Spongispora temasekensis, a new boletoid genus and species from Singapore.

Dipterocarp forests are a typical and widespread type of vegetation in tropical lowlands of southeast Asia that harbor a high diversity of ectomycorrhizal fungi, including boletes. Based on molecular and morphological characters, a unique bolete found in Singapore associated with the dipterocarp Hopea odorata was proven to represent a new species in the proposed new genus Spongispora. Phylogenetic analyses of five loci indicate that Spongispora is nested in the subfamily Leccinoideae of the Boletaceae, most closely related to an inclusive clade of Leccinum, Leccinellum, Octaviania, Rossbeevera, and Turmalinea. However, genetic distances between Spongispora and genera in Leccinoideae are mostly higher than that between any two known genera in this subfamily, which supports the proposal of a new genus. Spongispora temasekensis is characterized by a whitish to pale yellow hymenophore that stains brown where injured, coarsely reticulate stipe, interwoven trichodermial pileipellis, and broadly elliptical to ovoid basidiospores with sponge-like ornamentation perforated by irregular clefts, cracks, and warts under scanning electron microscopy. Morphological descriptions, illustrations, and comparisons with allied taxa are made, and a key to the genera of the subfamily Leccinoideae is provided.

Composition and Diversity of Soil Fungi in Dipterocarpaceae-Dominated Seasonal Tropical Forests in Thailand.

Although fungi play essential roles in nutrient cycles and plant growth in forest ecosystems, limited information is currently available on the community compositions of soil fungi in tropical forests. Few studies have examined fungal community structures in seasonal tropical forests, in which forest fires potentially have a large impact on above- and belowground community processes. Based on high-throughput sequencing technologies, we herein examined the diversity and community structures of soil fungi in dry seasonal tropical forests in Sakaerat, northeast Thailand. We found that fungal community compositions diverged among dry evergreen, dry deciduous, and fire-protected dry deciduous forests within the region. Although tree species diversity did not positively correlate with soil fungal diversity, the coverage of an understory bamboo species (Vietnamosasa pusilla) showed a strong relationship with fungal community structures. Our community ecological analysis also yielded a list of fungi showing habitat preferences for either of the neighboring evergreen and deciduous forests in Sakaerat. The present results provide a basis for managing soil fungal communities and aboveground plant communities in seasonal tropical forests in Southeast Asia.

Basidiospores attach to the seed of Shorea leprosula in lowland tropical dipterocarp forest and form functional ectomycorrhiza on seed germination.

This research aimed to study the ectomycorrhiza formed by basidiospores attached to the outer surface of Shorea leprosula (Dipterocarpaceae) seed collected from a lowland tropical dipterocarp forest. Two groups of seeds were collected: control seeds collected from plastic net hanging 2 m above the ground and forest floor seeds collected on the forest floor. Before planting, 15 seeds from each group were observed for basidiospores attached to the seed. Ten of the 3-week-old S. leprosula seedlings of each group were individually grown in 1.5 kg of sterile zeolite for 8 months in a greenhouse. Pots were fertilized with MMN solution containing half the strength of phosphate. Fungal identity, ectomycorrhizal root tip colonization and anatomy, plant growth, and phosphate uptake were measured. The control seeds did not have basidiospores attached, whereas the forest floor seeds had 2 × 105 basidiospores of Tomentella. Bioassay test results indicate that the seedlings from the control seeds did not form ectomycorrhiza, whereas those seedlings from the forest floor seeds formed 3 morphotypes of ectomycorrhizae. Based on ITS1, 5.8S, and ITS2 rDNA region analyses, the 3 morphotypes belonged to Tomentella sp. HBT2, Tomentella sp. HBT4, and Scleroderma sp. HBS3. Root colonization percentage was above 70% for all three morphotypes. Root colonization in general increased plant growth and phosphate uptake. This is the first report of Tomentella basidiospores attached on the seed surface as a functional inoculum and of Tomentella ectomycorrhiza from dipterocarps lowland tropical forest.

Two new species of Phylloporus from Bangladesh, with morphological and molecular evidence.

