Rice physical defenses and their role against insect herbivores.

MAIN CONCLUSION: Understanding surface defenses, a relatively unexplored area in rice can provide valuable insight into constitutive and induced defenses against herbivores. Plants have evolved a multi-layered defense system against the wide range of pests that constantly attack them. Physical defenses comprised of trichomes, wax, silica, callose, and lignin, and are considered as the first line of defense against herbivory that can directly affect herbivores by restricting or deterring them. Most studies on physical defenses against insect herbivores have been focused on dicots compared to monocots, although monocots include one of the most important crops, rice, which half of the global population is dependent on as their staple food. In rice, Silica is an important element stimulating plant growth, although Silica has also been found to impart resistance against herbivores. However, other physical defenses in rice including wax, trichomes, callose, and lignin are less explored. A detailed exploration of the morphological structures and functional consequences of physical defense structures in rice can assist in incorporating these resistance traits in plant breeding and genetic improvement programs, and thereby potentially reduce the use of chemicals in the field. This mini review addresses these points with a closer look at current literature and prospects on rice physical defenses.

Continuous mowing differentially affects floral defenses in the noxious and invasive weed Solanum elaeagnifolium in its native range.

In weeds, disturbance has been found to affect life history traits and mediate trophic interactions. In urban landscapes, mowing is an important disturbance, and we previously showed that continuous mowing leads to enhanced fitness and defense traits in Solanum elaeagnifolium, Silverleaf Nightshade (SLN). However, most studies have been focused on foliar defenses, ignoring floral defenses. In this study we examined whether continuous mowing affected floral defenses in SLN using mowed and unmowed populations in South Texas, their native range. We found flowers of mowed SLN plants larger but lighter than unmowed plants. Additionally, flowers on plants that were mowed frequently were both heavier and larger. Mowed plants had higher spine density and consequently unmowed flowers had higher herbivore damage. Additionally, early instar Manduca sexta fed on mowed flower-based artificial diets showed no difference in mass than the control and unmowed; however, later instars caterpillars on unmowed diets gained significantly more mass than the mowed treatment and control. Mowed plants had higher spine density which may shed light on why unmowed flowers experienced higher herbivore damage. We found caterpillars fed on high mowing frequency diets were heavier than those on low mowing frequency diets. Collectively, we show that mowing compromises floral traits and enhances plant defenses against herbivores and should be accounted for in management.

Sex and stress modulate pupal defense response in tobacco hornworm.

In insects, larval and adult defenses against predators have been well studied. However, pupal (also known as resting stage) defenses have been overlooked and not examined thoroughly. Although some pupa possess antipredator strategies such as hairs, spines, cryptic coloration, and exudation of chemicals, few studies have tested these responses and the factors affecting them. Here, we investigated the behavioral responses in tobacco hornworm Manduca sexta that pupates in soil by introducing an external stimulus using vibrations from an electric toothbrush to mimic predation. We observed that M. sexta made violent wriggling (twitching), followed by pulsating movements in response to the vibrational stimulus. Detailed examination showed that these twitches and pulsating events occurred more frequently and for longer periods of time in male pupa and were dependent on the magnitude of the stress (high and low frequency). However, when we estimated the angular force exerted by pupa using radian and angular momentum of twitches, it was found to be independent of pupal sex. A follow-up experiment on possible cascading effects of stress exposure on eclosion success revealed that low- and high-frequency stress exposure didn't cause any of the common defects in eclosed adults. Our study clearly demonstrates that the so-called defenseless pupal stage uses a wide range of measurable defense behaviors that can actively defend against predators and should be examined further-linking observed behavior with underlying mechanisms.

Sorghum defense responses to sequential attack by insect herbivores of different feeding guilds.

