Genetic architecture of heritable leaf microbes.

Host-associated microbiomes are shaped by both their environment and host genetics, and often impact host performance. The scale of host genetic variation important to microbes is largely unknown yet fundamental to the community assembly of host-associated microbiomes, with implications for the eco-evolutionary dynamics of microbes and hosts. Using Ipomoea hederacea, ivyleaf morning glory, we generated matrilines differing in quantitative genetic variation and leaf shape, which is controlled by a single Mendelian locus. We then investigated the relative roles of Mendelian and quantitative genetic variation in structuring the leaf microbiome and how these two sources of genetic variation contributed to microbe heritability. We found that despite large effects of the environment, both Mendelian and quantitative genetic host variation contribute to microbe heritability and that the cumulative small effect genomic differences due to matriline explained as much or more microbial variation than a single large effect Mendelian locus. Furthermore, our results are the first to suggest that leaf shape itself contributes to variation in the abundances of some phyllosphere microbes.IMPORTANCEWe investigated how host genetic variation affects the assembly of Ipomoea hederacea's natural microbiome. We found that the genetic architecture of leaf-associated microbiomes involves both quantitative genetic variation and Mendelian traits, with similar contributions to microbe heritability. The existence of Mendelian and quantitative genetic variation for host-associated microbes means that plant evolution at the leaf shape locus or other quantitative genetic loci has the potential to shape microbial abundance and community composition.

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.

Microbial community overlap between the phyllosphere and rhizosphere of three plants from Yongxing Island, South China Sea.

Phyllosphere and rhizosphere are unique and wide-ranging habitats that harbor various microbial communities, which influence plant growth and health, and the productivity of the ecosystems. In this study, we characterized the shared microbiome of the phyllosphere and rhizosphere among three plants (Ipomoea pes-caprae, Wedelia chinensis, and Cocos nucifera), to obtain an insight into the relationships between bacteria (including diazotrophic bacteria) and fungi, present on these host plants. Quantitative PCR showed that the abundances of the microbiome in the soil samples were significantly higher than those in the phyllosphere samples, though there was an extremely low abundance of fungi in bulk soil. High-throughput sequencing showed that the alpha-diversity of bacteria and fungi was higher in the rhizosphere than the phyllosphere samples associated with the same plant, while there was no obvious shift in the alpha-diversity of diazotrophic communities between all the tested phyllosphere and soil samples. Results of the microbial composition showed that sample-specific bacteria and fungi were found among the phyllosphere and rhizosphere of the different host plants. About 10%-27% of bacteria, including diazotrophs, and fungi overlapped between the phyllosphere and the rhizosphere of these host plants. No significant difference in microbial community structure was found among the tested rhizosphere samples, and soil properties had a higher influence on the soil microbial community structures than the host plant species.

Mixotrophic acidophiles increase cadmium soluble fraction and phytoextraction efficiency from cadmium contaminated soils.

A profound concern in developing microbially-assisted phytoextraction is that introduced microbes not only remove heavy metals from contaminated soils but also enhance metal uptake into plant tissues from the treated soils. Cadmium (Cd) removal efficiencies were compared after leaching with deionized water (CK), acidified basal salts medium (acid control), cell-free spent medium (spent bioleaching) and mixotrophic acidophiles (two-step bioleaching). Two-step bioleaching using the mixotrophic acidophiles removed 34% of total Cd and 87% of available Cd, significantly more than CK (3% and 4%), acid control (12% and 51%) and spent bioleaching (26% and 75%). Pot experiments of water spinach growing in four treated soils were conducted to evaluate the Cd uptake performance in plants. Notably, the mixotrophic acidophiles increased Cd concentration in plant tissues by 78% compared to the CK group. More interestingly, the mixotrophic acidophiles were not colonized in soils but caused the compositional increase of indigenous microbes such as the genera of Alicyclobacillus, Clostridium sensu strict and Streptacidiphilus. Meanwhile, two-step bioleaching had little effects on soil structure and physicochemical properties determined by the spectroscopy characteristics analysis. These results implied that the mixotrophic acidophiles facilitated the development of microbially-assisted phytoextraction technology.

Wickerhamiella dianesei f.a., sp. nov. and Wickerhamiella kurtzmanii f.a., sp. nov., two yeast species isolated from plants and insects.

