Toxigenic Aspergillus and Penicillium Isolates from Weevil-Damaged Chestnuts

Aspergillus and Penicillium were among the most common genera of fungi isolated on malt-salt agar from weevil-damaged Chinese chestnut kernels (16.8 and 40.7% occurrence, respectively). Chloroform extracts of 21 of 50 Aspergillus isolates and 18 of 50 representative Penicillium isolates, grown for 4 weeks at 21.1 C on artificial medium, were toxic to day-old cockerels. Tweleve of the toxic Aspergillus isolates were identified as A. wentii, eight as A. flavus, and one as A. flavus var. columnaris. Nine of the toxic Penicillium isolates were identified as P. terrestre, three as P. steckii, two each as P. citrinum and P. funiculosum, and one each as P. herquei (Series) and P. roqueforti (Series). Acute diarrhea was associated with the toxicity of A. wentii and muscular tremors with the toxicity of P. terrestre, one isolate of P. steckii, and one of P. funiculosum.     

Penicillium species endophytic in coffee plants and ochratoxin A production

Tissues from Coffea arabica, C. congensis, C. dewevrei and C. liberica collected in Colombia, Hawaii and at a local plant nursery in Maryland were sampled for the presence of fungal endophytes. Surface sterilized tissues including roots, leaves, stems and various berry parts were plated on yeast-malt agar. DNA was extracted from a set of isolates visually recognized as Penicillium, and the internal transcribed spacer region and partial LSU-rDNA was amplified and sequenced. Comparison of DNA sequences with GenBank and unpublished sequences revealed the presence of 11 known Penicillium species: P. brevicompactum, P. brocae, P. cecidicola, P. citrinum, P. coffeae, P. crustosum, P. janthinellum, P. olsonii, P. oxalicum, P. sclerotiorum and P. steckii as well as two possibly undescribed species near P. diversum and P. roseopurpureum. Ochratoxin A was produced by only four isolates, one isolate each of P. brevicompactum, P. crustosum, P. olsonii and P. oxalicum. The role these endophytes play in the biology of the coffee plant remains enigmatic.     

Antibacterial activity of marine-derived fungi

A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low molecular weight metabolites produced by various Penicillium species

Low-molecular weight volatile metabolites produced by Penicillium farinosum, P. citrinum, P. camemberti and P. chrysogenum were investigated. During first 40 days of cultivation the fungi produced mainly C-8 compounds, and later mainly 2-hexenal was synthesized. Addition of 0.1% linoleic acid significantly stimulated the secretion of volatile metabolites. P. citrinum and P. farenosum produced large quantities of geosmin.     

Degradation of organophosphonates by Penicillium citrinum

Utilization of organophosphonates as the sole source of phosphorus, carbon or nitrogen by a soil isolate of Penicillium citrinum was studied. Penicillium citrinum was found to utilize 2-aminoethylphosphonic and 2-oxoalkylphosphonic acids as the sole phosphorus source whereas 1-hydroxyalkylphosphonates as well as 1-aminoalkylphosphonates and their dipeptides did not support the growth of the fungus. The mould did not metabolize any of the phosphonates tested, when they served as the sole carbon or nitrogen source.
Penicillium citrinum is perhaps the first mould strain isolated from soil, shown to be capable of organophosphonate degradation.     

1-aminocyclopropane-1-carboxylate (ACC) deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces

We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70 % of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.     

Toxigenic species of Penicillium, Fusarium, and Aspergillus from weevil-damaged pecans.

Of a total of 2392 fungi isolated from weevil-damaged pecans, 46.4% were Alternaria and Epicoccum, 23.9% were Penicillium, 12.4% were Pestalotia and Monochaeta, 6.5% were Cladosporium, 6.4% were Fusarium, and less than 2% each were Phoma, Aspergillus, Rhizopus, Trichothecium, and miscellaneous. Chloroform extracts of 34 of 105 representative Penicillium isolates, 3 of 28 Fusarium isolates, and 3 of 23 Aspergillus isolates were toxic to day-old cockerels during three bioassays. Eight of the toxic extracts from Penicillium spp. were tremorgenic. One tremorgenic isolate was identified as P. paxilli, four were identified as P. lanoso-coeruleum, and three as P. cyclopium. Nine of the non-tremorgenic isolates were identified as P. citrinum, five as P. aurantio-virens, three as P. oxalicum, and two as P. meleagrinum. Others were identified as one each of P. brevi-compactum (Series), P. nigricans (Series), P. roqueforti, P. rugulosum (Series), P. terrestre, and P. stoloniferium. One was unidentified. Toxigenic Aspergillus isolates were all A. flavus. Two of the toxic Fusarium isolates were F. moniliforme, and one was unidentified.     

