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Byssochlamys species are responsible for spoilage and degradation of fruits and silages. Under specific conditions they are able to produce mycotoxins. The aim of this study was to evaluate the potential of 19 different strains of Byssochlamys nivea and Byssochlamys fulva to produce patulin in relation with the presence of two genes involved in the patulin biosynthesis pathways in the genome of these fungal strains. The strains were characterized by macroscopic, microscopic examinations, internal transcribed spacer (ITS) rRNA and beta-tubulin fragment amplification and sequencing. All of the 8 B. nivea strains tested produced patulin. By contrast, none of the 11 strains of B. fulva produce this toxin. Two genes of the patulin biosynthetic pathway, a polyketide synthase (pks) and the isoepoxydon dehydrogenase (idh) were cloned from B. nivea. The deduced amino acid sequence of the polyketide synthase was 74% identical to the 6-methylsalicylic acid synthase gene of Penicillium griseofulvum and had the five functional domains characteristic of fungal type I polyketide synthases (beta-ketosynthase, acyltransferase, dehydratase, beta-ketoreductase and acyl carrier protein). The complete coding sequence of idh gene displayed after translation 88% of identity with P. griseofulvum IDH and 85% with P. expansum IDH, respectively. Both pks and idh messengers were strongly co-expressed during the production of 6-methylsalicylic acid and patulin. The presence of these genes was then investigated in the genome of B. nivea and B. fulva strains by PCR. All B. nivea strains possess the two genes, by contrast none of the B. fulva strains display these genes. The absence of 6-methylsalicylic acid and isoepoxydon dehydrogenase genes can explain the inability of B. fulva to produce patulin. In conclusion, B. fulva don't seem to be responsible for the occurrence of patulin by lack of genes.

Understanding the fungi producing important mycotoxins.- Important mycotoxins and the fungi which produce them.- Recommendations concerning the chronic problem of misidentification of mycotoxigenic fungi associated with foods and feeds.- Media and method development in food mycology.- Comparison of hyphal length, ergosterol, mycelium dry weight, and colony diameter for quantifying growth of fungi from foods.- Evaluation of molecular methods for the analysis of yeasts in foods and beverages.- Standardization of methods for detecting heat resistant fungi.- Physiology and ecology of mycotoxigenic fungi.- Ecophysiology of fumonisin producers in Fusarium section Liseola.- Ecophysiology of Fusarium culmorum and mycotoxin production.- Food-borne fungi in fruit and cereals and their production of mycotoxins.- Black Aspergillus species in Australian vineyards: from soil to ochratoxin A in wine.- Ochratoxin A producing fungi from Spanish vineyards.- Fungi producing ochratoxin in dried fruits.- An update on ochratoxigenic fungi and ochratoxin A in coffee.- Mycobiota, mycotoxigenic fungi, and citrinin production in black olives.- Byssochlamys: significance of heat resistance and mycotoxin production.- Effect of water activity and temperature on production of aflatoxin and cyclopiazonic acid by Aspergillus flavus in peanuts.- Control of fungi and mycotoxins in foods.- Inactivation of fruit spoilage yeasts and moulds using high pressure processing.- Activation of ascospores by novel food preservation techniques.- Mixtures of natural and synthetic antifungal agents.- Probabilistic modelling of Aspergillus growth.- Antifungal activity of sourdough bread cultures.- Prevention of ochratoxin A in cereals in Europe.- Recommended methods for food mycology.

Most fungal survival structures can be regarded as heat resistant to some extent: sclerotia, conidia and ascospores can survive temperatures between 55 and 95°C. Byssochlamys, Neosartorya and Talaromyces are the most well known heat-resistant fungal genera. Ascospores of these fungi are the most resilient eukaryotic structures currently known. A decimal reduction time of 1.5-11 min at 90°C has been reported for some species (Scholte et al., 2004). Recently, Panagou et al. (2002) reported moderate heat resistance (D75 4.9-7.8 min) in ascospores of Monascus ruber isolated from brine of a commercial thermally processed can of green olives. There appeared to be a complex interaction between pH and salt content of the heating menstruum and decimal reduction time for this fungus. During recent work undertaken in our laboratory, moderate heat resistance has been observed for Talaromyces stipitatus and T. helicus (J. Dijksterhuis, unpublished results), and studies involving ascospores of Byssochlamys spectabilis have resulted in a calculated decimal reduction time at 85°C of 47-75 min (J. Houbraken, unpublished results). Table 1 shows a compilation of heat resistance data for many known heat resistant species and their D values in various heating menstrua (modified from Scholte et al., 2004).