Six yeast strains representing two novel Wickerhamiella species were isolated from plants and insects collected in Costa Rica, Brazil, and French Guiana. They belong to a subclade containing Wickerhamiella domercqiae and Wickerhamiella bombiphila, and differ by approximately 12 % in the D1/D2 sequences of the large subunit rRNA gene from these species. The intergenic spacer (ITS) regions of the two novel species differ by around 19 and 27 %, respectively, from those of W. domercqiae. The novel species exhibit 5 % divergence in the D1/D2 sequences among them (around 4 % in the ITS). The names Wickerhamiella dianesei f.a., sp. nov. and Wickerhamiella kurtzmanii f.a., sp. nov. are proposed to accommodate these species, for which a sexual cycle has not been observed. Wickerhamiella dianesei was isolated from the stingless bee, Trigona fulviventris, collected in an Asteraceae flower in Costa Rica, and from leaves of Sabicea brasiliensis (Rubiaceae) and a flower of Byrsonima crassifolia (Malpighiaceae) in Brazil. Wickerhamiellsa kurtzmanii was isolated from a flower of Ipomoea batatoides (Convolvulaceae) in Costa Rica, the surface of a fruit of B. crassifolia in Brazil, and flowers in French Guiana. The type strains are Wickerhamiella dianesei UWOPS 00-107.1T (=CBS 14185=NRRL Y-63789; Mycobank number MB 827008) and Wickerhamiella kurtzmanii UWOPS 00-192.1T (=CBS 15383=NRRL Y-63979; MB 827011).

Effect of inoculation with arbuscular mycorrhizal fungi and blanching on the bioaccessibility of heavy metals in water spinach (Ipomoea aquatica Forsk.).

A plant's tolerance to heavy metals (HMs) and its detoxification mechanisms are associated with the subcellular distribution of HMs and their chemical forms. In this study, water spinach (Ipomoea aquatica Forsk.) was grown in two soils contaminated with a single HM (cadmium, Cd) or combined HMs (Cd and nickel, Ni). Inoculation of arbuscular mycorrizal fungi (AMF) was conducted to increase the accumulation of phosphorus (P) in plants. One major exception was to decrease the migration and accumulation of HMs in edible parts by the formation of P-HM complexes. The effects of blanching and simulated digestion on bioaccessibility were also assessed. The experimental results showed that the water spinach species used in this study had a high capacity to accumulate HMs. AMF treatment improved water spinach growth and decreased the accumulation of Ni but not that of Cd. Soluble and inorganic Cd and Ni were the major subcellular fractions and chemical forms in water spinach; these two HMs also exhibited higher migration capacities in comparison to chromium (Cr). Relative to raw tissues, 45-84% of Cd, Cr, and Ni were leached after blanching. Approximately 32-55%, 16-50%, and 27-40% of Cd, Cr, and Ni, respectively, were bioaccessible and could be metabolized by in vitro digestive fluids.

Ergot Alkaloids and their Hallucinogenic Potential in Morning Glories.

Naturally occurring and semisynthetic ergot alkaloids play a role in health care or as recreational drugs in Western and indigenous Mexican societies. Evidence is summarized that ergot alkaloids present in Central American Convolvulaceae like Turbina corymbosa, Ipomoea violacea, and Ipomoea asarifolia are colonized by different species of a newly described clavicipitaceous fungal genus named Periglandula. The fungi are associated with peltate glandular trichomes on the adaxial leaf surface of its host plants. The Periglandula fungi are not yet culturable in vitro but were demonstrated to have the capacity to synthesize ergot alkaloids. The alkaloids do not remain in the fungal mycelium but are translocated via the glandular trichomes into their plant host. Both fungi and host benefit from a symbiotic lifestyle. In evolutionary terms the alkaloid biosynthetic gene cluster in the Periglandula/Ipomoea symbiosis is likely to have a conserved (basic) structure while biosynthetic ergot gene clusters within the genera Claviceps and Epichloe were under ecological selection for alkaloid diversification.

Evolution of host range in Coleosporium ipomoeae, a plant pathogen with multiple hosts.

