Anti-Pseudomonas aeruginosa activity of a C16-terpene dilactone isolated from the endophytic fungus Neofusicoccum luteum of Kigelia africana (Lam.).

Fungal endophytes have the capacity to biosynthesize secondary metabolites that are produced by their host plants. In this study, a dilactone terpenoid of C16 architecture was isolated from the fungal endophytes of Kigelia africana, in our attempt to identify anti-Pseudomonas aeruginosa metabolites. Thirty-eight fungal isolates were cultured for biomolecule production over a period of thirty days. Extracts from three (ZF 34, ZF 52 and ZF 91) of the fungi showed good anti-P. aeruginosa activity, with ZF 52 presenting the best MIC of 19.53 µg/mL and was accordingly subjected to chromatographic separation. Based on nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction (XRD) analyses, the isolated compound was identified as a C16-terpene dilactone, with a structure consistent with that of the known diterpene, CJ-14445. The isolated dilactone showed anti-P. aeruginosa activity with MIC of 0.61 µg/mL, signifying the antibacterial potential of the biomolecule. The bioactive fungal isolate (ZF 52) was identified as Neofusicoccum luteum based on genomic DNA sequencing. This is the first report of the endophyte N. luteum from K. africana and the first reported occurrence of CJ-14445 in the fungus.

Pulchranin A: First report of isolation from an endophytic fungus and its inhibitory activity on cyclin dependent kinases.

Fungal factories emerge as a promising source for the production of bioactive natural products. This study reports the isolation and structure elucidation of pulchranin A, for the first time, from an endophytic fungus (Aspergillus TRL1), which was cultured from Tabebuia rosea (Bignoniaceae) stems and identified by DNA ITS sequencing. Pulchranin A showed promising in-vitro cytotoxic effects against breast (MCF-7), liver (Hep-G2) and colorectal (HCT) cell lines, with IC50 values of 63, 80 and 91 µg/mL, respectively. In addition, it inhibited three cyclin-dependent kinases (CDK1, CDK2 and CDK4) in MCF-7 cells with IC50 values of 9.82, 15.6 and 2.7 µg/mL, respectively. Results were further supported by in-silico molecular docking of pulchranin A to CDK1, CDK2, and CDK4 crystal structures, where it demonstrated good interactions by H-bonding, hydrophobic and Pi-Pi interactions with different amino acid residues of these enzymes. Pulchranin A might be a potential CDK inhibitor in human breast cancer cells.[Figure: see text].

Antifungal activity of Bignoniaceae plants on Aspergillus carbonarius and Aspergillus niger.

Twenty four extracts from Bignoniaceae plants of northwest Argentina were tested for antifungal activity against Aspergillus species responsible of the grape black rot. Stems and leaves of Amphilophium cynanchoides, Macfadyena cynanchoides, Tecoma stans and Jacaranda mimosifolia were separately extracted with solvents of increasing polarity to obtain the dichloromethane (fCH2Cl2), ethyl acetate (fEtOAc) and methanol extracts (fMeOH). The fCH2Cl2 from stem of M. cynanchoides had the lowest IC50 (1.0-1.2 mg/mL) and MID values (0.6-1.2 mg) and the highest ID values (5.0-6.8 mm) on A. niger and A. carbonarius. The main contributors of the antifungal activity of fCH2Cl2 were identified as lapachol (MIC = 0.25-1.00 mg/ml) and 1-hydroxy-4-methylanthraquinone (MIC = 0.0625-0.125 mg/mL). These compounds synergized the antifungal activity of sodium metabisulfite and showed an additive effect in mixtures with propiconazol. They might be used as additives of commercial antifungals to protect grapes against A. niger and A. carbonarius.

Taxonomy, phylogeny, and divergence time estimation for Apiosphaeria guaranitica, a Neotropical parasite on bignoniaceous hosts.

Apiosphaeria guaranitica, the causal agent of brown crust disease of several bignoniaceous hosts, among them Handroanthus and Tabebuia species, has been traditionally placed in Phyllachoraceae, based exclusively on morphological studies, without supporting molecular evidence. Here, we provide molecular data for the link between sexual and asexual states of the fungus and elucidate the phylogeny of A. guaranitica. The multilocus phylogenetic analyses employed sequences from the 18S subunit (18S), 28S subunit (28S), and nuclear internal transcribed spacers (ITS1-5.8S-ITS2 = ITS) of the nuc rDNA, second-largest subunit of RNA polymerase II (RPB2), and translation elongation factor 1-α (TEF1) genetic loci. Estimates of the divergence time of this lineage were supported by fossil calibration (FC) and secondary calibration (SC) strategies. Our results indicate a natural placement of Apiosphaeria within Diaporthaceae (Diaporthales), where it represents an ancient lineage of the crown group of Diaporthaceae, diverging during the late Paleocene at 61.15 (FC) and 60.63 (SC) million years ago. This divergence time estimate within Diaporthales is based on Spataporthe taylori, a diaporthaceous fossil.

Antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of Markhamia tomentosa.

CONTEXT: Plants harbor endophytes with potential bioactivity. Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae) is reported to possess antioxidant, anti-inflammatory and anticancer activities. OBJECTIVE: The antifungal and antiproliferative properties of endophytic fungi extracts and fractions from M. tomentosa were evaluated. MATERIAL AND METHODS: Endophytic fungi were isolated from the leaves of M. tomentosa and identified by ITS-rDNA sequence analysis. The antagonistic effect of the fungal strains was investigated against pathogenic fungi viz, Fusarium oxysporum, Sclerotinia sclerotiorium, Rhizoctonia solani, and Botrytis cinerea using the dual culture assay for 5-7 days. Antiproliferative effect of the fungal extracts and fractions (3.91-250 µg/mL) on HeLa cancer cell line was tested and IC50 was calculated. Poisoning food assay and antifeedant activity against the pathogenic fungi and Spodoptera litura larvae, for 7 days and 2 h, respectively, was also tested at concentrations of 250, 500 and 1000 µg/mL. RESULTS: Fungal endophytes Trichoderma longibrachiatum and Syncephalastrum racemosum were isolated from the leaves of M. tomentosa. Isolated endophytic fungal strains and solvent extracts showed MIC value of 1000 µg/mL against tested pathogenic fungi in the dual culture and poisoning food assays. Methanol fraction of S. racemosum isolate showed the most effective antiproliferative activity with IC50 of 43.56 µg/mL. Minimal feeding deterrent activity against S. litura larvae was also observed. DISCUSSION AND CONCLUSION: These findings showed that the leaves of Markhamia tomentosa harbor strains of endophytic fungi with promising health benefits, and suggest their antifungal and antiproliferative effects against pathogenic fungi and HeLa cancer cell line.

Immunosuppressive potential of Botryosphaeria dothidea, an endophyte isolated from Kigelia africana.

CONTEXT: For years, natural products from microbes have been used as drugs. Endophytes are the most important fungi that produce many novel metabolites for potential use in pharmacology and agriculture. OBJECTIVE: The objective of the present study was to explore new endophytes for novel natural products. MATERIALS AND METHODS: An endophyte BAK-I was isolated from the bark of Kigelia africana (Lam.) Beneth (Bignoniaceae). BAK-I was characterized morphologically and on the basis of ITS-5.8S rDNA sequences. BAK-I was fermented to yield an extract, which was evaluated for its anticancer, antimicrobial, and immunomodulatory activities, using MTT, agar well-diffusion, tube dilution method, lymphocyte proliferation, and pro-inflammatory cytokines (TNF-α) (by macrophages) evaluation assays. For lymphocyte proliferation and pro-inflammatory cytokines studies, four concentrations were evaluated 10, 30, 100, and 1000 µg/mL and the experiments were conducted for 72 and 48 h, respectively. RESULTS AND DISCUSSION: The BAK-I showed pink cottony growth. SEM studies showed smooth fusoid-oblong conidia with a truncated base. Furthermore, ITS-5.8S rDNA sequence showed 99% homology with the Botryosphaeria dothidea strain suggesting that the endophyte is a strain of the genus Botryosphaeria. Less than 50% growth inhibition of SF295, Lung A-549, and THP-1 cancer cell lines after treatment with BAK-I extract suggested that it did not have significant cytotoxic potential, whereas it is bactericidal for Gram-positive pathogens MRSA and VRE with MIC value 200 and 250 µg/mL, respectively. To elucidate its immunomodulation potential, splenocyte proliferation studies showed that BAK-1 suppressed the T cell proliferation by 50%. TNF-α evaluation studies also showed that the extract inhibited TNF-α production in a concentration-dependent manner suggesting that it had immunosuppressive potential. Inhibition at 10 µg/mL was found to be 55% as against 48% using ß-methasone. CONCLUSION: The results suggested that BAK-I extract can be used as a potential immunosuppressive agent.

Effects of arbuscular mycorrhizal inoculation and fertilization on mycorrhizal Statute of Jacaranda mimosifolia D.Don cultivated in nurseries.

