The genomic attributes of Cd-resistant, hydrocarbonoclastic Bacillus subtilis SR1 for rhizodegradation of benzo(a)pyrene under co-contaminated conditions.

Bacillus subtilis SR1 is a metal resistant, polyaromatic hydrocarbon-degrading bacterium isolated from petroleum contaminated sites. This study reports the characteristics of the genome of the isolate containing one circular chromosome (4,093,698 bp) annotated into 4155 genes and 4095 proteins. The genome analysis confirmed the presence of multiple catabolic genes: aromatic ring-hydroxylating dioxygenase (COG2146), aromatic ring hydroxylase (COG2368), catechol 2, 3 dioxygenase (COG2514), 4-hydroxybenzoate decarboxylase (COG0043), carboxymuconolactone decarboxylase (COG0599) responsible for the catabolism of aromatic hydrocarbons along with the genes for biosurfactant production and functional genes (czcD and cadA) for resistance to cadmium, zinc, and cobalt. Gas Chromatography-Mass spectroscopy analysis revealed up to 35% in-vitro degradation of benzo(a)pyrene after 21 days of growth along with the production of different intermediate metabolites. The pot trial analysis in the greenhouse condition validated the rhizodegradation of BaP, which was significantly higher in the presence of plant-microbe association (85%) than degradation in bulk soil (68%).

Difference in the rhizosphere microbiome of Melia azedarach during removal of benzo(a)pyrene from cadmium co-contaminated soil.

Benzo(a)pyrene (BaP) is a highly persistent biohazard polyaromatic hydrocarbon and often reported to be present in soils co-contaminated with heavy metals. The present study explains the rhizodegradation of BaP using bacterial consortium in the rhizosphere of Melia azedarach, along with a change in taxonomical and functional properties of the rhizosphere microbiome. The relative abundance of most dominant phylum Proteobacteria was 2% higher with BaP, while in the presence of both BaP and Cd, its abundance was 2.2% lower. Functional metagenome analysis also revealed the shifting of microbial community and functional gene abundance in the favor of xenobiotic compound degradation upon augmentation of bacterial consortium. Interestingly, upon the addition of BaP the range of functional abundance for genes of PAH degradation (0.165-0.19%), was found to be decreasing. However, augmentation of a bacterial consortium led to an increase in its abundance including genes for degradation of benzoate (0.55-0.64%), toluene (0.2-0.22%), naphthalene (0.25-0.295%) irrespective of the addition of BaP and Cd. Moreover, under greenhouse condition, the application of M. azedarach-bacterial consortium enhanced the degradation of BaP in the rhizosphere (88%) after 60 days, significantly higher than degradation in bulk soil (68.22%). The analysis also showed an increase in degradation of BaP by 15% with plant-native microbe association than in bulk soil. Therefore, the association of M. azedarach-bacterial consortium enhanced the degradation of BaP in soil along with the taxonomical and functional attributes of the rhizosphere microbiome.

Isolation and Characterization of Antifungal Metabolites from the Melia azedarach-Associated Fungus Diaporthe eucalyptorum.

Two biosynthetically related new metabolites, eucalyptacid A (1) and eucalactam B (2), along with six known compounds (3-8), eugenitol (3), cytosporone C (4), 4-hydroxyphenethyl alcohol (5), 1-(4-hydroxyphenyl)ethane-1,2-diol (6), N-(2-hydroxy-2-phenylethyl)acetamide (7), and phomopene (8), were isolated from the solid rice cultures of the endophytic fungus Diaporthe eucalyptorum KY-9 that had been isolated from Melia azedarach. Also, two further new derivatives (2a, 2b) were prepared from 2. The structures were elucidated by exhaustive analysis of NMR and ESIMS data and chemical methods such as Marfey's protocol. Compound 1 was identified as a rare polyketide fatty acid, (8E)-3,5,11-trihydroxy-2,10,12-trimethyltetradecenoic acid, and 2 was determined to be the first cyclic depsipeptide containing the same fatty acid unit as 1 and a Gly-Gly-Thr tripeptide chain. Its N-terminal end is N-acylated by an 11-hydroxy fatty acid with a branch alkyl chain of 14:1. The 11-hydroxyl group connects to the carboxylic group of the C-terminal amino acid to form a 22-membered lactone ring. A hypothetical biosynthetic pathway for the new polyketides is proposed. The isolated compounds were assayed for their inhibition against four plant pathogenic fungi, Alternaria solani, Botrytis cinerea, Fusarium solani, and Gibberella saubinettii. Compounds 1, 4, 6, and 7 exhibited antifungal activities against Alternaria solani, with minimal inhibitory concentration (MIC) values from 6.25 to 50 µM. Thus, strain KY-9 represents an untapped source for the development of biological control agents to prevent the infection of pathogenic fungus A. solani.

