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Wearable plant sensors hold tremendous potential for smart agriculture. We report a lower leaf surface-attached multimodal wearable sensor for continuous monitoring of plant physiology by tracking both biochemical and biophysical signals of the plant and its microenvironment. Sensors for detecting volatile organic compounds (VOCs), temperature, and humidity are integrated into a single platform. The abaxial leaf attachment position is selected on the basis of the stomata density to improve the sensor signal strength. This versatile platform enables various stress monitoring applications, ranging from tracking plant water loss to early detection of plant pathogens. A machine learning model was also developed to analyze multichannel sensor data for quantitative detection of tomato spotted wilt virus as early as 4 days after inoculation. The model also evaluates different sensor combinations for early disease detection and predicts that minimally three sensors are required including the VOC sensors.

NASA is planning to launch robotic landers to the Moon as part of the Artemis lunar program. We have proposed sending a greenhouse housed in a 1U CubeSat as part of one of these robotic missions. A major issue with these small landers is the limited power resources that do not allow for a narrow temperature range that we had on previous spaceflight missions with plants. Thus, the goal of this project was to extend this temperature range, allowing for greater flexibility in terms of hardware development for growing plants on the Moon. Our working hypothesis was that a mixture of ecotypes of Arabidopsis thaliana from colder and warmer climates would allow us to have successful growth of seedlings. However, our results did not support this hypothesis as a single genotype, Columbia (Col-0), had the best seed germination, growth, and development at the widest temperature range (11–25 °C). Based on results to date, we plan on using the Columbia ecotype, which will allow engineers greater flexibility in designing a thermal system. We plan to establish the parameters of growing plants in the lunar environment, and this goal is important for using plants in a bioregenerative life support system needed for human exploration on the Moon.

35 p.-5 tab.-4 tab.supl., We performed a series of experiments to study the interaction between phototropism and gravitropism in Arabidopsis thaliana as part of the Seedling Growth Project on the International Space Station. Red-light-based and blue-light-based phototropism were examined in microgravity and at 1g, a control that was produced by an on-board centrifuge. At the end of the experiments, seedlings were frozen and brought back to Earth for gene profiling studies via RNASeq methods. In this paper, we focus on five genes identified in these space studies by their differential expression in space: one involved in auxin transport and four others encoding genes for: a methyltransferase subunit, a transmembrane protein, a transcription factor for endodermis formation, and a cytoskeletal element (an intermediate filament protein). Time course studies using mutant strains of these five genes were performed for blue-light and red-light phototropism studies as well as for gravitropism assays on ground. Interestingly, all five of the genes had some effects on all the tropisms under the conditions studied. In addition, RT-PCR analyses examined expression of the five genes in wild-type seedlings during blue-light-based phototropism. Previous studies have supported a role of both microfilaments and microtubules in tropism pathways. However, the most interesting finding of the present space studies is that NFL, a gene encoding an intermediate filament protein, plays a role in phototropism and gravitropism, which opens the possibility that this cytoskeletal element modulates signal transduction in plants., This work was supported by the National Aeronautics and Space Administration [grant 80NSSC17K0546 to John Z. Kiss] and by the Spanish Plan Estatal de Investigación Científica y Desarrollo Tecnológico [Grant RTI2018-099309-B-I00, co-funded by EU-ERDF, to F. Javier Medina].

Most sand flies and mosquitoes require a bloodmeal for egg production, but when blood-sources are scarce, some of them can reproduce without it, so called facultative autogeny. The evolution of autogenous reproduction is thought to involve a trade-off between the benefit of reproducing in the absence of bloodmeal hosts versus the quantitative cost of reduced fecundity and/or or qualitative effect on reduced offspring development and survivorship. We blood-fed (BF) some Phlebotomous papatasi (Scopoli) sand fly females on mice while keeping others (from the same cohort) not BF. We then compared the fecundity of BF and non-blood-fed (NBF) females and also evaluated their egg mass and hatching rate, larval development rate and survivorship, pupa mass and eclosion rates, and progeny fecundity. Among NBF females, only 55% became gravid and produced three times less mature oocytes than BF ones. Autogenous females laid 3.5 and 5.7 times fewer eggs in individual and multi-female bioassays, respectively. Egg mass and hatching rate were not affected by blood-feeding. Individual-larvae bioassays suggested reduced survival during larval stages in the autogenous group. In multi-larvae bioassays, overall and especially pupae survival was significantly reduced in the autogenous group. Development rate was slower and pupal mass was reduced in progeny from autogenous mothers. These effects were particularly apparent at high larval density. Mothers’ blood-feeding history did not affect daughter’s fecundity. Studies on the costs of autogeny provides insights on the evolution of blood feeding. Moreover, it also provides insights regarding potential implications of autogeny to the emergence of vector-borne diseases.

