Phoma Herbarum Research Articles

Neuraminidase Inhibitory Polyketides from the Marine-derived FungusPhoma herbarum

Two new polyketides, arthropsadiol C (1) and massarilactone H (2), together with six known derivatives (3-8) were isolated from the culture broth of the marine-derived fungus Phoma herbarum. Their structures were elucidated on the basis of spectroscopic methods, including 2D NMR techniques. Compounds 2, 4, 5, and 8 showed moderate neuraminidase inhibitory activity with IC(50) values ranging from 4.15 to 9.16 µM.

Cloning, expression and biochemical characterization of UDP-glucose 6-dehydrogenase, a key enzyme in the biosynthesis of an anti-tumor polysaccharide from the marine fungus Phoma herbarum YS4108

There is an urgent requirement for unraveling the pathway for biosynthesis of the polysaccharide YCP from the marine fungus Phoma herbarum YS4108 in order to exploit its potential as an anti-tumor agent. Here we present cloning and characterization of UDP-glucose 6-dehydrogenase (UDP-GlcDHase), a key enzyme for the biosynthesis of this glycan. A full-length cDNA encoding UDP-GlcDHase was obtained by PCR using degenerate primers and the RACE strategy. The cDNA was 1846bp in length with an open reading frame of 1560bp encoding a protein of 520 amino acids. The deduced amino acid sequence showed about 50% overall identity to its homologs and a high degree of conservation in the nucleotide binding and catalytic domains. The cDNA was cloned in the Pichia pastoris GS115 on the plasmid vector, pPIC3.5k, to allow inducible expression of the protein with an N-terminal histidine-tag. Recombinant UDP-GlcDHase was affinity purified from crude, cytosolic extracts of the host cells, and characterized in terms of the its substrate affinity and the optimum temperature and pH for its activity. The biochemical properties of the purified recombinant enzyme were comparable with those of its homologs. The present investigation provides a promising start for manipulating YCP biosynthesis in P. herbarum.

Anthraquinone pigment with herbicidal potential from Phoma herbarum FGCC#54

Metabolites from Phoma sp., a phytopathogenic fungus, were exploited for their herbicidal potential. The phytotoxic compound isolated was identified as anhydropseudophlegmacin-9,10-quinone-3′-amino-8′-O-methyl ether on the basis of spectral data. The isolated pigment was tested for its herbicidal potential against the prominent weeds of Central India, where a positive result was obtained.

Psychrotrophic microorganism communities in soils of Haswell Island, Antarctica, and their biosynthetic potential

Diversity of microorganisms plays an important role in the functioning of ecosystems and their response to large-scale environmental changes. Microbial soil communities from virgin Haswell Island, Antarctica, were studied using the serial dilution-spread plate method at two different temperatures. Microorganisms were identified by sporulation, morphological, chemotaxonomic assessment. Nitrogen-fixing cyanobacteria and microalgae prevailed in the samples. Representatives of Chamaesiphon, Dermocarpa, Fischerella, Oscillatoria, Prochlrococcus, Synechococcus, Chlorophyta were detected. Fungal assemblages comprised Aspergillus nidulans, Cladosporium cladosporioides, Alternaria alternata, Mortierella ramanniana, Penicillium verrucosum, Scopulariopsis fusca, Cladophialophora minutissima, Chaetomium gracile, Phoma herbarum, Phialophora fastigiata, Ulocladium consortiale and Candida sp. Actinomycetes were identified by morphological and chemotaxonomic assessment of cell-wall aminoacids and sugars as Streptomyces, Nocardia and Geodermatophilus. Some of the isolated microorganisms formed extracellular enzymes, others biosynthesized substances with antifungal and antibacterial activities. Single psychrotrophic strains have an ability to grow on n-paraffins and naphthalene, and thin-layer chromatographic analyses showed that they synthesized glycolipids. Assays for sugar moiety revealed that they contained different pentoses such as arabinose, xylose or deoxyhexose as rhamnose.

