Bioactive compounds guided diversity of endophytic fungi from Baliospermum montanum and their potential extracellular enzymes.

Baliospermum montanum (Willd.) Muell. Arg, a medicinal plant distributed throughout India from Kashmir to peninsular-Indian region is extensively used to treat jaundice, asthma, and constipation. In the current study, 203 endophytic fungi representing twenty-nine species were isolated from tissues of B. montanum. The colonization and isolation rate of endophytes were higher in stem followed by seed, root, leaf and flower. The phytochemical analysis revealed 70% endophytic isolates showed alkaloids and flavonoids, 13% were positive for phenols, saponins and terpenoids. Further, these endophytes produced remarkable extracellular enzymes such as amylase, cellulase, phosphates, protease and lipase. The most promisive three endophytic fungi were identified by ITS region and secreted metabolites were identified by gas chromatography-mass spectrometry (GC-MS/MS). The GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from flower exhibited nine bioactive compounds namely, 2-Cyclopentenone, 2-(4-chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl and 1-Acetyl-20a-hydroxy-16-methylene. The endophyte, Aspergillus brasiliensis isolated from root and Fusarium oxysporum isolated from seed produced nine and seven bioactive compounds, respectively. Overall, a significant contribution of bioactive compounds was noticed from the diverse endophytic fungi associated with B. montanum and could be explored for development of novel drug with commercial values.

Microbacterium excoecariae sp. nov., a novel endophytic actinobacterium isolated from bark of Excoecaria agallocha Linn.

A Gram-stain-positive, aerobic actinobacterium, designated strain CBS5P-1T, was isolated from bark of Excoecaria agallocha Linn collected from Guangxi Zhuang Autonomous Region, PR China. Cells were short rods. Colonies were light yellow, circular and had entire margins. Strain CBS5P-1T grew at 10-37 °C (optimum, 30 °C) and pH 6.0-12.0 (optimum, pH 7.0-8.0). Its nearest phylogenetic neighbour was Microbacterium amylolyticum DSM 24221T with 97.1 % 16S rRNA gene sequence similarity. The genomic DNA G+C content of strain CBS5P-1T was 71.8 mol%. Anteiso-C15  : 0, anteiso-C17 : 0, iso-C16 : 0 and C16:0 were predominant cellular fatty acids. Major menaquinones were MK-11 and MK-10. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and an unidentified phospholipid. The combination of chemotaxonomic, phylogenetic and phenotypic data clearly distinguished strain CBS5P-1T from its phylogenetic neighbour. Accordingly, the name Microbacterium excoecariae sp. nov. is proposed to accommodate this new member of the genus Microbacterium. The type strain is CBS5P-1T (=KCTC 49239T=CGMCC 1.13862T).

Combined Effects of Dissolved Nutrients and Oxygen on Plant Litter Decomposition and Associated Fungal Communities.

Aquatic ecosystems worldwide have been substantially altered by human activities, which often induce changes in multiple factors that can interact to produce complex effects. Here, we evaluated the combined effects of dissolved nutrients (nitrogen [N] and phosphorus [P]; three levels: concentration found in oligotrophic streams in the Cerrado biome, 10× and 100× enriched) and oxygen (O2; three levels: hypoxic [4% O2], depleted [55% O2], and saturated [96% O2]) on plant litter decomposition and associated fungal decomposers in laboratory microcosms simulating stream conditions under distinct scenarios of water quality deterioration. Senescent leaves of Maprounea guianensis were incubated for 10 days in an oligotrophic Cerrado stream to allow microbial colonization and subsequently incubated in microcosms for 21 days. Leaves lost 1.1-3.0% of their initial mass after 21 days, and this was not affected either by nutrients or oxygen levels. When considering simultaneous changes in nutrients and oxygen concentrations, simulating increased human pressure, fungal biomass accumulation, and sporulation rates were generally inhibited. Aquatic hyphomycete community structure was also affected by changes in nutrients and oxygen availability, with stronger effects found in hypoxic treatments than in depleted or saturated oxygen treatments. This study showed that the effects of simultaneous changes in the availability of dissolved nutrients and oxygen in aquatic environments can influence the activity and composition of fungal communities, although these effects were not translated into changes in litter decomposition rates.

3-Arylisoindolinone and sesquiterpene derivatives from the mangrove endophytic fungi Aspergillus versicolor SYSU-SKS025.

