First molecular detection of Borrelia sp. in tapirs (Tapirus terrestris).

Borrelia theileri is a tick-borne spirochete causative agent of fever, apathy and reduced food consumption in cattle. Molecular diagnosis has expanded the understanding of Borrelia theileri with new hosts and geographical locations being described. The present study aimed to describe the first molecular detection of B. theileri in wild tapirs (Tapirus terrestris) from South America. Blood DNA samples obtained from 99 tapirs sampled in Pantanal (n = 61) and Cerrado (n = 38) biomes were screened using a qPCR assay based on the 16 S rRNA gene of Borrelia sp. Positive samples in the qPCR assay were subjected to PCR assays to allow characterization of fragments from 16 S rRNA and flaB genes. Two (2/99; 2.0%) animals from Pantanal biome were positive in the qPCR and one sample presented bands of expected size for the flaB protocol. Amplicons from this sample were successfully cloned and sequenced. In the phylogenetic analysis, Borrelia sp. from T. terrestris grouped together with B. theileri sequences previously detected in Rhipicephalus microplus ticks and cattle from Minas Gerais State in Brazil, Rhipicephalus geigyi from Mali, and R. microplus and Haemaphysalis sulcata from Pakistan. This finding contributes to our knowledge regarding susceptible hosts species for B. theileri. More studies are necessary to understand the potential effects of B. theileri on tapir's health.

Latitudinal patterns in stabilizing density dependence of forest communities.

Numerous studies have shown reduced performance in plants that are surrounded by neighbours of the same species1,2, a phenomenon known as conspecific negative density dependence (CNDD)3. A long-held ecological hypothesis posits that CNDD is more pronounced in tropical than in temperate forests4,5, which increases community stabilization, species coexistence and the diversity of local tree species6,7. Previous analyses supporting such a latitudinal gradient in CNDD8,9 have suffered from methodological limitations related to the use of static data10-12. Here we present a comprehensive assessment of latitudinal CNDD patterns using dynamic mortality data to estimate species-site-specific CNDD across 23 sites. Averaged across species, we found that stabilizing CNDD was present at all except one site, but that average stabilizing CNDD was not stronger toward the tropics. However, in tropical tree communities, rare and intermediate abundant species experienced stronger stabilizing CNDD than did common species. This pattern was absent in temperate forests, which suggests that CNDD influences species abundances more strongly in tropical forests than it does in temperate ones13. We also found that interspecific variation in CNDD, which might attenuate its stabilizing effect on species diversity14,15, was high but not significantly different across latitudes. Although the consequences of these patterns for latitudinal diversity gradients are difficult to evaluate, we speculate that a more effective regulation of population abundances could translate into greater stabilization of tropical tree communities and thus contribute to the high local diversity of tropical forests.

Mycorrhizal feedbacks influence global forest structure and diversity.

One mechanism proposed to explain high species diversity in tropical systems is strong negative conspecific density dependence (CDD), which reduces recruitment of juveniles in proximity to conspecific adult plants. Although evidence shows that plant-specific soil pathogens can drive negative CDD, trees also form key mutualisms with mycorrhizal fungi, which may counteract these effects. Across 43 large-scale forest plots worldwide, we tested whether ectomycorrhizal tree species exhibit weaker negative CDD than arbuscular mycorrhizal tree species. We further tested for conmycorrhizal density dependence (CMDD) to test for benefit from shared mutualists. We found that the strength of CDD varies systematically with mycorrhizal type, with ectomycorrhizal tree species exhibiting higher sapling densities with increasing adult densities than arbuscular mycorrhizal tree species. Moreover, we found evidence of positive CMDD for tree species of both mycorrhizal types. Collectively, these findings indicate that mycorrhizal interactions likely play a foundational role in global forest diversity patterns and structure.

Phylogenetic patterns of bioactive secondary metabolites produced by fungal endosymbionts in morning glories (Ipomoeeae, Convolvulaceae).

