Phytopathogenic Fungus Cladosporium Research Articles

Metabolic engineering of Saccharomyces cerevisiae for the biosynthesis of a fungal pigment from the phytopathogenic fungus Cladosporium phlei

BackgroundCladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a photosensitizer that is a necessary component of photodynamic therapy. We applied synthetic biology to produce phleichrome using Saccharomyces cerevisiae.ResultsThe gene Cppks1, which encodes a non-reducing polyketide synthase (NR-PKS) responsible for the biosynthesis of phleichrome in C. phlei, was cloned into a yeast episomal vector and used to transform S. cerevisiae. In addition, a gene encoding a phosphopantetheinyl transferase (PPTase) of Aspergillus nidulans was cloned into a yeast integrative vector and also introduced into S. cerevisiae for the enzymatic activation of the protein product of Cppks1. Co-transformed yeasts were screened on a leucine/uracil-deficient selective medium and the presence of both integrative as well as episomal recombinant plasmids in the yeast were confirmed by colony PCR. The episomal vector for Cppks1 expression was so dramatically unstable during cultivation that most cells lost their episomal vector rapidly in nonselective media. This loss was also observed to a less degree in selective media. This data strongly suggests that the presence of the Cppks1 gene exerts a significant detrimental effect on the growth of transformed yeast cells and that selection pressure is required to maintain the Cppks1-expressing vector. The co-transformants on the selective medium showed the distinctive changes in pigmentation after a period of prolonged cultivation at 20 °C and 25 °C, but not at 30 °C. Furthermore, thin layer chromatography (TLC) revealed the presence of a spot corresponding with the purified phleichrome in the extract from the cells of the co-transformants. Liquid chromatography (LC/MS/MS) verified that the newly expressed pigment was indeed phleichrome.ConclusionOur results indicate that metabolic engineering by multiple gene expression is possible and capable of producing fungal pigment phleichrome in S. cerevisiae. This result adds to our understanding of the characteristics of fungal PKS genes, which exhibit complex structures and diverse biological activities.

Unique functional responses of fungal communities to various environments in the mangroves of the Maowei Sea in Guangxi, China

Fungi are important compartments of microbial communities of mangroves. Their diversity might be influenced by their habitat environment. This study analyzed the distribution and function of fungal communities in the sediments and plant samples from mangrove ecosystem of the Maowei Sea area in Guangxi, China. The results showed that phytopathogenic fungi Cladosporium (17.00%) was mainly observed in the sediments from the protected zone, while endophytic fungi Alternaria (9.22%) and Acremonium (6.09%) were only observed in the sediments from wharf. The fungi in the sediments from village and park were mainly consisted of high-activity endophytes and fungi related to lignin-degrading, respectively. Acaulospora and Aspergillus with higher relative abundance discovered in plant tissues could help plant growth. Cirrenalia (37.66%) and Lignincola (26.73%) with high-activity for lignin-degrading were discovered in decayed leaves. The distribution and function of fungi were highly dependent on the environment settings, thus the fungi can be used as indicators for monitoring the environmental change of mangrove ecosystems.

Acetylcholinesterase inhibition and antifungal activity of cyclohexanoids from the endophytic fungus Saccharicola sp.

Abstract Three new oxygenated cyclohexanoids, namely, speciosin U (1), speciosin V (2) and speciosin W (3), along with four known compounds consisting of 4-hydroxy-3-(3′-methylbut-3′-en-1′-ynyl)-benzoic acid (4), 4-hydroxy-3-prenyl-benzoic acid (5), 2,2-dimethyl-2H-chromene-6-carboxylic acid (6) and trans-3,4-dihydro-3,4-dihydroxy-anofinic acid (7) were isolated from scale-up cultures of Saccharicola sp., an endophyte of Eugenia jambolana. The structures were established by analysis of spectroscopic data, including 1D and 2D NMR. The absolute configuration of 1 was determined using Mosher’s method. Speciosin U (1) and trans-3,4-dihydro-3,4-dihydroxy-anofinic acid (7) exhibited a potent acetylcholinesterase (AChE) inhibitory activity comparable to reference inhibitor galantamine. Furthermore, speciosin U (1) presented antifungal activity invitro against the phytopathogenic fungi Cladosporium cladosporioides, while compounds 4-hydroxy-3-(3′-methylbut-3′-en-1′-ynyl)-benzoic acid (4) and 4-hydroxy-3-prenyl-benzoic acid (5) exhibited antifungal activity against C. sphaerospermum.

