See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/322908252

The Periglandula and Its Symbiotum with Morning Glory Plants ()

Chapter · February 2018 DOI: 10.1007/978-3-319-71740-1_5

CITATIONS READS 4 555

2 authors, including:

Eckhard Walter Leistner, My name is not Leister!!!!!!!!! University of Bonn

220 PUBLICATIONS 3,333 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Dear Suvarna, I am retired since 10 years. I cannot do any research anymore. I am reading a little bit and write reviews some time. Best wishes Eckhard Leistner View project

Ginkgo biloba View project

All content following this page was uploaded by Eckhard Walter Leistner, My name is not Leister!!!!!!!!! on 23 March 2018.

The user has requested enhancement of the downloaded file. The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae)

1 2 E. LEISTNER ,U.STEINER

CONTENTS characteristic of a limited amount of microbial or I. The Ecological Role of Natural plant taxa, e.g., an order, a family, a species, or Products ...... 131 even a subspecies only. Many of the natural pro- II. The Symbiosis Between Poaceae and ducts exhibit physiological activities which is the Clavicipitaceous Fungi ...... 132 basis for their use in medical applications III. Epibiotic Clavicipitaceous Fungi Associated with Convolvulaceae ...... 133 (Clardy and Walsh 2004). A. Identification of Genus Periglandula ...... 133 The high physiological activities of many 1. Microscopic and Electron Microscopic natural products had triggered a now historical Characterization ...... 133 dispute about the role of natural products in the 2. Phylogenetic Trees and . . . . . 136 producing organism. It was proposed that “the B. Seed Transmittance of Epibiotic Fungi Colonizing Convolvulaceae ...... 137 multiplicity of natural products is caused by ran- C. Plant Growth Under Germfree Conditions 139 dom processes of mutations, i.e. it reflects the D. Biosynthesis and Accumulation of gambling of nature rather than a sophisticated Alkaloids in the /Plant strategy” (Mothes 1981;Mothesetal.1985). Symbiotum ...... 141 This hypothesis, however, neglects the pos- E. Periglandula and the Evolution of the Ergot Biosynthetic Pathways ...... 142 sibility that mutations may turn out to be det- IV. Additional Fungus/Plant Symbiota in rimental or advantageous to the mutated Dicotyledonous Plants ...... 143 organism. In the former case, a mutated organ- V. Conclusions ...... 144 ism may be eliminated or in the latter case References ...... 145 benefit from an increased fitness and a better chance to survive in a certain ecological setting (Zenk 1967). Today the ecological role of natu- I. The Ecological Role of Natural ral products is well accepted in the scientific Products community (Harborne 2004; Eisner 2003; White Jr et al. 2003). Natural product research entered a new era Microorganisms and plants have one thing in when it was discovered that plants and fungi common: both are frequently equipped with an elaborated during evolution, another way to elaborate biosynthetic machinery responsible for acquire natural products. They may not only the formation of an almost unlimited variety of be formed in biosynthetic processes by one natural products. Typically, natural products— particular organism itself, but instead, a host which are also called secondary metabolites—are organism may harbor a natural product-pro- ducing microorganism: A plant may be asso- 1 Institut fu¨r Pharmazeutische Biologie, Rheinische Friedrich ciated with a bacterium (Piel 2004; Strobel et al. Wilhelm-Universita¨t Bonn, Bonn, Germany; e-mail: eleist- 2004; Gunatilaka 2006) or a fungus (Strobel et [email protected] 2 Institut fu¨r Nutzpflanzenwissenschaften und Ressour- al. 2004; Gunatilaka 2006), while a fungus may censchutz (INRES), Rheinische Friedrich Wilhelm-Universita¨t harbor a bacterium (Partida-Martinez and Bonn, Bonn, Germany; e-mail: [email protected] Hertweck 2005).

Physiology and Genetics, 2nd Edition The Mycota XV T. Anke, A. Schu¨ffler (Eds.) © Springer International Publishing AG 2018 132 E. Leistner and U. Steiner

In such associations both organisms may Steiner et al. 2006; Ahimsa-Mueller et al. 2007; form a symbiotum in which the associated Markert et al. 2008; Steiner et al. 2008, 2011; microorganism benefits by receiving nutrients, Beaulieu et al. 2015). This indicates that ergot protection, reproduction, and dissemination, alkaloids are components in a fungus/plant whereas the host takes advantage of physiolog- symbiotum characterized by mutual defense ically active compounds which may promote and interaction which constitutes a driving plant growth, herbivore deterrence, and/or force for evolutionary processes (Saikkonen et increased fitness (Arnold et al. 2003; White Jr al. 2015, Schardl et al. 2013). et al. 2003; Saikkonen et al. 2004). Symbiota and their contained natural pro- ducts play a decisive role in evolution as all species evolve in interactions with other species II. The Symbiosis Between Poaceae and (Saikkonen et al. 2015). A point in case is the Clavicipitaceous Fungi beneficial activity of ergot (syn. ) alka- loids which are products of clavicipitaceous A rather well-investigated experimental system fungi colonizing monocotyledonous plants consists of clavicipitaceous fungi colonizing like Poaceae, Juncaceae, and Cyperaceae (Clay Juncaceae, Cyperaceae, and Poaceae plants. In and Schardl 2002; White Jr et al. 2003; Schardl these symbiota ergoline alkaloids play an et al. 2006). Ergot alkaloids, however, are also important role (Keller and Tudzynski 2002). present in higher dicotyledonous plants of the The symbiotic fungi belong either to the tribe family Convolvulaceae (Hofmann 1961, 2006). Clavicipeae or Balanseae within the family This disjointed occurrence of a group of natural Clavicipitaceae (Bacon and Lyons 2005). The products in evolutionarily unrelated taxa (fungi morphological associations of the fungi with and Convolvulaceae plants) seemed to contra- grasses occur either epicuticular, epibiotic, or dict the generally accepted principle of chemo- endophytic (Bacon and Lyons 2005). In epi- taxonomy that similar or even identical natural phytic growth the fungal mycelium is concen- products are present in related taxa. It was trated on the surface of young leaves, buds, therefore assumed that during evolution, a hor- meristematic regions, and reproductive struc- izontal transfer of genes responsible for ergo- tures (Clay and Schardl 2002). The association line alkaloid biosynthesis might have occurred between fungi and their plant hosts is likely to from fungi to higher plants (Groeger and Floss be an example of host-symbiont codivergence 1998; Tudzynski et al. 2001; Clay and Schardl (Schardl et al. 2008). 2002). Alternatively, it was discussed that ergo- The fungus may be asexual belonging to the line alkaloid biosynthesis was repeatedly group of fungi imperfecti and shows a sexual invented during evolution (Mothes et al. lifestyle or switch between sexual and asexual 1985). In a recent review in this series, Keller propagation. In the sexual lifestyle, fungi para- and Tudzinsky (2002) dealt with the pharmaco- sitize a wide range of grasses where they form logical aspects, biochemistry, genetics, and bio- infections of single grass florets and replace the technology of ergot alkaloids in fungi seed with individual sclerotia (Clay and Schardl associated with Poaceae. We show in the pres- 2002). ent review that neither the horizontal transfer The asexual fungi are vertically transmitted of genes encoding the ergot alkaloid biosynthe- through seeds. They have never been known to sis nor the repeated invention of a rather com- produce infectious spores and rely entirely on plicated biosynthetic pathway took place seed transmission. Especially the asexual fungi during evolution but rather that clavicipitac- exhibit high host specificity. Most interesting, eous fungi not only live on different grasses sexual and asexual fungi may interact in para- but also colonize dicotyledonous plants of the sexual processes contributing to a high diversity family Convolvulaceae (Kucht et al. 2004; of fungal asexual endophytes (Tsai et al. 1994). The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 133