Heritable fungal endosymbiosis is underinvestigated in plant biology and documented in only three plant families (Convolvulaceae, Fabaceae, and Poaceae). An estimated 40% of morning glory species in the tribe Ipomoeeae (Convolvulaceae) have associations with one of two distinct heritable, endosymbiotic fungi (Periglandula and Chaetothyriales) that produce the bioactive metabolites ergot alkaloids, indole diterpene alkaloids, and swainsonine, which have been of interest for their toxic effects on animals and potential medical applications. Here, we report the occurrence of ergot alkaloids, indole diterpene alkaloids, and swainsonine in the Convolvulaceae; and the fungi that produce them based on synthesis of previous studies and new indole diterpene alkaloid data from 27 additional species in a phylogenetic, geographic, and life-history context. We find that individual morning glory species host no more than one metabolite-producing fungal endosymbiont (with one possible exception), possibly due to costs to the host and overlapping functions of the alkaloids. The symbiotic morning glory lineages occur in distinct phylogenetic clades, and host species have significantly larger seed size than nonsymbiotic species. The distinct and widely distributed endosymbiotic relationships in the morning glory family and their alkaloids provide an accessible study system for understanding heritable plant-fungal symbiosis evolution and their potential functions for host plants.

Phylogenetic Patterns of Swainsonine Presence in Morning Glories.

Endosymbionts play important roles in the life cycles of many macro-organisms. The indolizidine alkaloid swainsonine is produced by heritable fungi that occurs in diverse plant families, such as locoweeds (Fabaceae) and morning glories (Convolvulaceae) plus two species of Malvaceae. Swainsonine is known for its toxic effects on livestock following the ingestion of locoweeds and the potential for pharmaceutical applications. We sampled and tested herbarium seed samples (n = 983) from 244 morning glory species for the presence of swainsonine and built a phylogeny based on available internal transcribed spacer (ITS) sequences of the sampled species. We show that swainsonine occurs only in a single morning glory clade and host species are established on multiple continents. Our results further indicate that this symbiosis developed ∼5 mya and that swainsonine-positive species have larger seeds than their uninfected conspecifics.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Host blood meal identity modifies vector gene expression and competency.

A vector's susceptibility and ability to transmit a pathogen-termed vector competency-determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the tick vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. Rodents and birds are reservoirs for Bb and infect juvenile ticks, while lizards are refractory to Bb and cannot infect feeding ticks. Additionally, the lizard bloodmeal contains borreliacidal properties, clearing previously infected feeding ticks of their Bb infection. Despite I. pacificus feeding on a range of hosts, it is undetermined how the host identity of the larval bloodmeal affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb-infected mice. We found that lizard-fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse-fed larvae. We also conducted the first RNA-seq analysis on whole-bodied I. pacificus and found significant upregulation of tick antioxidants and antimicrobial peptides in the lizard-fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in vector-borne disease transmission and upends prior notions about the role of lizards in Lyme disease community ecology.

Novel genome characteristics contribute to the invasiveness of Phragmites australis (common reed).

The rapid invasion of the non-native Phragmites australis (Poaceae, subfamily Arundinoideae) is a major threat to native wetland ecosystems in North America and elsewhere. We describe the first reference genome for P. australis and compare invasive (ssp. australis) and native (ssp. americanus) genotypes collected from replicated populations across the Laurentian Great Lakes to deduce genomic bases driving its invasive success. Here, we report novel genomic features including a Phragmites lineage-specific whole genome duplication, followed by gene loss and preferential retention of genes associated with transcription factors and regulatory functions in the remaining duplicates. Comparative transcriptomic analyses revealed that genes associated with biotic stress and defence responses were expressed at a higher basal level in invasive genotypes, but native genotypes showed a stronger induction of defence responses when challenged by a fungal endophyte. The reference genome and transcriptomes, combined with previous ecological and environmental data, add to our understanding of mechanisms leading to invasiveness and support the development of novel, genomics-assisted management approaches for invasive Phragmites.

Distribution, Dynamics, and Diversity of Questing Ticks in the Lower Midwest.

We investigated the spatiotemporal patterns of medically important tick species in southcentral Indiana in 2018 and 2019. In recent decades, both Amblyomma americanum (Acari: Ixodidae) (Linnaeus) and Ixodes scapularis (Acari: Ixodidae) (Say) have been expanding their ranges into the Midwest. We report updates to the status of A. americanum, I. scapularis, and Dermacentor variabilis (Acari: Ixodidae) (Say) across 13 counties in southern and south-central Indiana. We found more northerly establishment of A. americanum and more widespread establishment of I. scapularis than previously reported in the literature. We also provide the first report of Amblyomma maculatum (Acari: Ixodidae) (Koch) in Indiana. Using negative binomial regression, we observed significant spatial variation among sampling sites in A. americanum adults and nymphs, D. variabilis adults, and I. scapularis nymphs. Further, we found higher densities of A. americanum nymphs and I. scapularis adults in more forested areas as opposed to less forested, more developed areas. We did not find significant interannual variation in tick densities for any species. More southern sites featured higher densities of A. americanum adults and nymphs, D. variabilis adults, and I. scapularis nymphs than more northern sites. Seasonally, we observed the highest peaks in tick activity in the spring and early summer, but I. scapularis adults were most common in the early spring and fall. Average adult A. americanum and adult D. variabilis densities were positively correlated at our sites. Our results reflect the northward expansion of A. americanum in the Lower Midwest, as well as the increasing geographic prevalence of I. scapularis in the region.