Pathogenicity and oxidative stress in Nile tilapia caused by Aphanomyces laevis and Phoma herbarum isolated from farmed fish

Identified (n = 17) and unidentified (n = 1) fish-pathogenic fungal species from 10 genera of Oomycetes and soil fungi were isolated from 40 infected freshwater fish samples of the species Oreochromis niloticus niloticus (Nile tilapia) and Clarias gariepinus (African catfish). Samples were collected from various fish farms in the Nile Delta, Egypt. Nile tilapia were tested in aquaria for their susceptibility to the commonest Oomycetes species, Aphanomyces laevis and Achlya klebsiana, and also against the 2 most prevalent pathogenic soil fungi, Paecilomyces lilacinus and Phoma herbarum. Two techniques were used: water bath exposure and intramuscular (subcutaneous) injection. Water bath exposure to the 2 species of Oomycetes caused greater mortalities of O. niloticus niloticus than intramuscular injection, but the reverse was true of the soil fungal species. Regardless of the infection method, the 2 Oomycetes species were more potent pathogens than the soil fungal species. In both gills and mytomal muscles of fish infected by A. laevis and P. herbarum, we measured and compared with controls the oxidative stress parameters total peroxide (TP), lipid peroxidation (LPO) and nitric oxide (NO), as well as levels of the antioxidants vitamin E and glutathione (GSH), and superoxide dismutase (SOD) and catalase (CAT) activities. Infection by these 2 fungal species through either spore suspension or spore injection significantly increased oxidative damage in gills and induced marked decrease in most studied antioxidants. In addition, both routes showed similar effects and A. laevis depressed the antioxidants CAT, vitamin E and GSH more than P. herbarum.

An approach to the study of the fungal deterioration of a classical art material: Mastic varnish

Mastic, one of the best natural varnishes, is frequently used as protective and finishing layer or as component of oleo-resinous media in paintings, both in the past and currently. However, this resin is affected by complex deterioration processes which can change its characteristics and thus the visual aspect of works of art. The alteration processes caused by radiation have been widely studied, but there is a lack of information on the biodeterioration of this natural product. In this paper, fungi from collections as well as from oil paintings of the Fine Arts Museum of Granada (Spain) were inoculated onto slides covered with mastic. The samples, after an incubation period of 15 days, were analysed by gas chromatography-mass spectrometry (GC-MS) to identify the chemical changes undergone, and a visual monitoring of the samples was performed to determine the formation of mycelia onto solidified resins. Major changes were detected in Chrysonilia sitophila, Phoma herbarum, and P. chrysogenum, showing evidence of alteration processes caused or favoured by these microorgamisms.

New production of haloquinones, bromochlorogentisylquinones A and B, by a halide salt from a marine isolate of the fungus Phoma herbarum

New production of haloquinones, bromochlorogentisylquinones A and B, by a halide salt from a marine isolate of the fungus Phoma herbarum

Non-dermatophyte moulds as skin and nail foot mycosis agents: Phoma herbarum, Chaetomium globosum and Microascus cinereus

The increased prevalence of dermatomycoses along with the wide range of organisms now recognized as potential pathogens needs accurate laboratory isolation and identification of the aetiological agents. In this report three cases of foot dermatomycoses due to filamentous fungi commonly present in the environment with ubiquitous distribution are described in immunocompetent subjects. Skin and nail samples were collected, suspended in 20 % KOH solution, examined under a light microscope and cultured in Mycobiotic agar and Sabouraud dextrose agar containing chloramphenicol to detect fungal growth. Phoma herbarum, Chaetomium globosum, and Microascus cinereus were isolated and identified.