Conjugation of antifungal benzoic acid derivatives as a path for detoxification in Penicillium brasilianum, an endophyte from Melia azedarach.

In this study, the consumption of 4-bromobenzoic acid and 4-chlorobenzoic acid by the fungus Penicillium brasilianum, an endophyte from Melia azedarach is evaluated. This fungus metabolizes these halobenzoic acids to produce three new brominated compounds, which have been isolated and characterized, and three new chlorinated derivatives identified by HRMS. Among these products, (4-bromobenzoyl)proline has been also chemically synthesized and employed in biological assays, thus providing insights for the elucidation of the defense mechanism of P. brasilianum towards these halobenzoic acids.

Actinobacteria associated with Chinaberry tree are diverse and show antimicrobial activity.

Many actinobacteria produce secondary metabolites that include antimicrobial compounds. Since most of the actinobacteria cannot be cultivated, their antimicrobial potential awaits to be revealed. We hypothesized that the actinobacterial endophyte communities inside Melia toosendan (Chinaberry) tree are diverse, include strains with antimicrobial activity, and that antimicrobial activity can be detected using a cultivation independent approach and co-occurrence analysis. We isolated and identified actinobacteria from Chinaberry, tested their antimicrobial activities, and characterized the communities using amplicon sequencing and denaturing gradient gel electrophoresis as cultivation independent methods. Most of the isolates were identified as Streptomyces spp., whereas based on amplicon sequencing the most abundant OTU was assigned to Rhodococcus, and Tomitella was the most diverse genus. Out of the 135 isolates, 113 inhibited the growth of at least one indicator organism. Six out of the 7577 operational taxonomic units (OTUs) matched 46 cultivated isolates. Only three OTUs, Streptomyces OTU4, OTU11, and OTU26, and their corresponding isolate groups were available for comparing co-occurrences and antimicrobial activity. Streptomyces OTU4 correlated negatively with a high number of OTUs, and the isolates corresponding to Streptomyces OTU4 had high antimicrobial activity. However, for the other two OTUs and their corresponding isolate groups there was no clear relation between the numbers of negative correlations and antimicrobial activity. Thus, the applicability of co-occurrence analysis in detecting antimicrobially active actinobacteria could not be proven.

Description of 'Candidatus Phytoplasma meliae', a phytoplasma associated with Chinaberry (Melia azedarach L.) yellowing in South America.

China tree yellows (ChTY) phytoplasma is associated with the yellowing disease of the China tree (Melia azedarach) in Argentina. According to partial 16S rRNA gene analysis, ChTY phytoplasma belongs to the 16Sr XIII group, subgroup G. Strains of species of ChTY have 98-99 % 16S rDNA gene sequence similarity with 16SrXIII-group phytoplasmas, and less than 97.5 % when compared to all 'CandidatusPhytoplasma' described so far, except for the novel 'CandidatusPhytoplasma hispanicum'. However, strains of species of ChTY are differentiated from the latter due to having additional molecular and biological attributes. The presence of unique features in the 16S rDNA sequence distinguishes ChTY from all species of 'CandidatusPhytoplasma' currently described. The in silico RFLP profile of 16S rDNA (1.2 kb) and rpLV-rpsC (1.3 kb) genes distinguished ChTY, as in the 16SrXIII-G subgroup within the 16SrXIIII group. The phylogenetic analyses, based on 16S rDNA, rpLV-rpsC and secA gene sequences, in addition to the restricted host range, characteristic symptoms and geographical distribution, confirm that the collective strains of the species ChTY represent a distinct lineage within the phytoplasma clade and support the description of a novel species of 'CandidatusPhytoplasma meliae' with the reference strain being ChTY-Mo3 (Montecarlo, Argentina).