Antimicrobial and Antitumor Photodynamic Effects of Phleichrome from the Phytopathogenic Fungus Cladosporium Phlei

Fungal perylenequinones have photodynamic activity and are promising photosensitizers for photodynamic therapy (PDT). Here, we investigated the bactericidal and antitumor activities of phleichrome from the fungal perylenequinone family in vitro. Photodynamic bactericidal activity of phleichrome was analyzed by agar-well diffusion method under dark and illuminated conditions. The photodynamic antitumor activity of phleichrome was analyzed in MCF-7, HeLa, SW480, and HepG2 human cancer cell lines using in vitro cytotoxicity assays. Photodynamic bactericidal activities against Gram-negative and Gram-positive bacteria were species-specific. Antitumor activity against all tumor cell lines increased under the illuminated condition. Depending on the results of the analyses, Phleichrome has potential for further drug development related to its antibacterial and antitumor activities.

Strains of soil microorganisms promising for the creation of a complex plant protection product against mycoses and harmful insects

We evaluated the antagonistic activity of 23 strains of Bacillus spр. against phytopathogenic fungi Fusarium oxysporum, F. culmorum, F. moniliforme, Cladosporium herbarum, Alternaria alternata and Aspergillus niger. The antagonistic activity was tested by agar diffusion (the method of blocks). For determining the influence of bacteria on barley plants , ardent seeds were treated by cultural liquid (dilution 1 : 10) for 2 hours and germinated in Petri dishes on moist filter paper. The fungistatic effect of Bacillus sрp. separately and in combination with entomopathogens (in equal ratio) was determined by the level of inhibition of the fungi Fusarium spp. on a solid nutrient medium with 5% of the culture liquid. Insecticidal activity of microorganisms was determined in the model experiments by the percentage of death of the caterpillar Archips podana Scop. Strains of Bacillus sp. KMB-3 and Bacillus sp. KMB-6 inhibited the growth of all test cultures (zones of growth inhibition 11.4–30.6 and 11.5–29.4 mm, respectively). We established the absence of antagonism between selected strains and entomopathogenic bacteria Bacillus thuringiensis IMB-7186, fungi Beauveria bassiana IMB-F-100043. We found that treatment of barley seeds with culture liquids of Bacillus sp. KMB-3 and Bacillus sp. KMB-6 didn’t have a negative effect on the morphometric indices and dry weight of seedlings. We established that the highest percentage of growth inhibition of F. culmorum IMB-F-50716 was provided by a complex of Bacillus sp. KMB-3, B. bassiana IMB-F-100043 and B. thuringiensis IMB-7186, whose action was at the same level as the action of monoculture Bacillus sp. KMB-3 (85.4% and 84.7%, respectively). The highest percentage inhibition of growth of F. oxysporum ІМВ-F-54201 was provided by a complex of strains of Bacillus sp. KMB-3 and B. bassiana IMB-F-100043, whose effect was slightly inferior to that of the monoculture Bacillus sp. KMB-3 (68.4% and 75.1%, respectively). The insecticidal activity of complexes Bacillus sp. KMB-3, B. bassiana IMB-F-100043, B. thuringiensis IMB-7186 or Bacillus sp. KMB-6, B. bassiana IMB-F-100043, B. thuringiensis IMB-7186 insignificantly differed from that of the complex entomopathogens B. bassiana IMB-F-100043 and B. thuringiensis IMB-7186 (71.1%, 73.3% death versus 80.0%). The selected microbial complexes can be considered as promising for the development of a preparation for the protection of plants against fungal diseases and harmful insects.