The European Union (EU) has introduced statutory limits for patulin in fruit products. Species definitions need to be unambiguous for this capability, especially to determine ‘weak spots’ in food, drink and feed production. Fungi were analysed for the isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway to indicate potential patulin production. In several cases inhibition of PCR product formation was indicated. Dilution as a means of overcoming PCR inhibition and to determine optimal concentrations was assessed and the ramifications for nucleic acid analysis in general are highlighted. The inhibitors may be secondary metabolites. However, inhibition was not involved obviously for Penicillium brevicompactum. The gene was detected frequently from Aspergillus section Clavati and Penicillium subgenus Penicillium species. Some strains within certain species were negative for the gene. Detection of the gene product in Byssochlamys is reported., UK Food Standards Agency - contract no. CO30005.

Ascopyrone P (APP), a novel antibacterial from fungi, was evaluated as a food preservative. Efficacy was generally assessed by comparing the time taken for test strains to grow to 10 6 CFU/g in food ±APP. In chilled chicken soup, 2000 mg kg −1 APP prevented Bacillus cereus, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella and Escherichia coli reaching this threshold for >60 days. Good activity was also observed at 500–1000 mg kg −1 but not against L. monocytogenes . No activity was observed against Saccharomyces cerevisiae . Activity was reduced at 20 °C, although 2000 mg kg −1 was still effective against B. cereus and P. fluorescens . APP was less effective in chilled cooked meat systems and ineffective in raw meat. In a cooked meat system at 8 °C, bacteriostatic effect was generally observed at 2000 mg kg −1 against Salmonella typhimurium , E. coli and P. fluorescens but not against L. monocytogenes or Lactobacillus sake . Activity against Gram-negative enteric bacteria was enhanced by low temperature. In milk, 2000 mg l −1 was effective against P. fluorescens at chilled but not ambient temperature. APP was ineffective against yeasts and the mould Byssochlamys in apple juice. A minimum of 2000 mg kg −1 APP would appear to be necessary for antibacterial efficacy in food, although low-temperature storage may help. Observed variations in sensitivity may be related to APP stability, which decreases >pH 5.5. Toxicology testing is needed before consideration of APP for food use.

The mycotoxin patulin causes gastroinstestinal distress, neurotoxic and immunotoxic effects in animals. It can be produced by several species of Penicillium, Aspergillus and Byssochlamys and it has been found in fruits, vegetables and cereals. Verruculogen is a toxin produced mainly by Penicillium and Aspergillus spp. and causes severe tremors in affected animals. Tomatoes are especially susceptible to fungi invasion and their products need to be investigated for possible mycotoxin contamination. A method for the determination of patulin and verruculogen in tomato products was developed involving an extraction with ethyl acetate, a cleanup by silica gel column and determination and confirmation by high performance liquid chromatography with diode array detector. The quantification limits of the method, defined as the minimum amount that allowed quantification and confirmation by the DAD detector, were 10 ng/g and 20 ng/g. The average recovery for patulin at five levels of addition (from 20 to 200 ng/g) was 75% and at the single level of 100 ng/g was 90 % .The average recovery for verruculogen at five levels of addition (from 50 to 300 ng/g) was 54% and at the single level of 100 ng/g was 52%. The processing of two tomato plants was followed during 1996, 1997, and 1998. Eighty-four samples of tomato pulp were analyzed for patulin and verruculogen. The toxins were not detected in any of the samples. A micotoxina patulina causa distúrbios gastrointestinais e efeitos neurotóxicos e imunotóxicos em animais. Pode ser produzida por várias espécies de Penicillium, Aspergillus e Byssochlamys e tem sido encontrada em frutas, verduras e cereais. Verruculogeno é uma toxina produzida principalmente por espécies de Penicillium e Aspergillus e causa fortes tremores em animais afetados. Tomates são especialmente susceptíveis a invasão fúngica e seus produtos precisam ser investigados com relação a possíveis contaminações por micotoxinas. Um método para determinação de patulina e verruculogeno em produtos de tomate foi desenvolvido envolvendo uma extração com acetato de etila, uma limpeza em coluna de sílica gel e determinação e confirmação por cromatografia de alta eficiência com detector de arranjo de diodos. Os limites de quantificação foram de 10 ng/g e 20 ng/g e as recuperações medias foram de 89% e 65% para patulina e verruculogeno, respectivamente. O processamento de duas fábricas de produtos de tomate foi amostrado durante 1996, 1997 e 1998. Oitenta e quatro amostras de polpa de tomate foram analisadas para patulina e verruculogeno. As toxinas não foram detectadas em nenhuma das amostras.