MAIN CONCLUSION: Insect herbivores of different feeding guilds induced sorghum defenses through differential mechanisms, regardless of the order of herbivore arrival on sorghum plants. Sorghum, one of the world's most important cereal crops, suffers severe yield losses due to attack by insects of different feeding guilds. In most instances, the emergence of these pests are not secluded incidents and are followed by another or can also co-infest host plants. Sugarcane aphid (SCA) and fall armyworm (FAW) are the two most important destructive pests of sorghum, which belongs to sap-sucking and chewing feeding guilds, respectively. While the order of the herbivore arriving on the plants has been found to alter the defense response to subsequent herbivores, this is seldom studied with herbivores from different feeding guilds. In this study, we investigated the effects of sequential herbivory of FAW and SCA on sorghum defense responses and their underlying mechanism(s). Sequential feeding on the sorghum RTx430 genotype by either FAW primed-SCA or SCA primed-FAW were monitored to unravel the mechanisms underlying defense priming, and its mode of action. Regardless of the order of herbivore arrival on sorghum RTx430 plants, significant defense induction was observed in the primed state compared to the non-primed condition, irrespective of their feeding guild. Additionally, gene expression and secondary metabolite analysis revealed differential modulation of the phenylpropanoid pathway upon insect attack by different feeding guilds. Our findings suggest that priming in sorghum plants upon sequential herbivory induces defense by the accumulation of the total flavonoids and lignin/salicylic acid in FAW primed-SCA and SCA primed-FAW interaction, respectively.

Trichomes mediate plant-herbivore interactions in two Cucurbitaceae species through pre- and post-ingestive ways.

Plant structural defenses such as trichomes exert a significant selection pressure on insect herbivores. However, whether variation in structural defense traits affects common herbivores in related plant species is less understood. Here, we examined the role of trichomes in plant-herbivore interactions in two commonly cultivated members in Cucurbitaceae: bottle gourd (Lagenaria siceraria) and cucumber (Cucumis sativa). In common garden experiments when the two species were grown together, we observed that they differed in their attractiveness to four major herbivore species (Trichoplusia ni, Acalymma vittatum, Diaphania indica, and Anasa tristis) and, consequently, their feeding behavior. We found that L. siceraria consistently harbored less herbivores, and the two lepidopteran herbivores (T. ni and D. indica) were found to take significantly longer time to commence feeding on them, a primary mode of pre-ingestive defense function of trichomes. To tease apart structural and chemical modes of defenses, we first used scanning electron microscopy to identify, quantify, and measure trichome traits including their morphology and density. We found that C. sativa has significantly lower number of trichomes compared to L. siceraria, regardless of trichome type and leaf surface. We then used artificial diet enriched with trichomes as caterpillar food and found that trichomes from these two species differentially affected growth and development of T. ni showing cascading effects of trichomes. Taken together, we show that trichomes, independent of chemical defenses, are an effective pre- and post-ingestive defense strategy against herbivores with negative consequences for their feeding, growth, and development. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-023-01611-x.

Morphological characterization of intraspecific variation for trichome traits in tomato (Solanum lycopersicum).

Trichomes, the hairlike protuberances in plants, have been well known to act as the first line of defense against herbivores, and abiotic stresses, along with other structural defenses such as spines, thorns, and waxes. We previously reported the tremendous variation in trichome traits among different wild and cultivated Solanum species and demonstrated that trichomes types and density are traditionally miscalculated and often misnamed. However, intraspecific variation in trichome traits is poorly understood, although this has implications for stress tolerance and resistance breeding programs in economically important crop species and can also mediate ecological interactions at multiple trophic levels in their wild congeners. In this study, using tomato as a model, we characterized the trichomes from 10 commonly grown varieties using a minimal sample prep desktop scanning electron microscopy, and followed up with estimating their dimensions across the varieties and trichome types. We hypothesized that although trichome number may vary, the varieties will have similar trichome types, based on current literature. Our results show that there is significant variation for trichome number as well as dimensions of trichome types among these varieties. Furthermore, when we separately analyzed the number and dimensions of commonly found glandular and non-glandular trichomes, the results were consistent with broad assessment of trichomes, showing consistent variation among varieties, suggesting that trichome studies should not be limited to basic classification into glandular and non-glandular, and should accommodate the sub-types and their dimensions.