A pair of 3-arylisoindolinone enantiomers: (+)-asperglactam A (1), (-)-asperglactam A (1) and a pair of nor-bisabolane enantiomers: (+)-1-hydroxyboivinianic acid (2), (-)-1-hydroxyboivinianic acid (2), along with seven known compounds (3-8) were obtained from the mangrove endophytic fungus Aspergillus versicolor SYSU-SKS025. Their structures were determined on the basis of HRESIMS and NMR spectroscopic data, and X-ray diffraction. (+)-Asperglactam A (1) and (-)-asperglactam A (1) are the first optically pure examples in the 3-arylisoindolinone family, which are rarely found in natural sources. All isolated compounds were evaluated for α-glucosidase inhibitory activity. The enantiomers of 1-3 showed moderate inhibitory activity against α-glucosidase with IC50 values ranging from 50 to 190µM. Compound 7 exhibited significant inhibitory activity against α-glucosidase with IC50 value of 7.5µM. In addition, compound 7 was found to inhibit nitric oxide production in RAW 264.7 macrophages with IC50 value of 12.5µM.

Effect of Pseudomonas putida on growth and anthocyanin pigment in two poinsettia (Euphorbia pulcherrima) cultivars.

Pseudomonas putida is plant growth promoting rhizobacteria (PGPR) that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble) using three strains of P. putida, as well as a mixture of the three (MIX). Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4), volume of roots (45 versus 3 cm(3)), number of leaves (78 versus 58), and area of leaf (1,788 versus 1,331 cm(2)), except for the number of flowers (8.8 versus 11.6). To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars.

New derivatives of nonactic and homononactic acids from Bacillus pumilus derived from Breynia fruticosa.

Six new nonactic and homononactic acid derivatives, ethyl homononactate (1), ethyl nonactate (2), homononactyl homononactate (6), ethyl homononactyl nonactate (7), ethyl homononactyl homononactate (8), and ethyl nonactyl nonactate (9), as well as four known compounds, homononactic acid (3), nonactic acid (4), homononactyl nonactate (5), and bishomononactic acid (10), were isolated from culture broth of Bacillus pumilus derived from Breynia fruticosa. The structures of new compounds were elucidated by spectroscopic analysis and chemical methods. The optical purities of 1-6 were determined by HPLC/MS after treatment with L-phenylalanine methyl ester. The dimeric compounds 5-9 showed weak cytotoxic activities against five human cancer cell lines (IC50 19-100 µg/ml).

Cytotoxic polyphenols from the fungus Penicillium expansum 091 006 endogenous with the mangrove plant Excoecaria agallocha.

As part of our ongoing chemical investigation of biologically active metabolites from marine-derived fungi, four new polyphenols containing both phenolic bisabolane and diphenyl ether units, expansols C-F (1-4), and one new diphenyl ether derivative, 3-O-methyldiorcinol (5), as well as twelve known compounds (6-17), were isolated from Penicillium expansum 091006 endogenous with the mangrove plant Excoecaria agallocha (Euphorbiaceae). The structures of the new metabolites were determined on the basis of NMR and mass spectroscopy. Among them, expansols C (1) and E (3) exhibited weak cytotoxicity against the HL-60 cell lines with IC50 values of 18.2 and 20.8 µM, respectively. The results showed that diphenyl ether substituted phenolic bisabolanes with a Δ7 double bond in the side chain are slightly less cytotoxic to HL-60 cell lines than the 7-OH or 7-OCH3 derivatives.

[Chemical-genetics based screening for furanonaphthoquinone producing endophytic actinomycetes from seeds of Trewia nudiflora].

OBJECTIVE: The seeds of Trewia nudiflora containing maytansine (an anticancer agent), was investigated to explore the endophytic actinomycetes diversity and screen for naphthoquinones producing strain. METHODS: The seeds of Trewia nudiflora were sliced and plated on different selective media after surface sterilization. Clones that looked like actinomycetes were selected, and classified according to the 16S rRNA sequences. Isolated strains were screened for furanonaphthoquinone biosynthesis gene by PCR, and tested for antibacterial and antifungal activity using Staphyloccocusaureus, Pseudomon-asaeruginosa, Bacillus subtilis, Rhizoctoniasolani and Gibberellasaubinetii. LC-MS and NMR were used to determine the structure of candidate compounds. RESULTS: More than 100 endophytic bacteria were isolated. Among them 66 were streptomycetes. FNQ6 (polyketide synthase Type III) and FNQ21 (carboxymuconate cycloisomerase) were only detected in Streptomyces sp. HTZ 27. We got 5 mg pure furanonaphthoquinone (FNQI) from 1 liter Streptomyces sp. HTZ 27 agar fermentation medium. CONCLUSION: The use of chemical-genetics method increased the efficiency of screening for target compound producing bacteria.