Plocoralides A-C, polyhalogenated monoterpenes from the marine alga Plocamium corallorhiza

Three new polyhalogenated monoterpenes, plocoralides A-C (1-3) along with three known compounds (4-6) have been isolated from the organic extract of the red alga P. corallorhiza. Structures of the new compounds were characterized as 4,8-dibromo-1,1-dichloro-3,7-dimethyl-2E,6E-octadiene (1), 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene (2) and 4,8-dibromo-1,1,7-trichloro-3,7-dimethyl-2E,5Z-octadiene (3) on the basis of one- and two-dimensional NMR spectroscopic data and MS analyses. Compounds 2-6 show moderate cytotoxicity toward esophageal cancer cells.     

1-Aminocyclopropane-1-carboxylate synthase of Penicillium citrinum: primary structure and expression in Escherichia coli and Saccharomyces cerevisiae

A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC). A fungus, Penicillium citrinum, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia. ACC synthase, responsible for ACC synthesis in P. citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA. The ACC synthase from P. citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme. The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P. citrinum. Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation. The sequence of ACC synthase from P. citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.     

Tanzawaic acid derivatives from a marine isolate of Penicillium sp. (SF-6013) with anti-inflammatory and PTP1B inhibitory activities

Chemical investigation of a marine-derived fungus Penicillium sp. SF-6013 resulted in the discovery of a new tanzawaic acid derivative, 2E,4Z-tanzawaic acid D (1), together with four known analogues, tanzawaic acids A (2) and D (3), a salt form of tanzawaic acid E (4), and tanzawaic acid B (5). Their structures were mainly determined by analysis of NMR and MS data, along with chemical methods. Preliminary screening for anti-inflammatory effects in lipopolysaccharide (LPS)-activated microglial BV-2 cells showed that compounds 1, 2, and 5 inhibited the production of nitric oxide (NO) with IC₅₀ values of 37.8, 7.1, and 42.5 μM, respectively. Compound 2 also inhibited NO production in LPS-stimulated RAW264.7 murine macrophages with an IC₅₀ value of 27.0 μM. Moreover, these inhibitory effects correlated with the suppressive effect of compound 2 on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 and BV2 cells. In addition, compounds 2 and 5 significantly inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with the same IC₅₀ value (8.2 μM).     

Oxygenated monoterpenoids from badea (Passiflora quadrangularis) fruit pulp

The new monoterpenoids (2E)-2,6-dimethyl-2,5-heptadienoic acid, (2E)-2,6-dimethyl-2,5-heptadienoic acid beta-D-glucopyranosyl ester, (5E)-2,6-dimethyl-5,7-octadiene-2,3-diol, and (3E)-3,7-dimethyl-3-octene-1,2,6,7-tetrol were isolated from the fruit pulp of Passiflora quadrangularis along with the known 2,5-dimethyl-4-hydroxy-3(2H)-furanone beta-D-glucopyranoside.     

Microbial lipids from cellulolytic oleaginous fungus Penicillium citrinum PKB20 as a potential feedstock for biodiesel production

Microbial lipids derived from oleaginous fungi are considered as an alternative feedstock for biodiesel production. We attempt to isolate a cellulolytic oleaginous fungi as a potential feedstock for biodiesel production. The fungus was identified by 5.8 S-ITS rRNA gene sequencing. The extracellular enzyme activities were recorded after every 24 h for 7 days. Nile red staining and fluorescence microscopy was used to visualise the lipid bodies within the fungal hyphae. A renewable heterogeneous base catalyst derived from Musa balbisiana cola peels was used for the transesterification of Penicillium citrinum PKB20 derived oil into biodiesel. GC-MS analysis was used to analyse the fatty acid methyl esters (FAME) profile of the transesterified lipids. Penicillium citrinum PKB20 was isolated from detritus rich soil of Assam, India. The endoglucanase, xylanase and β-glucosidase enzyme activities were found to be 292.83 ± 0.29, 111.72 ± 0.45 and 6.54 ± 0.13 U/mg respectively. The specific enzyme activity for extracellular lipase was found to be 3.12 ± 0.16 U/mg. It could accumulate up to 60.61% of lipids in nitrogen-limited medium (7.34 ± 0.45 g/L biomass production). The extracted lipids were converted to biodiesel with 89.3% conversion efficiency. The predominant fatty acids were oleic acid (30.09%), palmitic acid (20.25%) and linoleic acid (33.14%) suggesting a balance between oxidative stability and cold flow properties for suitable biodiesel quality. Penicillium citrinum PKB20 was found to be a potential feedstock for biodiesel production with desirable fuel properties. The cellulolytic nature could be utilised for simultaneous lipid production directly on cellulosic substrates.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

