Conidiogenesis and secondary metabolism in Penicillium urticae.

Submerged cultures of Penicillium urticae (NRRL 2159A) produced the antibiotics patulin and griseofulvin when grown in a glucose-nitrate medium. A high concentration of calcium (i.e., 68 mM) inhibited the production of both antibiotics while stimulating conidiogenesis. Conidial mutants that were defective in an early stage of conidiogenesis produced markedly less patulin, even under growth conditions that favored secondary metabolism. A mutant which lacked the ability to produce the patulin pathway metabolites m-cresol, toluquinol, m-hydroxybenzyl-alcohol, m-hydroxybenzaldehyde, gentisaldehyde, gentisyl alcohol, gentisic acid and patulin, as well as the pathway enzyme m-hydroxybenzyl-alcohol dehydrogenase, still produced yields of conidia that were equivalent to or greater than those of the parent strain. Other mutants which were blocked at later steps of the patulin pathway also produced conidia. These results indicate that patulin and the other related secondary metabolites noted above are not a prerequisite to conidiogenesis in P. urticae. Environmental and developmental factors such as calcium levels and conidiogenesis do, however, indirectly affect the production of patulin pathway metabolites.     

Manganese and antibiotic biosynthesis. I. A specific manganese requirement for patulin production in Penicillium urticae

The effect of trace metal nutrition on the functioning of the patulin biosynthetic pathway in submerged cultures of Penicillium urticae (NRRL 2159A) was examined by both chromatographic and enzymological means. Comprehensive metal ion analysis showed generally low levels of contaminating metal ions in media components. Of eight metal ions examined, only manganese strongly influenced secondary metabolite production. In control cultures or cultures deficient in calcium, iron, cobalt, copper, zinc, or molybdenum, pathway metabolites appeared in the medium at about 25 h after inoculation. The first pathway-specific metabolite, 6-methylsalicylic acid, accumulated only transiently before being converted to patulin whose concentration steadily increased. In manganese-deficient cultures, however, 6-methylsalicylic acid continued to accumulate, with only minor amounts of patulin being produced. Additionally, a marker enzyme for the pathway showed only 0-20% of control activity. Clear dose responses (patulin versus manganese) were found in different media, with no effect on growth yield. Addition of manganese to depleted cultures at 18, 26, or 36 h resulted in increasing marker enzyme activity and patulin concentrations. It is concluded that manganese exerts a specific, positive effect on patulin biosynthesis and may in some way control the section of the patulin pathway occurring after 6-methylsalicylic acid.     

Mycotoxin-Producing Potential of Mold Flora of Dried Beans

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.     

Identification of phyllostine as an intermediate of the patulin pathway in Penicillium urticae.

A patulin negative mutant (J1) of Penicillium urticae (NRRL 2159A) was found to accumulate large quantities (greater than 128 mg/L culture) of a reactive, photosensitive compound, which was isolated and identified as (-)-phyllostine (5,6-epoxygentisylquinone). This epoxyquinone possessed an antibiotic activity against Bacillus subtilis which was approximately 80% of that exhibited by patulin. In separate in vivo feeding experiments, [2-14C]acetate and [G-3H]gentisaldehyde were readily incorporated into phyllostine by mutant J1 and [14C]phyllostine was incorporated into patulin by the parent strain (NRRL 2159A). When fed to a washed-cell suspension of a second patulin negative mutant (J2) which produced gentisaldehyde but not phyllostine, unlabeled phyllostine was efficiently converted to patulin in yields of 33, 56, and 92% after 30 min, 1 and 5 h, respectively. The role of phyllostine as an intermediate of a new post-gentisaldehyde portion of the patulin biosynthetic pathway is discussed.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

Use of activated charcoal for the removal of patulin from cider.

Penicillium urticae (NRRL 2159A) was grown in culture broth containing 1 muCi of [1-14C-A1acetate to produce [14C]patulin. [14C]patulin was purified from the broth and added to apple cider. After the patulin concentration of the cider was adjusted to 30 mug/ml with unlabeled patulin, the cider was subjected to various charcoal treatments. [14C]patulin was completely removed by shaking the cider with 20 mg of activated charcoal per ml and by eluting the cider through a 40- to 60-mesh charcoal column. Activated charcola at 5 mg/ml reduced patulin in naturally contaminated cider to nondetectable levels.     