The growth and development of plants during spaceflight have important implications for both basic and applied research supported by NASA and other international space agencies. While there have been many reviews of plant space biology, this chapter attempts to fill a gap in the literature on the actual process and methods of performing plant research in the spaceflight environment. One of the authors (JZK) has been a principal investigator on eight spaceflight projects. These experiences include using the U.S. Space Shuttle, the former Russian Space Station Mir, and the International Space Station, utilizing the Space Shuttle and Space X as launch vehicles. While there are several ways to fly an experiment into space and to obtain a spaceflight opportunity, this review focuses on using the NASA peer-reviewed sciences approach to get an experiment manifested for flight. Three narratives for the implementation of plant space biology experiments are considered from rapid turn around of a few months to a project with new hardware development that lasted 6 years. The many challenges of spaceflight research include logistical and resource constraints such as crew time, power, cold stowage, data downlinks, among others. Additional issues considered are working at NASA centers, hardware development, safety concerns, and the engineering versus science culture in space agencies. The difficulties of publishing the results from spaceflight research based on such factors as the lack of controls, limited sample size, and the indirect effects of the spaceflight environment also are summarized. Lessons learned from these spaceflight experiences are discussed in the context of improvements for future space-based research projects with plants. We also will consider new opportunities for Moon-based research via NASA's Artemis lunar exploration program.

The growth and development of plants during spaceflight have important implications for both basic and applied research supported by NASA and other international space agencies. While there have been many reviews of plant space biology, this chapter attempts to fill a gap in the literature on the actual process and methods of performing plant research in the spaceflight environment. One of the authors (JZK) has been a principal investigator on eight spaceflight projects. These experiences include using the U.S. Space Shuttle, the former Russian Space Station Mir, and the International Space Station, utilizing the Space Shuttle and Space X as launch vehicles. While there are several ways to fly an experiment into space and to obtain a spaceflight opportunity, this review focuses on using the NASA peer-reviewed sciences approach to get an experiment manifested for flight. Three narratives for the implementation of plant space biology experiments are considered from rapid turn around of a few months to a project with new hardware development that lasted 6 years. The many challenges of spaceflight research include logistical and resource constraints such as crew time, power, cold stowage, data downlinks, among others. Additional issues considered are working at NASA centers, hardware development, safety concerns, and the engineering versus science culture in space agencies. The difficulties of publishing the results from spaceflight research based on such factors as the lack of controls, limited sample size, and the indirect effects of the spaceflight environment also are summarized. Lessons learned from these spaceflight experiences are discussed in the context of improvements for future space-based research projects with plants. We also will consider new opportunities for Moon-based research via NASA's Artemis lunar exploration program.