Physiological characteristics of 13 common fungal species in bathrooms

To clarify the factors affecting fungal contamination in bathrooms, the growth of 13 common fungal species (13 isolates) in bathrooms was studied under various environmental conditions. Most of the fungi examined grew on media of 0.01% and 0.05% sodium fatty acid and on media of 0.01% anion surfactant. On media of non-ion surfactant, however, growth varied from species to species. Fungi found commonly in bathrooms can be divided into two groups. The first group, including six species – Cladophialophora boppii, Exophiala spinifera, E. salmonis, Phialophora europaea, Phoma herbarum, and Scolecobasidium constrictum – grew on media of 0.01%, 0.05%, and 0.25% non-ion surfactant, with the latter five species also growing on alkali medium. Most of them did not grow at 33°C, or on media with 10%NaCl, however. Fungi of these six species, identified using DNA and morphological analysis, were common in bathrooms, but not in other indoor environments, for example, in house dust or on windows. The second group contained seven species including Aureobasidium sp., Cladosporium cladosporioides, and Fusarium sp., which were common both in house dust and in bathrooms; they did not grow on media of 0.05% or 0.25% non-ion surfactant, but most grew comparatively fast on normal medium (1/4PDA), and were able to grow on media with 10%NaCl and also at 33°C. The characteristic fungi found in bathrooms were able to exploit surfactant but were unable to grow well under comparatively dry or hightemperature conditions.

Fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole

Silver nanoparticles (Ag-NPs) are known to have inhibitory and bactericidal effects. Resistance of fungal infections has emerged in recent years and is a major health problem. Here, we report the extracellular biosynthesis of Ag-NPs using a common fungus, Alternaria alternata. Also in this study, these nanoparticles were evaluated for their part in increasing the antifungal activity of fluconazole against Phoma glomerata, Phoma herbarum, Fusarium semitectum, Trichoderma sp., and Candida albicans. The antifungal activity of fluconazole was enhanced against the test fungi in the presence of Ag-NPs. Fluconazole in combination with Ag-NPs showed the maximum inhibition against C. albicans, which was confirmed from the increase in fold area of inhibition, followed by P. glomerata and Trichoderma sp., which showed less increase in the fold area, whereas no significant enhancement of activity was found against P. herbarum and F. semitectum. From the Clinical Editor The antifungal activity of fluconazole was enhanced in presence of silver nanoparticles against the test fungi. Fluconazole in combination with Ag-NPs showed the maximum inhibition against C. albicans, followed by P. glomerata and Trichoderma sp. No significant enhancement of activity was found against P. herbarum and F. semitectum.

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation. Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production. YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation. We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Acta Pharmacologica Sinica (2009) 30: 1008-1014; doi: 10.1038/aps.2009.93.

In vitro Inhibition of Fungal Root-Rot Pathogens of Panax notoginseng by Rhizobacteria

The rhizobacteria of Panax notoginseng were isolated from six sites in Yanshan, Maguan and Wenshan Counties, Yunnan Province of China, and their antagonistic activity against P. notoginseng root-rot fungal pathogens was determined. Of the 574 rhizobacteria isolated, 5.8% isolates were antagonistic in vitro to at least one of the five pathogens, Cylindrocarpon didynum, Fusarium solani, Phytophthora cactorum, Phoma herbarum, and Rhizoctonia solani. The number of rhizo bacteria and the number that inhibited fungi differed depending on sampling sites and isolation methods. Rhizobacteria isolated from the site in Yanshan and Maguan showed more antagonistic effect than them in Wenshan. Heat treatment of rhizosphere soil at <TEX>$80^{\circ}C$</TEX> for 20 min scaled the antagonists up to 14.0%. Antagonistic bacteria in the roots proportioned 3.9% of the total isolates. The most antagonistic isolates 79-9 and 81-4 are Bacillus subtilis based on their 168 rDNA sequence and biochemical and physiological characteristics. Identification and evaluation of antagonistic bacteria against P. notoginseng root-rot pathogens in the main planting areas improved our understanding of their distribution in rhizosphere soil. Furthermore these results indicated that the interactions between biocontrol agent and soil microbes should be seriously considered for the successful survival and biocontrol efficacy of the agents in soil.

