Single strand conformation polymorphism (SSCP) of fungal DNA from IasaF02 ( lane 1 ), IasaF09, i.e. P. roquefortii ( lane 2 ), IasaF13 ( lane 3 ), total DNA extracted from leaves of an alkaloid- containing I. asarifolia plant ( lane 4 ), total DNA extracted from an I. asarifolia plant without alkaloids and without IasaF13 ( lane 5 ), total DNA extracted from seeds of I. asarifolia ( lane 6 ), total DNA extracted from a plant grown from surface-sterilized seeds under germfree conditions ( lane 7 ), total DNA extracted from an I. asarifolia plant regenerated from a callus culture under sterile conditions ( lane 8 ), total DNA extracted from an I. asarifolia callus culture ( lane 9 ), and total DNA extracted from an I. asarifolia cell suspension culture ( lane 10 ). Molecular weight marker ( lane 11 ) 

Contexts in source publication

Context 1

... samples were fixed in 2.5 glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.3 for 24 h (Karnovsky 1965). After rinsing with distilled water, specimens were dehydrated through a graded series of ethanol and critical point dried from CO 2 in eight cycles according to Svitkina et al. (1984) using a Balzers CPD 030 (BAL-TEC, Schalksmu ̈ hlen, Germany). Dried specimens were mounted on aluminium sample holders and sputter coated with 2 nm platinum/ palladium in a HR 208 coating device (Cressington, Watford, UK). Scanning electron microsopy (SEM) was performed using an XL 30 SFEG (Philips, Eindhoven, The Netherlands) equipped with a through lens secondary electron detector. Previous experiments suggested that a fungus on the upper leaf surface of I. asarifolia plants was responsible for the presence of ergot alkaloids. This fungus forms colonies on the upper leaf surface (Fig. 2). The hyphae often encircle oil glands (Kucht et al. 2004). In order to identify and characterize this fungus, culturable fungi were isolated from I. asarifolia plants using a published procedure (Petrini et al. 1992). Nineteen different fungal isolates were obtained from leaves, stems and flowers of plants kept in the greenhouse. It was to be expected that some fungi were repeatedly isolated. Therefore, all fungal isolates were grown in vitro and the DNA extracted from the mycelium. In every case, the DNA was subjected to PCR in which part of the small subunit rDNA gene (18S rDNA) was amplified using fungus-specific oligomers (UF 1 and S 3 ; Kappe et al. 1996). Comparison of the sequences of the amplified DNA stretches showed that the isolates belonged to 12 different fungi which were provisionally named IasaF01 to IasaF12. Database homology searches of the amplified sequences at the NCBI server using BLAST (Altschul et al. 1990) gave a first hint as to the possible taxonomic position of the fungal isolates. These fungal species may or may not be identical to our isolated fungi, however, they certainly bear a close taxonomic relationship to our isolates. Among the isolated fungi three belong to the genus Penicillium (Fig. 3), with Penicillium roquefortii known to be a producer of roquefortine (Fig. 1) and ergoline alkaloids (Scott et al. 1976). Thin-layer, HPLC and capillary electrophoresis showed that this strain (IasaF09) actually is a producer of roquefortine and of minor components which stain positive with van Urk’s reagent. This reaction indicates the possible presence of indole derivatives including ergoline alkaloids. Incuba- tion of each strain IasaF01 to IasaF12 in liquid medium (Bacon 1985; Schulz et al. 1993) and TLC analysis of the medium and the mycelium did not give any evidence, however, that ergoline alkaloids were present in strains other than IasaF09. While all fungi IasaF01 to IasaF12 were endophytic as judged from the isolation procedure (Petrini et al. 1992), microscopic inspection of the leaf surface had previously shown that an epibiotic fungus is associated with the leaf surface (Fig. 2). This fungus occurs notably on the surface of young leaves which are not yet unfolded. The fungus which apparently is epibiotic can be scraped off from the leaf surface with a spatula and was designated IasaF13. Attempts to grow this fungus on 15 different agar media designed for fungal growth were unsuccessful, however, even when the media contained leaf homogenates of the host plant. Seeds were also investigated for the presence of fungi because seeds of Convolvulaceae are known to contain alkaloids (Gro ̈ ger and Floss 1998). No fungus, however, grew within 8 weeks at 20 ° C from surface-sterilized seeds which were crushed after sterilization and put onto an agar medium (Bacon 1985; Schulz et al. 1993) allowing for fungal growth. This, however, does not mean that no fungus is present in the seeds ( vide infra ). After 18S rDNA gene amplification of strains Ia- saF01 to IasaF13 a phylogenetic tree was constructed (Fig. 3a) in which all fungi isolated from I. asarifolia are represented. In addition, sequencing data obtained from authentic clavicipitaceous fungi ( Balansia cyperi , Balansia obtecta , Atkinsonella hypoxylon , Claviceps purpurea , Neotyphodium coenophialum , Epichloe ̈ festucae ) were also entered. The phylogenetic tree (Fig. 3a) shows that our non-culturable strain (IasaF13) occurring on the leaf surface (Fig. 2) is related to representatives of the Clavicipitaceae family. It was desirable to confirm this observation. We therefore analysed the ITS which provide a better phylogenetic fine resolution for closely related taxa. These data are shown in Fig. 3b. In this case, clustering of our strain IasaF13 with authentic clavicipitaceous fungi was seen. These experiments indicated that there were two possible candidates responsible for the presence of ergoline alkaloids in the I. asarifolia plant, P. roquefortii (IasaF09) and the non-culturable epibiotic clavicipitaceous fungus IasaF13. P. roquefortii is a fungus known to produce roquefortine, a toxic diketopiperazine with an indole moiety and ergoline alkaloids such as isofumigaclavine A (Scott et al. 1976). In vitro culture of isolate IasaF09 ( P. roquefortii ) and analysis of its media and mycelium confirmed the presence of roquefortine and additional van Urk positive compounds. The isolated fungi IasaF09 ( P. roquefortii ) and Ia- saF13 were also characterized by PCR-SSCP as shown in Fig. 4. Comparison of the DNA of the isolated fungi (IasaF09 and IasaF13) with that of the total DNA obtained from the intact plant shows (Fig. 4) that both fungi (IasaF09 and IasaF13) are present but that ...