Two new species of Phylloporus from Bangladesh forests dominated by Shorea robusta are described and illustrated, based on morphological and molecular phylogenetic evidence. Phylloporus attenuatus is characterized by its wine-red to brownish-red pilei, decurrent lamellae that stain blue, downwards tapering stipe, a trichodermium pileipellis, and ellipsoidal-ovoid to ellipsoidal basidiospores (7-8 × 4-5 µm). Phylloporus catenulatus is characterized by its small-sized basidiomata with reddish-brown pilei, narrowly decurrent with distant lamellae that stain blue, a subepithelium pileipellis, and ellipsoidal to ellipsoidal-ovoid basidiospores (8-9.5 × 4-5 µm). Both new species of Phylloporus are distinctive from any known taxa based on phylogenetic analyses of partial sequences of the nuc rDNA internal transcribed spacer (ITS; including ITS1-ITS2 but not 5.8S) region, partial sequence of nuclear ribosomal large subunit (28S) domains D1 and D2, and translation elongation factor 1-α (TEF1α). Both species are compared with phenotypically similar taxa and illustrated with line drawings and photographs. A key to the species of Phylloporus known from Bangladesh is provided.

Sarcodon in the Neotropics II: four new species from Colombia and a key to the regional species.

This work reports on four species of the ectomycorrhizal (ECM) tooth fungus genus Sarcodon (Bankeraceae, Thelephorales, Basidiomycota) recently discovered in the Colombian Amazon. Sarcodon colombiensis sp. nov., Sarcodon rufobrunneus sp. nov., Sarcodon pallidogriseus sp. nov. and Sarcodon bairdii sp. nov. are described as new to science. These fungi occur in forests dominated by ECM trees in the genera Pseudomonotes (Dipterocarpaceae), Dicymbe (Fabaceae subfam. Caesalpinioideae) and Aldina (Fabaceae subfam. Papilionoideae). These records bring the number of Sarcodon species known from the Neotropics to 10. Each of the new species possesses the accepted diagnostic characters for the genus: pileate-stipitate stature, a dentate hymenophore, determinate basidiomata development, fleshy, non-zonate context, and brown, tuberculate basidiospores. Molecular phylogenetic analysis corroborated the generic placement of the species, and, in combination with morphological characters, confirmed that they are new to science. Macromorphological, micromorphological, habitat and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species. A key is provided that allows identification of all known Neotropical Sarcodon species and similar extralimital taxa.

Five new species of Inocybe (Agaricales) from tropical India.

Five new species of Inocybe, I. iringolkavensis, I. keralensis, I. kuruvensis, I. muthangensis and I. wayanadensis, are described from Kerala state, India, based on morphological and molecular data. All are associated with trees belonging to Dipterocarpaceae. Inocybe iringolkavensis is characterized by nodulose to somewhat stellate basidiospores, 1-4-spored basidia, and caulocystidia restricted to the stipe apex. Inocybe keralensis has a yellowish brown pileus, lamellae with whitish, serrate edges, smooth, ellipsoidal basidiospores and a duplex pileipellis with the superficial hyphae devoid of encrustations and encrusted hyphae beneath. The diagnostic features of I. kuruvensis include a dark brown pileus, stipe with a whitish base and grayish brown, floccose-fibrillose surface, nodulose basidiospores with saddle-shaped projections and faintly encrusted paracystidia with refractive contents. Violet basidiomata with a rimose, hygrophanous pileus, densely pruinose stipe with a marginate-bulbous base, and nodulose basidiospores are the major features of I. muthangensis. Inocybe wayanadensis is characterized by small, whitish basidiomata, a viscid pileus with a rimulose surface, a densely pruinose and fibrillose stipe with a marginate-bulbous base, nodulose basidiospores, thick-walled pleuro- cheilo- and caulocystidia and an ixotrichoderm-type pileipellis. The phylogenetic relationships of these new species are inferred from an analysis of nuc rDNA sequences of the internal transcribed spacers (ITS1-5.8S-ITS2) and the 28S gene. Except I. keralensis, which belongs to the Pseudosperma clade, all other species belong to the Inocybe clade. This study represents the second report of an Inocybe species (I. muthangensis) that combines violet basidiomata with nodulose basidiospores.

New Boletaceae taxa from Guyana: Binderoboletus segoi gen. and sp. nov., Guyanaporus albipodus gen. and sp. nov., Singerocomus rubriflavus gen. and sp. nov., and a new combination for Xerocomus inundabilis.

Binderoboletus segoi gen. and sp. nov., Guyanaporus albipodus gen. and sp. nov. and Singerocomus rubriflavus gen. and sp. nov. (Boletaceae, Boletales, Basidiomycota) are described from the Pakaraima Mountains and adjacent lowlands of Guyana. Xerocomus inundabilis, originally described from the central Brazilian Amazon and based solely on the type collection, is redescribed from numerous collections from Guyana and transferred into Singerocomus. These boletes occur in Neotropical forests dominated by ectomycorrhizal trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae), Aldina (Fabaceae subfam. Papilionoideae) and Pakaraimaea (Dipterocarpaceae). Three of the species were repeatedly found in a multiyear sporocarp survey in Dicymbe corymbosa-monodominant forest. Macromorphological, micromorphological, habitat and multilocus DNA sequence data are provided for each species. A molecular phylogenetic analysis based on a large taxon set across the Boletaceae justifies erection of the new genera.