In general, grasses are poor producers of III. Epibiotic Clavicipitaceous Fungi natural products that assist other plants in their Associated with Convolvulaceae long-term strategy to gain an ecological advan- tage. Grasses, however, have the ability to com- A. Identification of Genus Periglandula pensate for this deficiency by acquiring fungi notorious for their poisonous natural products. 1. Microscopic and Electron Microscopic In some cases fungi can be considered the live- Characterization stock of grasses. Fungi associated with plants may produce The infestation of the clavicipitaceous fungi on different classes of alkaloids among which toxic Ipomoea asarifolia and Turbina corymbosa, ergot alkaloids are an important group (Schardl members of the family Convolvulaceae, is sys- et al. 2004, 2007). The main ecological roles of temic. Evidence of systemic infection came from ergot alkaloids in nature are probably to protect demonstrations that the fungi are seed transmit- the fungi from consumption by vertebrate and ted, that surface-sterilized seeds grown in vitro invertebrate animals (Schardl et al. 2006). Ergot and under germfree conditions result in plant- alkaloids benefit the fungus by protecting the lets which are colonized exclusively by the health and productivity of the host (Schardl et respective clavicipitaceous fungi, and that they al. 2006). Other benefits include growth of the are transmitted through vegetative propagation plant, competitive abilities, and resistance to (Steiner et al. 2008). It is an unusual type of drought (Malinowski and Belesky 2000), pests, systemic infection under the aspect that there and fungal pathogens (Brem and Leuchtmann are no signs of penetration into the host tissue, 2002; White Jr et al. 2003). In some cases, cla- but the growth on the host plants is superficial. vicipitaceous fungi are culturable in vitro (Kel- Attempts made to visualize the fungus within the ler and Tudzynski 2002). This allowed to stem and leaf tissue, using methodologies com- identify the fungus as the producer of ergot monly employed to detect endophytes in grasses alkaloids and revealed that the host plant is (Bacon and White Jr 1994), were not successful. not the site of ergot alkaloid biosynthesis. Up to now the fungi proved to be non-detectable It was therefore somewhat unexpected using these procedures. Among the Clavicipita- when Hofmann (1961, 2006) found that dicoty- ceae, hypoxylon, cyperi, B. ledonous plants belonging to the family Con- pilulaeformis,andMyriogenospora atramentosa volvulaceae contained ergot alkaloids and that are examples of epibiotic species that grow on these alkaloids were responsible for the halluci- meristematic tissues of host plants (Leuchtmann nogenic properties enjoyed by Meso- and South and Clay 1988, 1989; Luttrell and Bacon 1977; American Indians in religious ceremonies Rykard et al. 1985; Clay and Frentz 1993). The (Schultes and Hofmann 1992). clavicipitaceous fungi colonizing members of The idea that a fungus could be responsible the Convolvulaceae inhabit an epibiotic niche for the alkaloid occurrence was discussed, but and thus seem most comparable to the epibiotic no evidence for the presence of such a fungus members of the grass borne Clavicipitaceae. The was found (Hofmann 2006). This seemed to be mutualistic endophyte Epichloe typhinum (for- in agreement with the notion that plant tissue merly typhinum) also forms a cultures which are believed to be germfree, i.e., stable external mycelial net on the leaves of the devoid of any microbes, were reported to pro- host plant (Moy et al. 2000). This suggested a duce ergot alkaloids (Dobberstein and Staba possible alternative pathway of fungal dispersal 1969), a report which, however, remained and transmission to hosts, i.e., through epiphyl- unsubstantiated (Kucht et al. 2004; Steiner lously produced conidia. et al. 2006). The clavicipitaceous fungi form colonies on the upper surfaces of young unfolded leaves 134 E. Leistner and U. Steiner which are visible to the naked eye as shown for lar trichomes (Fig. 1E), which consist of one T. corymbosa (Fig. 1A) as well as for Ipomoea basal cell, one stalk cell, up to eight glandular asarifolia.OnT. corymbosa colony distribution secretory cells, and a subcuticular oil storage mainly follows the veins of the leaves (Fig. 1A, cavity that is derived from the cuticle of the B), in contrast to the distribution on I. asarifo- secretory cells. Metabolites are released after lia which is more random. These colonies differ rupture of the cuticle. As indicated by staining in size and mycelium density, and depending with the lipophilic dye Nile red and chemical on the developmental stage, the fungi produce analysis, these specialized structures contain synnemata-like structures. No stromata with essential oils and palmitic acid (Kucht et al. perithecia and ascospores were detected in the 2004; Steiner et al. 2015). The secretory glands mycelium mats. Maybe the environmental con- and their specific metabolites may be the basis ditions are not suitable for the development of of a metabolic dialogue between the fungus and the sexual stage of the fungi, that they lost the the plant (Steiner et al. 2008, Steiner et al. 2015). ability to reproduce sexually or that the mating The fungi may feed on the volatile oil and type is lacking. On the lower (abaxial) side of derive precursors like terpenes and fatty acids the leaves, no mycelium was detected. Visual for the ergot alkaloid biosyntheses from the oil. inspection of leaf buds when opened by manip- The fungi inhabit the epibiotic niche of glandu- ulation showed that the fungus was well estab- lar cells on the upper surface of leaves. This lished as dense white mycelial layers on the observation is supported by showing hyphae adaxial leaf surfaces of both plants I. asarifolia of the clavicipitaceous fungus on T. corymbosa and T. corymbosa at this early stage of leaf outside of the subcuticular oil storage cavity as development. The mycelium is formed by well as inside of this compartment embedded in tightly packed hyphae in the cavity between an electron dense matrix (Fig. 1F). The locali- the leaf halves (Fig. 1C). Sections through colo- zation of mycelium with glandular cells ensures nized tissue revealed that fungal mycelium was the close association of the fungus with the host entirely superficial. The hyphae, measured tissues. A continuous maintenance of the sym- approx. 1.5 mm across, were hyaline, thin- biotic relationship requires that the fungus walled, and septated. Chlamydospore-like derives energy from the host plant. In clavici- structures and synnemata are produced (Stei- pitaceous epibiotic fungi, substrate utilization ner et al. 2011). Indicated by the intense myce- depends on the availability of organic material lium development, the space between the upper from the waxy cuticle covering the plant surface surfaces of folded leaves probably offers a ref- and exuded compounds, lipids, amino acids, uge of protection to the fungus. As leaves and vitamins. The main energy-yielding com- expand and mature, the hyphae are evident as pounds are simple sugars that in the case of isolated only microscopically visible clumps, endophytic mycelia are derived from the apo- often near or around peltate glandular tri- plasm through intercellular fungal hyphae chomes, and the ends of the hyphae often (White and Morgan-Jones 1996). In clavicipi- appeared broken (Fig. 1D). taceous fungi present on I. asarifolia and T. The epibiotic fungi of I. asarifolia (Steiner corymbosa, superficial fungal hyphae with tip and Leistner 2012; Steiner et al. 2015)andT. enlargements tightly adherent to the glandular corymbosa (Fig. 1D, E) are closely associated cells as well as to the cuticle have been observed with the secretory glands on the adaxial leaf (Steiner et al. 2015). It seems reasonable to surface, an anatomic feature which may be postulate a selective and efficient exchange of essential for the ergot alkaloid biosynthesis in metabolites between fungus and plant. the epibiotic fungus/plant association (Steiner Physiological changes paralleled by mor- et al. 2006, 2008, 2015). In cell cultures which phological adaptations of the host have been harbor the fungus, no ergot alkaloids are synthe- described for some endophytic associations sized and no secretory glands are developed. (Bacon and White Jr 2000). In M. atramentosa, Members of the Convolvulaceae like I. asar- plant host changes in the epidermal cell size ifolia and T. corymbosa formed peltate glandu- and shape suggest the activity of growth regu- The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 135