Background Gravid females assess the conditions of oviposition sites to secure the growth and survival of their offspring. Conspecific-occupied sites may signal suitable oviposition sites but may also impose risk due to competition or cannibalism at high population density or heterogeneous larval stage structure, respectively. Chemicals in the habitat, including chemicals emitted from other organisms, serve as cues for females to assess habitat conditions. Here, we investigated the attraction and oviposition preference of the Old World cutaneous leishmaniasis vector, Phlebotomus papatasi, to young and old conspecific stages, including eggs and evaluated the effect of a semiochemical associated with eggs and neonate larvae. Methods Attraction and oviposition preference of Ph. papatasi to each of various life stages (eggs, first-, second-, third-, fourth-instar larvae, pupae and male and female adults) was investigated using cage and oviposition jar behavioral assays. Identification of organic chemical compounds extracted from eggs was performed using GC-MS and chemicals were tested in the same behavioral assays in a dose-response manner. Behavioral responses were statistically analyzed using logistic models. Results Gravid Ph. papatasi females were significantly attracted to and preferred to oviposit on medium containing young life stages (eggs and first instars). This preference decreased towards older life stages. Dose effect of eggs indicated a hump-shaped response with respect to attraction but a concave-up pattern with respect to oviposition. Chemical analysis of semiochemicals from eggs and first-instar larvae revealed the presence of dodecanoic acid (DA) and isovaleric acid. Sand flies were attracted to and laid more eggs at the lowest DA dose tested followed by a negative dose-response. Conclusions Findings corroborated our hypothesis that gravid sand flies should prefer early colonized oviposition sites as indicators of site suitability but avoid sites containing older stages as indicators of potential competition. Findings also supported the predictions of our hump-shaped oviposition regulation (HSR) model, with attraction to conspecific eggs at low-medium densities and switching to repellence at high egg densities. This oviposition behavior is mediated by DA that was identified from surface extracts of both eggs and first-instar larvae. Isovaleric acid was also found in extracts of both stages.

Some cool-season pooid grasses partner with symbiotic fungal endophytes in the Epichloe genus for defense against insect herbivores via fungal alkaloids. Poa alsodes, North American woodland grass, independently hosts two species of Epichloe that vary by produced alkaloids. E. alsodes produces insecticidal N-acetylnorloline. E. schardlii var. pennsylvanica (E. schardlii hereafter) has the gene for peramine, an insect-deterring alkaloid, production, but peramine was not detected. We tested the effects of the two endophytes on survival, feeding preference, and plant damage by the generalist herbivore, Spodoptera frugiperda. No larvae survived when feeding on plants harboring E. alsodes. In contrast, survival was only slightly reduced by plants harboring E. schardlii. However, larvae that fed on E. schardlii infected plants experienced delayed development and reduced pupal mass. Uninfected plants and plants infected with E. schardlii were damaged severely when single larvae fed upon them, whereas larvae fed negligibly on plants infected with E. alsodes. Preference did not match performance. Larvae strongly avoided feeding on E. schardlii but not E. alsodes-infected leaves where survival was zero. When E. schardlii was experimentally removed, larval leaf choices suggested that this endophyte is responsible for deterrence. High levels of N-acetylnorloline were detected from E. alsodes infected plants. Peramine was not detected in the experimental plants harboring E. schardlii, so it remains unclear what mechanisms caused avoidance and developmental delays. The two endophytes may protect their common host in different ways: E. alsodes by larval mortality and E. schardlii by deterring feeding and negative effects on development.

The endophyte presence and diversity in natural populations of Poa alsodes were evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinct Epichloë hybrid taxa were identified from 23 populations. Each taxon could easily be distinguished by polymerase chain reaction (PCR) genotyping with primers designed to mating type genes and alkaloid biosynthesis genes that encode key pathway steps for ergot alkaloids, indole-diterpenes, lolines, and peramine. The most commonly found Epichloë taxon, Poa alsodes Taxonomic Group-1 (PalTG-1), was detected in 22 populations at high infection frequencies (72-100%), with the exception of one population at high elevation (26% infection). The second taxon, PalTG-2, was observed only in five populations in Pennsylvania constituting 12% of infected samples. Phylogenetic analyses placed PalTG-1 as an interspecific hybrid of E. amarillans and E. typhina subsp. poae ancestors, and it is considered a new hybrid species, which the authors name Epichloë alsodes. PalTG-2 is an intraspecific hybrid of two E. typhina subsp. poae ancestors, similar to E. schardlii from the host Cinna arundinacea, which the authors propose as a new variety, Epichloë schardlii var. pennsylvanica. Epichloë alsodes isolates were all mating type MTA MTB and tested positive for dmaW, easC, perA, and some LOL genes, but only the alkaloid N-acetylnorloline was detected in E. alsodes-infected plant material. Epichloë schardlii var. pennsylvanica isolates were all mating type MTB MTB and tested positive for perA, but peramine was not produced. Both E. alsodes and E. schardlii var. pennsylvanica appeared to have complete perA genes, but point mutations were identified in E. alsodes that would render the encoded perA gene nonfunctional.