In vivo antimalarial activity of the endophytic actinobacteria, Streptomyces SUK 10.

Endophytic bacteria, such as Streptomyces, have the potential to act as a source for novel bioactive molecules with medicinal properties. The present study was aimed at assessing the antimalarial activity of crude extract isolated from various strains of actinobacteria living endophytically in some Malaysian medicinal plants. Using the four day suppression test method on male ICR strain mice, compounds produced from three strains of Streptomyces (SUK8, SUK10, and SUK27) were tested in vivo against Plasmodium berghei PZZ1/100 in an antimalarial screen using crude extracts at four different concentrations. One of these extracts, isolated from Streptomyces SUK10 obtained from the bark of Shorea ovalis tree, showed inhibition of the test organism and was further tested against P. berghei-infected mice for antimalarial activity at different concentrations. There was a positive relationship between the survival of the infected mouse group treated with 50 µg/kg body weight (bw) of ethyl acetate-SUK10 crude extract and the ability to inhibit the parasites growth. The parasite inhibition percentage for this group showed that 50% of the mice survived for more than 90 days after infection with the parasite. The nucleotide sequence and phylogenetic tree suggested that Streptomyces SUK10 may constitute a new species within the Streptomyces genus. As part of the drug discovery process, these promising finding may contribute to the medicinal and pharmaceutical field for malarial treatment.

New neotropical sebacinales species from a Pakaraimaea dipterocarpacea forest in the Guayana Region, Southern Venezuela: structural diversity and phylogeography.

Pakaraimaea dipterocarpacea, a member of the Dipterocarpaceae endemic in the Guayana region, is associated with a diverse community of ectomycorrhizal (ECM) fungi. Amongst the 41 ECM fungal species detected in a 400 m2 P. dipterocarpacea ssp. nitida plot in Southern Venezuela, three species belonged to the Sebacinales. We tested whether ECM anatomotype characterization can be used as a feasible element in an integrative taxonomy in this diverse fungal group, where the relevance of fruitbody morphology for species delimitation seems limited. Using a combination of ECM morpho-anatomical characterizations and phylogenetic analyses based on nuclear ITS and LSU sequences, we report three new species. The main distinguishing features of Sebacina guayanensis are the yellowish cell walls together with conspicuous undifferentiated, uniform compact (type B) rhizomorphs. Staghorn-like hyphae are characteristic of S. tomentosa. The combination of clusters of thick-walled emanating hyphae, including hyphae similar to awl-shaped cystidia with basal dichotomous or trichotomous ramifications, and the presence of type B rhizomorphs were characteristic of a third, yet unnamed species. The three species belong to three different, possibly specifically tropical clades in Sebacinales Group A. The geographic distribution of phylogenetically related strains was wide, including a Dicymbe forest in Guyana and an Ecuadorian rainforest with Coccoloba species. We show that ECM morpho-anatomy can be used, in combination with other analyses, to delineate species within Sebacinales Group A. In addition to phylogenetic information, type B rhizomorphs observed in different Sebacinales clades have important ecological implications for this fungal group. The phylogeography of Sebacinales suggests that dispersion and host jump are important radiation mechanisms that shaped P. dipterocarpacea ECM fungal community. This study emphasizes the need for more sequence data to evaluate the hypothesis that phylogeographic relationships between neo- and paleotropical ECM fungal species could be attributed to the vicariance of cross-continental hosts such as the Dipterocarpacae.

Austroboletus amazonicus sp. nov. and Fistulinella campinaranae var. scrobiculata, two commonly occurring boletes from a forest dominated by Pseudomonotes tropenbosii (Dipterocarpaceae) in Colombian Amazonia.

Two boletes that frequently form fruiting bodies in Pseudomonotes tropenbosii forests are described from Colombian Amazonia. One is a new species of Austroboletus here described as A. amazonicus and the other one is Fistulinella campinaranae var. scrobiculata Singer, which is a new record for Colombia. Macromorphological, micromorphological and habitat data for these species are provided as well as DNA sequence data of the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) ribosomal DNA.

Accelerated degradation of exogenous indole by Burkholderia unamae strain CK43B exposed to pyrogallol-type polyphenols.