Context 2

... samples were fixed in 2.5 glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.3 for 24 h (Karnovsky 1965). After rinsing with distilled water, specimens were dehydrated through a graded series of ethanol and critical point dried from CO 2 in eight cycles according to Svitkina et al. (1984) using a Balzers CPD 030 (BAL-TEC, Schalksmu ̈ hlen, Germany). Dried specimens were mounted on aluminium sample holders and sputter coated with 2 nm platinum/ palladium in a HR 208 coating device (Cressington, Watford, UK). Scanning electron microsopy (SEM) was performed using an XL 30 SFEG (Philips, Eindhoven, The Netherlands) equipped with a through lens secondary electron detector. Previous experiments suggested that a fungus on the upper leaf surface of I. asarifolia plants was responsible for the presence of ergot alkaloids. This fungus forms colonies on the upper leaf surface (Fig. 2). The hyphae often encircle oil glands (Kucht et al. 2004). In order to identify and characterize this fungus, culturable fungi were isolated from I. asarifolia plants using a published procedure (Petrini et al. 1992). Nineteen different fungal isolates were obtained from leaves, stems and flowers of plants kept in the greenhouse. It was to be expected that some fungi were repeatedly isolated. Therefore, all fungal isolates were grown in vitro and the DNA extracted from the mycelium. In every case, the DNA was subjected to PCR in which part of the small subunit rDNA gene (18S rDNA) was amplified using fungus-specific oligomers (UF 1 and S 3 ; Kappe et al. 1996). Comparison of the sequences of the amplified DNA stretches showed that the isolates belonged to 12 different fungi which were provisionally named IasaF01 to IasaF12. Database homology searches of the amplified sequences at the NCBI server using BLAST (Altschul et al. 1990) gave a first hint as to the possible taxonomic position of the fungal isolates. These fungal species may or may not be identical to our isolated fungi, however, they certainly bear a close taxonomic relationship to our isolates. Among the isolated fungi three belong to the genus Penicillium (Fig. 3), with Penicillium roquefortii known to be a producer of roquefortine (Fig. 1) and ergoline alkaloids (Scott et al. 1976). Thin-layer, HPLC and capillary electrophoresis showed that this strain (IasaF09) actually is a producer of roquefortine and of minor components which stain positive with van Urk’s reagent. This reaction indicates the possible presence of indole derivatives including ergoline alkaloids. Incuba- tion of each strain IasaF01 to IasaF12 in liquid medium (Bacon 1985; Schulz et al. 1993) and TLC analysis of the medium and the mycelium did not give any evidence, however, that ergoline alkaloids were present in strains other than IasaF09. While all fungi IasaF01 to IasaF12 were endophytic as judged from the isolation procedure (Petrini et al. 1992), microscopic inspection of the leaf surface had previously shown that an epibiotic fungus is associated with the leaf surface (Fig. 2). This fungus occurs notably on the surface of young leaves which are not yet unfolded. The fungus which apparently is epibiotic can be scraped off from the leaf surface with a spatula and was designated IasaF13. Attempts to grow this fungus on 15 different agar media designed for fungal growth were unsuccessful, however, even when the media contained leaf homogenates of the host plant. Seeds were also investigated for the presence of fungi because seeds of Convolvulaceae are known to contain alkaloids (Gro ̈ ger and Floss 1998). No fungus, however, grew within 8 weeks at 20 ° C from surface-sterilized seeds which were crushed after sterilization and put onto an agar medium (Bacon 1985; Schulz et al. 1993) allowing for fungal growth. This, however, does not mean that no fungus is present in the seeds ( vide infra ). After 18S rDNA gene amplification of strains Ia- saF01 to IasaF13 a phylogenetic tree was constructed (Fig. 3a) in which all fungi isolated from I. asarifolia are represented. In addition, sequencing data obtained from authentic clavicipitaceous fungi ( Balansia cyperi , Balansia obtecta , Atkinsonella hypoxylon , Claviceps purpurea , Neotyphodium coenophialum , Epichloe ̈ festucae ) were also entered. The phylogenetic tree (Fig. 3a) shows that our non-culturable strain (IasaF13) occurring on the leaf surface (Fig. 2) is related to representatives of the Clavicipitaceae family. It was desirable to confirm this observation. We therefore analysed the ITS which provide a better phylogenetic fine resolution for closely related taxa. These data are shown in Fig. 3b. In this case, clustering of our strain IasaF13 with authentic clavicipitaceous fungi was seen. These experiments indicated that there were two possible candidates responsible for the presence of ergoline alkaloids in the I. asarifolia plant, P. roquefortii (IasaF09) and the non-culturable epibiotic clavicipitaceous fungus IasaF13. P. roquefortii is a fungus known to produce roquefortine, a toxic diketopiperazine with an indole moiety and ergoline alkaloids such as isofumigaclavine A (Scott et al. 1976). In vitro culture of isolate IasaF09 ( P. roquefortii ) and analysis of its media and mycelium confirmed the presence of roquefortine and additional van Urk positive compounds. The isolated fungi IasaF09 ( P. roquefortii ) and Ia- saF13 were also characterized by PCR-SSCP as shown in Fig. 4. Comparison of the DNA of the isolated fungi (IasaF09 and IasaF13) with that of the total DNA obtained from the intact plant shows (Fig. 4) that both fungi (IasaF09 and IasaF13) are present but that ...