Can trap color affect arthropod community attraction in agroecosystems? A test using yellow vane and colorless traps.

Vane trapping is one of the most effective methods for sampling flower-visiting arthropods. Despite its importance in pollinator studies, the effects of trap color on the abundance and richness of pollinators are less understood. To test this, we conducted a 3-season field experiment over 2 years with two types of vane traps: yellow and colorless. We set up twelve traps each in three field sites within the Lower Rio Grande Valley in south Texas, planted with Vigna unguiculata, Crotalaria juncea, Raphanus raphanistrum, and Sorghum drummondii. At each site, six colorless vane and six yellow vane traps were placed equidistant from each other. The experiment was replicated three times across three seasons, first during the pre-flowering season, when the crops were in full bloom, and when there was no crop on the field. In total, we collected 1912 insects, out of which 76.7% were pollinators. Generalized Linear Regression analyses showed that yellow traps consistently attracted significantly more arthropods and pollinators, but these differences were also season dependent. Furthermore, we noticed that Hymenoptera, followed by Coleoptera, were the most prevalent orders in both the yellow vane and colorless vane traps. Interestingly, although there was no significant difference in species richness of the arthropods in the yellow and colorless vane traps, our results suggest that trap color plays a significant role in capturing pollinators, including non-target arthropods. Our data add another line of evidence suggesting that trap color should be accounted for designing experiments that estimate pollinator and arthropod community diversity.

Cascading Effects of Cover Crops on the Subsequent Cash Crop Defense against the Polyphagous Herbivore Fall Armyworm (Spodoptera frugiperda).

Recent studies have started to show that the benefits of cover crops can cascade to the cash crop growing seasons. However, the impact of cover crops on the subsequent cash crop defense against herbivores is not well understood. To test this, we conducted a field and laboratory study to assess the possible cascading effects of cover crops such as Vigna unguiculata, Sorghum drummondii, Raphanus sativus, and Crotalaria juncea on the subsequent cash crop (Sorghum bicolor) defense against the notorious polyphagous herbivore fall armyworm (Spodoptera frugiperda) across three farms in the Lower Rio Grande Valley. Our field and laboratory studies showed that the cash crop planted in the cover crop treatment differentially affected S. frugiperda. More specifically, we found that cover crops have positive effects on the growth and development of S. frugiperda on the subsequent cash crop, including both larval and pupal parameters. However, our experiments on physical and chemical defenses in cash crops failed to show any significant differences between cover and control. Collectively, our results add an additional line of evidence on the effects of cover crops on pest dynamics outside the cash crop season, a key consideration for the selection and management of cover crops and cash crops, whose underlying mechanisms need to be examined further.

Desktop scanning electron microscopy in plant-insect interactions research: a fast and effective way to capture electron micrographs with minimal sample preparation.

The ability to visualize cell and tissue morphology at a high magnification using scanning electron microscopy (SEM) has revolutionized plant sciences research. In plant-insect interactions studies, SEM-based imaging has been of immense assistance to understand plant surface morphology including trichomes [plant hairs; physical defense structures against herbivores], spines, waxes, and insect morphological characteristics such as mouth parts, antennae, and legs, that they interact with. While SEM provides finer details of samples, and the imaging process is simpler now with advanced image acquisition and processing, sample preparation methodology has lagged. The need to undergo elaborate sample preparation with cryogenic freezing, multiple alcohol washes, and sputter coating makes SEM imaging expensive, time consuming, and warrants skilled professionals, making it inaccessible to majority of scientists. Here, using a desktop version of SEM (SNE- 4500 Plus Tabletop), we show that the "plug and play" method can efficiently produce SEM images with sufficient details for most morphological studies in plant-insect interactions. We used leaf trichomes of Solanum genus as our primary model, and oviposition by tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) and fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae), and leaf surface wax imaging as additional examples to show the effectiveness of this instrument and present a detailed methodology to produce the best results with this instrument. While traditional sample preparation can still produce better resolved images with less distortion, we show that even at a higher magnification, the desktop SEM can deliver quality images. Overall, this study provides detailed methodology with a simpler "no sample preparation" technique for scanning fresh biological samples without the use of any additional chemicals and machinery.