In modified Winogradsky's (MW) medium supplemented with excessive indole (1), Burkholderia unamae strain CK43B isolated from polyphenol-rich Shorea rhizosphere showed almost no cell growth, but it showed drastic cell growth given further supplementation of gallic acid, a simple plant polyphenol. This active cell growth of B. unamae CK43B was due to the stimulating effect of gallic acid on 1-degradation of bacterial cells, which acquired a nitrogen source in 1. Under aerobic culture conditions with appropriate concentrations (0.5-2.0 mM) of gallic acid, B. unamae CK43B started to decompose exogenous 1 in a dose-dependent manner, and finally accumulated catechol (5) via anthranilic acid (4). Pyrogallol also showed a cometabolic effect on decarboxylation-coupled oxidative deamination of B. unamae CK43B, producing 5 from 4, as gallic acid did. These results suggest that pyrogallol-type plant polyphenols act as stimulators on B. unamae CK43B, causing it to degrade an N-heterocyclic aromatic compound (NHAC) including nitrogen-containing humic substances.

Distinct metabolites for photoreactive L-phenylalanine derivatives in Klebsiella sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling.

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores--aryl azide, benzophenone and trifluoromethyldiazirine--are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing L-phenylalanine derivatives.

Effectiveness of sal deoiled seed cake as an inducer for protease production from Aeromonas sp. S1 for its application in kitchen wastewater treatment.

The present study is an attempt to demonstrate the feasibility of sal (Shorea robusta) deoiled cake--a forest-based industrial by-product--as a cheaper media supplement for augmented protease production from Aeromonas sp. S1 and application of protease in the treatment of kitchen wastewater. Under optimized conditions, protease production could successfully be enhanced to 5.13-fold (527.5 U mL(-1)) on using sal deoiled seed cake extract (SDOCE), as medium additive, compared to an initial production of 102.7 U mL(-1) in its absence. The culture parameters for optimum production of protease were determined to be incubation time (48 h), pH (7.0), SDOCE concentration (3 % (v/v)), inoculum size (0.3-0.6 % (v/v)), and agitation rate (100 rpm). The enzyme was found to have an optimum pH and temperature of 8.0 and 60 °C, respectively. The protease preparation was tested for treatment of organic-laden kitchen wastewater. After 96 h of wastewater treatment under static condition, enzyme preparation was able to reduce 74 % biological oxygen demand, 37 % total suspended solids, and 41 % oil and grease. The higher and improved level of protease obtained using sal deoiled seed cake-based media hence offers a new approach for value addition to this underutilized biomass through industrial enzyme production. The protease produced using this biomass could also be used as pretreatment tool for remediation of organic-rich food wastewater.

Induction of biofilm formation in the betaproteobacterium Burkholderia unamae CK43B exposed to exogenous indole and gallic acid.

Burkholderia unamae CK43B, a member of the Betaproteobacteria that was isolated from the rhizosphere of a Shorea balangeran sapling in a tropical peat swamp forest, produces neither indole nor extracellular polymeric substances associated with biofilm formation. When cultured in a modified Winogradsky's medium supplemented with up to 1.7 mM indole, B. unamae CK43B maintains its planktonic state by cell swelling and effectively degrades exogenous indole. However, in medium supplemented with 1.7 mM exogenous indole and 1.0 mM gallic acid, B. unamae CK43B produced extracellular polymeric substances and formed a biofilm. The concentration indicated above of gallic acid alone had no effect on either the growth or the differentiation of B. unamae CK43B cells above a certain concentration threshold, whereas it inhibited indole degradation by B. unamae CK43B to 3-hydroxyindoxyl. In addition, coculture of B. unamae CK43B with indole-producing Escherichia coli in nutrient-rich Luria-Bertani medium supplemented with 1.0 mM gallic acid led to the formation of mixed cell aggregates. The viability and active growth of B. unamae CK43B cells in a coculture system with Escherichia coli were evidenced by fluorescence in situ hybridization. Our data thus suggest that indole facilitates intergenus communication between indole-producing gammaproteobacteria and some indole-degrading bacteria, particularly in gallic acid-rich environments.

The ectomycorrhizal fungal community in a neotropical forest dominated by the endemic dipterocarp Pakaraimaea dipterocarpacea.