Context 3

... clearly predominates among all the plant-associated fungi suggesting that it is identical to the abun- dantly visible fungus attached to the upper leaf surface ( Fig. 2). In contrast, an Ipomoea plant devoid of both fungi and alkaloids does not show the pattern of bands observed for both isolated fungi IasaF09 and IasaF13 (Fig. 4). If one of the isolated fungi is a producer of ergoline alkaloids in the Ipomoea plant, it should contain genes responsible for ergoline alkaloid biosynthesis. Oligonu- cleotides (deg1 and deg4) targeted to conserved regions of the dmaW (or cpd) dimethylallyl-tryptophan-synthase gene (Wang et al. 2004), known to be involved in the introduction of a dimethylallyl residue into tryptophan (Fig. 1) were synthesized and employed in a PCR reaction with DNA from the epibiotic fungus IasaF13 as a template. A PCR product of 939 bp was obtained and its deduced protein sequence showed very high similarity with the available dmaW sequences of Clavicipitaceaen fungi, being most similar to a Balansia obtecta homologue with 76% identical amino acids (Fig. 3c). The clavicipitaceous fungi live on grasses and are known producers of ergoline alkaloids. The same experiment was carried out with DNA isolated from IasaF09 (P. roquefortii ) revealing a dmaW homologue with 63% sequence identity to an Aspergillus fumigatus sequence. Hence, a dimethylallyl-tryptophan-synthase appears also to be present in IasaF09 (P. roquefortii ), however, the corresponding gene in the epibiotic fungus IasaF13 is expectedly much more closely related to the genes detected in Clavicipitaceae. A phylogenetic analysis of the Cpd1 protein sequences, also including several predicted protein sequences form different fungi, indicates several distant homologues in Aspergillus and more recent, independent gene duplications in the Clavicipitaceae , at least in Claviceps purpurea and Neotyphodium coenophialum (Fig. 3c). Subsequently both candidate fungi were used to inoculate I. asarifolia plants free of alkaloids and fungi. The fungus IasaF09 ( P. roquefortii ) was grown on a defined solid medium, a mixture of hyphae and spores was suspended in water following a method used by Latch and Christensen (1985) and the suspension injected into leaves with a syringe and in addition spread onto leaves of the I. asarifolia plant. The inoculated plants were kept in the greenhouse. Microscopic exam- ination of the plants 6, 18 and 26 weeks after inoculation showed that the fungus was well established on the plant. Analysis of the plant 26 weeks after inoculation did not show the presence of roquefortine or of any ergoline alkaloid. Thus, P. roquefortii appears not to be the candidate fungus responsible for the accumulation of ergoline alkaloids in I. asarifolia . The same experiment was carried out with hyphae of IasaF13 isolated from the unfolded leaves of an I. asarifolia plant and spread onto and injected into I. asarifolia leaves of a plant devoid of alkaloids and fungi. As opposed to the experiment with P. roquefortii , however, no fungal growth was observed. This observation was not unexpected (see later). The inability to establish the epibiotic fungus IasaF13 on I. asarifolia was also experienced in the so-called ‘‘attachment experiment’’, in which a normal plant and a plant devoid of fungi and alkaloids were kept in close contact in a cylindrical plastic glass container in the green house with the upper leaf surfaces of both plants attached to each other. After 18 weeks no spread of fungal hyphae to the plant devoid of IasaF13 was observed. In addition, this plant did not contain any alkaloids. Spread of fungal hyphae and presence of ergoline alkaloids, however, were observed when externally sterilized seeds were germinated in a sterile environment on an artificial agar medium in a closed Erlenmeyer flask. Plants grown under these conditions contained both fungus IasaF13 and alkaloids indicating that I. asarifolia seeds harbour fungal propagules of IasaF13 that were spread to the growing plant. Indeed, SSCP (Fig. 4) and microscopic investigation of seeds showed that seeds clearly contain the fungus IasaF13. Evidently, this fungus is spread to the shoot of the plant during growth (Fig. 4). Moreover, the plantlets contained the full spectrum of alkaloids (TLC, HPLC-MS) known to be present in the untreated intact plant. In a similar experiment, a surface-sterilized piece of a stem was placed on an agar medium. After 2–4 weeks a callus was formed. Stem and callus were then transferred into a liquid medium. Shoots regenerated from the plant material. The shoots of these plantlets were cut-off and the cuttings placed into solid agar medium. Roots developed within 2 weeks . Microscopic inspection, SSCP and chemical analysis showed that both fungus IasaF13 and the complete spectrum of alkaloids were again present. We conclude that both seeds and plant cell cultures of the I. asarifolia plant contain the clavicipitaceous fungus and that this fungus is involved in the accumulation of alkaloids in I. asarifolia . Absence and presence of fungus IasaF13 was also investigated microscopically, by SSCP and also by sequencing of DNA after PCR amplification using oligomers (ITS1F and ITS4) (White et al. 1990; Gardes and Bruns 1993) targeted to the ITS region. These ...