Fig. 1 Colonization of Turbina corymbosa with the leaf bud showing that the fungus is well established clavicipitaceous fungus Periglandula turbinae:(A) on the adaxial leaf surfaces at this early stage of plant Colonies formed by white mycelium on the adaxial development. The mycelium is formed by tightly surface of a young unfolded leaf. Preferential develop- packed hyphae as a mycelium layer (ml, arrows) in ment on the veins is visible with the naked eye. (B) the cavity between the halves of the leaf. (D) Close Aggregated hyphae differentiating typical mycelium association of secretory cells (gsc) on the adaxial leaf mats (mm) consisting of several layers which cover surface with hyphae (hy) which often encircle the pel- leaf areas with peltate glandular trichomes and are tate glandular trichomes of the plants. (E) Cross- adhered to the cuticle. (C) Cross-section of a folded section of a peltate glandular trichome composed of 136 E. Leistner and U. Steiner latory substances which are either produced by erance to drought, it is shown that such associ- the fungus or secreted into the host or that are ation has not only in grass hosts but also in produced by the host in response to the fungal dicots evolutionary value. The colonization of a symbiont (Bacon and White Jr 2000). The epi- unique plant niche, the clavicipitaceous fungi phytic proliferation of hyphae on the cuticle on Convolvulaceae, represents a novel finding may be additionally enabled through degrada- among beneficial plant-fungus symbioses in tion of the cuticular layers of the leaf surfaces. non-graminaceous plants. Previous ultrastructural studies of the host-fun- gus interfaces of the clavicipitaceous fungi on I. asarifolia and T. corymbosa revealed progres- 2. Phylogenetic Trees and Taxonomy sive cuticular disintegration. Substrate utiliza- tion studies showed that epiphytic Atkinsonella Among a broad spectrum of plant-associated hypoxylon possess the capacity to colonize and fungi isolated from the plant I. asarifolia, only degrade paraffin wax droplets (White Jr et al. the epiphytic leaf-associated fungus belonged 1991). A. hypoxylon grows superficially on to the family Clavicipitaceae within the order young leaves of grasses as an epiphyte, perhaps (Steiner et al. 2006). Conventional degrading wax in the cuticle to obtain nutrients techniques to continuously cultivate the epi- for epiphytic growth (White Jr et al. 1991). phytic fungi from I. asarifolia or T. corymbosa Leaves and inflorescence primordia within the on synthetic media usually supporting fungal stroma never develop a cuticular layer that growth turned out to be negative indicating that would impede flow of nutrients and moisture the leaf material contains factors or structures to the fungus. Through these modifications of essential for a prolonged growth of the fungus. the host tissues, the endophyte removes bar- On potato dextrose agar, a very limited growth riers to nutrient flow into the mycelium. Very which soon discontinued after inoculation was similar to this situation, the cuticle covering the observed, and some morphological features glandular cells of the Convolvulaceae appears such as synnemata and chlamydospore-like thinner and therefore more permeable than the structures were noted (Steiner et al. 2011). A cuticle on epidermal cells (Steiner et al. 2015). morphological differentiation of fungi on I. Clavicipitaceous fungi have evolved to sur- asarifolia from those on T. corymbosa was not vive as saprophytes, degrading organic mate- possible. All experiments to characterize these rial, as well as biotrophs of plants, fungi, fungi in terms of taxonomy are therefore based nematodes, and insects. They are described to on molecular biological techniques (Steiner have become particularly successful as endo- et al. 2006, 2011). phytes and epibionts of grasses. The association Removal of fungal mycelium from the leaf between clavicipitaceous fungi and their hosts surface of convolvulaceous plants was inter alia constitutes unique biotrophic symbioses where possible by ultrasonic treatment. This method the stages of physiological adaptation to the gave access to extraction and analysis of DNA plant host may yield an understanding of how derived from the externally plant-associated evolution among these fungi and their hosts mycelium (Markert et al. 2008). One of the (Schardl et al. 2008, 2013; Young et al. 2015) important observations was detection of the has progressed. With the detection of Periglan- whole set of genes necessary for the biosynthe- dula species on Convolvulaceae able to synthe- sis of ergot alkaloids in the fungus present on I. size ergot alkaloid known to play a role in asarifolia which was highly suggestive of the enhanced resistance to diseases, pests, and tol- presence of a clavicipitaceous fungus (Markert

Fig. 1 (continued) basal cell (bc), stalk cell (sc), and tory cells with hyphae outside and inside of the sub- secretory cells (gsc) showing the epiphytic develop- cuticular oil storage cavity (scc) bordered by the cuticle ment of mycelium embedded in a mucilage matrix (c, arrow). No evidence for direct penetration of the concentrated on the cuticle over a subcuticular oil plant cells is visible storage cavity. (F) Electron microscopic view of secre- The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 137 et al. 2008; Schardl et al. 2013). In-depth inves- their host specificity and their alkaloid spectra tigation of the enzyme (DmaW) initiating the (Ahimsa-Mueller et al. 2007) but also in the biosynthetic pathway leading to ergot alkaloids Atp6, actG, dmaW, rpbA,andtefA genes. All provided further evidence for the clavicipitac- sequences obtained from the fungus associated eous nature of the epibiotic fungus on I. asar- with the red blooming I. asarifolia plant were ifolia (Markert et al. 2008). identical to those from the fungus living on the Construction of phylogenetic trees has been white blooming host variety. Only the rpbA repeatedly and successfully employed in the sequences of both fungi living on the two plant systematic classification of grass borne clavici- varieties of I. asarifolia differed in one base pair pitaceous fungi (Spatafora and Blackwell 1993; of the rpbA gene (Steiner et al. 2011). This, Glenn et al. 1996; Kuldau et al. 1997; Reddy however, cannot be taken as evidence for the et al. 1998; Lewis et al. 2002; Bischoff and presence of two different fungal species. White Jr 2005; Sung et al. 2007). The fungi on I. asarifolia and T. corymbosa Sequencing of various genes and construc- are considered separate species and named tion of phylogenetic trees from 18S rDNA, Periglandula ipomoeae U. Steiner, E. Leistner internal transcribed spacer, and 4-[g,g- et Schardl and Periglandula turbinae U. Steiner, dimethylallyl]tryptophan synthase (DmaW) E. Leistner et Schardl, respectively. Since the red catalyzing the committed step in ergot alkaloid blooming Ipomoea asarifolia plant is associated biosynthesis pointed to a clavicipitaceous ori- with a fungus which by molecular techniques gin of the fungus on I. asarifolia (Ahimsa-Muel- cannot be discerned from the one on the white ler et al. 2007). Essentially the same results were blooming I. asarifolia plant, the former was also observed when the fungi associated with I. asar- named Periglandula ipomoeae U. Steiner, E. ifolia (red variety), T. corymbosa, and Ipomoea Leistner et Schardl (Steiner et al. 2011). tricolor were investigated (Ahimsa-Mueller While I. asarifolia and T. corymbosa are et al. 2007; Leistner and Steiner 2009) indicating New World tropical vines, eight Convolvula- that our observations are not restricted to the ceae plants from Asia, Australia, Africa, and white blooming I. asarifolia plant and its asso- North America were recently demonstrated to ciated fungus but are of a broader significance. be ergot alkaloid positive (Beaulieu et al. 2015). The fungi formed a monophyletic clade within The alkaloid content coincided in each case the family Clavicipitaceae in PCR-generated with the presence of a leaf-associated epibiotic partial sequences of B-tubulin (tubB) , RNA fungal Periglandula symbiont. A phylogenetic polymerase II large subunit (rpbA), and tree calculated from sequences of the transla- mitochondrial ATP synthase subunit 6 (Atp6) tion elongation factor 1-alpha (tefA) showed a (Steiner et al. 2011). Based on these observa- monophyletic clade with the above-described tions, they were placed into a new genus which Periglandula fungi P. ipomoeae and P. turbinae was named Periglandula with reference to its hosted by I. asarifolia and T. corymbosa, close association of fungal hyphae with peltate respectively (Fig. 3). The newly described glandular trichomes (Fig. 2A; Steiner et al. 2006; fungi (Beaulieu et al. 2015) fall into four differ- Steiner and Leistner 2012). ent chemotypes. It may be an intriguing ques- In addition, these experiments showed that tion how this relates to the architecture and the fungi on T. corymbosa and I. asarifolia could evolution of ergot alkaloid biosynthetic gene be distinguished by the sequences from rpbA, clusters within the genus Periglandula. Atp6, g-actin (atgG), translation elongation fac- tor 1-alpha (tefA), and the 4-[g,g-dimethylallyl] tryptophan synthase gene (dmaW). Thus, the B. Seed Transmittance of Epibiotic Fungi fungi on T. corymbosa and I. asarifolia belong Colonizing Convolvulaceae to the same family, Clavicipitaceae, and the same genus, Periglandula, but are not identical in A freshly harvested and surface-sterilized seed every aspect. They clearly differ not only in grown under germfree conditions gives a plant 138 E. Leistner and U. Steiner