Morphological characterization of trichomes shows enormous variation in shape, density and dimensions across the leaves of 14 Solanum species.

Trichomes are the epidermal appendages commonly observed on plant surfaces including leaves, stem and fruits. Plant trichomes have been well studied as a structural plant defence designed to protect plants against abiotic and biotic stressors such as UV rays, temperature extremities and herbivores. Trichomes are primarily classified into glandular and non-glandular trichomes, based on the presence or absence of a glandular head. The plant genus Solanum is the largest genus of family Solanaceae that houses ~3500 species of ecological and economic importance have a diverse set of trichomes that vary in density and morphology. However, due to the incomplete and contradictory classification system, trichomes have subjective names and have been largely limited to be grouped into glandular or non-glandular types. Through this study, we did a complete workup to classify and characterize trichomes on both adaxial and abaxial leaf surface of 14 wild and domesticated species of the genus Solanum. Using electron microscopy, statistical analyses and artistic rendition, we examined finer details of trichomes and measured their density and dimensions to compile a detailed data set which can be of use for estimating the variation in trichome types, and their density, with consequences for understanding their functional roles. Our study is the first of its kind that provides us with a better and well-defined classification, density and dimension analysis to complete the morphological classification of trichomes on both leaf surfaces of a diverse range of members in Solanum genus.

Examining the Role of Buzzing Time and Acoustics on Pollen Extraction of Solanum elaeagnifolium.

Buzz pollination is a specialized pollination syndrome that requires vibrational energy to extract concealed pollen grains from poricidal anthers. Although a large body of work has examined the ecology of buzz pollination, whether acoustic properties of buzz pollinators affect pollen extraction is less understood, especially in weeds and invasive species. We examined the pollination biology of Silverleaf nightshade (Solanum elaeagnifolium), a worldwide invasive weed, in its native range in the Lower Rio Grande Valley (LRGV) in south Texas. Over two years, we documented the floral visitors on S. elaeagnifolium, their acoustic parameters (buzzing amplitude, frequency, and duration of buzzing) and estimated the effects of the latter two factors on pollen extraction. We found five major bee genera: Exomalopsis, Halictus, Megachile, Bombus, and Xylocopa, as the most common floral visitors on S. elaeagnifolium in the LRGV. Bee genera varied in their duration of total buzzing time, duration of each visit, and mass. While we did not find any significant differences in buzzing frequency among different genera, an artificial pollen collection experiment using an electric toothbrush showed that the amount of pollen extracted is significantly affected by the duration of buzzing. We conclude that regardless of buzzing frequency, buzzing duration is the most critical factor in pollen removal in this species.

Pre-Sowing Treatments Improve Germinability of South Texas Native Plant Seeds.

The incorporation of native plant species is central to restoration efforts, but this is often limited by both the availability of seeds and the relatively low viability and germination rates of commercially available seeds. Although pre-sowing treatments are commonly used to improve germination rates of seeds, the efficacy of these treatments is found to vary across species. In this study, we tested how four pre-sow treatments (physical scarification, acid scarification, cold stratification, and aerated hydropriming) affected the viability and seed germination rates of 12 commercially available plant species native to south Texas and commonly used in restoration efforts. Our results show that the viability of the seeds have a wide range, from 78% to 1.25%. Similarly, the total germination rate ranged from 62% to 0%. We found that pre-sowing treatments accelerated the germination rate in 9 of 12 plant species tested, but the effect varied by treatment. Collectively, our results identify various methods to achieve the best germination rates for native plants of south Texas, to help improve restoration efforts across the region.