Context 4

... reaction with DNA from the epibiotic fungus IasaF13 as a template. A PCR product of 939 bp was obtained and its deduced protein sequence showed very high similarity with the available dmaW sequences of Clavicipitaceaen fungi, being most similar to a Balansia obtecta homologue with 76% identical amino acids (Fig. 3c). The clavicipitaceous fungi live on grasses and are known producers of ergoline alkaloids. The same experiment was carried out with DNA isolated from IasaF09 (P. roquefortii ) revealing a dmaW homologue with 63% sequence identity to an Aspergillus fumigatus sequence. Hence, a dimethylallyl-tryptophan-synthase appears also to be present in IasaF09 (P. roquefortii ), however, the corresponding gene in the epibiotic fungus IasaF13 is expectedly much more closely related to the genes detected in Clavicipitaceae. A phylogenetic analysis of the Cpd1 protein sequences, also including several predicted protein sequences form different fungi, indicates several distant homologues in Aspergillus and more recent, independent gene duplications in the Clavicipitaceae , at least in Claviceps purpurea and Neotyphodium coenophialum (Fig. 3c). Subsequently both candidate fungi were used to inoculate I. asarifolia plants free of alkaloids and fungi. The fungus IasaF09 ( P. roquefortii ) was grown on a defined solid medium, a mixture of hyphae and spores was suspended in water following a method used by Latch and Christensen (1985) and the suspension injected into leaves with a syringe and in addition spread onto leaves of the I. asarifolia plant. The inoculated plants were kept in the greenhouse. Microscopic exam- ination of the plants 6, 18 and 26 weeks after inoculation showed that the fungus was well established on the plant. Analysis of the plant 26 weeks after inoculation did not show the presence of roquefortine or of any ergoline alkaloid. Thus, P. roquefortii appears not to be the candidate fungus responsible for the accumulation of ergoline alkaloids in I. asarifolia . The same experiment was carried out with hyphae of IasaF13 isolated from the unfolded leaves of an I. asarifolia plant and spread onto and injected into I. asarifolia leaves of a plant devoid of alkaloids and fungi. As opposed to the experiment with P. roquefortii , however, no fungal growth was observed. This observation was not unexpected (see later). The inability to establish the epibiotic fungus IasaF13 on I. asarifolia was also experienced in the so-called ‘‘attachment experiment’’, in which a normal plant and a plant devoid of fungi and alkaloids were kept in close contact in a cylindrical plastic glass container in the green house with the upper leaf surfaces of both plants attached to each other. After 18 weeks no spread of fungal hyphae to the plant devoid of IasaF13 was observed. In addition, this plant did not contain any alkaloids. Spread of fungal hyphae and presence of ergoline alkaloids, however, were observed when externally sterilized seeds were germinated in a sterile environment on an artificial agar medium in a closed Erlenmeyer flask. Plants grown under these conditions contained both fungus IasaF13 and alkaloids indicating that I. asarifolia seeds harbour fungal propagules of IasaF13 that were spread to the growing plant. Indeed, SSCP (Fig. 4) and microscopic investigation of seeds showed that seeds clearly contain the fungus IasaF13. Evidently, this fungus is spread to the shoot of the plant during growth (Fig. 4). Moreover, the plantlets contained the full spectrum of alkaloids (TLC, HPLC-MS) known to be present in the untreated intact plant. In a similar experiment, a surface-sterilized piece of a stem was placed on an agar medium. After 2–4 weeks a callus was formed. Stem and callus were then transferred into a liquid medium. Shoots regenerated from the plant material. The shoots of these plantlets were cut-off and the cuttings placed into solid agar medium. Roots developed within 2 weeks . Microscopic inspection, SSCP and chemical analysis showed that both fungus IasaF13 and the complete spectrum of alkaloids were again present. We conclude that both seeds and plant cell cultures of the I. asarifolia plant contain the clavicipitaceous fungus and that this fungus is involved in the accumulation of alkaloids in I. asarifolia . Absence and presence of fungus IasaF13 was also investigated microscopically, by SSCP and also by sequencing of DNA after PCR amplification using oligomers (ITS1F and ITS4) (White et al. 1990; Gardes and Bruns 1993) targeted to the ITS region. These experiments fully confirmed the presence of IasaF13 in the intact control plant, seeds, plants grown under sterile conditions from seeds, regenerated plants, plant callus and cell suspension cultures. The ITS sequencing was repeated 35 times (cf. Materials and methods). Each time the sequence of IasaF13 but never that of IasaF09 ( P. roquefortii ) was found. The fungal ITS sequence again was not found in plants which are devoid of alkaloids (see Fig. 4). Whenever ITS and SSCP (Fig. 4) were positive with respect to the presence of fungus Ia- saF13 microscopic inspection of the plant material confirmed the presence of this fungus, including the plant cell culture material (Fig. 5) and in spite of the fact that plant cell cultures are considered to be sterile. An epibiotic fungus was also detected on another plant species, Turbina corymbosa (L.) Hall. This plant also belongs to the family Convolvulaceae and contains ergoline alkaloids. T. corymbosa and I. asarifolia are indigenous to Central or South America, respectively. The epibiotic fungus from T. corymbosa was submitted to 18S rDNA (DQ127 278) and ITS analysis (AY995219). It turned out that the sequences were 100% identical when compared with those of the epibiotic fungus IasaF13 on I. asarifolia . This result was obtained independently in the laboratories of U.S. and E.L. The alkaloid spectrum of aerial parts of both plant species was investigated quantitatively and qualitatively using a high pressure liquid chromatograph connected to a mass spectrometer (Fig. 6). The compounds were identified by comparison with authentic standards. Both plants contain chanoclavine, lysergic acid a -hydroxy- ethyl amide (including its isoform), lysergic acid amide (including its isoform) and ergonovine. In addition, elymoclavine and agroclavine are present in T. corymbosa but were not detectable in I. asarifolia (Fig. 6). The total amount of alkaloids in the T. corymbosa plant amounted to roughly twice as much as found in the I. asarifolia plant (Fig. 6). The latter contained 7.0 l g alkaloids expressed as ergonovine per gram fresh weight (Kucht et al. 2004) whereas the former contained 14.6 l g alkaloids per gram fresh weight expressed as ergonovine. Twelve culturable fungi and one unculturable fungus were isolated from the I. asarifolia plant. Phylogenetic analysis of these organisms resulted in essentially con- gruent observations for the 18S rDNA and the ITS data set with respect to confidently identified nodes (Fig. 3a, b). In three cases, sequences from the new isolates have identical counterparts in the databases both for 18 SrDNA and for ITS, respectively: IasaF05 ( Cladosporium cladosporioides ) Iasa F10 ( Glomerella cingulata , anamorph: Colletotrichum gloeosporioides ) and IasaF12 ( Sclerotinia sclerotiorum ). Isolates IasaF01, IasaF04, IasaF07 and IasaF11 clearly fall into the Homobasidiomycetes, and are related to available Agaricales ( Collybia , Lepista ) or Aphyllophorales ( Athelia , Phanerochaete , Sistotrema ) sequence entries, respectively. Generally, the ITS data set (Fig. 3b) provides better phylogenetic fine resolution for closely related taxa. The ITS sequence of isolate IasaF04 is identical to the corresponding sequence of Thanatephorus cucumeris (anamorph: Rhizoctonia solani) . Sequences from isolates IasaF06, IsaF08 and IasaF09 are identical to those of different Penicillium species. Isolate IasaF13 is clearly identified as a member of the family Clavicipitaceae (Hypocreales). Other genera and those of the sister families in the Hypocreales, the Bionectriaceae ( Myrothecium ), Ceratostomataceae ( Melanospora ), Hypocrea- ceae ( Hypomyces ), Nectriaceae ( Calonectria ) and Niessliaceae ( Melanopsamma ) branch more distantly. Thus, both trees distinguish between fungi belonging to the family of Clavicipitaceae and those which do not. In both phylogenetic trees IasaF13 groups together with ergoline alkaloid-producing clavicipitaceous fungi. This is an important observation because it strongly suggests that the nonculturable epibiotic fungus IasaF13 (Fig. 2) is responsible for the production of ergoline alkaloids as is the case for clavicipitaceous fungi occurring on plants belonging to grasses. Epibiotic clavicipitaceous fungi are also found within the genus Balansia (Reddy et al. 1998), relatives of our epibiotic strain IasaF13 (Fig. 3b). As expected, the fungus IasaF13 has a gene with significant similarity to the gene encoding a protein responsible for catalysing the synthesis of 4-( c , c -dim- ethylallyl)tryptophan (Tsai et al. 1995; Tudzynski et al. 1999, 2001; Unso ̈ ld and Li 2005) a precursor of ergoline alkaloids. This gene is present in C. purpurea (Tudzynski et al. 1999, 2001), C. fusiformis (Wang et al. 2004), Neotyphodium sp. isolate Lp1 (Wang et al. 2004), and Aspergillus fumigatus (Unso ̈ ld and Li 2005) and is known to be responsible for the first committed step in ergoline alkaloid biosynthesis. At present we cannot fully exclude the possibility that P. roquefortii contributes to the spectrum of ergoline alkaloids in I. asarifolia but we did not find any evidence that supports this view: 1. Alkaloids occurring in I. asarifolia are chanoclavine- I, elymoclavine, lysergic acid amide, isolysergic acid amide (Kucht et al. 2004) as well as ergobalansine and ergobalansinine (Jenett-Siems et al. 1994). Isofumigaclavine A, the ergoline alkaloid present in P. roquefortii ...