Secretion of ligninperoxidase by Penicillium citrinum,Fusarium oxysporum and Aspergillus terreus

Secretion of ligninperoxidase [E.C.1.11.1.7] by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus in liquid culture growth medium has been demonstrated. Enzymatic characteristics like Km, pH and temperature optima using veratryl alcohol as the organic substrate of ligninperoxidases from above sources have been determined. Km values using veratryl alcohol as substrate for enzymes from P. citrinum, F. oxysporum and A. terreus were 69, 64 and 60 microM respectively. Km values using H2O2 as the variable substrate were 64, 72 and 80 microM.The pH optima were 4.0, 2.3 and 2.0 respectively. The values of temperature optima were 30 degrees, 25 degrees and 22 degrees C for the enzymes from P. citrinum, F. oxysporum and A. terreus respectively.     

Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum

Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described.     

Concentration on Diapak C 16 capsules of lovastatin, mevinolinic acid and other inhibitors of biosynthesis of sterins produced by Penicillium citrinum 89]

The method of lovastatine and mevinolinic acid known as competitive inhibitors of HMG-CoA-reductase and produced by micromycetes was elaborated. The inhibitors from diluted water solutions were fully absorbed on Diapak C16 patrons. The rate of inhibitors elution from the patrones was more than 95 per cent. Patrons may be used for concentration of lovastatine group inhibitors from the culture media. Inhibitors synthesis by the Penicillium citrinum 89 was investigated in dynamics with the use of Diapak C16 patrones. It was shown that UV-spectrum of inhibitor produced by P. citrinum 89 was identical with compactin spectrum and had absorbance maximum at 230, 237 and 247 nm.     

UV-guided screening of benzodiazepine producing species in Penicillium

The benzodiazepine sclerotigenin (auranthine B) recently described as a metabolite of Penicillium sclerotigenum, has been isolated as the major metabolite from an isolate of P. commune. The structure of sclerotigenin was established by a single-crystal X-ray diffraction study and by NMR spectroscopy. UV-guided screening for benzodiazepine production by other penicillia revealed that sclerotigenin was also produced by isolates of P. clavigerum, P. lanosum, P. melanoconidium, P. sclerotigenum and P. verrucosum. Sclerotigenin was detected both intra- and extracellularly. Apparently, P. aurantiogriseum is the only auranthine producing species in genus Penicillium.     

A new species from tropical soils,Eupenicillium tropicum

Forty-three strains of Eupenicillium tropicum sp. nov. were isolated from soils collected in India, Costa Rica and Galapagos, Ecuador. The species is characterized by biverticillate penicilli, slightly rough, subglobose to ovate conidia, brownish cleistothecia that become brown-gray with age, and ascospores with two equatorial flanges and slightly roughened valves. All strains produced a large number of indole alkaloids, and many types of unknown secondary metabolites with characteristic chromophores were produced by a majority of strains. Eupenicillium tropicum is morphologically most similar to E. shearii, but based on ITS-LSU sequences, is most closely related to Penicillium citrinum, P. sartoryi and P. westlingii. Eupenicillium shearii strains consistently produce paxillin, paspalinine and shearinins, while the latter three penicillia all produce citrinin consistently.     

Diagnostic characterization of Penicillium palitans, P. commune and P. solitum

A total of 98 isolates of Penicillium commune and P. solitum were analysed and it was shown that these isolates produced three unique combinations of secondary metabolites. Penicillium commune produced cyclopiazonic acid, cyclopaldic acid and rugulovasine A and B; P. solitum produced compactin and a new group including the ex-type culture of P. palitans produced cyclopiazonic acid and fumigaclavine A. Isolates in all three groups were able to produce cyclopenin, cyclopenol and viridicatin. An optimal thin layer chromatography system to detect the secondary metabolites from P. commune, P. palitans and P. solitum was developed using an agar plug method. The results were confirmed by high performance liquid chromatography with diode array detection. The chemotaxonomic allocations were backed up by differences in conidial colour on Czapek yeast autolysate agar, reverse colour on yeast extract sucrose agar and origin of the isolates. Even though P. palitans previously has been considered synonymous with P. commune or P. solitum it was concluded that P. palitans is a distinct species.