Plants and their pathogens coevolve locally. Previous investigations of one host-one pathogen systems have demonstrated that natural selection favors pathogen genotypes that are virulent on a broad range of host genotypes. In the present study, we examine a system consisting of one pathogen species that infects three host species in the morning glory genus Ipomoea. We show that many pathogen genotypes can infect two or three of the host species when tested on plants from nonlocal communities. By contrast, pathogen genotypes are highly host-specific, infecting only one host species, when tested on host species from the local community. This pattern indicates that within-community evolution narrows the host breadth of pathogen genotypes. Possible evolutionary mechanisms include direct selection for narrow host breadth due to costs of virulence and evolution of ipomoea resistance in the host species.

Remediation of textile azo dye acid red 114 by hairy roots of Ipomoea carnea Jacq. and assessment of degraded dye toxicity with human keratinocyte cell line.

Bioremediation has proven to be the most desirable and cost effective method to counter textile dye pollution. Hairy roots (HRs) of Ipomoea carnea J. were tested for decolourization of 25 textile azo dyes, out of which >90% decolourization was observed in 15 dyes. A diazo dye, Acid Red 114 was decolourized to >98% and hence, was chosen as the model dye. A significant increase in the activities of oxidoreductive enzymes was observed during decolourization of AR114. The phytodegradation of AR114 was confirmed by HPLC, UV-vis and FTIR spectroscopy. The possible metabolites were identified by GCMS as 4- aminobenzene sulfonic acid 2-methylaniline and 4- aminophenyl 4-ethyl benzene sulfonate and a probable pathway for the biodegradation of AR114 has been proposed. The nontoxic nature of the metabolites and toxicity of AR114 was confirmed by cytotoxicity tests on human keratinocyte cell line (HaCaT). When HaCaT cells were treated separately with 150 µg mL(-1) of AR114 and metabolites, MTT assay showed 50% and ≈100% viability respectively. Furthermore, flow cytometry data showed that, as compared to control, the cells in G2-M and death phase increased by 2.4 and 3.6 folds respectively on treatment with AR114 but remained unaltered in cells treated with metabolites.

Kodamaea transpacifica f.a., sp. nov., a yeast species isolated from ephemeral flowers and insects in the Galapagos Islands and Malaysia: further evidence for ancient human transpacific contacts.

Twenty-four yeast strains were isolated from ephemeral flowers of Ipomoea spp. and Datura sp. and their associated insects in the Galápagos Archipelago, Ecuador, and from Ipomoea spp. and associated insects in the Cameron Highlands, Malaysia. Sequences of the D1/D2 domains of the large subunit rRNA gene indicated that these strains belong to a novel yeast species of the Kodamaea clade, although the formation of ascospores was not observed. The closest relative is Candida restingae. The human-mediated dispersion of this species by transpacific contacts in ancient times is suggested. The name Kodamaea transpacifica f.a., sp. nov. is proposed to accommodate these isolates. The type strain is CLQCA-24i-070(T) ( = CBS 12823(T) = NCYC 3852(T)); MycoBank number MB 803609.

Differential allocation of seed-borne ergot alkaloids during early ontogeny of morning glories (Convolvulaceae).

Ergot alkaloids are mycotoxins that can increase host plant resistance to above- and below-ground herbivores. Some morning glories (Convolvulaceae) are infected by clavicipitaceous fungi (Periglandula spp.) that produce high concentrations of ergot alkaloids in seeds-up to 1000-fold greater than endophyte-infected grasses. Here, we evaluated the diversity and distribution of alkaloids in seeds and seedlings and variation in alkaloid distribution among species. We treated half the plants with fungicide to differentiate seed-borne alkaloids from alkaloids produced de novo post-germination and sampled seedling tissues at the cotyledon and first-leaf stages. Seed-borne alkaloids in Ipomoea amnicola, I. argillicola, and I. hildebrandtii remained primarily in the cotyledons, whereas I. tricolor allocated lysergic acid amides to the roots while retaining clavines in the cotyledons. In I. hildebrandtii, almost all festuclavine was found in the cotyledons. These observations suggest differential allocation of individual alkaloids. Intraspecific patterns of alkaloid distribution did not vary between fungicide-treated and control seedlings. Each species contained four to six unique ergot alkaloids and two species had the ergopeptine ergobalansine. De novo production of alkaloids did not begin immediately, as total alkaloids in fungicide-treated and control seedlings did not differ through the first-leaf stage, except in I. argillicola. In an extended time-course experiment with I. tricolor, de novo production was detected after the first-leaf stage. Our results demonstrate that allocation of seed-borne ergot alkaloids varies among species and tissues but is not altered by fungicide treatment. This variation may reflect a response to selection for defense against natural enemies.