Context 5

... strain IasaF13 (Fig. 3b). As expected, the fungus IasaF13 has a gene with significant similarity to the gene encoding a protein responsible for catalysing the synthesis of 4-( c , c -dim- ethylallyl)tryptophan (Tsai et al. 1995; Tudzynski et al. 1999, 2001; Unso ̈ ld and Li 2005) a precursor of ergoline alkaloids. This gene is present in C. purpurea (Tudzynski et al. 1999, 2001), C. fusiformis (Wang et al. 2004), Neotyphodium sp. isolate Lp1 (Wang et al. 2004), and Aspergillus fumigatus (Unso ̈ ld and Li 2005) and is known to be responsible for the first committed step in ergoline alkaloid biosynthesis. At present we cannot fully exclude the possibility that P. roquefortii contributes to the spectrum of ergoline alkaloids in I. asarifolia but we did not find any evidence that supports this view: 1. Alkaloids occurring in I. asarifolia are chanoclavine- I, elymoclavine, lysergic acid amide, isolysergic acid amide (Kucht et al. 2004) as well as ergobalansine and ergobalansinine (Jenett-Siems et al. 1994). Isofumigaclavine A, the ergoline alkaloid present in P. roquefortii (Scott et al. 1976), has so far not been reported to be a constituent of I. asarifolia . 2. Inoculation of I. asarifolia plants with P. roquefortii showed that the fungus was well established on the plant which, however, was devoid of ergoline alkaloids and roquefortine. 3. Growth of I. asarifolia plants (either from seeds or after regeneration) in a sterile environment gave a full spectrum of alkaloids. 4. These plants as well as the plant cell cultures contained the ITS sequence of the clavicipitaceous fungal isolate IasaF13 alone: Among 36 (see Materials and methods) cloned ITS sequences none was identical to that of IasaF09 ( P. roquefortii ) but all were identical to those of IasaF13. 5. Throughout these experiments, it was found that the fungus IasaF13 was always detected when alkaloids were present: Thus, treatment of the plant with sys- temic fungicides ‘‘Folicur’’ and ‘‘Pronto Plus’’ gave plants devoid of both fungus and alkaloids. Treat- ment of the plants with fungicides ‘‘Benomyl’’ and ‘‘Switch’’ neither removed the fungus IasaF13 nor the alkaloids (Kucht et al. 2004). The fungus P. roquefortii may be a non-specific associate of I. asarifolia . It has been isolated from var- ious substrates such as soil samples (Ohomo et al. 1975) and from livestock feed (Boysen 1999). The fungus grows even on cheese (Carlile et al. 2001). It is conceivable that P. roquefortii is horizontally transmitted as is often the case among endophytes (Arnold et al. 2003). The fact that the clavicipitaceous fungus IasaF13 is non-culturable in defined media shows that it heavily depends on the plant for growth and vegetative repro- duction and that there is a highly specific interaction between both organisms. It is remarkable that the fungus never spread to I. asarifolia plants devoid of fungus and alkaloids although the plant carrying the fungus was kept in the immediate neighbourhood in the same green house. This is in line with the result of the ‘‘attachment- experiment’’ in which no inoculation of the plant devoid of fungi was observed ( vide supra ) although both plants were kept in close contact in an environment of high humidity. Our observations are in agreement with results from experiments on clavicipitaceous fungi colonizing grasses: Infection of host plants with asexual propagules (conidia and mycelia) is very difficult and unlikely to occur in nature (Gentile et al. 2005). Thus, the fungus IasaF13 behaves in a similar way experienced for clavicipitaceous fungi living on Poaceae plants, including the fact that they are often seed transmitted (Clay and Schardl 2002). Plant callus and cell suspension cultures are believed to be sterile. This, however, was not experienced with the I. asarifolia cell culture derived from surface-sterilized stems. Microscopic inspection (Fig. 5), SSCP analysis and ITS sequencing of DNA obtained from a callus and a cell suspension culture of I. asarifolia showed that the fungus was also present in cell cultures. Fungal hyphae typically consisting of up to 20 compartments which stained with calcofluor were clearly and microscopically visible (Fig. 5). Thus, during establishment of the cell culture, the process of surface sterilization of a stem segment of the plant does not remove the fungus Ia- saF13 which even in cell cultures is able to live in association with the plant cells (cf. Figs. 4, 5). The presence of fungal hyphae in the cultured cells is not visible to the naked eye and plant cells seem to grow unaffected by the fungus. This may indicate that plant cells and the fungus keep each other in check during a balanced growth. Intensive studies using different culture media, however, did not give any indication that undifferentiated cultured plant cells contained any trace of ergoline alkaloids (Kucht et al. 2004). Alkaloids and fungal colonies (Fig. 2) appeared only during the regeneration process (Fig. 4). This shows that for the successful production of ergoline alkaloids the fungus IasaF13 and a morphologically differentiated I. asarifolia plant are essential. We observed that a fungus like IasaF13 is not only present on I. asarifolia but also on T. corymbosa . Again, the epibiotic fungus can be removed by fungicide treatment. Loss of the fungus from the plant again (Kucht et al. 2004) occurs concomitantly with elimination of ergoline alkaloids (data not shown). The fungus on T. corymbosa is identical to the epibiotic fungus IasaF13 as far as ITS and 18S rDNA sequences are concerned. The alkaloid spectrum of both plants, however, differs qualitatively (Fig. 6) and quantitatively (Fig. 6 and Results). This is in agreement with data obtained from experiments with Neotyphodium lolii , an endophyte of the grass Lolium perenne : Although it is clearly the fungus that is responsible for the synthesis of alkaloids, accumulation of alkaloids is affected and modulated by the plant genotype (Lane et al. 2000; Spiering et al. 2005). We conclude that the presence of alkaloids in the family Convolvulaceae very likely is not due to a horizontal gene transfer which occurred during evolution, or a repeated ‘‘invention’’ of the same biosynthetic path- way in two different taxa Ascomycota and Convolvulaceae, or due to an ancestral trait that was eliminated during evolution in most taxa except a few but rather that clavicipitaceous fungi not only colonize monoco- telydonous plants such as Poaceae but also dicotyledonous plants belonging to the family ...