Lactone Derivatives Produced by a Phaeoacremonium sp., an Endophytic Fungus from Senna spectabilis.

Three new isoaigialones, A, B, and C (1-3), along with aigialone (4), were isolated from the crude EtOAc extract of a Phaeoacremonium sp., an endophytic fungus obtained from the leaves of Senna spectabilis. The structures of these compounds were elucidated based on the analysis of spectroscopic data. Compounds 2 and 4 were active against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum. This is the first report of metabolites produced by an Phaeoacremonium sp., associated with S.spectabilis.

Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

Two different diketopiperazines, cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers. No further significant induction was observed by supplementing inducers over the concentration of maximum yield. Among the two inducers, cyclo-(L-Pro-L-Phe) showed better inducing capability than cyclo-(L-Pro-L-Leu). The maximum yield was observed from the M0035 strain grown on V8 juice media supplemented with 150 μM cyclo-(L-Pro-L-Phe), which was estimated to be 232.6 mg of phleichrome per gram of mycelia and 10.2 mg of secreted phleichrome per 20 agar-plugs. Interestingly, growth inhibition was observed on V8 juice agar media with 100, 150, and 200 μM cyclo-(L-Pro-L-Phe) but not on PDA with the same amount of inducer, which suggests that the inhibitory effect might be through the overproduction of phleichrome rather than the toxic effect of the inducer itself. Superoxide production by purified phleichrome was dramatically stimulated upon illumination, thus demonstrating photodynamic production of superoxide in vitro by phleichrome.

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.

Sesquiterpenos produzidos pelo fungo endofítico Phomopsis cassiae com atividade antifúngica e inibidora de acetilcolinesterase

SESQUITERPENES PRODUCED BY ENDOPHYTIC FUNGUS Phomopsis cassiae WITH ANTIFUNGAL AND ACETYLCHOLINESTERASE INHIBITION ACTIVITIES. Two new diastereoisomeric cadinanes sesquiterpenes 3,9-dihydroxycalamenene (1-2), along with the known 3-hydroxycalamen-8-one (3) and aristelegone-A (4), were isolated from ethyl acetate extract of Phomopsis cassiae, an endophytic fungus in Cassia spectabilis. Their structures, including relative stereochemistry, were determined on the basis of detailed interpretation of 2D NMR spectra and comparison with related known compounds. Compounds 1-4 displayed antifungal activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum, as well as inhibition of acetylcholinesterase.

Helicascolide C, a new lactone from an Indonesian marine algicolous strain of Daldinia eschscholzii (Xylariaceae, Ascomycota)

From an endophytic Daldinia eschscholzii strain isolated from the agar-producing red alga Gracilaria sp. SGR-1, collected from the coast of South Sulawesi, Indonesia, a new lactone helicascolide C (1) was obtained as colourless crystals from the ethyl acetate extract together with the related structurally known compound helicascolide A (2). The structure of the new compound 1 reveals a carbonyl group replacing an alcohol group of compound 2. The structure of 1 was elucidated by X-ray diffraction and spectral analyses. Compound 1 showed fungistatic activity against the phytopathogenic fungus Cladosporium cucumerinum.

Patagonicosides B and C, Two Antifungal Sulfated Triterpene Glycosides from the Sea Cucumber Psolus patagonicus

Two new triterpene glycosides, patagonicosides B and C (2 and 3, resp.), together with the known patagonicoside A (1), have been isolated from the EtOH extract of the sea cucumber Psolus patagonicus. The structures of the new compounds were established on the basis of extensive NMR spectroscopic analysis ((1)H- and (13)C-NMR, (1)H,(1)H-COSY, HMBC, HSQC, TOCSY, and NOESY), HR-ESI-MS data, and chemical transformations. Compounds 1-3 and their desulfated analogs showed antifungal activities against the phytopathogenic fungus Cladosporium cladosporoides in a dose-dependent fashion.