Phoma herbarum as a New Gibberellin-Producing and Plant Growth-Promoting Fungus

Endophytic fungi are known for the production of valuable metabolites, but information on the gibberellin production capacity of this group is limited. We isolated 9 endophytic fungi from the roots of salt-stressed soybean plants and screened them on waito-c rice, in order to identify plant growth promoting fungal strains. The fungal isolate TK- 2-4 gave maximum plant length (20.35 cm) promotion in comparison with wild-type Gibberella fujikuroi (19.5 cm). In a separate experiment, bioassay of TK-2-4 promoted plant length and biomass of soybean cultivar Taegwangkong. The TK-2-4 culture filtrate was analyzed for the presence of gibberellins, and it was found that all physiologically active gibberellins, especially GA(4) and GA(7), were present in higher amounts (GA(1), 0.11 ng/ml; GA(3), 2.91 ng/ml; GA(4), 3.21 ng/ml; and GA(7), 1.4 ng/ml) in conjunction with physiologically inactive GA(9) (0.05 ng/ml), GA(12) (0.23 ng/ ml), GA(15) (0.42 ng/ml), GA(19) (0.53 ng/ml), and GA(20) (0.06 ng/ml). The fungal isolate TK-2-4 was later identified as a new strain of Phoma herbarum, through the phylogenetic analysis of 28S rDNA sequence.

Physiological characteristics of 13 common fungal species in bathrooms

To clarify the factors affecting fungal contamination in bathrooms, the growth of 13 common fungal species (13 isolates) in bathrooms was studied under various environmental conditions. Most of the fungi examined grew on media of 0.01% and 0.05% sodium fatty acid and on media of 0.01% anion surfactant. On media of non-ion surfactant, however, growth varied from species to species. Fungi found commonly in bathrooms can be divided into two groups. The first group, including six species – Cladophialophora boppii, Exophiala spinifera, E. salmonis, Phialophora europaea, Phoma herbarum, and Scolecobasidium constrictum – grew on media of 0.01%, 0.05%, and 0.25% non-ion surfactant, with the latter five species also growing on alkali medium. Most of them did not grow at 33°C, or on media with 10%NaCl, however. Fungi of these six species, identified using DNA and morphological analysis, were common in bathrooms, but not in other indoor environments, for example, in house dust or on windows. The second group contained seven species including Aureobasidium sp., Cladosporium cladosporioides, and Fusarium sp., which were common both in house dust and in bathrooms; they did not grow on media of 0.05% or 0.25% non-ion surfactant, but most grew comparatively fast on normal medium (1/4PDA), and were able to grow on media with 10%NaCl and also at 33°C. The characteristic fungi found in bathrooms were able to exploit surfactant but were unable to grow well under comparatively dry or hightemperature conditions.

Tn5 transposase-assisted high-efficiency transformation of filamentous fungus Phoma herbarum YS4108

Conventionally, filamentous fungi are transformed by using conidia or protoplasts as recipients. However, induction of sporulation is difficult in some fungi, and protoplasting is an awing, frequently frustrating, and batch-dependent work. In this study, we established a simple and convenient method to prepare single cells from mycelia without enzymatic protoplasting. As a case study on the pathogenic fungus Phoma herbarum YS4108, the single cells could be directly and highly efficiently transformed with the aid of Tn5 transposase. The optimal electric pulse delivery parameters were 25 microF in capacitance, 0.75 kV (0.2-cm cuvette) in voltage, and 400 Omega in resistance, under which the efficiency of transposase-assisted transformation (TNAT) was enhanced to two to threefold compared to that of non-TNAT method, resulting in >230 transformants/cuvette (10(6) recipients). Further cell wall weakening of the single cells by lytic enzymes and linearization of the plasmid were found to have no effects on transformation efficiency, but vector linearization apparently lowered the background growth. The present study for the first time explained that Tn5 transposase could be used to increase transformation efficiency in filamentous fungi, and the method presented here may be of wide applicability in different studies and may be the first choice when transformation efficiency and convenience are priorities and mycelia have to be used as transformation recipients.