An alkali-extractable and water-soluble fraction (F1S) was obtained from cell walls of Paecilomyces variotii and species of the related genera Talaromyces, Byssochlamys and Thermoascus. The structure of the main polysaccharide of these fractions was studied and found to consist of a core of (1→6)-α-mannopyranose partially substituted at 0-2 by chains of galactofuranose and shorter chains of mannopyranose. The differences in the regularity of the branching points and the length of the galactofuranose side chains are useful to distinguish between species. These differences were detected by immunological methods, since highly specific polyclonal antibodies were raised against these polysaccharides. Mycelium of P. variotii CBS 990.73A was stained by indirect immunofluorescence. The polysaccharides studied in this work differ from the one described for species from section Isarioidea, and this is another indication of the heterogeneity of the genus Paecilomyces., This work was supported by Grants PB 95}0078 and 93}1027 from Dirección General de Investigación Científica y Técnica.

The rapid increase in the global production of apples has led to improved monitoring and control of toxic substances present in apples and their derivatives. One of these toxic substances is patulin, a mycotoxin and secondary metabolite produced by different fungi species of the genera Penicillium, Aspergillus, Gymnoascus, Paecilomyces and Byssochlamys. This study evaluated patulin levels in juice produced from Fuji apples subjected to different temperature conditions and storage times in southern Brazil. The apples were divided into groups weighing 1 kg and stored at the following temperatures: 0ºC, 5ºC, 6.7ºC, 19.5ºC and 25ºC. Sub-samples of each group were processed to determine their patulin level by HPLC at the following intervals: 0 (control group), 7, 14 and 21 days. After 28 days of storage at all temperatures, the apples could not be analyzed due to their deterioration. The results indicated that at all temperatures; there was variation in the patulin levels. The smallest patulin value was observed in apple juice stored at 19.5°C and 25°C.

A strain of Byssochlamys sp. produced β-hydroxypropionic acid when grown on media containing high concentrations of acrylic acid. The maximal production of β-hydroxypropionic acid was 4.8% when the initial culture medium contained 7% acrylic acid and 2% glucose, and the initial culture pH was adjusted to 7.0. β-Hydroxypropionic acid production from acrylic acid depended greatly on the pH of the culture medium. Calcium hydroxide was the best neutralizer.

Patulin and cyclopiazonic acid are fungal metabolites produced by certain species of Penicillium and Aspergillus. Patulin is also produced by certain Byssochlamys species. Both compounds are known to contaminate numerous agricultural products at low levels and are commonly consumed by both animals and man. Although food and feed safety is a prime concern, we are also interested in these compounds because understanding their interaction with biological systems can reveal useful information that could be the basis for future technological advances in controlling agricultural productivity. For the past several years we have studied these two compounds with regards to their effects on membrane function and in particular to their abilities to alter ion transport.6,7,9,30,31,32,33 Basic information about the mode of action of mycotoxins at the level of cell membranes could lead to a better understanding of the role these chemicals may play in 1) the growth and development of the fungus and competing species, 2) the fungal-plant relationship, and 3) the fungal-plant-animal relationship. Additionally, these studies can identify new applications for fungal products as biochemical probes of membrane function and potential therapeutic applications in the areas of plant and animal health and productivity. The U.S. Department of Agriculture/Agricultural Research Service is well aware of the potential of membrane research in developing new agricultural frontiers11 and thus we have chosen as a point of departure the study of the mechanisms by which fungal products alter membrane activity.