Hybridization is common among plants, animals and microbes. However, the ecological consequences of hybridization for microbes are far less understood than for plants and animals. For symbiotic Epichloë fungi, hybridization is widespread and may augment the well-known benefits of the endophytes to their grass hosts, especially in stressful environments. We tested the hybrid fitness hypothesis (HFH) that hybrid endophytes enhance fitness in stressful environments relative to non-hybrid endophytes. In a long-term field experiment, we monitored growth and reproduction of hybrid-infected (H+), non-hybrid infected (NH+), naturally endophyte free (E-) plants and those plants from which the endophyte had been experimentally removed (H- and NH-) in resource-rich and resource-poor environments. Infection by both endophyte species enhanced growth and reproduction. H+ plants outperformed NH+ plants in terms of growth by the end of the experiment, supporting HFH. However, H+ plants only outperformed NH+ plants in the resource-rich treatment, contrary to HFH. Plant genotypes associated with each endophyte species had strong effects on growth and reproduction. Our results provide some support the HFH hypothesis but not based upon adaptation to stressful environments. Our results reinforce the notion of a complex interplay between endophyte and plant genotype and environmental factors that determine fitness of the symbiotum.

Asexual Epichloë endophytes are prevalent in cool season grasses, and many are of hybrid origin. Hybridization of asexual endophytes is thought to provide a rapid influx of genetic variation that may be adaptive to endophyte-host grass symbiota in stressful environments. For Arizona fescue (Festuca arizonica), hybrid symbiota are commonly found in resource-poor environments, whereas non-hybrid symbiota are more common in resource-rich environments. There have been very few experimental tests where infection, hybrid and non-hybrid status, and plant genotype have been controlled to tease apart their effects on host phenotype and fitness in different environments. We conducted a greenhouse experiment where hybrid (H) and non-hybrid (NH) endophytes were inoculated into plant genotypes that were originally uninfected (E-) or once infected with either the H or NH endophytes. Nine endophyte and plant genotypic group combinations were grown under low and high water and nutrient treatments. Inoculation with the resident H endophyte enhanced growth and altered allocation to roots and shoots, but these effects were greatest in resource-rich environments, contrary to expectations. We found no evidence of co-adaptation between endophyte species and their associated host genotypes. However, naturally E- plants performed better when inoculated with the hybrid endophyte, suggesting these plants were derived from H infected lineages. Our results show complex interactions between endophyte species of hybrid and non-hybrid origin with their host plant genotypes and environmental factors.

Traditional tall fescue cultivar, Kentucky 31, possesses a wild-type endophyte strain that produces several bioactive compounds including the ergot alkaloid, ergovaline, known to cause cattle toxicity and effects on insects. Novel cultivars, BarOptima PLUS E34, Jesup MaxQ, and Texoma MaxQ II, possess different endophyte strains that do not have negative effects on cattle but still protect from insect grazing. Our study investigated if different cultivars have different effects on insect dung decomposers such as dung beetles and determined if ergovaline could be detected in cow dung. Ergovaline at 0.04 and 0.27 μg/g was detected only from Kentucky 31 dung samples from 2017 and 2018 collections, respectively. From the 2017 dung collection, we tested Onthophagus taurus oviposition substrate preferences for each cultivar versus dung from uninfected pasture and also larval survival and development for each dung-type. From the 2018 dung collection, O. taurus and Digitonthophagus gazella oviposition substrate preferences, larval survival and development were tested for Texoma MaxQ II versus Kentucky 31 dung-types. Among the four cultivars, for making brood-balls O. taurus preferred dung from Texoma MaxQ II while dung from Kentucky 31 and BarOptima PLUS E34 were avoided. Both beetle species preferred dung from Texoma MaxQ II versus Kentucky 31 pasture. Larval survival was not affected by dung-type with the 2017 samples. However, with the 2018 samples both beetle species had reduced larval survival on Kentucky 31 than on Texoma MaxQ II brood-balls. Development time for O. taurus was shorter for larvae from Texoma MaxQ II versus Kentucky 31 or uninfected dung brood-balls. Adult mass was not affected in the 2017 collection but was reduced in 2018 Kentucky 31 samples when compared with Texoma MaxQ II. Finally, dung beetles can differentiate dung from pastures with different tall fescue cultivars. Novel cultivar, Texoma MaxQ II, provides more benefits for dung beetles. Pasture renovations with Texoma MaxQ II may improve pasture ecology by enhancing dung beetle populations.