Deciphering the Role of Ion Channels in Early Defense Signaling against Herbivorous Insects.

Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (Vm) fluctuation, intracellular calcium (Ca2+), and reactive oxygen species (ROS) generation, followed by defense gene expression, and, ultimately, plant defense responses. In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.

Exploring the role of soil types on defense and fitness traits of silverleaf nightshade (Solanum elaeagnifolium), a worldwide invasive species through a field survey in the native range.

Silverleaf nightshade (Solanum elaeagnifolium) is a highly successful invasive weed that has caused agricultural losses both in its home and invaded ranges. Surveying 50 sub-populations over 36,000 km2 in its native range in South Texas, we investigated the interactions among soil type, population size, plant height, herbivory, and plant defenses in its home range with the expectation that populations growing in the plant's preferred sandier soils would host larger colonies of healthier and better defended plants. At each sampling location, on randomly selected plants, we measured height, insect herbivore damage, and presence, and density of internode spines. Soil type was determined using the NRCS Web Soil Survey and primarily grouped into sand, clay, or urban. Our results show a tradeoff between growth and defense with larger colonies and taller plants in clay soils, but smaller colonies of shorter, spinier plants in sandy soils. We also observed decreased herbivory in urban soils, further confirming the plant's ability to survive and even be strengthened by highly disturbed conditions. This study is a starting point for a better understanding of silverleaf nightshade's ecology in its home range and complicates the assumption that it thrives best in sandy soils.

Picking sides: feeding on the abaxial leaf surface is costly for caterpillars.

MAIN CONCLUSION: The study provides us with the evidence that caterpillars tend to feed on the abaxial leaf surface despite the damage caused to them because of higher trichome density. To defend against herbivory, plants have evolved physical and chemical defense mechanisms, including trichomes (hair like appendages on leaves and stem) being one of them. Caterpillars, a major group of insect herbivores are generally found to occupy the abaxial (underside) leaf surface, considered as an avoidance mechanism from biotic and abiotic stresses. Since trichomes are a first line of defense, we examined the correlation between abaxial vs adaxial (above side) trichomes and caterpillar feeding, behavior, and growth. A combination of field, lab and microscopy experiments were performed using tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae), a Solanaceae specialist caterpillar, and multiple host species. We found that M. sexta caterpillars overwhelmingly preferred to stay and feed on the abaxial leaf surface, but the abaxial leaf surface also had significantly more trichomes, and consequently, caterpillars took significantly longer to commence feeding. In addition, lab-based diet experiment containing shaved trichomes showed that feeding on the abaxial leaf surface with more trichomes also affected caterpillar growth. Taken together, our study shows that although caterpillars prefer to feed on the abaxial leaf surface, they accrue feeding delays and developmental constraints, indicating tradeoffs affecting performance, and exposure to predation and abiotic stressors.

Local adaptation to continuous mowing makes the noxious weed Solanum elaeagnifolium a superweed candidate by improving fitness and defense traits.

The role of disturbance in accelerating weed growth is well understood. While most studies have focused on soil mediated disturbance, mowing can also impact weed traits. Using silverleaf nightshade (Solanum elaeagnifolium), a noxious and invasive weed, through a series of field, laboratory, and greenhouse experiments, we asked whether continuous mowing influences growth and plant defense traits, expressed via different avenues, and whether they cascade into offspring. We found that mowed plants produced significantly less number of fruits, and less number of total seeds per plant, but had higher seed mass, and germinated more and faster. When three herbivores were allowed to feed, tobacco hornworm (Manduca sexta) caterpillars, gained more mass on seedlings from unmowed plants, while cow pea aphid (Aphis craccivora), a generalist, established better on mowed seedlings; however, leaf trichome density was higher on unmowed seedlings, suggesting possible negative cross talk in defense traits. Texas potato beetle (Leptinotarsa texana), a co-evolved specialist on S. elaeagnifolium, did not show any differential feeding effects. We also found that specific root length, an indicator of nutrient acquisition, was significantly higher in first generation seedlings from mowed plants. Taken together, we show that mowing is a selective pressure that enhances some fitness and defense traits and can contribute to producing superweeds.