A simplified method for screening ethylene-producing phytopathogenic fungi

An easy and reliable method for measuring ethylene production is a prerequisite for research on fungal ethylene biosynthesis. In the present study, Botrytis cinerea was used as a model fungus to develop a method to detect in vitro production of ethylene. Spore suspensions were cultured in methionine-supplemented PDA. Small plates were excised from the medium and placed in a sealed ampoule bottle for the ethylene production assay. Gaseous samples were obtained from the sealed bottle for gas chromatography analysis. Using this method, eight species of phytopathogenic fungi (Alternaria alternate, Aspergillus niger, B. cinerea, Cladosporium sp., Fusarium oxysporum, Rhizopus stolonifer, Pestalotiopsis spp. and Penicillium spp.) were shown to produce significant levels of ethylene. Ethylene production by these fungi varied markedly during the seven day incubation period. In addition to the facilitation of fungal ethylene analyses, the present method allowed the observation of morphological characteristics of the test fungi during the assay. This spore-plate method is a simple, convenient, and broadly applicable method for assessing fungal ethylene production in vitro. This method may improve screening of fungi with high ethylene production rates, analysis of fungal growth and development, and be useful in crop protection.

Ampullosine, a new Isoquinoline Alkaloid from Sepedonium ampullosporum (Ascomycetes)

A new isoquinoline alkaloid, ampullosine (3-methyl-isoquinoline-6-carboxylic acid, 1), was isolated from Sepedonium ampullosporum and characterized by spectroscopic analysis and chemical reactions. This compound is responsible for the deep yellow color of the culture fluid of this species. Moreover, the known compounds sepedonin (2) and anhydrosepedonin (3) were detected. Twelve strains belonging to eight species of Sepedonium have been screened for these three metabolites by LC/ESI-SRM (selected reaction monitoring). Ampullosine (1) could be detected in almost all species in Sepedonium, but not in the phylogenetically more distant species S. brunneum and S. tulasneanum. Anhydrosepedonin (3) showed antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum.

A highly selective ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

Abstract G-I, a highly selective β-glucosidase, was purified from phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-I was a monomer with native molecular weight of 85 kDa and pI value of 4.2. The maximal activity to p-nitrophenyl-β- d -glucopyranoside (pNPG) occurred at pH 6.0 and 45 °C at which the Km against pNPG was 0.18 mM and Vmax was 46.7 μmol nitrophenol/min/mg. G-I was highly stable within pH 4.0–11.0 and below 40 °C. It was inhibited by Co2+, Cu2+ and Zn2+ (50 mM), but showed resistance to sodium dodecyl sulfonate (SDS, 250 mM). G-I was highly active against β-linked disaccharide cellobiose, gentiobiose and sophorose, but exhibited very low activities against other aryl-glycosides, methyl-α-glycosides and disaccharides trehalose and sucrose. Moreover, G-I specifically hydrolyzed β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb1 to produce ginsenoside Rd, without attack on other β- d -glucosidic linkages. The oligopeptide fragments of G-I were sequenced by nanoESI-MS/MS and showed similarity to the sequences from the glycoside hydrolase family 3. G-I is different to G-II (a glycosidase previously purified from the same fungus) in composition and molecular weight. It shows more stable and higher selectivity than G-II.

Trypanocidal, leishmanicidal and antifungal potential from marine red alga Bostrychia tenella J. Agardh (Rhodomelaceae, Ceramiales)