Fig. 2 The glandular trichome as interface of the plant structure of the plant cell wall (pcw) at the contact fungus symbiotum Periglandula/Ipomoea: (A) Myce- site with the fungus; accumulation of mitochondria, lium encircle glandular secretory cells (gsc) embedded membrane systems, and ribosomes as indicators of in a dense matrix. (B) Contact site of hyphae (hy) high metabolic activity at the plant/fungus contact forming an appressorium-like structure (ap) on the site; (E) release of lipids (stained with Nile red) by cuticle (c) of the secretory cell covering the subcuticu- secretory glandular cells encircled by the fungus lar oil storage cavity (scc). (C) Hyphae colonizing the (stained green with WGA); (F) lipid vacuoles (stained outer side of the cuticle and the subcuticular cavity with Nile red) in hyphae encircle glandular cell; (G) after disruption of the cuticle; (D) alteration in the uptake of 6(5)carboxyfluorescein diacetate (green fluo- The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 139 colonized by the epibiotic clavicipitaceous fun- therefore be used as a test system to probe the gus. This plant contains ergot alkaloids. The biosynthetic capacities of plant cells. epibiotic fungus is the only fungus that is Numerous attempts, however, to reproduce detectable by SSCP on this particular plant. this result (Dobberstein and Staba 1969) and to Such a fungus is detectable in seeds of Ipomoea find a plant cell culture raised from I. asarifolia, asarifolia and I. violacea (Steiner et al. 2006; T. corymbosa, and Ipomoea tricolor (L) (Con- Ahimsa-Mueller et al. 2007). This shows that volvulaceae) showing ergot alkaloid production the fungus is seed transmitted and points to were unsuccessful in our hands (Hussein 2004; the host specificity typical of asexual clavicipi- Kucht et al. 2004). Indeed, thin-layer chroma- taceous fungi (see below). tography combined with vanUrk’s spray The viability of the seed-transmitted fun- reagent were used by Dobberstein and Staba gus very likely is limited and depends on age (1969) to detect ergot alkaloids, techniques and storage (Schardl 1994) as well as moisture which are of limited reliability in the identifica- and storage temperature (Welty et al. 1987)of tion of natural products (Jenett-Siems et al. the seed. An Ipomoea violacea plant devoid of 1994, 2004; Kucht et al. 2004). ergot alkaloids derived from an alkaloid and Again, it was a surprise when we found that clavicipitaceous fungus containing seed has the epibiotic fungus lived together with the recently been described (Ahimsa-Mueller et plant cells in the callus and cell suspension al. 2007). In this particular case, the viability culture. Microscopic examination, single- of the seed exceeds the viability of the inhabit- strand conformation polymorphism (SSCP), ing fungus. This may be a reason for contra- and sequencing of the internal transcribed dictory reports on the occurrence of ergot spacer revealed the presence in the cell culture alkaloids in the seeds of Ipomoea species of the epibiotic fungus P. ipomoeae previously (Eich 2008). detected on the leaf surface of I. asarifolia. It follows that the presence or absence of Other endophytic fungi which had been ergot alkaloids in a convolvulaceous plant or isolated from the intact I. asarifolia plants seed may be an unsuitable character for taxo- were not detectable by SSCP within the callus nomic classifications, but instead the ability of and cell suspension culture (Steiner et al. 2006). a plant to host an ergot alkaloid-produc- When a callus culture was subjected to a ing clavicipitaceous fungus should be the char- new hormone regime (the amount of benzyla- acter in question. minopurine was lowered from 2 mg/l to 0.01 mg/l), a plantlet regenerated from the cal- lus. This plantlet was colonized by the fungus C. Plant Growth Under Germfree Conditions and contained ergot alkaloids (Steiner et al. 2006, 2008). The notion that fungicides eliminate ergot alka- These observations show also that an intact loids from the plant was a clear indication that I. asarifolia plant colonized by the fungus P. ergot alkaloids in Convolvulaceae plants are of ipomoeae is required for the successful synthe- fungal origin (Kucht et al. 2004). This observa- sis of ergot alkaloids and gives an idea about tion was somewhat unusual because it had been the extreme specificity between the epibiotic reported that ergot alkaloids are produced by fungus and the I. asarifolia plant (Steiner et al. plant cell cultures established from different 2008). It is in line with these conclusions that Convolvulaceae plants (Dobberstein and Staba we were hitherto unable to grow the fungi P. 1969). Plant cell cultures are usually germfree; ipomoeae or P. turbinae in vitro (Steiner et al.

they should not contain any microbes and can 2006). Apparently the plant contains some kind

⁄ ⁄ ä ä

Fig. 2 (continued) rescence) into the mycelium of the cells remained unstained (modified from Steiner et al. fungus and into glandular trichomes; (H) epidermal 2015) 140 E. Leistner and U. Steiner