Context 6

... clearly predominates among all the plant-associated fungi suggesting that it is identical to the abun- dantly visible fungus attached to the upper leaf surface ( Fig. 2). In contrast, an Ipomoea plant devoid of both fungi and alkaloids does not show the pattern of bands observed for both isolated fungi IasaF09 and IasaF13 (Fig. 4). If one of the isolated fungi is a producer of ergoline alkaloids in the Ipomoea plant, it should contain genes responsible for ergoline alkaloid biosynthesis. Oligonu- cleotides (deg1 and deg4) targeted to conserved regions of the dmaW (or cpd) dimethylallyl-tryptophan-synthase gene (Wang et al. 2004), known to be involved in the introduction of a dimethylallyl residue into tryptophan (Fig. 1) were synthesized and employed in a PCR reaction with DNA from the epibiotic fungus IasaF13 as a template. A PCR product of 939 bp was obtained and its deduced protein sequence showed very high similarity with the available dmaW sequences of Clavicipitaceaen fungi, being most similar to a Balansia obtecta homologue with 76% identical amino acids (Fig. 3c). The clavicipitaceous fungi live on grasses and are known producers of ergoline alkaloids. The same experiment was carried out with DNA isolated from IasaF09 (P. roquefortii ) revealing a dmaW homologue with 63% sequence identity to an Aspergillus fumigatus sequence. Hence, a dimethylallyl-tryptophan-synthase appears also to be present in IasaF09 (P. roquefortii ), however, the corresponding gene in the epibiotic fungus IasaF13 is expectedly much more closely related to the genes detected in Clavicipitaceae. A phylogenetic analysis of the Cpd1 protein sequences, also including several predicted protein sequences form different fungi, indicates several distant homologues in Aspergillus and more recent, independent gene duplications in the Clavicipitaceae , at least in Claviceps purpurea and Neotyphodium coenophialum (Fig. 3c). Subsequently both candidate fungi were used to inoculate I. asarifolia plants free of alkaloids and fungi. The fungus IasaF09 ( P. roquefortii ) was grown on a defined solid medium, a mixture of hyphae and spores was suspended in water following a method used by Latch and Christensen (1985) and the suspension injected into leaves with a syringe and in addition spread onto leaves of the I. asarifolia plant. The inoculated plants were kept in the greenhouse. Microscopic exam- ination of the plants 6, 18 and 26 weeks after inoculation showed that the fungus was well established on the plant. Analysis of the plant 26 weeks after inoculation did not show the presence of roquefortine or of any ergoline alkaloid. Thus, P. roquefortii appears not to be the candidate fungus responsible for the accumulation of ergoline alkaloids in I. asarifolia . The same experiment was carried out with hyphae of IasaF13 isolated from the unfolded leaves of an I. asarifolia plant and spread onto and injected into I. asarifolia leaves of a plant devoid of alkaloids and fungi. As opposed to the experiment with P. roquefortii , however, no fungal growth was observed. This observation was not unexpected (see later). The inability to establish the epibiotic fungus IasaF13 on I. asarifolia was also experienced in the so-called ‘‘attachment experiment’’, in which a normal plant and a plant devoid of fungi and alkaloids were kept in close contact in a cylindrical plastic glass container in the green house with the upper leaf surfaces of both plants attached to each other. After 18 weeks no spread of fungal hyphae to the plant devoid of IasaF13 was observed. In addition, this plant did not contain any alkaloids. Spread of fungal hyphae and presence of ergoline alkaloids, however, were observed when externally sterilized seeds were germinated in a sterile environment on an artificial agar medium in a closed Erlenmeyer flask. Plants grown under these conditions contained both fungus IasaF13 and alkaloids indicating that I. asarifolia seeds harbour fungal propagules of IasaF13 that were spread to the growing plant. Indeed, SSCP (Fig. 4) and microscopic investigation of seeds showed that seeds clearly contain the fungus IasaF13. Evidently, this fungus is spread to the shoot of the plant during growth (Fig. 4). Moreover, the plantlets contained the full spectrum of alkaloids (TLC, HPLC-MS) known to be present in the untreated intact plant. In a similar experiment, a surface-sterilized piece of a stem was placed on an agar medium. After 2–4 weeks a callus was formed. Stem and callus were then transferred into a liquid medium. Shoots regenerated from the plant material. The shoots of these plantlets were cut-off and the cuttings placed into solid agar medium. Roots developed within 2 weeks . Microscopic inspection, SSCP and chemical analysis showed that both fungus IasaF13 and the complete spectrum of alkaloids were again present. We conclude that both seeds and plant cell cultures of the I. asarifolia plant contain the clavicipitaceous fungus and that this fungus is involved in the accumulation of alkaloids in I. asarifolia . Absence and presence of fungus IasaF13 was also investigated microscopically, by SSCP and also by sequencing of DNA after PCR amplification using oligomers (ITS1F and ITS4) (White et al. 1990; Gardes and Bruns 1993) targeted to the ITS region. These experiments fully confirmed the presence of IasaF13 in the intact control plant, seeds, plants grown under sterile conditions from seeds, regenerated plants, plant callus and cell suspension cultures. The ITS sequencing was repeated 35 times (cf. Materials and methods). Each time the sequence of IasaF13 but never that of IasaF09 ( P. roquefortii ) was found. The fungal ITS sequence again was not found in plants which are devoid of alkaloids (see Fig. 4). Whenever ITS and SSCP (Fig. 4) were positive with respect to the presence of fungus Ia- saF13 microscopic inspection of the plant material confirmed the presence of this fungus, including the plant cell culture material (Fig. 5) and in spite of the fact that plant cell cultures are considered to be sterile. An epibiotic fungus was also detected on another plant species, Turbina corymbosa (L.) Hall. This plant also belongs to the family Convolvulaceae and contains ergoline alkaloids. T. corymbosa and I. asarifolia are indigenous to Central or South America, respectively. The epibiotic fungus from T. corymbosa was submitted to 18S rDNA (DQ127 278) and ITS analysis (AY995219). It turned out that the sequences were 100% identical when compared with those of the epibiotic fungus IasaF13 on I. asarifolia . This result was obtained independently in the laboratories of U.S. and E.L. The alkaloid spectrum of aerial parts of both plant species was investigated quantitatively and qualitatively using a high pressure liquid chromatograph connected to a mass spectrometer (Fig. 6). The compounds were identified by comparison with authentic standards. Both plants contain chanoclavine, lysergic acid a -hydroxy- ethyl amide (including its isoform), lysergic acid amide (including its isoform) and ergonovine. In addition, elymoclavine and agroclavine are present in T. corymbosa but were not detectable in I. asarifolia (Fig. 6). The total amount of alkaloids in the T. corymbosa plant amounted to roughly twice as much as found in the I. asarifolia plant (Fig. 6). The latter contained 7.0 l g alkaloids expressed as ergonovine per gram fresh weight (Kucht et al. 2004) whereas the former contained 14.6 l g alkaloids per gram fresh weight expressed as ergonovine. Twelve culturable fungi and one unculturable fungus were isolated from the I. asarifolia plant. Phylogenetic analysis of these organisms resulted in essentially con- gruent observations for the 18S rDNA and the ITS data set with respect to confidently identified nodes (Fig. 3a, b). In three cases, sequences from the new isolates have identical counterparts in the databases both for 18 SrDNA and for ITS, respectively: IasaF05 ( Cladosporium cladosporioides ) Iasa F10 ( Glomerella cingulata , anamorph: Colletotrichum gloeosporioides ) and IasaF12 ( Sclerotinia sclerotiorum ). Isolates IasaF01, IasaF04, IasaF07 and IasaF11 clearly fall into the Homobasidiomycetes, and are related to available Agaricales ( Collybia , Lepista ) or Aphyllophorales ( Athelia , Phanerochaete , Sistotrema ) sequence entries, respectively. Generally, the ITS data set (Fig. 3b) provides better phylogenetic fine resolution for closely related taxa. The ITS sequence of isolate IasaF04 is identical to the corresponding sequence of Thanatephorus cucumeris (anamorph: Rhizoctonia solani) . Sequences from isolates IasaF06, IsaF08 and IasaF09 are identical to those of different Penicillium species. Isolate IasaF13 is clearly identified as a member of the family Clavicipitaceae (Hypocreales). Other genera and those of the sister families in the Hypocreales, the Bionectriaceae ( Myrothecium ), Ceratostomataceae ( Melanospora ), Hypocrea- ceae ( Hypomyces ), Nectriaceae ( Calonectria ) and Niessliaceae ( Melanopsamma ) branch more distantly. Thus, both trees distinguish between fungi belonging to the family of Clavicipitaceae and those which do not. In both phylogenetic trees IasaF13 ...