Following the discovery of penicillin by Fleming in 1929 a flurry of work ensued in which similar approaches were applied to the search for other antibiotics. One of the products of this search was the compound patulin, initially isolated from filtrates of a strain of Penicillium claviforme by Chain et al. (1942). The compound, originally referred to as clariformin and possessing potent antibacterial activity, was the subject of numerous chemical and biological studies soon after its isolation from a strain of Penicillium patulum Painier and characterization by Birkinshaw et al. (1943). Elucidation of the structure was made by Woodward and Singh (1949). It has since been shown to be produced by several species of Aspergillus and Penicillium and at least one species of Byssochlamys, as reviewed by Ciegler (1977) and Stott and Bullerman (1975).

Ascospores of three strains each of Talaromyces flavus, Neosartorya fischeri and Byssochlamys fulva/nivea were analyzed for resistance to thermal inactivation in five fruit-based (blueberry, cherry, peach, raspberry and strawberry) products. D91°C values for two strains of T. flavus ranged from 2.9–5.4 min; D88°C values ranged from 7.1–22.3 min. Ascospores of N. fischeri were somewhat less heat resistant; D91°C values were < 2.0 min and D88°C were 4.2–16.2 min. Ascospores of Byssochlamys spp. were considerably less heat resistant. The type of fruit product did not appear to substantially influence rates of thermal inactivation. No heat-resistant ascospores of T. flavus or N. fischeri, i.e., ascospores capable of surviving 15 min at 75°C, were formed on fruit products stored at 10°C for 137 days. However, T. flavus and N. fischeri formed ascospores on cherry substrate stored at 25°C within 65 and 137 days, respectively, that survived 15 min at 88°C.

Ten strains of Byssochlamys fulva and three strains of B. nivea were cultured in a laboratory medium and tested for their ability to produce patulin. Two strains of B. fulva and all three strains of B. nivea produced the mycotoxin. One strain of B. fulva produced patulin in 11 of 13 processed fruit juices, with greatest amounts being produced in blueberry, red raspberry, and boysenberry juices, whereas no patulin was detected in prune or tomato juices. Grown in Concord grape juice at 18, 25, 30, and 38 degrees C, this strain produced the highest patulin concentration at 18 degrees C after 25 days, whereas biomass production was greatest at 25 and 30 degrees C after 20 and 25 days.

A study was conducted to determine whether the difference in heat resistance of the ascospores produced by two strains of the mold Byssochlamys fulva was related to their chemical composition. Cleaned spore preparations were analyzed for proteins, amino acids, lipids and minerals. The most significant difference was a greater quantity of fatty acids longer than C20, in the more heat resistant spores. Some of the longer chained fatty acids recovered from both strains had not previously been detected in fungal spores and it was concluded that they may be a factor in explaining the high heat resistance of Byssochlamys ascospores.

A strain of Byssochlamys nivea, isolated from altered heat processed strawberries, produces an antibiotic substance which is efficient against several species of gram (-) bacteria and a few species of gram (+).

Studies have been conducted to determine growth requirements for fungi belonging to the genus Byssochlamys. Incubation temperature significantly affected growth, with visual detection in 2 days at 30° C and 4 days at 20° C. Sugar concentration was important in liquid culture techniques, with no growth observed in 30° Brix apple drink base after 2,640 hr vs growth in 15° Brix apple drink base after 48 hr. Thermal death studies of three Byssochlamys isolates (G-5, G-52, and M-78) demonstrated wide variation in heat resistance with z-values of 7°, 9.5°, and 14°F respectively. Excellent electron micrographs were obtained of conidia, aleuriospores, and ascospores using a JEOL JSM 35 scanning electron microscope. The micrographs also revealed asci with thicker walls in older (90 days vs 30 days) M-78 cultures. A modification of the Shaeffer-Fulton Spore Stain Technique was used to stain the ascospores within an intact ascus.