Fig. 3 Phylogenetic diversity of Periglandula species dence values (calculated with the approximate likeli- (Clavicipitaceae) forming a symbiotum on 11 different hood ratio) greater than 75% are shown. The branch morning glory hosts (Convolvulaceae). The plants lengths are in the units of number of base substitutions occur on four different continents and comprise mainly per site. Taxon names are followed by the strain ID and tropical and subtropical vines but also shrub growth when applicable GenBank accession number. Periglan- forms (e.g., T. corymbosa). The fungi form a monophy- dula spp. from new host species in the Convolvulaceae letic group within the Clavicipitaceae. The maximum are listed with their host plant’s name (Beaulieu et al. likelihood phylogeny was calculated from sequences of 2015; reprinted with permission from Mycologia. the translation factor 1-gene (tefA locus). Node confi- #The Mycological Society of America) The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 141 of component essential for fungal growth. The the genes nor the enzymatic machinery were specificity between the plant and its associated detectable in the shoots. The complete genetic fungus is also evident from the fact that differ- material responsible for ergot alkaloid biosyn- ent plant taxa within the Convolvulaceae (e.g., thesis was clearly found in the associated fungi Ipomoea or Turbina) are colonized by related present on I. asarifolia and T. corymbosa (Mar- but different clavicipitaceous fungi (Ahimsa- kert et al. 2008; Schardl et al. 2013). The deter- Mueller et al. 2007; Beaulieu et al. 2015). minant step in ergot alkaloid biosynthesis is the This raises the question as to how the speci- prenylation in 4 position of tryptophan cata- ficity between the fungus and the host plant is lyzed by 4-[g,g-dimethylallyl] tryptophan brought about (Steiner et al. 2008). Interesting, synthase (DmaW) (Groeger and Floss 1998; the fungus apparently has a very high affinity to Keller and Tudzinski 2002). The encoding the secretory glands on the adaxial leaf surface gene—which has different synonyms, i.e., (Kucht et al. 2004; Steiner and Leistner 2012). dmaW or cpd1 (Schardl et al. 2006) or fgaPT2 This seems to be unusual because essential oils (Unso¨ld and Li 2005; Gerhards et al. 2014)—is may have an antifungal activity (Chang et al. clearly present in the fungus and is part of a 2008). It is conceivable that during evolution, cluster (Schardl et al. 2013) in which the ergot clavicipitaceous fungi were able to overcome alkaloid genes are oriented as is found in Cla- this barrier and to take advantage of oil compo- viceps but different from Aspergillus and Epi- nents using these compounds as mediators of chloe species (Markert et al. 2008; Gerhards et specificity and even as substrates to feed upon. al. 2014). A reverse genetics experiment showed The volatile oil of I. asarifolia consists of that the fungus is also the site of transcription many minor but five major components, the of the dmaW gene (Markert et al. 2008). A latter of which are sesquiterpenes (Kucht et al. polyclonal antibody directed against the 2004) and palmitic acid (Steiner et al. 2015). DmaW enzyme locates the target antigen to Sesquiterpenes play an important role in eco- the fungal hyphae but not to the glandular logical interactions between plants and insects trichomes which are devoid of the enzyme. It (Schnee et al. 2006; Gershenzon and Dudareva follows that not only transcription but also 2007). Our observations raise the question if translation in ergot alkaloid biosynthesis are this class of terpenoids is also essential for the the capacity of the fungus (Steiner et al. 2015). interaction between different Convolvulaceae Initial attempts to detect ergot alkaloids in species and their associated clavicipitaceous the fungal mycelium present on I. asarifolia and fungi. T. corymbosa failed although two different ana- lytical approaches were used (Markert et al. 2008). When a sample of the mycelium found D. Biosynthesis and Accumulation of Ergot on T. corymbosa was directly placed into the Alkaloids in the Fungus/Plant Symbiotum injection port of a GC/MS system, a trace of agroclavine was detectable and clearly identi- Ergot alkaloids are natural products of high fied by comparison with an authentic sample. physiological activity. They are described to No alkaloid was detectable when a mycelial confer drought resistance, herbivore deter- sample from I. asarifolia was checked in the rence, and fitness to the host plant (Malinowski same way (W. Boland 2008, personal commu- and Belesky 2000; White Jr et al. 2003; Bacon nication). When the leaf material was analyzed and Lyons 2005; Gershenzon and Dudareva for ergot alkaloids after removal by ultrasonic 2007). This raises the question as to how this treatment of the mycelium, alkaloids were qual- may be brought about when plant-associated itatively and quantitatively detected in the plant clavicipitaceous fungi are the site of ergot alka- material showing that the plant leaf material loid biosynthesis. Indeed, Convolvulaceae contains almost all alkaloids whereas the pro- plants do not seem to have the biosynthetic ducing fungus P. turbinae contained only a capacity to produce ergot alkaloids: neither trace of agroclavine (Markert et al. 2008). 142 E. Leistner and U. Steiner

Thus, biosynthesis of alkaloids takes place in uptake of low-molecular-weight compounds, the mycelium; however, ergot alkaloids accu- and it may indicate that a secretion of low- mulate in the host plant. We therefore postulate molecular compounds like ergot alkaloids a transport system that translocates ergot alka- from the fungal hyphae and specific uptake loids from the mycelium into the plant tissue into the peltate glandular trichomes is likely. (Steiner et al. 2015). In an experimental system Thus, glandular trichomes may have a dual similar to the one discussed here, transport was function for they secrete oil which attracts and postulated to occur through the apparently sustains the fungus but also absorb ergot alka- intact cuticle (Smith et al. 1985). loids which play an ecological role in the life of The hyphae of Periglandula spp. character- the plant (Steiner et al. 2015). It is remarkable istically showed a close association with the that a two-way transport system in glandular peltate glandular trichomes of the host plants trichomes of Drosera rotundifolia leaves has being concentrated on the cuticle above the already been postulated by Charles Darwin subcuticular oil storage cavity (Fig. 2A-D) (1875). This close symbiotum establishment appar- ently starts with appressorium-like hyphal tips attaching to the cuticle covering oil cavities E. Periglandula and the Evolution of the Ergot (Fig. 2B). Cross sections of glandular trichomes Biosynthetic Pathways showed hyphae on top and underneath the cuticle after disruption where the fungus is in The process of ergot alkaloid biosynthesis may close contact with the cell walls of the host plant be divided into initial steps leading to a basic (Fig. 2C,D). These cells show a high density of core structure like chanoclavine present in all organelles, mitochondria, and ribosomes indi- ergot alkaloid-producing fungi and subsequent cating their high metabolic activity, while the steps in which the core structure is decorated plant cell wall features a reduced thickness and by a series of metabolic reactions encoded by appears somewhat translucent. The peltate mid- and late pathway genes and enzymes. Dif- glandular trichomes involved in this process ferent evolutionary processes such as integra- secrete a mixture of volatile and fat oil in tion of AT-rich and transposon-derived repeat which the fungal hyphae are immersed at a blocks into ergot alkaloid gene clusters may later stage of development (Fig. 2C,E). Hyphae result in gene losses, gains, or neofunctionali- encircling the glandular trichomes contained a zations (Schardl et al. 2013; Young et al. 2015). huge number of globular structures (Fig. 2F). Clavicipitaceous strains that produce only cha- Since these structures stain with “Nile red,” noclavine are derived by losses of genes as they are likely to be functional equivalents of evidenced by remnants and pseudogenes often lipid vesicles. The oil secreted by the plant very remaining in the genome (Young et al. 2015). likely plays a nutritional role for the fungus. Thus, the biosynthetic process may have come Besides their secretion capacity, the glandular to a halt at chanoclavine due to a vanishing trichomes additionally may provide a site of biosynthetic capacity because a once complete entry into the plant for metabolites like the biosynthetic pathway was truncated during alkaloids produced by Periglandula species. evolution by multiple frameshifts and stop The transport capabilities of the glandular tri- codons or both (Young et al. 2015). Gene losses chomes were tested using 6(5)carboxyfluores- are often present in the Clavicipitaceae but are cein diacetate. The uptake of this low- absent from P. ipomoeae which is not only molecular-weight compound into glandular tri- equipped with a complete set of genes respon- chomes and underlying mesophyll cells as well sible also for mid-pathway and late steps in as into fungal cells was observed (Fig. 2G,H). alkaloid biosynthesis but produces also natural This indicates that the hydrophobic quality of products like simple amides (Ahimsa-Mu¨ller the cuticle on the epidermal cells restricts et al. 2007; Eich 2008) and an ergopeptine like The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 143 ergobalansine (Eich 2008; Jennett-Siems et al. IV. Additional Fungus/Plant Symbiota 1994, 2004). Both types of compounds are end in Dicotyledonous Plants products of the ergot biosynthetic pathway (Young et al. 2015). For this reason the P. ipo- Convolvulaceae are a family which are not moeae gene cluster is considered a basal cluster only notorious for the presence of ergot alka- with an ancient character. It is doubtful if this loids but may also contain simple tropane applies also to the newly described Periglan- alkaloids such as calystegines and the indoli- dula strains (Beaulieu et al. 2015). At least zidine alkaloid swainsonine. The latter com- some of them seem to lack end pathway alka- pound is remarkable as it occurs also in plants loids like simple amides and ergobalansine. of the Fabaceae and Malvaceae. The alkaloid is Diversification of ergot biosynthetic pathways toxic and causes a lysosomal storage disease in may also occur as a result of a relaxed specific- grazing animals resulting in a staggering walk ity in ergot alkaloid peptide synthases (Robin- and lack of muscular coordination. The tro- son and Panaccione 2015). pane alkaloids and swainsonine are constitu- Plants of the tribus Ipomoeeae, the largest ents of Ipomoea carnea. This plant is equipped tribe within the Convolvulaceae, are host to the with two types of trichomes on the adaxial leaf Periglandula fungi. Based on whole plastome surface: simple trichomes and peltate glandu- sequences, the tribe was shown to be monophy- lar trichomes as seen on the adaxial leaf sur- letic, and the subtribal classification identifies face of I. asarifolia and T. corymbosa.The two major clades: the Astripomoeinae and the peltate trichomes are associated with a verti- Argyreiinae with the former diverging into five cally transmitted fungal symbiont belonging and the latter into two subclades. The genus to the Ascomycete order Chaetothyriales Ipomoea is present in each of these subclades which is responsible for the presence of swain- demonstrating it is not monophyletic as tradi- sonine in I. carnea, while calystegines are pro- tionally recognized. In the phylogenetic ducts of the host plant. It is striking that description of the Ipomoeeae results based on distantly related fungi (Hypocreales and Chae- DNA sequences, morphology, and RFLP ana- tothyriales) that produce different alkaloids lyses are largely congruent (Eserman et al. (ergot alkaloids and swainsonine) converged 2014). The tribe comprises an estimate of 650– upon a similar life history within the same 900 plant species. Following Eich’s (2008) ana- plant family (Convolvulaceae) (Cook et al. lyses of the occurrence of alkaloids in Convol- 2013). vulaceae plants, it was assumed that 50% of all Another interesting fungus/plant associa- plants in this tribe may be ergot alkaloid posi- tion has been described for locoweed plants tive meaning that up to 450 clavicipitaceous belonging to the family Fabaceae. Astragalus Periglandula fungi, each one living on its own mollissimus, Oxytropis lambertii, and Oxytropis convolvulaceous host, may exist (Eserman et al. sericea are collectively called locoweed and are 2014). The association with ergot alkaloid-pro- colonized by endophytes which seem to be ducing fungi is the ancestral condition in the closely related to the genus Embellisia. Locoism Ipomoeeae. But ergot alkaloid biosynthesis has is observed in cattle intoxicated by locoweed been lost four times during evolution from the plants. The causative agent seems to be again plant/fungus symbiota within the tribe Ipo- the indolizidine alkaloid swainsonine. This moeeae diverging into different clades and sub- alkaloid is also known to be a product of in clades (Eserman et al. 2014). Apparently, not vitro grown Rhizoctonia leguminicola cultures only loss of individual alkaloids must have (Braun et al. 2003). occurred during evolution but also loss of com- An interesting association consisting of plete clavicipitaceous fungal strains from the Ipomoea batatas (L.) Lam. (i.e., sweet potato) symbiota. 144 E. Leistner and U. Steiner