Diversification of ergot alkaloids and heritable fungal symbionts in morning glories.

Heritable microorganisms play critical roles in life cycles of many macro-organisms but their prevalence and functional roles are unknown for most plants. Bioactive ergot alkaloids produced by heritable Periglandula fungi occur in some morning glories (Convolvulaceae), similar to ergot alkaloids in grasses infected with related fungi. Ergot alkaloids have been of longstanding interest given their toxic effects, psychoactive properties, and medical applications. Here we show that ergot alkaloids are concentrated in four morning glory clades exhibiting differences in alkaloid profiles and are more prevalent in species with larger seeds than those with smaller seeds. Further, we found a phylogenetically-independent, positive correlation between seed mass and alkaloid concentrations in symbiotic species. Our findings suggest that heritable symbiosis has diversified among particular clades by vertical transmission through seeds combined with host speciation, and that ergot alkaloids are particularly beneficial to species with larger seeds. Our results are consistent with the defensive symbiosis hypothesis where bioactive ergot alkaloids from Periglandula symbionts protect seeds and seedlings from natural enemies, and provide a framework for exploring microbial chemistry in other plant-microbe interactions.

Conspecific leaf litter induces negative feedbacks in Asteraceae seedlings.

The plant soil feedback (PSF) framework has been instrumental in understanding the impacts of soil microbes on plant fitness and species coexistence. PSFs develop when soil microbial communities are altered due to the identity and density of a particular plant species, which can then enhance or inhibit the local survival and growth of that plant species as well as different plant species. The recent extension of the PSF framework to aboveground microbiota, termed here as plant phyllosphere feedbacks (PPFs), can also help to determine the impact of aboveground microbes on plant fitness and species interactions. However, experimental tests of PPFs during early plant growth are nascent and the prevalence of PPFs across diverse plant species remains unknown. Additionally, it is unclear whether plant host characteristics, such as functional traits or phylogenetic distance, may help to predict the strength and direction of PPFs. To test for the prevalence of litter-mediated PPFs, recently senesced plant litter from 10 native Asteraceae species spanning a range of life history strategies was used to inoculate seedlings of both conspecific and heterospecific species. We found that exposure to conspecific litter significantly reduced the growth of four species relative to exposure to heterospecific litter (i.e., significant negative PPFs), three species experienced marginally significant negative PPFs, and the PPF estimates for all 10 species were negative. However, neither plant functional traits, nor phylogenetic distance were predictive of litter feedbacks across plant species pairs, suggesting that other mechanisms or traits not measured may be driving conspecific negative PPFs. Our results indicate that negative, litter-mediated PPFs are common among native Asteraceae species and that they may have substantial impacts on plant growth and plant species interactions, particularly during early plant growth.

Using convolutional neural networks for tick image recognition - a preliminary exploration.

Smartphone cameras and digital devices are increasingly used in the capture of tick images by the public as citizen scientists, and rapid advances in deep learning and computer vision has enabled brand new image recognition models to be trained. However, there is currently no web-based or mobile application that supports automated classification of tick images. The purpose of this study was to compare the accuracy of a deep learning model pre-trained with millions of annotated images in Imagenet, against a shallow custom-build convolutional neural network (CNN) model for the classification of common hard ticks present in anthropic areas from northeastern USA. We created a dataset of approximately 2000 images of four tick species (Ixodes scapularis, Dermacentor variabilis, Amblyomma americanum and Haemaphysalis sp.), two sexes (male, female) and two life stages (adult, nymph). We used these tick images to train two separate CNN models - ResNet-50 and a simple shallow custom-built. We evaluated our models' performance on an independent subset of tick images not seen during training. Compared to the ResNet-50 model, the small shallow custom-built model had higher training (99.7%) and validation (99.1%) accuracies. When tested with new tick image data, the shallow custom-built model yielded higher mean prediction accuracy (80%), greater confidence of true detection (88.7%) and lower mean response time (3.64 s). These results demonstrate that, with limited data size for model training, a simple shallow custom-built CNN model has great prospects for use in the classification of common hard ticks present in anthropic areas from northeastern USA.