While it has previously been suspected that Byssochlamys spp. may grow in canned fruits and produce the mycotoxin patulin, direct evidence for this occurrence is not available: The effects of different initial static oxygen levels, and temperatures on the formation of patulin by B. nivea in Concord grape juice packed in glass jars was examined. The greatest amount of patulin(0.31 mg/SO ml) was formed after 28 days in jars with a 5.1 cm headspace incubated at 20°C. Patulin was formed more rapidly at 25° and 30°C, but declined in a mounts after reaching maximum values. Cultures of B. fulva and B. nivea produced less patulin in grape juice packed in glass jars with headspaces of 2.5 and 1.3 cm than in glass jars with a 5.1 cm headspace. More favorable conditions were used in this study to produce patulin than would be found in the commercial production of Concord grape juice.

SUMMARY The type culture ATCC 26215 of Aphanoascus cinnabarinus, type species of the genus, is found for the first time to produce enteroblasticphialidic conidia, and Paecilomyces cinnabarinus is described to accommodate this conidial state. Light is essential for production of the Paecilomyces state but not for the Aphanoascus state. The fungus is proven to be homothallic and nonkeratinophilic. The separation between Aphanoascus and its closely related genera Byssochlamys, Talaromyces, Dactylomyces, Eupenicillium, and Hemicarpenteles is discussed. The genus Aphanoascus was erected by Zukal (1890) to accommodate a single species, A. cinnabarinus Zukal, which was isolated from alligator dung in Vienna in 1889. The fungus has recently been rediscovered by Udagawa and Takada (1973). Their isolates were forwarded to the American Type Culture Collection (ATCC) for conservation as ATCC 26215, ATCC 26216, and ATCC 26696. Apparently these collections are the only extant representatives of the species. Since none of Zukal's specimens exist, the ATCC 26215 was designated as the neotype. Udagawa and Takada placed Aphanoascus in the family Eurotiaceae of which, according to Malloch and Cain (1971, 1972), the conidial state is of the enteroblastic-phialidic type. However, no conidial state of A. cinnabarinus was reported. It is therefore of particular interest to know if Udagawa and Takada's isolates show a conidial state developmentally akin to the type now expected for the members of Eurotiaceae. The cultures of ATCC 26215, ATCC 26216, and ATCC 26696 were run through a series of tests on different media and substrates, including horse hair, under various environmental conditions. The results showed that all the cultures tested produced enteroblastic-phialidic conidia belonging to Paecilomyces Bainier on rabbit food agar under controlled temperature and light conditions and were not capable of colonizing and

Methods for the detection of low numbers of heat-resistant molds on fruits were studied by using cultures of Byssochlamys and a number of unidentified mold isolates. Ascospore dormancy had a marked effect on viable recoveries, and the medium in which ascospores were heated influenced activation rates. Best results were obtained when fruit homogenates were heated for 60 min at 70 C in Concord grape juice, followed by culturing on acidified Potato Dextrose Agar.

Summary: Heat resistant moulds of the genera Byssochlamys, Aspergillus, Penicillium and Paecilomyces can be cultivated selectively in the presence of Bacillus spp. when at least 10 p/m of chloramphenicol or chlortetracycline is added to the growth medium. The inhibitory agents sulphafurazole, penicillin, streptomycin, oxytetracycline and furazolidone, do not give completely selective and optimal growth of these moulds. Selective cultivation of heat resistant moulds from 200 samples of Dutch soil showed that Byssochlamys nivea occurs more frequently than was expected, whereas Byss. fulva occurs by way of exception. Other heat resistant moulds observed in soil are, in order of frequency, species of the genera Aspergillus, Paecilomyces and Penicillium.