Production of the alkaloid swainsonine by a fungal endosymbiont of the Ascomycete order Chaetothyriales in the host Ipomoea carnea.

Some plant species within the Convolvulaceae (morning glory family) from South America, Africa, and Australia cause a neurologic disease in grazing livestock caused by swainsonine. These convolvulaceous species including Ipomoea carnea contain the indolizidine alkaloid swainsonine, an inhibitor of α-mannosidase and mannosidase II, and polyhydroxy nortropane alkaloids, the calystegines which are glycosidase inhibitors. Swainsonine has been shown to be produced by a fungal endosymbiont in legumes of the Astragalus and Oxytropis genera, where it causes a similar neurologic disease in grazing livestock called locoism. Here we demonstrate that I. carnea plants are infected with a fungal endosymbiont that was cultured from its seeds and which produced swainsonine in pure culture but not the calystegines. The same fungal endosymbiont was detected by PCR and by culturing in I. carnea plants containing swainsonine. The fungal endosymbiont belongs to the Ascomycete order Chaetothyriales. Plants derived from fungicide-treated seeds lacked swainsonine, but calystegine concentrations were unaltered.

Structural elucidation of the exopolysaccharide produced by fungus Fusarium oxysporum Y24-2.

The extracellular polysaccharide FO1 was isolated from the fermentation broth of an endophytic fungus (Fusarium oxysporum) of Ipomoea pes-caprae. Its structural characteristics were studied by chemical and methylation analyses, and 1D and 2D (1)H and (13)C NMR spectroscopy. Results indicated that this exopolysaccharide consists of a disaccharide repeating unit with the following structure (n≈111): [→2)-ß-D-Galf(1→6)-α-D-Glcp(1→](n).

Occurrence of white rust (Albugo ipomoeae-panduratae) on Ipomoea acuminata in the brazilian mid-west

Spontaneous plants of Ipomoea acuminata ("morning glory") exhibiting white rust pustules were found in a field crop area of Planaltina, DF, in the fall season of 2010 and the disease causal agent was identified as Albugo ipomoea-panduratae (Oomycota). No reports of the association between A. ipomoea-panduratae and I. acuminata were known in Brazil previously to 2010. A reference specimen was deposited at the University of Brasilia Mycological Reference Collection.

Genetics of resistance to the rust fungus Coleosporium ipomoeae in three species of morning glory (Ipomoea).

We examined the genetic basis of resistance to the rust pathogen Coleosporium ipomoea in three host species: Ipomoea purpurea, I. hederacea, and I. coccinea (Convolvulaceae). In crosses between resistant and susceptible individuals, second-generation selfed offspring segregated in ratios that did not differ statistically from the 3:1 ratio indicative of single-gene resistance with the resistant allele dominant. One out of three crosses between resistant individuals from two different populations revealed that resistance loci differed in the two populations, as evidenced by the production of susceptible individuals among the S(2) generation. These results suggest that gene-for-gene interactions contribute substantially to the dynamics of coevolution in this natural pathosystem. They also suggest that evolution of resistance to the same pathogen strain may involve different loci in different Ipomoea populations.

Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae.

We describe two newly discovered fungi living on the adaxial leaf surface of plants belonging to the Convolvulaceae, Ipomoea asarifolia and Turbina corymbosa. The fungi apparently are epibionts because hyphae were never observed to penetrate epidermal cells or stomata of their respective host plants, and most remarkably are intimately associated with secretory glands on the leaf surface. Hyphae and structures resembling chlamydospores and synnemata (but lacking conidia), formed by both fungal species are phenotypically nearly indistinguishable after in vitro growth or when examined in vivo on the leaf surface. Phylogenetic trees based on aligned DNA sequences from nuclear genes for ß-tubulin (tubB) and RNA Polymerase II subunit 1 (rpbA), and the mitochondrial gene for ATP synthase F0 subunit A (atp6), grouped the fungal species in a clade within the Clavicipitaceae. Clavicipitaceous fungi isolated from the two different plant species could be distinguished by their atp6 and rpbA sequences, and nuclear genes for γ-actin (actG), translation elongation factor 1-α (tefA), and 4-(γ,γ-dimethylallyl)tryptophan synthase (dmaW), the determinant step in ergoline (i.e. ergot) alkaloid biosynthesis. Based on these findings we propose a new fungal genus, Periglandula, gen. nov., and describe two new species, Periglandula ipomoeae sp. nov., from host plant I. asarifolia, and Periglandula turbinae sp. nov., from T. corymbosa.