The Multifunctional Roles of Polyphenols in Plant-Herbivore Interactions.

There is no argument to the fact that insect herbivores cause significant losses to plant productivity in both natural and agricultural ecosystems. To counter this continuous onslaught, plants have evolved a suite of direct and indirect, constitutive and induced, chemical and physical defenses, and secondary metabolites are a key group that facilitates these defenses. Polyphenols-widely distributed in flowering plants-are the major group of such biologically active secondary metabolites. Recent advances in analytical chemistry and metabolomics have provided an opportunity to dig deep into extraction and quantification of plant-based natural products with insecticidal/insect deterrent activity, a potential sustainable pest management strategy. However, we currently lack an updated review of their multifunctional roles in insect-plant interactions, especially focusing on their insect deterrent or antifeedant properties. This review focuses on the role of polyphenols in plant-insect interactions and plant defenses including their structure, induction, regulation, and their anti-feeding and toxicity effects. Details on mechanisms underlying these interactions and localization of these compounds are discussed in the context of insect-plant interactions, current findings, and potential avenues for future research in this area.

Inbreeding in Solanum carolinense alters floral attractants and rewards and adversely affects pollinator visitation.

PREMISE: Inbreeding depression is well documented in flowering plants and adversely affects a wide range of fitness-related traits. Recent work has begun to explore the effects of inbreeding on ecological interactions among plants and other organisms, including insect herbivores and pathogens. However, the effects of inbreeding on floral traits, floral scents, and pollinator visitation are less well studied. METHODS: Using inbred and outbred maternal families of horsenettle (Solanum carolinense, Solanaceae), we examined the effects of inbreeding on traits associated with pollinator attraction and floral rewards. Specifically, we measured corolla size, counted pollen grains per flower, and analyzed floral volatile emissions via gas chromatography and mass spectrometry. We also examined pollinator visitation to experimental arrays of flowering inbred and outbred plants under field conditions. RESULTS: Compared to those of outbred plants, flowers of inbred plants exhibited reduced corolla size and pollen production, as well as significantly reduced emission of the two most abundant volatile compounds in the floral blend. Furthermore, bumblebees-the main pollinators of horsenettle-discriminated against inbred flowers in the field: bees were more likely to make initial visits to flowers on outbred plants, visited outbred flowers more often overall, and spent more time on outbred flowers. CONCLUSIONS: These results show that inbreeding can (1) alter floral traits that are known to mediate pollinator attraction; (2) reduce the production of floral rewards (pollen is the sole reward in horsenettle); and (3) adversely affect pollinator visitation under field conditions.

Tobacco Hornworm (Manduca sexta) Oral Secretion Elicits Reactive Oxygen Species in Isolated Tomato Protoplasts.

Plants are under constant attack by a suite of insect herbivores. Over millions of years of coexistence, plants have evolved the ability to sense insect feeding via herbivore-associated elicitors in oral secretions, which can mobilize defense responses. However, herbivore-associated elicitors and the intrinsic downstream modulator of such interactions remain less understood. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces reactive oxygen species (ROS) in tomato (Solanum lycopersicum) protoplasts. By using a dye-based ROS imaging approach, our study shows that application of plant-fed (PF) M. sexta OS generates significantly higher ROS while artificial diet-fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. Elevation in ROS generation was saturated after ~140 s of PF OS application. ROS production was also suppressed in the presence of an antioxidant NAC (N-acetyl-L-cysteine). Interestingly, PF OS-induced ROS increase was abolished in the presence of a Ca2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate a potential signaling cascade involving herbivore-associated elicitors, Ca2+, and ROS in plants during insect feeding. In summary, our results demonstrate that plants incorporate a variety of independent signals connected with their herbivores to regulate and mount their defense responses.