Summary 1A survey of various fruit-producing areas in England showed that fruit and foliage were liable to be contaminated with ascospores of Byssochlamys fulva. Contamination was most pronounced on mummified plums and on certain fruit refuse. 2The thermal death point of ascospores is about 96°C., but only a small proportion of spores show this maximum heat resistance. Conidia are killed by exposure to relatively low temperatures. 3The reaction and the sucrose concentration of the heating medium, and the strain of the fungus, are factors influencing the heat resistance of ascospores. 4Ascospores germinate slowly and irregularly, but their germination is stimulated by previous heating to about 70°C. Ascospore germination and mycelial growth take place at relatively high temperatures, in high sucrose concentrations, in acid media, and in very high concentrations of carbon dioxide. 5Ascospores are relatively insensitive to antiseptics and to desiccation. 6Growth is accompanied by the production of a pectin-destroying enzyme. 7The results of laboratory experiments were applied to canning experiments and good agreement was obtained. Natural contamination on fruits could be overcome in cans by heating the contents to 195°F. 8The amount of mycelial growth developing in an infected can depends upon the size of head space and the storage temperature.

A mycological survey of fungi, present in several stages of the manufacturing of cork discs for champagne stoppers in Portugal, was made. Sixty-nine strains belonging to the Glabra series of the genus Penicillium were isolated and subsequently grouped according to their partial β-tubulin gene sequences. Six groups with different partial β-tubulin gene sequences were observed, and a selection of isolates of each group was made. These selected isolates and various related ex-type strains were subjected to a taxonomical study using a polyphasic approach. This approach included analysis of macro- and microscopic features, the comparison of extrolite profiles and sequencing a part of the β-tubulin and calmodulin gene. The six β-tubulin types were reduced to three different species. One group of isolates was centred on the ex-type strain of P. glabrum, a second group accommodated the type strain of P. spinulosum and a third group contained isolates which were unique in their β-tubulin and calmodulin sequences, extrolite profiles and growth characteristics. This group of isolates is described as the new species Penicillium subericola. The type strain of P. subericola CBS 125096T was isolated from Portuguese raw cork, but additional isolates were found from soil, air and lumen.

Mechanically-harvested tart cherries and Concord grapes were not more heavily contaminated with Byssochlamys ascospores than were hand-picked fruit. The data suggested, however, that both harvest procedures may be sources of contamination, probably because of soiled surfaces. Studies on various Byssochlamys isolates revealed marked differences in heat resistance.

When Byssochlamys is grown for production of ascospores, some of the asci break up into their constituent ascospores, whereas others do not. For heat resistance studies, it is desirable to prepare a uniform suspension of free ascospores. This was accomplished with the aid of a pressure cell, from which a suspension of asci under high pressure was released to atmospheric pressure through a small orifice. Spores so treated had about the same heat resistance as untreated spores.

Publisher Summary This chapter discusses that spoilage of canned and bottled fruits by Byssochlamys spp., first recognized in Great Britain in the 1930s and has since been documented in several countries around the world. The first documented fruit spoilage outbreaks attributed to Byssochlamys occurred in England and it was thought at that time the organism was confined to that country. The mold isolated from spoiled processed fruits in many instances is Paecilomyces. It has been long recognized that a large percentage of any given crop of ascospores of Byssochlamys possesses a dormancy that can be broken by heat treatment. Like bacterial spores, ungerminated ascospores are highly refractile when viewed under the phase microscope, but lose this characteristic on germination before the germ tube(s) emerges. Byssochlamys is a problem in fruit spoilage mainly because of the exceptionally high heat resistance exhibited by its ascospores. In the course of studying the chief metabolic products of B. fulva cultured on a synthetic medium containing glucose as a sole source of carbon, Raistrick and Smith noted that in preliminary tests an ether-soluble precipitate formed upon the addition of hydrochloric acid to 4-week-old Czapek-Dox culturing fluid. Foods, feeds, and grains that become moist during storage may subsequently overheat and provide thermophilic fungi with a competitive growth advantage over other microorganisms. The chapter discusses that it does not seem feasible to eliminate Byssochlamys spp. from fruit refuse and soils which contaminate fruits as they arrive from the field.