Caryophyllene sesquiterpenoids from the endophytic fungus, Pestalotiopsis sp.

Eight new caryophyllene sesquiterpenoids named pestaloporinates A-G (1-7) and 14-acetylhumulane (8) have been isolated from the solid cultures of an endophytic fungus Pestalotiopsis sp., which was obtained from the fresh stem bark of Melia azedarach Linn. Their structures as well as absolute configurations were determined by spectroscopic data, ECD experimentation, and single-crystal X-ray diffraction. Among all the isolates, compound 2 displayed potent inhibitory activity with IC50 value of 19.0 µM during the evaluation of nitric oxide (NO) inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells.

Secondary metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal, antibacterial, antioxidant, and cytotoxic activities.

Two new metabolites, an α-pyridone derivative, 3-hydroxy-2-methoxy-5-methylpyridin-2(1H)-one (1), and a ceramide derivative, 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide (2), and a new natural product, 3-hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide (3), along with 15 known compounds including chaetoglobosin C (7) and chaetoglobosin F (8) were isolated from the solid culture of the endophytic fungus Botryosphaeria dothidea KJ-1, collected from the stems of white cedar (Melia azedarach L). The structures were elucidated on the basis of spectroscopic analysis (1D and 2D NMR experiments and by mass spectrometric measurements), and the structure of 1 was confirmed by X-ray single-crystal diffraction. These metabolites were evaluated in vitro for antimicrobial, antioxidant, and cytotoxicity activities. Pycnophorin (4) significantly inhibited the growth of Bacillus subtilis and Staphyloccocus aureus with equal minimum inhibitory concentration (MIC) values of 25 µM. Stemphyperylenol (5) displayed a potent antifungal activity against the plant pathogen Alternaria solani with MIC of 1.57 µM comparable to the commonly used fungicide carbendazim. Both altenusin (9) and djalonensone (10) showed markedly DPPH radical scavenging activities. In addition, stemphyperylenol (5) and altenuene (6) exhibited strong cytotoxicity against HCT116 cancer cell line with a median inhibitory concentration (IC50) value of 3.13 µM in comparison with the positive control etoposide (IC50 = 2.13 µM). This is the first report of the isolation of these compounds from the endophytic B. dothidea.

Antifungal and antibacterial metabolites from an endophytic Aspergillus sp. associated with Melia azedarach.

Seven known metabolites, dianhydro-aurasperone C (1), isoaurasperone A (2), fonsecinone A (3), asperpyrone A (4), asperazine (5), rubrofusarin B (6) and (R)-3-hydroxybutanonitrile (7), were isolated from the culture of Aspergillus sp. KJ-9, a fungal endophyte isolated from Melia azedarach and identified by spectroscopic methods. All isolates were evaluated in vitro against several phytopathogenic fungi (Gibberella saubinetti, Magnaporthe grisea, Botrytis cinerea, Colletotrichum gloeosporioides and Alternaria solani) and pathogenic bacteria (Escherichia coli, Bacillus subtilis, Staphyloccocus aureus and Bacillus cereus). Compounds 3 and 7 were active against almost all phytopathogenic fungi tested with minimum inhibitory concentration (MIC) range of 6.25-50 µM. Moreover, compound 3 was active against all pathogenic bacteria with MIC in the range of 25-100 µM. Compound 7 is a rare new natural product isolated from a natural source for the first time, and the detailed NMR data of 1 were first assigned.