Microbispora hainanensis sp. nov., isolated from rhizosphere soil of Excoecaria agallocha in a mangrove.

Strain 211020(T) was isolated from rhizosphere soil of Excoecaria agallocha in a mangrove in Hainan, China. The strain produced longitudinal pair spores branching from aerial hyphae. 16S rRNA gene sequence analysis showed that the isolate belonged to the genus Microbispora, exhibiting the highest 16S rRNA gene sequence similarity (98.75 %) to Microbispora corallina JCM 10267(T) with a low DNA-DNA relatedness value (13 ± 0.6 %). The isolate contained meso-diaminopimelic acid as the diagnostic diamino acid but madurose was not detected. The predominant menaquinones were MK-9(H(4)), MK-9(H(2)) and MK-9(H(0)), and the major fatty acids were iso-C(16 : 0), iso-C(15 : 0) and C(17 : 0). The phospholipid profile of strain 211020(T) comprised phosphatidylinositol mannoside, phosphatidylethanolamine, diphosphatidylglycerol and phospholipids of unknown structure containing glucosamine. The DNA G+C content was 70.8 mol%. On the basis of phenotypic and genotypic data, strain 211020(T) can be distinguished as a novel species of the genus Microbispora, for which the name Microbispora hainanensis sp. nov., is proposed. The type strain is 211020(T) ( = CGMCC 4.5595(T) = DSM 45428(T)).

New Meliolaceae from the Brazilian Atlantic forest 1. Species on hosts in the families Asteraceae, Burseraceae, Euphorbiaceae, Fabaceae and Sapindaceae.

Five new species, two new varieties and three newly reported taxa belonging to the Meliolaceae were collected in fragments of Atlantic forest from Minas Gerais, Brazil, in association with native plants and are described and illustrated herein. The newly described species are Appendiculella eupatorii, Meliola cassiae-ferrugineae, M. mutisiae, M. peruiferae, M. vernaliae. The new varieties are M. garugae var. protii and M. paullinifolii var. rubiginosae. These taxa are reported in Brazil for the first time: Asteridiella cyclopoda, A. entebbeensis var. codiaei and Meliola pazschkeana var. macropoda. We studied other species belonging in Meliolaceae collected on hosts belonging to the Asteraceae, Burseraceae, Euphorbiaceae, Fabaceae (Caesalpinioideae and Papilionoideae) and Sapindaceae in Brazil.

Micromonospora rhizosphaerae sp. nov., isolated from mangrove rhizosphere soil.

Strain 211018(T) was isolated from mangrove Excocaria agallocha rhizosphere soil. 16S rRNA gene sequence analysis showed the highest similarity to the type strains of Micromonospora olivasterospora DSM 43868(T) (98.6 %) and Micromonospora pattaloongensis TJ2-2(T) (98.4 %). gyrB gene sequence analysis also indicated that strain 211018(T) should be assigned to the genus Micromonospora. The characteristic whole-cell sugars are xylose, mannose and arabinose. The predominant menaquinone is MK-9(H(4)) and the major fatty acids are iso-C(15 : 0) (27.5 %), 10-methyl C(17 : 0) (14.2 %), C(17 : 1)ω8c (12.8 %), iso-C(16 : 0) (12.6 %), anteiso-C(15 : 0) (6.1 %), iso-C(17 : 0) (4.1 %) and anteiso-C(17 : 0) (4.0 %). The phospholipid profile comprises phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The DNA G+C content is 70.8 mol%. The chemotaxonomic data of the strain coincided with those of the genus Micromonospora. Furthermore, a combination of DNA-DNA hybridization results and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain 211018(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora rhizosphaerae sp. nov. is proposed. The type strain is 211018(T) (=CGMCC 4.5599(T) =DSM 45431(T)).

Testing the Janzen-Connell mechanism: pathogens cause overcompensating density dependence in a tropical tree.