(Convolvulaceae) and Fusarium lateritium The same conclusion was drawn for a Nees:Fr has also been reported. As described Mentha piperita L. plant colonized by a Pyre- for our clavicipitaceous fungi (Sect. III), F. nomycete which is also associated with glandu- lateritium is primarily located between the lar trichomes (Mucciarelli et al. 2002), a halves of young unfolded leaves of the I. batatas striking observation which led to speculations plant (Hyun and Clark 1998). Yet there is about the possible function of the secretory another feature of this fungus/plant association glands and trichomes in the establishment of a which we also observed in our system (Sect. symbiotic association. It is possible,that also in III): The fungus is associated on the phyllo- this case the glandular trichomes are entry plane with pearl glands and is located around gates for the fungus in its attempt to establish the bases of trichomes (Clark 1992). The fungus a molecular dialogue with the host plant (com- apparently produces trichothecenes and pro- pare Steiner et al. 2008, 2015). tects the host plant against infection by patho- genic Fusarium oxysporum f. sp. batatas (Wollenw.) W.C. Snyder and H.N. Hans. How- ever, the associated F. lateritium may also be V. Conclusions the cause for the chlorotic leaf distortion (CLD) disease mediated by trichothecenes (Clark The data described in Sect. III solve a historical 1994). After light activation of trichothecenes mystery and explain why ergot alkaloids occur in during prolonged exposure of the plant to sun- disjointed taxa, clavicipitaceous fungi, and con- light, CLD occurs. Plants usually recover when volvulaceous plants. They dispute the possibility cloudy weather prevails. Thus, the associated that during evolution a horizontal transfer of fungus may exert a beneficial and a detrimental genes responsible for the synthesis of ergot alka- effect on the host plant, and in both cases tri- loids occurred from fungi to plants. They also chothecenes are likely to be the causative agent. show that there is no necessity to invoke a Two new clavicipitaceous fungi belonging repeated invention of the ergot alkaloid biosyn- to a newly established genus (Hypodermium) thetic pathway during evolution. In fact, genes were isolated from an unidentified Asteraceae present in P. ipomoeae, P. turbinae, Claviceps plant (genus Bernonia). The fungi were named purpurea, , Balansia obtecta, Hypodermium bertonii (Speg.) J. White, R. Sul- Epichloe coenophialum,aswellasAspergillus livan, G. Bills et N. Hywel-Jones and Hyperder- fumigatus involved in the biosynthesis of ergot mium pulvinatum J. White, R. Sullivan, G. Bills alkaloids are orthologs which share a high simi- et Hywel-Jones. As with the clavicipitaceous larity (Markert et al. 2008; Schardl et al. 2013). fungi described in Sect. III, the fungi are epi- The gene cluster responsible for ergot alkaloid biotic. They belong to the subfamily Cordycipi- biosynthesis in P. ipomoeae is likely to have a toideae (Sullivan et al. 2000). An entirely conserved character which may represent an superficial mycelium was observed on a South early point in the evolutionary processes that American Asteraceae plant, Baccharis coridifo- diversified the gene clusters and molecular struc- lia DC. The endophyte belongs to the Hypo- tures of ergot alkaloids in symbiota consisting of creales, an order which accommodates also the different higher plant species and their epibiotic family Clavicipitaceae. The fungus occurs not clavicipitaceous fungi (Schardl et al. 2013). only epibiotic but also in meristematic tissue of It is also clear that the association between leaf primordia. No reproductive structures fungus and convolvulaceous plant is asymp- were detectable. The plant is toxic, and it was tomatic and that a molecular dialogue occurs assumed that the epibiont is a trichothecene between associated fungi and convolvulaceous producer. Since this fungus and graminaceous plants indicating that both are members of a Clavicipitaceae (Chap. II) are not closely symbiotum in which biosynthesis and accumu- related, colonizations (that must have occurred lation of ergot alkaloids are spatially separated during evolution) were assumed to be distinct and sequestered in different organisms. The events (Bertoni et al. 1997). molecular dialogue between both organisms The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 145 requires a subcuticular interaction between organisation and role in the ecosystems (3rd ed.), fungal hyphae and cell walls of peltate glandular CRC Taylor and Francis, Boca Raton, pp 505-518 trichomes. It is likely that the oil secreted by the Braun U, Romero J, Liddell C, Creamer R (2003) Pro- duction of swainsonine by fungal endophytes of trichomes is of nutritional value to the fungus locoweed. Mycol Res 107:980–988 and mediates the contact between both organ- Brem D, Leuchtmann A (2002) Intraspecific competi- isms. As shown here, however, secretion of oil tion of endophyte infected vs uninfected plants in is not the only function of the trichomes, but two woodland grass species. Oikos 96:281–290 rather they seem to have a second function by Chang H-T, Cheng Y-H, C-L W, Chang S-T, Chang T-T, Su Y-C (2008) Antifungal activity of essential oil absorbing ergot alkaloids. We believe that pel- and its constituents from Calocedrus macrolepis tate glandular trichomes are two-way systems var. formosana Florin leaf against plant pathogenic in this symbiotum (Steiner et al. 2015). fungi. Bioresour Technol 99:6266–6270 One of the unsolved questions is if there are Clardy J, Walsh C (2004) Lessons from natural mole- also sexual forms of these vertically transmitted cules. Nature 432:829–837 Clark CA (1992) Histological evidence that Fusarium Periglandula fungi. It is also unknown if and lateritium is an exopathogen on sweet potato with how the plant-associated fungi spread in the chlorotic leaf distortion. Phytopathology 82:656– symbiotum. Despite repeated attempts to local- 663 ize hyphae, spores, or propagules within the Clark CA (1994) The chlorotic leaf distortion pathogen, host plants, structures of the fungi inside the Fusarium lateritium, cross protects sweet potato against Fusarium wilt caused by Fusarium oxy- host remained undetected until now. sporum f.sp. batatas. Biol Contr 4:59–66 Clay K, Frentz IC (1993) Balansia pilulaeformis,an epiphytic species. Mycologia 85:527–534 Clay K, Schardl C (2002) Evolutionary origins and eco- References logical consequences of endophyte symbiosis with grasses. Am Nat 160:S99–S127 Ahimsa-Mueller MA, Markert A, Hellwig S, Knoop V, Cook D, Beaulieu WT, Mott IW, Riet-Correa F, Gardner Steiner U, Drewke C, Leistner E (2007) Clavicipi- DR, Grum D, Pfister JA, Clay K, Marcolongo-Per- taceous fungi associated with ergoline alkaloid- eira C (2013) Production of the alkaloid Swainso- containing Convolvulaceae. J Nat Prod 70:1955– nine by a fungal endosymbiont of the Ascomycete 1960 order Chaetothyriales in the host Ipomoea carnea. Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, J Agric Food Chem 61:3797–3803 Robbins N, Herre EA (2003) Fungal endophytes Darwin C (1875) Insectivorous plants. John Murray, limit pathogen damage in a tropical tree. Proc Natl London Acad Sci U S A 100:15649–15654 Dobberstein RH, Staba EJ (1969) Ipomoea, Rivea and Bacon CW, Lyons P (2005) Ecological fitness factors for Argyreia tissue cultures: influence of various fungi within the and Clavicipiteae. In: chemical factors on indole alkaloid production Dighton J, White JF, Oudemans P (eds) The fungal and growth. Lloydia 32:141–177 community, its organisation and role in the eco- Eich E (2008) Solanaceae and Convolvulaceae: second- system, 3rd edn. CRC Taylor and Francis, Boca ary metabolites- biosynthesis, chemotaxonomy, Raton, pp 519–532 biological and economic significance (a hand- Bacon CW, White JF Jr (1994) Stains, media, and pro- book). Springer, Berlin Heidelberg cedures for analyzing endophytes. In: Bacon CW, Eisner T (2003) For love of insects. Harvard University White JF Jr (eds) Biotechnology of endophytic Press, Cambridge fungi of grasses. CRC, Boca Raton, FL, pp 47–58 Eserman LA, Tiley GP, Jarret RL, Leebens-Mack JL, Bacon CW, White JF Jr (2000) Microbial endophytes. Miller RE (2014) Phylogenetics and diversification Marcel-Dekker, New York, pp 341–388 of morning glories (tribe ipomoeeae, convolvula- Beaulieu WT, Panaccione DG, Ryan KL, Kaonongbua ceae) based on whole plastome sequences. Am J W, Clay K (2015) Phylogenetic and chemotypic Bot. https://doi.org/10.3732/ajb.1300207 diversity of Periglandula species in eight new Gerhards N, Neubauer L, Tudzynski P, Li SM (2014) morning glory hosts (Convolvulaceae). Mycologia Biosynthetic pathways of ergot alkaloids. Toxins 107:667–678 6:3281–3295 Bertoni MD, Romero N, Reddy PV, White JF Jr (1997) A Gershenzon J, Dudareva N (2007) The function of ter- hypocralean epibiont on meristems of Baccharis pene natural products in the natural world. Nat coridifolia. Mycologia 89:375–382 Chem Biol 3:408–414 Bischoff JF, White JF Jr (2005) Evolutionary develop- Glenn AE, Bacon CW, Price R, Hanlin RT (1996) Molec- ment of the Clavicipitaceae. In: Dighton J, White JF ular phylogeny of Acremonium and its taxonomic Jr, Oudemans P (eds) The fungal community – its implications. Mycologia 88:369–383 146 E. Leistner and U. Steiner