Ectomycorrhizal (ECM) plants and fungi can be diverse and abundant in certain tropical ecosystems. For example, the primarily paleotropical ECM plant family Dipterocarpaceae is one of the most speciose and ecologically important tree families in Southeast Asia. Pakaraimaea dipterocarpacea is one of two species of dipterocarp known from the Neotropics, and is also the only known member of the monotypic Dipterocarpaceae subfamily Pakaraimoideae. This Guiana Shield endemic is only known from the sandstone highlands of Guyana and Venezuela. Despite its unique phylogenetic position and unusual geographical distribution, the ECM fungal associations of P. dipterocarpacea are understudied throughout the tree's range. In December 2010 we sampled ECM fungi on roots of P. dipterocarpacea and the co-occurring ECM tree Dicymbe jenmanii (Fabaceae subfamily Caesalpinioideae) in the Upper Mazaruni River Basin of Guyana. Based on ITS rDNA sequencing we documented 52 ECM species from 11 independent fungal lineages. Due to the phylogenetic distance between the two host tree species, we hypothesized that P. dipterocarpacea would harbor unique ECM fungi not found on the roots of D. jenmanii. Although statistical tests suggested that several ECM fungal species did exhibit host preferences for either P. dipterocarpacea or D. jenmanii, most of the ECM fungi were multi-host generalists. We also detected several ECM fungi that have never been found in long-term studies of nearby rainforests dominated by other Dicymbe species. One particular mushroom-forming fungus appears to be unique and may represent a new ECM lineage of Agaricales that is endemic to the Neotropics.

Fully substituted unsaturated lactones from endophytic Myrothecium sp.

Two new α,ß-unsaturated γ-lactones, myrolactones A (1) and B (2), were characterized from the culture broth of the Myrothecium sp. IFB-E106 isolated from the roots of Vatica mangachapoi Blauco. The absolute configuration was determined by the computational electronic circular dichroism approach. Myolactone B showed neuraminidase inhibitory activity with the IC(50) value of 13.95 µM.

Salkowski's reagent test as a primary screening index for functionalities of rhizobacteria isolated from wild dipterocarp saplings growing naturally on medium-strongly acidic tropical peat soil.

Rhizobacteria isolated from wild dipterocarp saplings in Central Kalimantan, Indonesia, were subjected to Salkowski's reagent test, which is often used in detecting indolic substances. Among 69 isolates grown in a low-nitrogen medium supplemented with L-tryptophan (TRP), culture fluids of 29 strains were positive to the test, in which 17 bacteria turned red and other 10 pink. All the red type rhizobacteria actively converted TRP into tryptophol (TOL), while some yielded indole-3-acetic acid (IAA) with TOL production. They also showed a capacity to decompose gallotannin into pyrogallol via gallic acid. On the other hand, an active IAA-producing Serratia sp. CK67, and three Fe-solubilizing Burkholderia spp. CK28, CK43, and Citrobacter sp. CK42, were all involved in pink type rhizobacteria, which were more effective, oxidative TRP-degraders than the red type rhizobacteria. Thus, Salkowski's reagent test should be a useful primary index in the screening of functional rhizobacteria in peatland ecosystem.

Benzophenones from Guignardia sp. IFB-E028, an endophyte on Hopea hainanensis.

The first natural S-containing benzophenone dimer, named guignasulfide (3), was isolated from the culture of Guignardia sp. IFB-E028, an endophytic fungus residing in healthy leaves of Hopea hainanensis. Its structure was determined through correlative analyses of its MS, 1D- and 2D-NMR spectroscopic data. Two other known benzophenone derivatives, monomethylsulochrin and rhizoctonic acid (1 and 2, resp.) were also isolated. Guignasulfide (3) was more active against the human liver cancer cell line HepG2 (IC(50) value: 5.2+/-0.4 microM) than metabolites 1 and 2 (IC(50) values: 63.5+/-0.6 and 60.2+/-0.5 microM); compounds 1-3 showed also moderately inhibitory effects on the human bacterial pathogen Helicobacter pylori with MIC values of 28.9+/-0.1, 60.2+/-0.4, and 42.9+/-0.5 microM, respectively.

Cytotoxic cytochalasin metabolites of endophytic Endothia gyrosa.

In addition to the known metabolites cytochalasin H (1), cytochalasin J (2), and epoxycytochalasin H (3), two new 10-phenyl-(11)-cytochalasans, named cytochalasin Z10 and Z11 (4 and 5, resp.) were isolated from the solid substrate culture of Endothia gyrosa IFB-E023, an endophytic fungus residing inside the healthy leaf of Vatica mangachapo (Dipterocarpaceae). The structure determination of 4 and 5 was accomplished through correlative analyses of their spectral data (UV, ESI-MS, IR, (1)H- and (13)C-NMR, COSY, NOESY, HMQC, and HMBC). Metabolites 1-5 were demonstrated to be substantially cytotoxic to the human leukaemia K562 cell line with the IC(50) values of 10.1, 1.5, 24.5, 28.3, and 24.4 microM, respectively, which are comparable to that of 5-fluorouracil (33.0 microM), co-assayed as the positive reference.