The Janzen-Connell hypothesis is a leading explanation for plant-species diversity in tropical forests. It suggests that specialized natural enemies decrease offspring survival at high densities beneath parents, giving locally rarer species an advantage. This mechanism, in its original form, assumes that density dependence is overcompensating: mortality must be disproportionately high at the highest densities, with few offspring recruiting below their parents. We tested this assumption using parallel shadehouse and field density-series experiments on seedlings of a tropical tree, Pleradenophora longicuspis. We found strong, overcompensating mortality driven by fungal pathogens, causing 90% (shadehouse) or 100% (field) mortality within 4 weeks of germination, and generating a negative relationship between initial and final seedling densities. Fungicide treatment led to much lower, density-independent, mortality. Overcompensating mortality was extremely rapid, and could be missed without detailed monitoring. Such dynamics may prevent dead trees from being replaced by conspecifics, promoting coexistence as envisioned by the Janzen-Connell hypothesis.

Selective isolation and ansamycin-targeted screenings of commensal actinomycetes from the "maytansinoids-producing" arboreal Trewia nudiflora.

To verify the hypothesis on the involvement of commensal actinomycetes in the biosynthesis of plant maytansinoids that belong to the ansamycin family, selective isolation and targeted screenings were conducted. In total, 164 endophytic actinomycetes isolates were obtained from the roots, twigs, seeds, callus, and germ-free seedlings of "maytansinoids-producing" Trewia nudiflora Linn. by selective isolation methods. Crude extracts of the isolates were screened by antifungal bioassay against Penicillium avellaneum UC-4376, thin-layer chromatography, and liquid chromatography-mass spectrometry detection according to the bioactivity as well as structural characteristics of maytansinoids. Respectively, 25.0%, 64.8%, and 26.7% of the extracts were positive in corresponding screenings. Ten isolates showed positive results in all three different screenings. To confirm on a genetic level, 28 representative isolates from 20 morphological groups were screened by polymerase chain reaction with 3-amino-5-hydroxybenzoic acid synthase gene and carbamoyltransferase gene primers, both of which were involved in the biosynthesis of ansamycins. According to the results of the targeted screenings, two isolates Streptomyces sp. 5B and Streptomyces sp. M27m3 might have the potential of producing ansamycins, which further enhanced the hypothesis that endophyte(s) might be involved in the biosynthesis of plant maytansinoids.

Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves.

Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited "Arum" type colonization with highly branched arbuscules, whereas C. tagal showed "Paris" type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.

Negative plant-soil feedbacks may limit persistence of an invasive tree due to rapid accumulation of soil pathogens.

Soil organisms influence plant species coexistence and invasion potential. Plant-soil feedbacks occur when plants change soil community composition such that interactions with that soil community in turn may positively or negatively affect the performance of conspecifics. Theories predict and studies show that invasions may be promoted by stronger negative soil feedbacks for native compared with exotic species. We present a counter-example of a successful invader with strong negative soil feedbacks apparently caused by host-specific, pathogenic soil fungi. Using a feedback experiment in pots, we investigated whether the relative strength of plant-soil feedbacks experienced by a non-native woody invader, Sapium sebiferum, differed from several native tree species by examining their performance in soils collected near conspecifics ('home soils') or heterospecifics ('away soils') in the introduced range. Sapium seedlings, but no native seedlings, had lower survival and biomass in its home soils compared with soils of other species (negative feedback'). To investigate biotic agents potentially responsible for the observed negative feedbacks, we conducted two additional experiments designed to eliminate different soil taxa ('rescue experiments'). We found that soil sterilization (pot experiment ) or soil fungicide applications (pot and field experiments) restored Sapium performance in home soil thereby eliminating the negative feedbacks we observed in the original experiment. Such negative feedbacks apparently mediated by soil fungi could have important effects on persistence of this invader by limiting Sapium seedling success in Sapium dominated forests (home soils) though their weak effects in heterospecific (away) soils suggest a weak role in limiting initial establishment.

Arbuscular mycorrhizas and ectomycorrhizas of Uapaca bojeri L. (Euphorbiaceae): sporophore diversity, patterns of root colonization, and effects on seedling growth and soil microbial catabolic diversity.