Context 7

... clearly predominates among all the plant-associated fungi suggesting that it is identical to the abun- dantly visible fungus attached to the upper leaf surface ( Fig. 2). In contrast, an Ipomoea plant devoid of both fungi and alkaloids does not show the pattern of bands observed for both isolated fungi IasaF09 and IasaF13 (Fig. 4). If one of the isolated fungi is a producer of ergoline alkaloids in the Ipomoea plant, it should contain genes responsible for ergoline alkaloid biosynthesis. Oligonu- cleotides (deg1 and deg4) targeted to conserved regions of the dmaW (or cpd) dimethylallyl-tryptophan-synthase gene (Wang et al. 2004), known to be involved in the introduction of a dimethylallyl residue into tryptophan (Fig. 1) were synthesized and employed in a PCR reaction with DNA from the epibiotic fungus IasaF13 as a template. A PCR product of 939 bp was obtained and its deduced protein sequence showed very high similarity with the available dmaW sequences of Clavicipitaceaen fungi, being most similar to a Balansia obtecta homologue with 76% identical amino acids (Fig. 3c). The clavicipitaceous fungi live on grasses and are known producers of ergoline alkaloids. The same experiment was carried out with DNA isolated from IasaF09 (P. roquefortii ) revealing a dmaW homologue with 63% sequence identity to an Aspergillus fumigatus sequence. Hence, a dimethylallyl-tryptophan-synthase appears also to be present in IasaF09 (P. roquefortii ), however, the corresponding gene in the epibiotic fungus IasaF13 is expectedly much more closely related to the genes detected in Clavicipitaceae. A phylogenetic analysis of the Cpd1 protein sequences, also including several predicted protein sequences form different fungi, indicates several distant homologues in Aspergillus and more recent, independent gene duplications in the Clavicipitaceae , at least in Claviceps purpurea and Neotyphodium coenophialum (Fig. 3c). Subsequently both candidate fungi were used to inoculate I. asarifolia plants free of alkaloids and fungi. The fungus IasaF09 ( P. roquefortii ) was grown on a defined solid medium, a mixture of hyphae and spores was suspended in water following a method used by Latch and Christensen (1985) and the suspension injected into leaves with a syringe and in addition spread onto leaves of the I. asarifolia plant. The inoculated plants were kept in the greenhouse. Microscopic exam- ination of the plants 6, 18 and 26 weeks after inoculation showed that the fungus was well established on the plant. Analysis of the plant 26 weeks after inoculation did not show the presence of roquefortine or of any ergoline alkaloid. Thus, P. roquefortii appears not to be the candidate fungus responsible for the accumulation of ergoline alkaloids in I. asarifolia . The same experiment was carried out with hyphae of IasaF13 isolated from the unfolded leaves of an I. asarifolia plant and spread onto and injected into I. asarifolia leaves of a plant devoid of alkaloids and fungi. As opposed to the experiment with P. roquefortii , however, no fungal growth was observed. This observation was not unexpected (see later). The inability to establish the epibiotic fungus IasaF13 on I. asarifolia was also experienced in the so-called ‘‘attachment experiment’’, in which a normal plant and a plant devoid of fungi and alkaloids were kept in close contact in a cylindrical plastic glass container in the green house with the upper leaf surfaces of both plants attached to each other. After 18 weeks no spread of fungal hyphae to the plant devoid of IasaF13 was observed. In addition, this plant did not contain any alkaloids. Spread of fungal hyphae and presence of ergoline alkaloids, however, were observed when externally sterilized seeds were germinated in a sterile environment on an artificial agar medium in a closed Erlenmeyer flask. Plants grown under these conditions contained both fungus IasaF13 and alkaloids indicating that I. asarifolia seeds harbour fungal propagules of IasaF13 that were spread to the growing plant. Indeed, SSCP (Fig. 4) and microscopic investigation of seeds showed that seeds clearly contain the fungus IasaF13. Evidently, this fungus is spread to the shoot of the plant during growth (Fig. 4). Moreover, the plantlets contained the full spectrum of alkaloids (TLC, HPLC-MS) known to be present in the untreated intact plant. In a similar experiment, a surface-sterilized piece of a stem was placed on an agar medium. After 2–4 weeks a callus was formed. Stem and callus were then transferred into a liquid medium. Shoots regenerated from the plant material. The shoots of these plantlets were cut-off and the cuttings placed into solid agar medium. Roots developed within 2 weeks . Microscopic inspection, SSCP and chemical analysis showed that both fungus IasaF13 and the complete spectrum of alkaloids were again present. We conclude that both seeds and plant cell cultures of the I. asarifolia plant contain the clavicipitaceous fungus and that this fungus is involved in the accumulation of alkaloids in I. asarifolia . Absence and presence of fungus IasaF13 was also investigated microscopically, by SSCP and also by sequencing of DNA after PCR amplification using oligomers (ITS1F and ITS4) (White et al. 1990; Gardes and Bruns 1993) targeted to the ITS region. These experiments fully confirmed the presence of IasaF13 in the intact control plant, seeds, plants grown under sterile conditions from seeds, regenerated plants, plant callus and cell suspension cultures. The ITS sequencing was repeated 35 times (cf. Materials and methods). Each time the sequence of IasaF13 but never that of IasaF09 ( P. roquefortii ) was found. The fungal ITS sequence again was not found in plants which are devoid of alkaloids (see Fig. 4). Whenever ITS and SSCP (Fig. 4) were positive with respect to the presence of fungus Ia- saF13 microscopic inspection of the plant material confirmed the presence of this fungus, including the plant cell culture material (Fig. 5) and in spite of the fact that plant cell cultures are considered to be sterile. An epibiotic fungus was also detected on another plant species, Turbina corymbosa (L.) Hall. This plant also belongs to the family Convolvulaceae and contains ergoline alkaloids. T. corymbosa and I. asarifolia are indigenous to Central or South America, respectively. The epibiotic fungus from T. corymbosa was submitted to 18S rDNA (DQ127 278) and ITS analysis (AY995219). It turned out that the sequences were 100% identical when compared with those of the epibiotic fungus IasaF13 on I. asarifolia . This result was obtained independently in the laboratories of U.S. and E.L. The alkaloid spectrum of aerial parts of both plant species was investigated quantitatively and qualitatively using a high pressure liquid chromatograph connected to a mass spectrometer (Fig. 6). The compounds were identified by comparison with authentic standards. Both plants contain chanoclavine, lysergic acid a -hydroxy- ethyl amide (including its isoform), lysergic acid amide (including its isoform) and ergonovine. In addition, elymoclavine and agroclavine are present in T. corymbosa but were not detectable in I. asarifolia (Fig. 6). The total amount of alkaloids in the T. corymbosa plant amounted to roughly twice as much as found in the I. asarifolia plant (Fig. 6). The latter contained 7.0 l g alkaloids expressed as ergonovine per gram fresh weight (Kucht et al. 2004) whereas the former contained 14.6 l g alkaloids per gram fresh weight expressed as ergonovine. Twelve culturable fungi and one unculturable fungus were isolated from the I. asarifolia plant. Phylogenetic analysis of these organisms resulted in essentially con- gruent observations for the 18S rDNA and the ITS data set with respect to confidently identified nodes (Fig. 3a, b). In three cases, sequences from the new isolates have identical counterparts in the databases both for 18 SrDNA and for ITS, respectively: IasaF05 ( Cladosporium cladosporioides ) Iasa F10 ( Glomerella cingulata , anamorph: Colletotrichum gloeosporioides ) and IasaF12 ( Sclerotinia sclerotiorum ). Isolates IasaF01, IasaF04, IasaF07 and IasaF11 clearly fall into the Homobasidiomycetes, and are related to available Agaricales ( Collybia , Lepista ) or Aphyllophorales ( Athelia , Phanerochaete , Sistotrema ) sequence entries, respectively. Generally, the ITS data set (Fig. 3b) provides better phylogenetic fine resolution for closely related taxa. The ITS sequence of isolate IasaF04 is identical to the corresponding sequence of Thanatephorus cucumeris (anamorph: Rhizoctonia solani) . Sequences from isolates IasaF06, IsaF08 and IasaF09 are identical to those of different Penicillium species. Isolate IasaF13 is clearly identified as a member of the family Clavicipitaceae (Hypocreales). Other genera and those of the sister families in the Hypocreales, the Bionectriaceae ( Myrothecium ), Ceratostomataceae ( Melanospora ), Hypocrea- ceae ( Hypomyces ), Nectriaceae ( Calonectria ) and Niessliaceae ( Melanopsamma ...