Morning glory as a powerful model in ecological genomics: tracing adaptation through both natural and artificial selection.

Many diverse questions in ecology and evolution have been addressed using species belonging to the genus Ipomoea, commonly referred to as the morning glory genus. Ipomoea exhibits a wide range of diversity in floral color, growth form, mating system and tolerance to environmental factors, both within and among species, and as such has been a focal group of many investigations in the last 80 years. In this review, we highlight recent work to which Ipomoea species have contributed-from studies of the mating system, molecular evolution, plant-herbivore and plant-parasite interactions to their impact on and importance to agriculture. Genomic resources for this group are currently under development, and given the breadth of studies and history of this group, combined with an expanding genetics toolkit, we argue that Ipomoea should provide the next model organism for ecological genomics.

Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes.

Some common floating aquatic macrophytes could remove nutrients, such as nitrogen, from eutrophic water. However, the relationship between these macrophytes and the ammonia-oxidizing microorganisms on their rhizoplanes is still unknown. In this study, we examined communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) on the rhizoplanes of common floating aquatic macrophytes (Eichhornia crassipes, Pistia stratiotes and Ipomoea aquatic) in a eutrophic reservoir.The results show that AOB were the predominant ammonia-oxidizer on the three rhizoplanes. The principal AOB were Nitrosomonas europaea and Nitrosomonas ureae clades. The principal group of AOA was most similar to the clone from activated sludge. The ratio of AOB amoA gene copies to AOA varied from 1.36 (on E. crassipes) to 41.90 (on P. stratiotes). Diversity of AOA was much lower than that of AOB in most samples, with the exception of P. stratiotes.

Candida golubevii sp. nov., an asexual yeast related to Metschnikowia lunata.

Two strains of a novel yeast species were isolated from insect frass and a flower in Thailand and Brazil, respectively. The strain from Thailand was isolated from insect frass collected in Than-Tong waterfall, Nong Khai Province, whereas the strain from Brazil was recovered from a flower of Ipomoea sp. collected on the banks of the Paraguai River in the state of Mato Grosso do Sul. The sequences of the D1/D2 domains of the large subunit of the rDNA of both strains were identical. This novel species belonged to the Metschnikowia clade and was related to Metschnikowia lunata. No signs of sporulation were observed for the two strains on various culture media. The novel species, Candida golubevii sp. nov., is proposed to accommodate these isolates. The type strain is BCC 8332(T) (=CBS 11362(T)=NBRC 105679(T)).

Community of arbuscular mycorrhizal fungi in a coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum under salt-treated conditions.

Community of arbuscular mycorrhizal (AM) fungi in a coastal vegetation on Okinawa island in Japan was examined. A sampling plot was established in a colony of Ipomoea pes-caprae (Convolvulaceae) on the beach in Tamagusuku, Okinawa Pref, in which eight root samples of I. pes-caprae and three root samples each of Vigna marina (Leguminosae) and Paspalum distichum (Poaceae) were collected. Partial 18S rDNA of AM fungi was amplified from the root samples by polymerase chain reaction (PCR) with primers NS31 and AM1. Restriction fragment length polymorphism analysis with HinfI and RsaI for cloned PCR products revealed that two types of Glomus sp., type A and type B, were dominant in the colony. Among them, the fungi of type A were especially dominant near the edge of the colony facing the sea. A phylogenetic analysis showed that the AM fungi of type B are closely related to Glomus intraradices and those of type A are nearly related to type B. From the sequence data, it was also found that type A was further divided into two types, type A1 and A2. One representative strain each of the three types, type A1, A2, and B, propagated from single spore each, was examined for the growth of sorghum (Sorghum bicolor) at three different salinity levels, 0, 100, and 200 mM NaCl. At the non-salt-treated condition, the type B fungus was the most effective on shoot growth enhancement of the host plant, whereas at the salt-treated conditions, the type A2 fungus was the most effective. An efficient suppression of Na + translocation into the shoot by the examined AM fungi was found. These results suggested that the AM fungi dominant near the sea are adapted to salt-stressed environment to alleviate the salt stress of host plants.