Isoepoxydon, a new metabolite of the patulin pathway in Penicillium urticae

A patulin-negative mutant (J1) of Penicillium urticae (N.R.R.L. 2159A) was known to accumulate about 100mg per litre quantities of the 5,6-epoxygentisyl quinone, (-)-phyllostine and another metabolite (UIII). Both were derived from acetate and hence were polyketides. Purified UIII (m.p. 53 degrees C, [alpha](32) (D)+206 degrees , lambda(methanol) (max.) 240nm; epsilon 3806 litre.mol(-1).cm(-1)) was characterized as a partially reduced derivative of (-)-phyllostine and was found to be a diastereoisomer of the known phytotoxin, (+)-epoxydon. Hence its designation as (+)-iso- or epi-epoxydon. From (1)H n.m.r. and c.d. data the stereochemistry of the epoxide ring in (+)-isoepoxydon was determined to be identical with that in (+)-epoxydon (i.e. R,R) but the configuration of the secondary alcohol at C-4 was S rather than R as in (+)-epoxydon. Isoepoxydon (compound UIII) is therefore (4S,5R,6R)-5,6-epoxy-4-hydroxy-2-hydroxymethylcyclohex-2-en-1-one. The boat conformation in which the C-4 hydroxy group is axial is preferred. In the range of 1mm to 5mm, the antibiotic activity of (+)-isoepoxydon against Bacillus subtilis sp. was 56% of that obtained with patulin. Over a period of 1 to 3h, [(14)C]isoepoxydon was efficiently converted into patulin by a shake culture of the parent strain of P. urticae. The precursor relationship of isoepoxydon to patulin was confirmed by feeding unlabelled isoepoxydon (1mm) to a washed-cell suspension of a mutant (J2) in which, over a period of 3 to 5h, a better than 60% conversion into patulin was attained. The enzymic relationship between isoepoxydon and phyllostine and their positions in the late portion of the patulin biosynthetic pathway are discussed.     

Manganese and antibiotic biosynthesis. II. Cellular levels of manganese during the transition to patulin production in Penicillium urticae

The radionuclide 54MnCl2 was used to examine the cellular manganese content of submerged cultures of Penicillium urticae NRRL 2159A. Liquid-scintillation spectroscopy allowed sensitive detection of isotopic manganese in both normally supplemented and manganese-deficient cultures. The cellular manganese content in supplemented cultures showed three distinct phases, including a period of uptake that coincided with the time of transition to antibiotic biosynthesis. Such an uptake was not seen for manganese-deficient cultures, but addition of normal quantities of unlabelled manganese to the media appeared to stimulate uptake. Preliminary characterization shows this manganese uptake is not inhibited by other metal ions, does not require metabolic energy or a protein component, but is disrupted by changes in incubation temperature. The significance of these observations is discussed in the light of recent work on the requirement for manganese for antibiotic biosynthesis in this organism.     

Effects of Patulin and Method of Application on Growth Stages of Wheat

When a single, 100-mug/ml application of patulin, produced by Penicillium urticae Bainier, was applied to growth stages 7, 9, 10, and 10.1 (Feekes scale) of Lee spring wheat (Triticum aestivum L.), decreases in internodal elongation, floret number, seed weight, and seed number were observed. Yields were reduced according to the proximity of application prior to heading. Application of patulin to the soil in crystalline form and dissolved in aqueous solution were also investigated, and the solution method of application was found to be the treatment of choice. A single exposure of growing wheat plants to patulin can produce yield reductions similar to those observed in stubble-mulch farming.     

Antibiotics in the chemical communication of fungi

In dual cultures Oudemansiella mucida and Xerula melanotricha (basidiomycetes) react to the presence of living Penicillium notatum or P. turbatum with an increased production of strobilurin A (1) or X (2). P. notatum in turn reacts to the two basidiomycetes or their antibiotic strobilurin A alone with the production of N-(2-hydroxypropanoyl)-2-aminobenzoic acid amide (3) or chrysogine (4). P. melinii and P. urticae overgrow O. mucida due to complete resistance to strobilurin A. P. brevicompactum, P. citrinum, P. janczewskii and the other Penicillium strains are all sensitive but apparently do not induce O. mucida to produce the amounts of strobilurin A needed to inhibit their growth.     

De novo biosynthesis of secondary metabolism enzymes in homogeneous cultures of Penicillium urticae.

The initiation of patulin biosynthesis in submerged batch cultures of Penicillium urticae NRRL 2159A was investigated at the enzyme level. In contrast to earlier studies, this study achieved a clear temporal separation of growing cells devoid of secondary metabolism-specific enzymes from nongrowing cells, which rapidly produce these enzymes. A spore inoculum, silicone-treated flasks, and two new media which supported a rapid, pellet-free, filamentous type of growth were used. In yeast extract-glucose-buffer medium, a marked drop in the specific growth rate (approximately equal to 0.26 h-1) coincided with the appearance of the first pathway-specific enzyme, 6-methylsalicylic acid synthetase, at about 19 h after inoculation. About 3 h later, when replicatory growth had ceased entirely, the sparsely branched mycelia (length, approximately equal to 550 microns) began the rapid synthesis of a later pathway enzyme, m-hydroxybenzyl alcohol dehydrogenase. A similar sequence of events occurred in a defined nitrate-glucose-buffer medium; 12 other strains or isolates of P. urticae, as well as some patulin-producing aspergilli, behaved in a similar manner. The age at which a culture produced m-hydroxybenzyl alcohol dehydrogenase was increased by increasing the nutrient nitrogen content of the medium or by decreasing the size of the spore inoculum. In each instance the appearance of enzyme was determined by the nutritional status of the culture and not by its age. A similar appearance of patulin pathway enzymes occurred when a growing culture was resuspended in a nitrogen-free 4% glucose solution with or without 0.1 M phosphate (pH 6.5). The appearance of both the synthetase and the dehydrogenase was arrested by the addition of cycloheximide (0.4 to 5 micrograms/ml) or actinomycin D (20 to 80 micrograms/ml). This requirement for de novo protein and ribonucleic acid syntheses was confirmed by the incorporation of labeled leucine into the dehydrogenase, and the possibility that latent or preformed proteins were being activated was eliminated.     