Large-spored Drechslera gigantea is a Bipolaris species causing disease on the invasive grass Microstegium vimineum.

Environmentally damaging invasive plants can also serve as reservoir hosts for agricultural pathogens. Microstegium vimineum is an invasive C4 annual grass that is present throughout the midwestern and eastern United States. It can reach high densities in disturbed areas such as crop-forest interfaces, which creates the potential for pathogen spillover from M. vimineum to agricultural crops and native plants. A previous study that surveyed disease on M. vimineum found a large-spored Bipolaris species that was widespread on M. vimineum and also isolated from co-occurring native grasses. Here, we report that the large-spored fungus isolated from M. vimineum and the native grass Elymus virginicus is Drechslera gigantea, based on comparison with published descriptions of morphological traits, and establish that D. gigantea is a pathogen of M. vimineum and E. virginicus. We review the phylogenetic placement and taxonomic history of D. gigantea and propose that it be reassigned to the genus Bipolaris as Bipolaris gigantea.

Disease in Invasive Plant Populations.

Non-native invasive plants can establish in natural areas, where they can be ecologically damaging and costly to manage. Like cultivated plants, invasive plants can experience a relatively disease-free period upon introduction and accumulate pathogens over time. Diseases of invasive plant populations are infrequently studied compared to diseases of agriculture, forestry, and even native plant populations. We evaluated similarities and differences in the processes that are likely to affect pathogen accumulation and disease in invasive plants compared to cultivated plants, which are the dominant focus of the field of plant pathology. Invasive plants experience more genetic, biotic, and abiotic variation across space and over time than cultivated plants, which is expected to stabilize the ecological and evolutionary dynamics of interactions with pathogens and possibly weaken the efficacy of infectious disease in their control. Although disease is expected to be context dependent, the widespread distribution of invasive plants makes them important pathogen reservoirs. Research on invasive plant diseases can both protect crops and help manage invasive plant populations.

Foliar fungal endophyte community structure is independent of phylogenetic relatedness in an Asteraceae common garden.

Phylogenetic distance among host species represents a proxy for host traits that act as biotic filters to shape host-associated microbiome community structure. However, teasing apart potential biotic assembly mechanisms, such as host specificity or local species interactions, from abiotic factors, such as environmental specificity or dispersal barriers, in hyperdiverse, horizontally transmitted microbiomes remains a challenge. In this study, we tested whether host phylogenetic relatedness among 18 native Asteraceae plant species and spatial distance between replicated plots in a common garden affects foliar fungal endophyte (FFE) community structure. We found that FFE community structure varied significantly among host species, as well as host tribes, but not among host subfamilies. However, FFE community dissimilarity between host individuals was not significantly correlated with phylogenetic distance between host species. There was a significant effect of spatial distance among host individuals on FFE community dissimilarity within the common garden. The significant differences in FFE community structure among host species, but lack of a significant host phylogenetic effect, suggest functional differences among host species not accounted for by host phylogenetic distance, such as metabolic traits or phenology, may drive FFE community dissimilarity. Overall, our results indicate that host species identity and the spatial distance between plants can determine the similarity of their microbiomes, even across a single experimental field, but that host phylogeny is not closely tied to FFE community divergence in native Asteraceae.

Decreased Root-Knot Nematode Gall Formation in Roots of the Morning Glory Ipomoea tricolor Symbiotic with Ergot Alkaloid-Producing Fungal Periglandula Sp.

Many species of morning glories (Convolvulaceae) form symbioses with seed-transmitted Periglandula fungal endosymbionts, which produce ergot alkaloids and may contribute to defensive mutualism. Allocation of seed-borne ergot alkaloids to various tissues of several Ipomoea species has been demonstrated, including roots of I. tricolor. The goal of this study was to determine if infection of I. tricolor by the Periglandula sp. endosymbiont affects Southern root-knot nematode (Meloidogyne incognita) gall formation and host plant biomass. We hypothesized that I. tricolor plants infected by Periglandula (E+) would develop fewer nematode-induced galls compared to non-symbiotic plants (E-). E+ or E- status of plant lines was confirmed by testing methanol extracts from individual seeds for endosymbiont-produced ergot alkaloids. To test the effects of Periglandula on nematode colonization, E+ and E- I. tricolor seedlings were grown in soil infested with high densities of M. incognita nematodes (N+) or no nematodes (N-) for four weeks in the greenhouse before harvesting. After harvest, nematode colonization of roots was visualized microscopically, and total gall number and plant biomass were quantified. Four ergot alkaloids were detected in roots of E+ plants, but no alkaloids were found in E- plants. Gall formation was reduced by 50% in E+ plants compared to E- plants, independent of root biomass. Both N+ plants and E+ plants had significantly reduced biomass compared to N- and E- plants, respectively. These results demonstrate Periglandula's defensive role against biotic enemies, albeit with a potential trade-off with host plant growth.

