß-actin (Actin), ß-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.

Fall armyworm (Spodoptera frugiperda Smith) feeding elicits differential defense responses in upland and lowland switchgrass.

Switchgrass (Panicum virgatum L.) is a low input, high biomass perennial grass being developed for the bioenergy sector. Upland and lowland cultivars can differ in their responses to insect herbivory. Fall armyworm [FAW; Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae)] is a generalist pest of many plant species and can feed on switchgrass as well. Here, in two different trials, FAW larval mass were significantly reduced when fed on lowland cultivar Kanlow relative to larvae fed on upland cultivar Summer plants after 10 days. Hormone content of plants indicated elevated levels of the plant defense hormone jasmonic acid (JA) and its bioactive conjugate JA-Ile although significant differences were not observed. Conversely, the precursor to JA, 12-oxo-phytodienoic acid (OPDA) levels were significantly different between FAW fed Summer and Kanlow plants raising the possibility of differential signaling by OPDA in the two cultivars. Global transcriptome analysis revealed a stronger response in Kanlow plant relative to Summer plants. Among these changes were a preferential upregulation of several branches of terpenoid and phenylpropanoid biosynthesis in Kanlow plants suggesting that enhanced biosynthesis or accumulation of antifeedants could have negatively impacted FAW larval mass gain on Kanlow plants relative to Summer plants. A comparison of the switchgrass-FAW RNA-Seq dataset to those from maize-FAW and switchgrass-aphid interactions revealed that key components of plant responses to herbivory, including induction of JA biosynthesis, key transcription factors and JA-inducible genes were apparently conserved in switchgrass and maize. In addition, these data affirm earlier studies with FAW and aphids that the cultivar Kanlow can provide useful genetics for the breeding of switchgrass germplasm with improved insect resistance.

Dynamics of a geminivirus-encoded pre-coat protein and host RNA-dependent RNA polymerase 1 in regulating symptom recovery in tobacco.

RNA silencing is an integral part of the cellular defense mechanisms in plants that act against virus infection. However, the specific role of RNA silencing and the interplay between host and virus components during recovery from geminivirus infection remains unknown. Hence, in this study we aimed to examine the mechanism behind the host-specific recovery of Nicotiana tabacum infected with Tomato leaf curl Gujarat virus (ToLCGV). Unlike Tomato leaf curl New Delhi virus (ToLCNDV), ToLCGV infection resulted in symptom remission in N. tabacum, and we found that this was mainly due to cross-talk between the pre-coat protein (encoded by the AV2 ORF) of the virus and the host RNA-silencing component RNA-dependent RNA polymerase 1 (encoded by NtRDR1) of N. tabacum. Moreover, apart from the AV2 mutant, other mutants of ToLCNDV developed severe symptoms on a transgenic NtRDR1-overexpression line of N. benthamiana. In contrast, inoculation with ToLCGV resulted in symptom remission, which was due to enhanced methylation of the ToLCGV promoter. Our study reveals a novel 'arms race' in which the pre-coat protein of ToLCNDV selectively blocks the recovery process through inhibiting host-specific RDR1-mediated antiviral silencing in tobacco.

Altering Plant Defenses: Herbivore-Associated Molecular Patterns and Effector Arsenal of Chewing Herbivores.

Chewing herbivores, such as caterpillars and beetles, while feeding on the host plant, cause extensive tissue damage and release a wide array of cues to alter plant defenses. Consequently, the cues can have both beneficial and detrimental impacts on the chewing herbivores. Herbivore-associated molecular patterns (HAMPs) are molecules produced by herbivorous insects that aid them to elicit plant defenses leading to impairment of insect growth, while effectors suppress plant defenses and contribute to increased susceptibility to subsequent feeding by chewing herbivores. Besides secretions that originate from glands (e.g., saliva) and fore- and midgut regions (e.g., oral secretions) of chewing herbivores, recent studies have shown that insect frass and herbivore-associated endosymbionts also play a critical role in modulating plant defenses. In this review, we provide an update on a growing body of literature that discusses the chewing insect HAMPs and effectors and the mechanisms by which they modulate host defenses. Novel "omic" approaches and availability of new tools will help researchers to move forward this discipline by identifying and characterizing novel insect HAMPs and effectors and how these herbivore-associated cues are perceived by host plant receptors.

Lessons from the Far End: Caterpillar FRASS-Induced Defenses in Maize, Rice, Cabbage, and Tomato.

Plant defenses to insect herbivores have been studied in response to several insect behaviors on plants such as feeding, crawling, and oviposition. However, we have only scratched the surface about how insect feces induce plant defenses. In this study, we measured frass-induced plant defenses in maize, rice, cabbage, and tomato by chewing herbivores such as European corn borer (ECB), fall armyworm (FAW), cabbage looper (CL), and tomato fruit worm (TFW). We observed that caterpillar frass induced plant defenses are specific to each host-herbivore system, and they may induce herbivore or pathogen defense responses in the host plant depending on the composition of the frass deposited on the plant, the plant organ where it is deposited, and the species of insect. This study adds another layer of complexity in plant-insect interactions where analysis of frass-induced defenses has been neglected even in host-herbivore systems where naturally frass accumulates in enclosed feeding sites over extended periods of time.

Intraplant communication in maize contributes to defense against insects.

The vasculature of plants act as a channel for transport of signal(s) that facilitate long-distance intraplant communication. In maize, Maize insect resistance1-Cysteine Protease (Mir1-CP), which has homology to papain-like proteases, provides defense to different feeding guilds of insect pests. Furthermore, accumulation of Mir1-CP in the vasculature suggests that Mir1-CP can potentially function as a phloem-mobile protein. In a recent study, we provided evidence that Mir1-CP can curtail the growth of phloem-sap sucking insect, corn leaf aphid (CLA; Rhopalosiphum maidis). Our current study further examined whether aboveground feeding by CLA can induce resistance to subsequent herbivory by belowground feeding western corn rootworm (WCR; Diabrotica virgifera virgifera). Aboveground feeding by CLA systemically induced the accumulation of Mir1-CP in the roots. Furthermore, foliage feeding by CLA provided enhanced resistance to subsequent herbivory by belowground feeding of WCR. Taken together, our previous findings and results presented here indicate that long-distance transport of Mir1-CP is critical for providing enhanced resistance to insect attack in maize.

Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India.

Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report on a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60 × 10- 3 substitutions site- 1 year- 1) and the ßC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57 × 10- 4 substitution site- 1 year- 1; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22 × 10- 4 substitution site- 1 year- 1]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.