Dissection of patulin biosynthesis,spatial control and regulation mechanism in Penicillium expansum

The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.     

Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum.

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.     

Patulin biosynthesis: enzymatic and nonenzymatic transformations of the mycotoxin (E)-ascladiol.

A mycotoxin, (E)-ascladiol, was established as a direct precursor of patulin in cell-free preparations of Penicillium urticae patulin-minus mutants J1 and S11, but not S15. Isomerization to a side product, (Z)-ascladiol, was nonenzymatically catalyzed by sulfhydryl compounds.     

Effect of nitrogen supplementation on the longevity of antibiotic production by immobilized cells of Penicillium urticae

Summary Conidia of Penicillium urticae were immobilized in Kappa-Carrageenan beads (2–3 mm) by a previously described procedure to yield an in situ grown immobilized cell population which could be induced to produce the antibiotic and mycotoxin, patulin. When repeatedly transferred into a nitrogen-free production medium every 2 days, the patulin productivity of these cells gradually decreased to 50% within 14 days while the total cell protein remained constant. This decline was due to the gradual loss of the cells' catalytic capacity for converting glucose to 6-methylsalicylic acid (6-MSA), the first metabolite of the patulin pathway, as well as for converting 6-MSA to patulin. When these 14 day-old cells were incubated in a nutrient rich growth medium for 2 days their patulin producing activity increased from 50% to 130%. On the other hand the addition of a protein synthesis inhibitor, cycloheximide, to the N-free production medium drastically reduced the patulin producing activity of the immobilized cells; in particular, their capacity for converting 6-MSA to patulin. The cells' patulin producing activity was maintained at >100% for longer than 15 days when the cells were repeatedly transferred into a yeast extract supplemented production medium or when they were occasionally transferred into 10 or 20% strength growth medium. Repeated transfers to a 10% strength growth medium appeared to stabilize the cells' capacity for converting 6-MSA to patulin.     

Patulin is a cultivar‐dependent aggressiveness factor favouring the colonization of apples by Penicillium expansum

The blue mould decay of apples is caused by Penicillium expansum and is associated with contamination by patulin, a worldwide regulated mycotoxin. Recently, a cluster of 15 genes (patA–patO) involved in patulin biosynthesis was identified in P. expansum. blast analysis revealed that patL encodes a Cys₆ zinc finger regulatory factor. The deletion of patL caused a drastic decrease in the expression of all pat genes, leading to an absence of patulin production. Pathogenicity studies performed on 13 apple varieties indicated that the PeΔpatL strain could still infect apples, but the intensity of symptoms was weaker compared with the wild‐type strain. A lower growth rate was observed in the PeΔpatL strain when this strain was grown on nine of the 13 apple varieties tested. In the complemented PeΔpatL:patL strain, the ability to grow normally in apple and the production of patulin were restored. Our results clearly demonstrate that patulin is not indispensable in the initiation of the disease, but acts as a cultivar‐dependent aggressiveness factor for P. expansum. This conclusion was strengthened by the fact that the addition of patulin to apple infected by the PeΔpatL mutant restored the normal fungal colonization in apple.     

A practical approach for identifications based on mycotoxin characters of Penicillium

The taxonomy of the penicillia is unstable particularly in the important antibiotic and mycotoxin-producing subgenus Penicillium. There are difficulties relating identifications to mycotoxin production. Also, the validity of dual nomenclature for pleomorphic fungi is under discussion increasingly. Patulin is an important mycotoxin produced by various fungi and has strict limits in the European Union. The mycotoxin and/or the isoepoxydon dehydrogenase (IDH) gene of the metabolic pathway have been assessed in 318 strains predominately of subgenus Penicillium. These data were used to classify the isolates. Subgenus Penicillium contained most of the IDH and patulin positives. The species and varieties in subgenus Penicillium which were associated with patulin detection can be reduced to one name, viz. Penicillium Pen p+ (p = patulin). This has been extended to other mycotoxin producing penicillia to indicate the scope of the scheme. The classification will lead to the number of taxa being reduced, while avoiding species names and hence dual nomenclature. Culture independent analysis of environmental samples is mentioned. The scheme could be used with advantage for other fungi.     

Patulin Production in Apples Decayed by Penicillium expansum

Sixty isolates of Penicillium expansum were tested for patulin production in decaying apples. All the isolates were found to produce the mycotoxin patulin as determined by thin-layer chromatography. Since patulin is known to be stable in many apple products, the results indicate that apple products made partially from apples decayed by P. expansum will contain patulin which may present a health hazard. The results also suggest that patulin may be important in the decay of apples by P. expansum.