Background Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. Methodology/principal findings To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. Conclusion and significance Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies’ sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed., Author summary Sand flies are vectors of human leishmaniases, an important neglected tropical disease. An alternative approach to the conventional delivery of an insecticide to the vector is to bring the vector to the insecticide using oviposition (egg-laying)-site attractants. Olfactory cues originating from organic matter have been identified as important sources of oviposition attractants. However, nothing is known regarding visual assessment of oviposition sites by sand flies. Also, little is understood about diel egg-laying patterns of sand flies. Finally, it is not known if visual cues and time-of-day may affect their sensitivity to olfactory cues. In this study, we investigated these questions in a series of lab experiments using free-flight cage arenas, with Phlebotomus papatasi (vector of cutaneous leishmaniasis). We showed that peak oviposition-site search and sensitivity to olfactory cues occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. We demonstrated that only gravid females, but not any other stage or sex, were attracted to dark, round oviposition jars resembling burrow openings. Finally, we showed that sensitivity to olfactory cues is reduced in the presence of strong visual cues and in the absence of natural diel photoperiod.

Ergot alkaloids are mycotoxins with an array of biological effects. With this study, we investigated for the first time the application of atmospheric pressure photoionization (APPI) as an ionization method for LC-MS analysis of ergot alkaloids, and compared its performance to that of the more established technique of electrospray ionization (ESI). Samples of the grass Achnatherum robustum infected with the ergot producing Epichloe fungus were extracted using cold methanol and subjected to reserved-phase HPLC-ESI-MS and HPLC-APPI-MS analysis. The ergot alkaloids ergonovine and lysergic acid amide were detected in these samples, and quantified via external calibration. Validation parameters were recorded in accordance with ICH guidelines. A triple quadrupole MS operated in multiple reaction monitoring yielded the lowest detection limits. The performance of APPI and ESI methods was comparable. Both methods were subject to very little matrix interference, with percent recoveries ranging from 82% to 100%. As determined with HPLC-APPI-MS quantification, lysergic acid amide and ergonovine were extracted from an A. robustum sample infected with the Epichloe fungus at concentrations of 1.143±0.051 ppm and 0.2822±0.0071 ppm, respectively. There was no statistically significant difference between these concentrations and those determined using ESI for the same samples.

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloe funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

Aims: In cool-season grasses, systemic and vertically transmitted Epichloe infections often provide a suite of benefits including increased growth, reproduction and competitive abilities. However, these effects of Epichloe endophytes on their hosts often depend upon host and endophyte genotype and environmental factors. Methods: Achnatherum robustum (sleepygrass) harbors at least two Epichloe species within natural populations in the Southwest USA. We tested the effects of endophyte infection and species, host population and plant genotype (by experimentally removing the endophyte), and soil moisture (a key limiting factor) on growth and drought stress response of infected A. robustum plants from two populations (Weed and Cloudcroft) in the Sacremento Mountains of New Mexico, USA). Important Findings: Although the two populations harbor distinct Epichloe species each with very different chemoprofiles, neither endophyte status (infected vs. uninfected) nor endophyte species affected most growth parameters at 8 or 25 weeks of the experiment, except for leaf length. In high water treatment, infected plants from the Weed population had longer leaf length compared with uninfected plants. In contrast, the population of origin affected all growth parameters, including plant height, leaf number, length and width, tiller number and shoot and root biomass, as well as wilting time. Grasses from the Cloudcroft population generally showed greater growth than grasses from the Weed population. Endophyte infection did affect wilting time, with infection in the Weed population generally reducing time to wilting under low and high water, whereas infection in the Cloudcroft population reduced time to wilting only under high water conditions. Our results suggest that plant population and their associated plant genotypes may play a much larger role in endophyte–host grass interactions in varying environments than previously thought. Asexual Epichloe species may be compatible with only specific host genotypes within populations such that the phenotypic effects due to population may be greater than phenotypic changes influenced by variation in the endophyte.