The effects of fertilization and the nature of the inoculum as well as the variation of the dose intake of the latter on the level of Jacaranda mimosifolia D.Don mycorhization were tested. Young plants were treated with two inoculums presenting different origins, compositions and modes of application: one is a commercial product containing Glomus irregulare, and the other is a composite indigenous inoculum resulting from trapping five species of genus Glomus and also from multiplication on mycotrophic plants: leek (Allium porrum L.) and vetch (Vicia sativa L.). For each inoculum, two doses were tested and for each dose of inoculum, four levels of fertilization based on a complete commercial fertilizer (Osmocote) were tested: 0 g/plant, 2 g/plant, 4 g/plant, and 6g/plant. Three repetitions were performed for each combination treatment of inoculum/fertilizer. One-year-old young Jacaranda plants, being about 40 cm high, were cultured under greenhouse in 10/12 cm caliber pots. After six months, all the inoculated plants were mycorrhized. According to endomycorrhizal structures found on their roots, plants receiving doses of composite indigenous inoculum reached a more advanced stage of mycorrhization than those treated with the commercial inoculum. The existence of an interaction effect between the inoculum dose and the level of fertilization on Jacaranda mycorhization rate was excluded. These two parameters of variation were studied as simple effects. The increase in commercial inoculum dose had a significant positive influence on the level of Jacaranda plants mycorrhization (P=0.05). The rate of mycorrhization jumped from 12.69% to 21.92%. Nonetheless, for plants receiving increasing doses of composite indigenous inoculum, the level of mycorrhization has varied randomly. In both instances of inoculum treatments, increasing the dose of fertilizer significantly inhibited endomycorrhizal colonization of Jacaranda roots (P=0.01). Thus, the rate of root colonization decreased from 47.43% to 2.41% for plants receiving the composite indigenous inoculums. It decreased from 32.35% to 3.95% for those treated with the commercial inoculum. Mycorrhization had a positive effect on root dry biomass of Jacaranda, as in the case of unfertilize ave the highest rates of colonization.

Influence of arbuscular mycorrhizal fungi (AMF) on zinc biogeochemistry in the rhizosphere of Lindenbergia philippensis growing in zinc-contaminated sediment.

The association of arbuscular mycorrhizal fungi (AMF) with the roots of Lindenbergia philippensis (Cham.) Benth., sampled from a Zn-contaminated settling pond at a zinc smelter, significantly enhanced Zn accumulation (72,540 ± 5,092 mg kg⁻¹ dry weight) in rhizosphere sediment amended with 1,000 mg L⁻¹ of Zn sulfate solution compared to fungicide-treatments that suppressed AMF colonization. This can be explained by a significant proportion of Zn being found in rectangular crystals that were associated with the root mucilaginous sheath. Despite this, all treatments maintained the same Zn coordination geometry in both Zn oxidation state and the coordinated neighbouring atoms. X-ray absorption spectroscopy (XAS) showed a Zn(II) oxidation state as a core atom and associated with six oxygen atoms symmetrically arranged in an octahedral coordination and coordinated with sulfur. The results may indicate a role for AMF in enhancing Zn immobilization in the rhizosphere of indigenous plants that successfully colonize Zn mining and smelting disposal sites.

Paenibacillus catalpae sp. nov., isolated from the rhizosphere soil of Catalpa speciosa.

A bacterial strain, designated D75(T), was isolated from the rhizosphere soil of Catalpa speciosa. Phylogenetic analysis based on the complete 16S rRNA gene sequence revealed that strain D75(T) was a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain D75(T) and Paenibacillus glycanilyticus DS-1(T) (99.2 %), Paenibacillus xinjiangensis B538(T) (97.5 %) and Paenibacillus castaneae Ch-32(T) (97.2 %). The chemotaxonomic properties of strain D75(T) were consistent with those of the genus Paenibacillus: the cell-wall peptidoglycan type was based on meso-diaminopimelic acid (A1γ), the predominant menaquinone was MK-7, and the major fatty acids were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. However, levels of DNA-DNA relatedness between strain D75(T) and P. glycanilyticus NBRC 16618(T), P. xinjiangensis DSM 16970(T) and P. castaneae DSM 19417(T) were 35, 20 and 18 %, respectively. On the basis of phenotypic and chemotaxonomic analyses, phylogenetic data and DNA-DNA relatedness values, strain D75(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus catalpae sp. nov. is proposed. The type strain is D75(T) ( = DSM 24714(T) = CGMCC 1.10784(T)).

Genetic analysis of a novel Xylella fastidiosa subspecies found in the southwestern United States.

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

Morphological and phylogenetic comparisons amongst powdery mildews on Catalpa in the UK.

Three species of powdery mildew, Erysiphe elevata, E. catalpae, and Neoerysiphe galeopsidis were identified on Catalpa species in England in 2004. A new disease record, N. galeopsidis was the first Catalpa mildew to appear (in June), but it was later out-competed by E. elevata that caused the most serious damage. Both mildews also attacked C. speciosa, C. xerubescens and a new host, xChitalpa tashkentensis, a Chilopsis xCatalpa hybrid. No powdery mildew was detected on C. bungei, C. ovata, or C. fargesii. Identifications of the pathogens using morphological data were fully supported by DNA analysis yielding characteristic rDNA ITS sequences. The sequences placed E. catalpae within the E. aquilegiae clade. The sequences for E. elevata from southern England and France closely matched those from Hungary and North America, confirming the recent spread of this pathogen from the USA. It eventually overran N. galeopsidis and its sudden appearance in the UK could be due to greater aggressiveness and to the production of more ascomata especially during autumns with delayed leaf fall as in 2001. A further species, Oidium hiratae (i.e. Podosphaera sp.), though described from a 1978 UK collection on C. bignonioides, was not detected in the field.