Colonization of barley roots by endophytic fungi and their reduction of take-all caused by Gaeumannomyces graminis var. tritici

Fungal root endophytes obtained from natural vegetation were tested for antifungal activity in dual culture tests against the root pathogen Gaeumannomyces graminis var. tritici. Fifteen isolates, including Acremonium blochii, Acremonium furcatum, Aspergillus fumigatus, Cylindrocarpon sp., Cylindrocarpon destructans, Dactylaria sp., Fusarium equiseti, Phoma herbarum, Phoma leveillei, and a sterile mycelium, selected based on the dual culture test, were inoculated on barley roots in growth tubes under axenic conditions, both in the absence and presence of G. graminis var. tritici. All isolates colonized the rhizosphere and very often the root cortex without causing disease symptoms and without affecting plant growth. Eight isolates significantly reduced the symptoms caused by G. graminis var. tritici, and 6 of them reduced its presence in the roots.

Cloning of actin Promoter and Transformation of the Marine-Sourced Filamentous Fungus Phoma herbarum YS4108

AIM: To establish a convenient and efficient transformation system of the marine-sourced filamentous fungus Phoma herbarum YS4108. METHODS: The endogenous actin promoter was cloned with degenerate primers and genome walking strategy, and utilized in the electroporation of P. herbarum YS4108. Using hygromycin B phosphotransferase gene as a selection marker and single cells prepared from mycelia as recipients, the effect of electroporation parameters on transformation efficiency was examined. RESULTS: The actin promoter was successfully cloned. The optimum parameters for electroporation were found to be 0.75 kV (0.2 cm cuvette) in voltage and 400Ω in resistance with 9μg of plasmid, under which more than 100 transformants per cuvette (10^6 recipients) could be obtained. CONCLUSION: By using the endogenous actin promoter, the erectric transformation system of P. herbarum YS4108 was established, which served as a practical approach for transgenic study on this fungus.

First Report of Spring Black Stem and Leaf Spot of Alfalfa in Washington State Caused by Phoma medicaginis.

Lesions were observed on leaves and stems of alfalfa (Medicago sativa L.) growing as weeds in Pullman, Washington in June of 2001. Lesions appeared similar to those described for spring black stem and leaf spot caused by Phoma medicaginis Malbr. & Roum. in Roum. var. medicaginis Boerema (synonyms Phoma herbarum Westend. var. medicaginis Fckl. and Ascochyta imperfecta Peck). Sporulation was induced by placing surface-disinfested pieces of infected tissue on 3% water agar (WA) for 24 h under fluorescent light with a 12-h photoperiod. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia after 18 h. Isolates had cultural and conidial morphology similar to descriptions of P. medicaginis and isolate ATCC52798 when grown on V8 agar and PDA at room temperature (3). Distinction between P. medicaginis var. medicaginis and P. medicaginis var. macrospora was not attempted. Conidial suspensions (1 × 106 conidia/ml) of isolates AS1, AS2, AS3, and AS4 were spray inoculated to runoff onto 3-week-old plants. PI lines 536535 and 536534 of M. sativa subsp. sativa (4-trifolate stage) and PI lines 442896 and 577609 of M. truncatula (5- to 7-trifolate stage) from the USDA Western Region Plant Introduction Station, Pullman, Washington were inoculated, with at least two replicate plants inoculated per isolate. Plants were incubated in a dew chamber at 20°C in the dark for 24 h to promote infection and then transferred to a growth chamber at 18°C with a 12-h photoperiod. Lesions were apparent on M. sativa subsp. sativa plants 4 days postinoculation (dpi) and 7 dpi on M. truncatula plants. At 12 dpi, many dark brown lesions with chlorotic halos were noted on leaves of M. sativa subsp. sativa, occasionally killing the entire trifoliate leaf and progressing approximately 1 cm down the stem. According to the previously published 1-to-5 visual rating scale for this disease (4), disease scores on both genotypes of M. sativa subsp. sativa were 4 (susceptible), while disease ratings on M. truncatula were 1-2 (resistant) with a few dark brown lesions noted on leaves and stems generally restricted to less than 2 mm in diameter. DNA was extracted from isolates AS1 and AS4, and PCR was performed using gpd-1 and gpd-2 primers for the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) (1), and EF1-728F and EF1-986R primers for the translation elongation factor 1-alpha gene (EF) (2), resulting in amplification of an approximately 600-bp fragment from each primer set. Amplicons were direct-sequenced on both strands, and BLAST searches of the NCBI nucleotide database were conducted with consensus G3PD and EF sequences of both isolates AS1 and AS4. Closest matches obtained for the G3PD and EF sequences were P. medicaginis isolate ATCC52798 (Accession No. DQ525740) and P. medicaginis var. medicaginis CBS316.90 (Accession No. AY831548), respectively. The G3PD and EF sequences for these isolates have been deposited in GenBank database (Accession Nos. EU394712-EU394715). To our knowledge, this is the first confirmed report of spring black stem and leaf spot of alfalfa in Washington State supported by Koch's postulates, cultural morphology, and multigene sequencing.