Specimens of the red alga Bostrychia tenella J. Agardh (Rhodomelaceae, Ceramiales) were collected from the São Paulo coast and submitted to room temperature solvent extraction. The resulting extract was fractionated by partitioning with organic solvent. The n-hexane (BT-H) and dichloromethane (BT-D) fractions showed antiprotozoal potential in biological tests with Trypanosoma cruzi and Leishmania amazonensis and presented high activity in an antifungal assay with the phytopathogenic fungi Cladosporium cladosporioides and Cladosporium sphaerospermum. Chromatography methods were used to generate subfractions from BT-H (H01 to H11) and from BT-D (D01 to D19). The subfractions were analyzed by gas chromatography–mass spectrometry (GC/MS), and the substances were identified by retention index (Kovats) and by comparison to databases of commercial mass spectra. The volatile compounds found in marine algae were identified as fatty acids, low molecular mass hydrocarbons, esters and steroids; some of these have been previously described in the literature based on other biological activities. Moreover, uncommon substances, such as neophytadiene were also identified. In a trypanocidal assay, fractions BT-H and BT-D showed IC 50 values of 16.8 and 19.1 μg/mL, respectively, and were more active than the gentian violet standard (31 μg/mL); subfractions H02, H03, D01 and D02 were active against L. amasonensis, exhibiting IC 50 values of 1.5, 2.7, 4.4, and 4.3 μg/mL, respectively (standard amphotericin B: IC 50 = 13 μg/mL). All fractions showed antifungal potential. This work reports the biological activity and identification of compounds by GC/MS for the marine red alga B. tenella for the first time.

Streptomycetes antagonism against Cladosporium fulvum Cooke and Fusarium oxysporium f.sp. lycopersici

This research aimed to evaluate the secondary effects of secondary metabolites produced by streptomycetes on spore germination and mycelial growth of the phytopathogenic fungi Cladosporium fulvum Cooke and Fusarium oxysporium f. sp. lycopersici from tomato plants. Metabolites produced by streptomycete isolates codified as AC-147 and AC-92 caused 94.1% inhibition of C. fulvum while AC-95 isolate caused 33.9% inhibition. AC-92 was the most efficient for F. oxysporum f. sp. lycopersici, causing 94.2% inhibition of spore germination. For mycelial growth, AC-26 and AC-92 were the most efficient in inhibiting C. fulvum growth by 46.6% and F. oxysporum f. sp. lycopersici by 29.9%. These streptomycetes are potential agents for biocontrol development methods of these tomato plant pathogenic fungi.

Caldensinic acid, a prenylated benzoic acid from Piper caldense

The CH 2Cl 2 and MeOH extracts from leaves of Piper caldense were subjected to chromatographic separation procedures to afford the new prenylated benzoic acid, caldensinic acid (3-[(2′ E,6′ E,10′ E)-11′-carboxy-3′,7′,15′-trimethylhexadeca-2′,6′,10′,14′-tetraenyl]-4,5-dihydroxybenzoic acid) whose structure was determined by spectral analysis, mainly NMR ( 1H, 13C, HSQC, HMBC) and ESI-MS. The natural compound and derivatives displayed antifungal activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum by direct bioautography.

Highly selective biotransformation of ginsenoside Rb1 to Rd by the phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb(1) to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml(-1); temperature, 37 degrees C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb(1) by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.

A novel ginsenoside Rb 1-hydrolyzing β- d-glucosidase from Cladosporium fulvum

A novel β-glucosidase (G-II) was purified to homogeneity from a culture filtrate of the phytopathogenic fungus Cladosporium fulvum ( syn. Fulvia fulva). G-II specifically cleaved the β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb 1 to produce ginsenoside Rd, but did not hydrolyze the other β- d-glucosidic linkages in protopanaxadiol-type ginsenosides. In specificity tests, G-II was active against pNPG and disaccharides such as cellobiose and gentiobiose, but exhibited very low activities against other aryl-glycosides and methyl-α-glycosides. G-II consisted of two identical subunits with a native molecular mass of 180 kDa and a p I of 4.4. The optimal pH of G-II was pH 5.5, and the enzyme was highly stable over a range of pH 5.0–11.0. The optimal temperature was 45 °C, and the enzyme became unstable at temperatures above 40 °C. The K m and V max values against pNPG were 0.19 mM and 57.7 μmol/(min mg), respectively. The enzyme was inhibited by Zn 2+, Cu 2+ (over 50 mM) and SDS (250 mM). However, the inhibition by SDS was partially reversed by 10 mM dithiothreitol. Three oligopeptide fragments obtained after enzymatic digestion of G-II were sequenced by nanoESI-MS/MS. The amino acid sequence homology analysis showed that G-II possessed significant homology with the family 3 β-glucosidases.