Groeger D, Floss HG (1998) Biochemistry of ergot alka- Luttrell ES, Bacon CW (1977) Classification of Myrio- loids-achievements and challenges. In: Cordell GA genospora in the Clavicipitaceae. Can J Bot (ed) The alkaloids: chemistry and biology, vol 50. 55:2090–2097 Academic, New York, pp 171–218 Malinowski DP, Belesky DP (2000) Adaptations of Gunatilaka AAL (2006) Natural products from plant- endophyte-infected cool-season grasses to envi- associated microorganisms: distribution, struc- ronmental stresses. Crop Sci 40:923–940 tural diversity, bioactivity, and implications of Markert A, Steffan N, Ploss K, Hellwig S, Steiner U, their occurrence. J Nat Prod 69:509–526 Drewke C, Li S-M, Boland W, Leistner E (2008) Harborne JB (2004) Introduction to ecological bio- Biosynthesis and accumulation of ergoline alka- chemistry, 4th edn. Academic, London loids in a mutualistic association between Ipomoea Hofmann A (1961) Die Wirkstoffe der mexikanischen asarifolia (Convolvulaceae) and a clavicipitalean Zauberdroge “Ololuiqui”. Planta Med 9:354–367 fungus. Plant Physiol 147:296–305 Hofmann A (2006) LSD-mein Sorgenkind, die Entdeck- Mothes K (1981) The problem of chemical convergence ung einer Wunderdroge. Deutscher Taschenbuch- in secondary metabolism. In: Kageyama M, Naka- verlag, Munich mura K, Oshima T, Uchida T (eds) Science and Hussein YHA (2004) Biochemical analysis of Convol- scientists. Japan Scientific Societies Press, Tokyo, vulaceae plant tissue cultures for the presence of pp 323–326 ergoline alkaloids. PhD thesis, Botany Depart- Mothes K, Schu¨tte HR, Luckner M (1985) Biochemistry ment, Faculty of Science, Zagazig University, of alkaloids. VEB Deutscher Verlag der Wis- Zagazig, Egypt senschaften, Berlin Hyun J-W, Clark CA (1998) Analysis of Fusarium later- Moy M, Belanger F, Duncan R, Freehoff A, Leary C, itium using RAPD and rDNA RFLP techniques. Meyer W, Sullivan R, White JF Jr (2000) Identifi- Mycol Res 102:1259–1264 cation of epiphyllous mycelial nets on leaves of Jenett-Siems K, Kaloga M, Eich E (1994) Ergobalansine/ grasses infected by clavicipitaceous endophytes. ergobalansinine, a proline-free peptide type alka- Symbiosis 28(4):291–302 loid of the fungal genus Balansia is a constituent of Mucciarelli M, Scannerini S, Bertea CM, Maffei M Ipomoea piurensis. J Nat Prod 57:1304–1306 (2002) An ascomycetous endophyte isolated from Jennett-Siems K, Kaloga M, Eich E (2004) Ergobalan- Mentha piperita L.: biological features and molec- sine/ergobalansinine, a proline-free peptide-type ular studies. Mycologia 94:28–39 alkaloid of the fungal genus Balansia is a constitu- Partida-Martinez LP, Hertweck C (2005) Pathogenic ent of Ipomoea piurensis. J Nat Prod 67:2160 fungus harbours endosymbiotic bacteria for toxin Keller U, Tudzynski P (2002) Ergot alkaloids. In: Osie- production. Nature 437:884–888 wacz HD (ed) The Mycota, industrial applications, Piel J (2004) Metabolites from symbiotic bacteria. Nat vol X. Springer, Berlin, pp 157–181 Prod Rep 21:519–538 Kucht S, Gross J, Hussein Y, Grothe T, Keller U, Basar S, Reddy PV, Bergen MS, Patel R, White JF Jr (1998) An Koenig WA, Steiner U, Leistner E (2004) Elimina- examination of molecular phylogeny and mor- tion of ergoline alkaloids following treatment of phology of the grass endophyte Balansia claviceps Ipomoea asarifolia (Convolvulaceae) with fungi- and similar species. Mycologia 90:108–117 cides. Planta 219:619–625 Robinson SL, Panaccione DG (2015) Diversification of Kuldau GA, Liu JS, White JF Jr, Siegel MR, Schardl CL ergot alkaloids in natural and modified fungi. Tox- (1997) Molecular systematics of Clavicipitaceae ins 7:201–218 supporting monophyly of genus Epichloe¨ and Rykard DM, Bacon CW, Luttrell ES (1985) Host rela- form genus Ephelis. Mycologia 89:431–441 tions of atramentosa and Balan- Leistner E, Steiner U (2009) Fungal origin of ergoline sia epichloe¨ (Clavicipitaceae). Phytopathology alkaloids present in Dicotyledonous plants (Con- 75:950–956 volvulaceae). In: Anke T, Weber D (eds) The Saikkonen K, Wa¨li P, Helander M, Feath SH (2004) Mycota. Physiology and Genetics, vol XV. Evolution of endophyte-plant symbiosis. Trends Springer, Berlin, pp 197–208 Plant Sci 9:275–280 Leuchtmann A, Clay K (1988) Atkinsonella hypoxylon Saikkonen K, Young CA, Helander M, Schardl CL and Balansia cyperi, epiphytic members of the (2015) Endophytic Epichloe species and their Balansiae. Mycologia 80:192–199 grass hosts: from evolution to applications. Plant Leuchtmann A, Clay K (1989) Morphological, cultural Mol Biol. https://doi.org/10.1007//s11103-015- and mating studies on Atkinsonella, including A. 0399-6 texensis. Mycologia 81:692–701 Schardl CL (1994) Molecular and genetic methodolo- Lewis EA, Bills GF, Heredia G, Reyes M, Arias RM, gies and transformation of grass endophytes. In: White JF Jr (2002) A new species of endophytic Bacon CW, White JF Jr (eds) Biotechnology of balansia from Veracruz, Mexico. Mycologia endophytic fungi of grasses. CRC, Taylor and 94:1066–1070 Francis, Boca Raton, pp 151-166 The Genus Periglandula and Its Symbiotum with Morning Glory Plants (Convolvulaceae) 147