Production of Monoclonal Antibodies for Detecting Marine Fungal Polysaccharide Isolated from the Phoma herbarum YS4108

Seven monoclonal antibodies (MAbs) specific for marine fungal polysaccharide YCP isolated from the Phoma herbarum YS4108 were obtained after immunization of BALB/c mice with the conjugate of YCP coupled to bovine serum albumin (BSA). Their epitope mapping and binding specificity characterized by blocking and inhibition Enzyme-linked immunosorbent assay (ELISA) indicated that these specific MAbs have similar binding patterns. An immuno-capture ELISA has been developed on the basis of employing the MAbs N3F and P12B specific for YCP as the capture antibody and the detecting antibody, respectively. The working range for YCP in aqueous solution was 1–10,000 ng/ml with a good reproducibility (relative standard deviation ≤7.2%). Subsequently, the extraction process of YCP from Phoma herbarum YS4108 was successfully optimized by the method.

Asymmetric Total Synthesis of Herbarumin III: Introduction of the syn-1,3-Diol Moiety from an Optically Pure Hydroxy Epoxide Resolved by HKR

Herbarumin III (1) isolated from the fermentation broth and mycelium of the fungus Phoma herbarum displays significant phytotoxic effects against seedlings of A. hypochondriacus. The herbarumin macrolides (1-3, Figure 1) interact with bovin-brain calmodulin and inhibit the activation of the calmodulin-dependent enzyme cAMP phosphodiesterase. Construction of the 10-membered lactone ring and the streocontrol of syn-1,3-diol unit are two major issues in the total synthesis of herbarumin III (1). In the previous total synthesis of 1, the 10-membered lactone ring was synthesized by ring-closing metathesis (RCM) reaction and Yamaguchi’s lactonization method. Asymmetric synthesis of the syn-1,3-diol moiety have been achieved using chiral pool methods, chemoenzymatic method, and asymmetric allylation/Sharpless epoxidation method. Herein, we would like to report an asymmetric total synthesis of herbarumin III (1) starting from an enantiomerically pure hydroxy epoxide generated by using Jacobsen’s hydrolytic kinetic resolution (HKR) method to install the syn-1,3-diol moiety. Retrosynthetically (Scheme 1), the macrolactone ring of 1 could be constructed by RCM reaction at the final stage and the corresponding diene 4 would be prepared from alcohol 6 and 5-hexenoic acid (5). The syn-1,3-diol moiety of 6 could be introduced by using a nucleophilic epoxide opening reaction of epoxide 7. The starting epoxide 7 is prepared from (–)-8 that we have prepared previously by using Jacobsen’s hydrolytic kinetic resolution (HKR) method. Removal of double bond in 8 by catalytic hydrogenation followed by treatment with dimethylsulfonium methylide yields the syn-1,3-diol moiety 9 in good yield (Scheme 2). Protection of the allylic alcohol using ethyl vinyl ether (EVE) in the presence of PPTS (pyridinium para-toulenesulfonate) in dichloromethane gives 10