Biodiversity of Convolvulaceous species that contain Ergot Alkaloids, Indole Diterpene Alkaloids, and Swainsonine.

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n=30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.

Foliar endophytic fungi alter patterns of nitrogen uptake and distribution in Theobroma cacao.

Colonization by foliar endophytic fungi can affect the expression of host plant defenses and other ecologically important traits. However, whether endophyte colonization affects the uptake or redistribution of resources within and among host plant tissues remains unstudied. We inoculated leaves of Theobroma cacao with four common colonizers that range in their effect from protective to pathogenic (Colletotrichum tropicale, Pestalotiopsis sp., Colletotrichum theobromicola, or Phytophthora palmivora). We pulsed the soil with nitrogen-15 (15 N) and then traced 15 N uptake and its subsequent distribution to whole plants and individual leaves. At a whole-plant level, C. tropicale-inoculated plants showed significantly greater 15 N uptake than endophyte-free plants did in the same pot. Among leaves within plants, younger leaves were particularly enriched in 15 N, but endophyte inoculation at the individual leaf level did not alter 15 N distribution within plants. However, leaves co-inoculated with pathogenic Phytophthora and protective C. tropicale experienced significantly elevated 15 N content as pathogen damage increased, compared with leaves inoculated only with the pathogen. Further, endophyte-pathogen co-infection also increased total plant biomass. Our results indicate that colonization by foliar endophytes significantly affects N uptake and distribution among and within host plants in ways that appear to be context dependent on other microbiome components.

Foliar fungal endophyte communities are structured by environment but not host ecotype in Panicum virgatum (switchgrass).

Experimental tests of community assembly mechanisms for host-associated microbiomes in nature are lacking. Asymptomatic foliar fungal endophytes are a major component of the plant microbiome and are increasingly recognized for their impacts on plant performance, including pathogen defense, hormonal manipulation, and drought tolerance. However, it remains unclear whether fungal endophytes preferentially colonize certain host ecotypes or genotypes, reflecting some degree of biotic adaptation in the symbioses, or whether colonization is simply a function of spore type and abundance within the local environment. Whether host ecotype, local environment, or some combination of both controls the pattern of microbiome formation across hosts represents a new dimension to the age-old debate of nature versus nurture. Here, we used a reciprocal transplant design to explore the extent of host specificity and biotic adaptation in the plant microbiome, as evidenced by differential colonization of host genetic types by endophytes. Specifically, replicate plants from three locally-adapted ecotypes of the native grass Panicum virgatum (switchgrass) were transplanted at three geographically distinct field sites (one home and two away) in the Midwestern US. At the end of the growing season, plant leaves were harvested and the fungal microbiome characterized using culture-dependent sequencing techniques. Our results demonstrated that fungal endophyte community structure was determined by local environment (i.e., site), but not by host ecotype. Fungal richness and diversity also strongly differed by site, with lower fungal diversity at a riparian field site, whereas host ecotype had no effect. By contrast, there were significant differences in plant phenotypes across all ecotypes and sites, indicating ecotypic differentiation of host phenotype. Overall, our results indicate that environmental factors are the primary drivers of community structure in the switchgrass fungal microbiome.

Response to Comment on "Plant diversity increases with the strength of negative density dependence at the global scale".

Hülsmann and Hartig suggest that ecological mechanisms other than specialized natural enemies or intraspecific competition contribute to our estimates of conspecific negative density dependence (CNDD). To address their concern, we show that our results are not the result of a methodological artifact and present a null-model analysis that demonstrates that our original findings-(i) stronger CNDD at tropical relative to temperate latitudes and (ii) a latitudinal shift in the relationship between CNDD and species abundance-persist even after controlling for other processes that might influence spatial relationships between adults and recruits.