Schardl CL, Leuchtmann A, Spiering MJ (2004) Symbi- within the Clavicipitaceae and its association osis of grasses with seedborn fungal endophytes. with Convolvulaceae. Mycologia 103:1133–1145 Annu Rev Plant Biol 55:315–340 Steiner U, Hellwig S, Ahimsa-Mu¨ller MA, Grundmann N, Schardl CL, Panaccione DG, Tudzynski P (2006) Ergot Li SM, Drewke C, Leistner E (2015) The key role of alkaloids-biology and molecular biology. In: Cor- peltate glandular trichomes in symbiota comprising dell GA (ed) The alkaloids: chemistry and biology, Clavicipitaceous fungi of the genus Periglandula vol 63. Academic, New York, pp 45–86 and their host plants. Toxins 7:1355–1373 Schardl CL, Grossman RB, Nagabhyru P, Faulkner JR, Strobel G, Daisy B, Castillo U, Harper J (2004) Natural Mallik UP (2007) Loline alkaloids: currencies of products from endophytic microorganisms. J Nat mutualism. Phytochemistry 68:980–996 Prod 67:257–268 Schardl CL, Craven KD, Speakman S, Stromberg A, Sullivan RF, Bills GF, Hywel-Jones NL, White JF Jr Lindstrom A, Yoshida R (2008) A novel test for (2000) Hypodermium: a new clavicipitalean host-symbiotum codivergence indicates ancient genus for some tropical epibionts of dicotyledon- origin of fungal endophytes in grasses. Syst Biol ous plants. Mycologia 92:908–918 57:483–498 Sung GH, Sung JM, Hywel-Jones NL, Spatafora JW Schardl CL, Young CA, Hesse U, Amyotte SG, Andreeva (2007) A multi-gene phylogeny of Clavicipitaceae K et al (2013) Plant-symbiotic fungi as chemical (, Fungi): identification of localized engineers: multi-genome analysis of the Clavicipi- incongruence using a combinational bootstrap taceae reveals dynamics of alkaloid loci. PLoS approach. Mol Phylogenet Evol 44:1204–1223 Genet 9(2):e1003323. https://doi.org/10.1371/jour- Tsai H-F, Liu J-S, Staben C, Christensen MJ, Latch CM, nal.pgen.1003323 Siegel MR, Schardl CL (1994) Evolutionary diver- Schnee C, Ko¨llner TG, Held M, Turlings TCJ, Gershen- sification of fungal endophytes of tall fescue grass zon J, Degenhardt J (2006) The products of a single by hybridisation with Epichloe¨ species. Proc Natl maize sesquiterpene synthase form a volatile Acad Sci U S A 91:2542–2546 defense signal that attracts natural enemies of Tudzynski P, Correia T, Keller U (2001) Biotechnology maize herbivores. Proc Natl Acad Sci U S A and genetics of ergot alkaloids. Appl Microbiol 103:1129–1134 Biotechnol 57:593–605 Schultes RE, Hofmann A (1992) Plants of the gods, their Unso¨ld IA, Li S-M (2005) Overproduction, purification sacred, healing and hallucinogenic powers. Heal- and characterization of FgaPT2, a dimethylallyl- ing Arts, Vermont tryptophan synthase from Aspergillus fumigatus. Smith KT, Bacon CW, Luttrell ES (1985) Reciprocal Microbiology 151:1499–1505 translocation of carbohydrates between host and Welty RE, Azevedo MD, Cooper TM (1987) Influence of fungus in Bahiagrass infected with Myriogenos- moisture content, temperature, and length of stor- pora atramentosa. Phytopathology 75:407–411 age on seed germination and survival of endo- Spatafora JW, Blackwell M (1993) Molecular systemat- phytic fungi in seeds of tall fescue and perennial ics of unitunicate perithecial ascomycetes: the Cla- ryegrass. Phytopathology 77:893–900 vicipitales-Hypocreales connection. Mycologia White JF Jr, Morgan-Jones G (1996) Morphological and 85:912–922 physiological adaptations of Balansieae and trends Steiner U, Leistner E (2012) Ergoline alkaloids in con- in the evolution of grass endophytes. In: Redlin volvulaceous host plants originate from epibiotic SC, Carris LM (eds) Endophytic Fungi in Grasses clavicipitaceous fungi of the genus Periglandula. and Woody Plants. APS Press, St. Paul, pp 133–154 Fungal Ecol 5:316–321 White JF Jr, Bacon CW, Hinton DM (1991) Substrate Steiner U, Ahimsa-Mueller MA, Markert A, Kucht S, utilization in selected Acremonium, Atkinosella, Gross J, Kauf N, Kuzma M, Zych M, Lamshoeft M, and Balansia species. Mycologia 83:601–610 Furmanowa M, Knoop V, Drewke C, Leistner E White JF Jr, Bacon CW, Hywel-Jones NL, Spatafora JW (2006) Molecular characterisation of a seed trans- (2003) In: Bennet JW (ed) Clavicipitalean fungi, mitted clavicipitaceous fungus occurring on dicot- evolutionary biology, chemistry, biocontrol, and yledonous plants (Convolvulaceae). Planta cultural impacts, Mycology series, vol 19. Marcel 224:533–544 Dekker, New York Steiner U, Hellwig S, Leistner E (2008) Specificity in the Young CA, Schardl CL, Panaccione DG, Florea S, interaction between an epibiotic clavicipitalean Takach JE, Charlton ND, Moore N, Webb JS, Jar- fungus and its convolvulaceous host in a fungus/ omczyk J (2015) Genetics, genomics and evolution plant symbiotum. Plant Signal Behav 3:704–706 of ergot alkaloid diversity. Toxins 7:1273–1302 Steiner U, Leibner S, Schardl CL, Leuchtmann A, Leist- Zenk MH (1967) Biochemie und Physiologie sekun- ner E (2011) Periglandula a new fungal genus da¨rer Pflanzenstoffe. Ber Dt Bot Ges 80:573–591

View publication stats