Context 8

... fungus IasaF13 as a template. A PCR product of 939 bp was obtained and its deduced protein sequence showed very high similarity with the available dmaW sequences of Clavicipitaceaen fungi, being most similar to a Balansia obtecta homologue with 76% identical amino acids (Fig. 3c). The clavicipitaceous fungi live on grasses and are known producers of ergoline alkaloids. The same experiment was carried out with DNA isolated from IasaF09 (P. roquefortii ) revealing a dmaW homologue with 63% sequence identity to an Aspergillus fumigatus sequence. Hence, a dimethylallyl-tryptophan-synthase appears also to be present in IasaF09 (P. roquefortii ), however, the corresponding gene in the epibiotic fungus IasaF13 is expectedly much more closely related to the genes detected in Clavicipitaceae. A phylogenetic analysis of the Cpd1 protein sequences, also including several predicted protein sequences form different fungi, indicates several distant homologues in Aspergillus and more recent, independent gene duplications in the Clavicipitaceae , at least in Claviceps purpurea and Neotyphodium coenophialum (Fig. 3c). Subsequently both candidate fungi were used to inoculate I. asarifolia plants free of alkaloids and fungi. The fungus IasaF09 ( P. roquefortii ) was grown on a defined solid medium, a mixture of hyphae and spores was suspended in water following a method used by Latch and Christensen (1985) and the suspension injected into leaves with a syringe and in addition spread onto leaves of the I. asarifolia plant. The inoculated plants were kept in the greenhouse. Microscopic exam- ination of the plants 6, 18 and 26 weeks after inoculation showed that the fungus was well established on the plant. Analysis of the plant 26 weeks after inoculation did not show the presence of roquefortine or of any ergoline alkaloid. Thus, P. roquefortii appears not to be the candidate fungus responsible for the accumulation of ergoline alkaloids in I. asarifolia . The same experiment was carried out with hyphae of IasaF13 isolated from the unfolded leaves of an I. asarifolia plant and spread onto and injected into I. asarifolia leaves of a plant devoid of alkaloids and fungi. As opposed to the experiment with P. roquefortii , however, no fungal growth was observed. This observation was not unexpected (see later). The inability to establish the epibiotic fungus IasaF13 on I. asarifolia was also experienced in the so-called ‘‘attachment experiment’’, in which a normal plant and a plant devoid of fungi and alkaloids were kept in close contact in a cylindrical plastic glass container in the green house with the upper leaf surfaces of both plants attached to each other. After 18 weeks no spread of fungal hyphae to the plant devoid of IasaF13 was observed. In addition, this plant did not contain any alkaloids. Spread of fungal hyphae and presence of ergoline alkaloids, however, were observed when externally sterilized seeds were germinated in a sterile environment on an artificial agar medium in a closed Erlenmeyer flask. Plants grown under these conditions contained both fungus IasaF13 and alkaloids indicating that I. asarifolia seeds harbour fungal propagules of IasaF13 that were spread to the growing plant. Indeed, SSCP (Fig. 4) and microscopic investigation of seeds showed that seeds clearly contain the fungus IasaF13. Evidently, this fungus is spread to the shoot of the plant during growth (Fig. 4). Moreover, the plantlets contained the full spectrum of alkaloids (TLC, HPLC-MS) known to be present in the untreated intact plant. In a similar experiment, a surface-sterilized piece of a stem was placed on an agar medium. After 2–4 weeks a callus was formed. Stem and callus were then transferred into a liquid medium. Shoots regenerated from the plant material. The shoots of these plantlets were cut-off and the cuttings placed into solid agar medium. Roots developed within 2 weeks . Microscopic inspection, SSCP and chemical analysis showed that both fungus IasaF13 and the complete spectrum of alkaloids were again present. We conclude that both seeds and plant cell cultures of the I. asarifolia plant contain the clavicipitaceous fungus and that this fungus is involved in the accumulation of alkaloids in I. asarifolia . Absence and presence of fungus IasaF13 was also investigated microscopically, by SSCP and also by sequencing of DNA after PCR amplification using oligomers (ITS1F and ITS4) (White et al. 1990; Gardes and Bruns 1993) targeted to the ITS region. These experiments fully confirmed the presence of IasaF13 in the intact control plant, seeds, plants grown under sterile conditions from seeds, regenerated plants, plant callus and cell suspension cultures. The ITS sequencing was repeated 35 times (cf. Materials and methods). Each time the sequence of IasaF13 but never that of IasaF09 ( P. roquefortii ) was found. The fungal ITS sequence again was not found in plants which are devoid of alkaloids (see Fig. 4). Whenever ITS and SSCP (Fig. 4) were positive with respect to the presence of fungus Ia- saF13 microscopic inspection of the plant material confirmed the presence of this fungus, including the plant cell culture material (Fig. 5) and in spite of the fact that plant cell cultures are considered to be sterile. An epibiotic fungus was also detected on another plant species, Turbina corymbosa (L.) Hall. This plant also belongs to the family Convolvulaceae and contains ergoline alkaloids. T. corymbosa and I. asarifolia are indigenous to Central or South America, respectively. The epibiotic fungus from T. corymbosa was submitted to 18S rDNA (DQ127 278) and ITS analysis (AY995219). It turned out that the sequences were 100% identical when compared with those of the epibiotic fungus IasaF13 on I. asarifolia . This result was obtained independently in the laboratories of U.S. and E.L. The alkaloid spectrum of aerial parts of both plant species was investigated quantitatively and qualitatively using a high pressure liquid chromatograph connected to a mass spectrometer (Fig. 6). The compounds were identified by comparison with authentic standards. Both plants contain chanoclavine, lysergic acid a -hydroxy- ethyl amide (including its isoform), lysergic acid amide (including its isoform) and ergonovine. In addition, elymoclavine and agroclavine are present in T. corymbosa but were not detectable in I. asarifolia (Fig. 6). The total amount of alkaloids in the T. corymbosa plant amounted to roughly twice as much as found in the I. asarifolia plant (Fig. 6). The latter contained 7.0 l g alkaloids expressed as ergonovine per gram fresh weight (Kucht et al. 2004) whereas the former contained 14.6 l g alkaloids per gram fresh weight expressed as ergonovine. Twelve culturable fungi and one unculturable fungus were isolated from the I. asarifolia plant. Phylogenetic analysis of these organisms resulted in essentially con- gruent observations for the 18S rDNA and the ITS data set with respect to confidently identified nodes (Fig. 3a, b). In three cases, sequences from the new isolates have identical counterparts in the databases both for 18 SrDNA and for ITS, respectively: IasaF05 ( Cladosporium cladosporioides ) Iasa F10 ( Glomerella cingulata , anamorph: Colletotrichum gloeosporioides ) and IasaF12 ( Sclerotinia sclerotiorum ). Isolates IasaF01, IasaF04, IasaF07 and IasaF11 clearly fall into the Homobasidiomycetes, and are related to available Agaricales ( Collybia , Lepista ) or Aphyllophorales ( Athelia , Phanerochaete , Sistotrema ) sequence entries, respectively. Generally, the ITS data set (Fig. 3b) provides better phylogenetic fine resolution for closely related taxa. The ITS sequence of isolate IasaF04 is identical to the corresponding sequence of Thanatephorus cucumeris (anamorph: Rhizoctonia solani) . Sequences from isolates IasaF06, IsaF08 and IasaF09 are identical to those of different Penicillium species. Isolate IasaF13 is clearly identified as a member of the family Clavicipitaceae (Hypocreales). Other genera and those of the sister families in the Hypocreales, the Bionectriaceae ( Myrothecium ), Ceratostomataceae ( Melanospora ), Hypocrea- ceae ( Hypomyces ), Nectriaceae ( Calonectria ) and Niessliaceae ( Melanopsamma ) branch more distantly. Thus, both trees distinguish between fungi belonging to the family of Clavicipitaceae and those which do not. In both phylogenetic trees IasaF13 groups together with ergoline alkaloid-producing clavicipitaceous fungi. This is an important observation because it strongly suggests that the nonculturable epibiotic fungus IasaF13 (Fig. 2) is responsible for the production of ergoline alkaloids as is the case for clavicipitaceous fungi occurring on plants belonging to grasses. Epibiotic clavicipitaceous fungi are also found within the genus Balansia (Reddy et al. 1998), relatives of our epibiotic strain IasaF13 (Fig. 3b). As expected, the fungus IasaF13 has a gene with significant similarity to the gene encoding a protein responsible for catalysing the synthesis of 4-( c , c -dim- ethylallyl)tryptophan (Tsai et al. 1995; Tudzynski et al. 1999, 2001; Unso ̈ ld and Li 2005) a precursor of ergoline alkaloids. This gene is present in C. purpurea (Tudzynski et al. 1999, 2001), C. fusiformis (Wang et al. 2004), Neotyphodium sp. isolate Lp1 (Wang et al. 2004), and Aspergillus fumigatus (Unso ̈ ld and Li 2005) and is known to be responsible for the first committed step in ergoline alkaloid biosynthesis. At present we cannot fully exclude the possibility that P. roquefortii contributes to the spectrum of ergoline alkaloids in I. asarifolia but we did not find any evidence that supports this view: 1. Alkaloids occurring in I. asarifolia are chanoclavine- I, elymoclavine, lysergic acid amide, isolysergic acid amide (Kucht et al. 2004) as well as ergobalansine and ergobalansinine (Jenett-Siems et al. 1994). Isofumigaclavine A, the ergoline alkaloid present in P. roquefortii (Scott et al. 1976), has so far ...