Absolute configuration of fusarone, a new azaphilone from the endophytic fungus Fusarium sp. isolated from Melia azedarach, and of related azaphilones.

A new azaphilone derivative, named fusarone (1), has been isolated from the ethyl acetate soluble extract of the fermentation broth of an endophytic fungus, Fusarium sp. LN-12, isolated from the leaves of Melia azedarach Linn. The structure of the new compound was established on the basis of extensive spectroscopic analysis, including 1D-NMR and 2D-NMR ((1) H-(1)H COSY, TOCSY, HSQC, HMBC, and NOESY) experiments. The absolute configurations of fusarone (1) and of a second related azaphilone were determined by means of electronic circular dichroism spectroscopy and optical rotation calculations.

Oxidative potential of some endophytic fungi using 1-indanone as a substrate.

The oxidative potential of the fungus Penicillium brasilianum, a strain isolated as an endophyte from a Meliaceae plant (Melia azedarach), was investigated using 1-indanone as a substrate to track the production of monooxygenases. The fungus produced the dihydrocoumarin from 1-indanone with the classical Baeyer-Villiger reaction regiochemistry, and (-)-(R)-3-hydroxy-1-indanone with 78% ee. Minor compounds resulting from lipase and SAM activities were also detected. The biotransformation procedures were also applied to a collection of Penicillium and Aspergillus fungi obtained from M. azedarach and Murraya paniculata. The results showed that Baeyer-Villiger were mostly active in fungi isolated from M. azedarach. Almost all of the fungi tested produced 3-hydroxy-1-indanone..

Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities.

Thirty-nine fungal metabolites 1-39, including two new alkaloids, 12ß-hydroxy-13α-methoxyverruculogen TR-2 (6) and 3-hydroxyfumiquinazoline A (16), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach. Their structures were elucidated on the basis of detailed spectroscopic analysis (mass spectrometry and one- and two-dimensional NMR experiments) and by comparison of their NMR data with those reported in the literature. These isolated compounds were evaluated for in vitro antifungal activities against some phytopathogenic fungi, toxicity against brine shrimps, and antifeedant activities against armyworm larvae (Mythimna separata Walker). Among them, sixteen compounds showed potent antifungal activities against phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii), and four of them, 12ß-hydroxy-13α-methoxyverruculogen TR-2 (6), fumitremorgin B (7), verruculogen (8), and helvolic acid (39), exhibited antifungal activities with MIC values of 6.25-50 µg/mL, which were comparable to the two positive controls carbendazim and hymexazol. In addition, of eighteen that exerted moderate lethality toward brine shrimps, compounds 7 and 8 both showed significant toxicities with median lethal concentration (LC(50)) values of 13.6 and 15.8 µg/mL, respectively. Furthermore, among nine metabolites that were found to possess antifeedant activity against armyworm larvae, compounds 7 and 8 gave the best activity with antifeedant indexes (AFI) of 50.0% and 55.0%, respectively. Structure-activity relationships of the metabolites were also discussed.

Fusaroside, a unique glycolipid from Fusarium sp., an endophytic fungus isolated from Melia azedarach.

Fusaroside (1), a unique trehalose-containing glycolipid composed of the 4-hydroxyl group of a trehalose unit attached to the carboxylic carbon of a long-chain fatty acid, was isolated from the organic extract of fermentation broths of an endophytic fungus, Fusarium sp. LN-11 isolated from the leaves of Melia azedarach. Six known compounds, phalluside (2), (9R*,10R*,7E)-6, 9,10-trihydroxyoctadec-7-enoic acid (3), porrigenic acid (4), (9Z)-2,3-dihydroxypropyl octadeca-9-enoate (5), cerevisterol (6) and ergokonin B (7), were also isolated from this fungus. The glycolipid contains a rare branched long-chain fatty acid (C(20:4)) with a conjugated diene moiety and a conjugated ketone moiety. The structure of the new compound 1 was elucidated by spectroscopic methods (1D and 2D NMR experiments, MS) and chemical degradations. The metabolites 1-5 were shown to have moderate to weak active against the brine shrimp larvae. To our knowledge, this is the first report of isolation of the first representative of a new family of glycolipids from natural sources.

[Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry (Melia azedarach Z) witches'-broom phytoplasma].

OBJECTIVE: To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization. METHODS: Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe. RESULTS: One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3, 2, 1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6% -85.6% , however, the homology of different SSB proteins was between 74.0% - 89.4%. Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy), whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc). CONCLUSION: The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB), which were for the replication of plasmid. Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localized to the membrane. The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation, among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids. CWBFq, PaWBNy, PeVHn, CWBFz and MDPy possessed distinct plasmids in terms of number and size, whereas there was no plasmid detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.

Biosynthesis of phenylpropanoid amides by an endophytic Penicillium brasilianum found in root bark of Melia azedarach.

Biosynthetic studies on brasiliamides, potently convulsive and bacteriostatic compounds from an endophytic Penicillium brasilianum isolated from Melia azedarach (Meliaceae), confirms their phenylpropanoid origin, which is very uncommon in fungi. Feeding experiments with [2-(13)C]- phenylalanine indicated the incorporation of two units of this amino acid on brasiliamide structures. The first step in the phenylpropanoid pathway to those compounds was evaluated through enzymatic bioassays and confirmed the phenylalanine ammonia-lyase (PAL) participation. The metabolism of phenylalanine in this fungus is discussed.

Co-production of bisphenylpropanoid amides and meroterpenes by an endophytic Penicillium brasilianum found in the root bark of Melia azedarach.

A fungus, isolated from the root bark of Melia azedarach (Meliaceae), from which a series of meroterpenes have been reported, was identified as Penicillium brasilianum based on analysis of the ITS region of ribosomal DNA. From a rice culture of this fungus, the known phenylpropanoid amides brasiliamide A and B were obtained together with and a new, slightly modified congener, along with the meroterpenoids preaustinoid A1, preaustinoid B2 and austinolide. The compounds were isolated by the use of combined chromatographic procedures and identified by physical methods, mainly 1D and 2D NMR experiments, with distinction for 1H{15N} HMBC applied to brasiliamide A. The amides were tested for their antimicrobial activity and showed only weak inhibitory effects, against a set of pathogenic bacteria.

Metabolites from an endophytic fungus sphaceloma sp. LN-15 isolated from the leaves of Melia azedarach.

Two new natural compounds, a symmetrical disulfide dimer didodecyl 3,3''-dithiodipropionate (1) and a pregnane steroid 5,16-pregnadien-3beta-ol-20-one acetate (2), were isolated together with two known compounds, ergosta-4,6,8(14),22-tetraen-3-one (3) and ergosterol peroxide (4), from the ethyl acetate soluble extract of fermentation broth of an endophytic fungus, Sphaceloma sp. LN-15 isolated from the leaves of Melia azedarach L. and grown in pure culture. Their structures were determined on the basis of spectroscopic methods including 1D and 2D nuclear magnetic resonance spectroscopy (NMR) experiments and by mass spectrometric measurements (MS). These fungal metabolites were isolated for the first time from the genus Sphaceloma. The structure of 1 was also confirmed by chemical synthesis.

Further meroterpenes produced by Penicillium sp., an endophyte obtained from Melia azedarach.

From the methanol extract of Penicillium sp. cultivated on sterilized rice for three weeks we isolated three new meroterpenes preaustinoid A1, A2 and B1. The fungus was isolated from the root bark of Melia azedarach after surface sterilization. The structures of these compounds were identified by intensive spectroscopic studies.

Meroterpenes from Penicillium sp found in association with Melia azedarach.

A Penicillium sp was isolated from the root bark of Melia azedarach and cultivated over sterilized rice. After chromatographic procedures, two meroterpenes, named preaustinoid A and B, were obtained in addition to the known alkaloid verruculogen. Their structures were identified by extensive spectroscopic studies, and they exhibited moderate bacteriostatic effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus sp.