The main objectives of this study were (1) to describe the diversity of mycorrhizal fungal communities associated with Uapaca bojeri, an endemic Euphorbiaceae of Madagascar, and (2) to determine the potential benefits of inoculation with mycorrhizal fungi [ectomycorrhizal and/or arbuscular mycorrhizal (AM) fungi] on the growth of this tree species and on the functional diversity of soil microflora. Ninety-four sporophores were collected from three survey sites. They were identified as belonging to the ectomycorrhizal genera Afroboletus, Amanita, Boletus, Cantharellus, Lactarius, Leccinum, Rubinoboletus, Scleroderma, Tricholoma, and Xerocomus. Russula was the most frequent ectomycorrhizal genus recorded under U. bojeri. AM structures (vesicles and hyphae) were detected from the roots in all surveyed sites. In addition, this study showed that this tree species is highly dependent on both types of mycorrhiza, and controlled ectomycorrhization of this Uapaca species strongly influences soil microbial catabolic diversity. These results showed that the complex symbiotic status of U. bojeri could be managed to optimize its development in degraded areas. The use of selected mycorrhizal fungi such the Scleroderma Sc1 isolate in nursery conditions could be of great interest as (1) this fungal strain is very competitive against native symbiotic microflora, and (2) the fungal inoculation improves the catabolic potentialities of the soil microflora.

Plant pathogens drive density-dependent seedling mortality in a tropical tree.

One explanation for the extraordinary diversity of tropical forest trees is that density-dependent mortality from herbivores or pathogens puts locally rare species at an advantage. Density-dependent mortality of seeds and small seedlings is particularly intense in tropical forests, but its causes remain uncertain. Here, we show experimentally that pathogens from the Oomycota are associated with intense mortality in seedlings of a neotropical tree, Sebastiana longicuspis. Seedlings in untreated plots experienced eight times higher mortality compared with seedlings in plots treated with fungicide. Mortality was strongly density dependent: in fungicide-treated plots survival was unaffected by density, but survival in unsprayed plots was over three times higher at low density. Density-dependent mortality observed in a simultaneous, non-manipulative study was highly transient, suggesting that short-term observational studies may underestimate the intensity and form of pathogen-induced mortality. If such effects are widespread, plant pathogens may play a key role in maintaining and structuring tropical diversity.

Enrichment for microbes living in association with plant tissues.

AIMS: To investigate a cultivation-independent method of enrichment for microbes living in association with plant tissues. METHODS AND RESULTS: A large quantity of leaves or seeds was enzymatically hydrolyzed, and the pellets were collected by differential centrifugation. Enzyme concentration, buffer and incubation time were optimized for release of plant-associated microbes. The relative abundance of plant nuclear DNA and bacterial DNA in the enriched sample was estimated by PCR amplification of genome-specific marker genes. The efficiency of microbe enrichment was estimated from the proportion of bacterium-derived clones and their restriction fragment length polymorphism (RFLP) types as detected by 16S rRNA gene-based techniques. With a higher ratio of bacterial to plant nuclear DNA, the enriched samples showed a considerably enhanced proportion of bacterium-derived clones and a wider sequence diversity of those clones. CONCLUSIONS: The method described here proved to be remarkably effective in enriching for bacteria living in association with plant tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to study plant-associated microbes in the field of environmental molecular ecology and environmental metagenomics.

The use of high pressure freezing and freeze substitution to study host-pathogen interactions in fungal diseases of plants.

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host-pathogen interactions in fungal diseases of plants. The specific host-pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host-pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host-pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.

[Comparative studies on the fatty acids contained in four species of medicinal plants from family Euphorbiaceae and their endophytic fungi].

OBJECTIVE: The relation of four species of medicinal plants from family Euphorbiaceae and their endophytic fungi was studied to find the source of active substances for developing new pharmaceutical resources. METHOD: The main fatty acids contained in Sapium sebiferum, Euphorbia pekinensis, Euphorbia helioscopia, Bischofia polycarpam and their 28 strains of endophytic fungi were compared and analysed by GC. RESULT: The main fatty acids of the plants are: alpha-linolenic acid, palmitic acid, linolenic acid and oleic acid. Linolenic acid, palmitic acid and oleic acid are the main fatty acids of the endophytic fungi. CONCLUSION: The fatty acids could be produced by the endophytic fungi, which could be used as a factor for identification. There are great differences at the contents of alpha-linolenic acid between the plants and their endophytic fungi, which were suggested to be related with the nutrition absorption and the relationship between the endophytes and the host plant.