Patulin, a mycotoxin produced by some Aspergillus, Penicillium, and Byssochlamys species, is found in rotten fruits and fruit products. By its structure, an unsaturated α,β-lactone, it reacts with sulfhydryl groups. Patulin inhibits the growth rate of the ciliate Tetrahymena at a concentration of 3.2 μg/ml by 50%. Using the same patulin concentration, the protein synthesis of this organism is inhibited by 85% and the RNA synthesis by 86% compared with the control. Four hours after addition of the mycotoxin (3.2 μg/ml), DNA synthesis is reduced to 20%; it rises to the value of the control after an additional 2 h. An in vitro system could be developed consisting of permeabilized cells of Tetrahymena. This system allows the separation of regulatory and secondary effects induced by patulin. The mycotoxin reduces DNA synthesis by 50%, whereas RNA and protein synthesis are less inhibited than in the in vivo system. A further characterization of these results could be obtained by experiments with the large fragment of DNA polymerase I. Patulin inhibits the enzyme at rather high concentrations.

Patulin is a mycotoxin produced by various Penicillium, Aspergillus and Byssochlamys species. To evaluate its inhibitory effect on cells, hepatoma tissue culture cells in suspension were incubated in presence of 30 μM of patulin for 7 h and investigated by transmission and scanning electron microscopy. By transmission electron microscopy, the most significant difference observed between treated and control cells was the disorganization of the cytoplasmic microfilaments in the treated cells. The disappearance of superficial membrane microvilli which contain microfibrillar material was visualized by scanning electron microscopy; the cells also presented protrusions. The effect of this toxin on the cytoskeleton can be compared to that exerted by colchicine or by cytochalasines.

Ascospores from 25 strains of Byssochlamys were studied for their ability to resist heat treatment in a standard defined medium. Seven of these were able to survive heating at 90 degrees C for 25 min or longer, when initial numbers were frequently near 10(6)/ml. Ascospores from five resistant strains suspended in the medium at pH 5.0 were usually more resistant than those at pH 3.6. Rapid heat inactivation occurred for one strain at pH 6.6. Nonlogarithmic heat death rate was observed in all strains tested.

“Baseline” counts are defined as the populations of molds and yeasts that normally occur in wholesome food or feed ingredients. Enumeration of fungal propagules is justified for the following reasons: 1. They provide an indication of compliance with Good Manufacturing Practice (GMP) conditions during the growing, processing or storage of the food 2. High numbers of particular groups or species may indicate that mycotoxins are present 3. Even low numbers of propagules of some species may be highly significant and undesirable in some instances (e.g., Zygosaccharomyces bailii in soft drinks or Byssochlamys species in fruit juice).

Heat-resistant strains of Byssochlamys fulva, B. nivea , and other heat-resistant fungi were isolated from vineyard soil, grapes, grape-processing lines, and waste pomace. They are known to remain in grape juice occasionally and to grow in grape juice products. Ascospores of these fungi have a D value (decimal reduction time) of about 10 min at 190 F (88 C), but in the presence of 90 μliters of SO 2 per liter (normally added to the juice) the D value was cut in half. Filtration through a commercial diatomaceous filter aid (also a common processing step) entrapped all but about 0.001% of experimentally added spores. Thus, heat in the presence of SO 2 and filtration together can reduce the population of these spores by several orders of magnitude. Growth was also prevented by benzoate or sorbate in low concentrations. Oxygen must be reduced to extremely low levels before lack of oxygen limits growth.

Patulin (4-hydroxy-4H-furo [3,2c] pyran-2 (6H)-one), a mycotoxin produced by several species in the genera Aspergillus, Penicillium, and Byssochlamys, was produced in gram/liter quantities in poultry feed by P. patulum. The patulin LD50 dosage for White Leghorn cockerels was 170 mg./kg. Sublethal feeding produced hemorrhaging, limited to the digestive tract.

Le projet MYCO-TOX doit permettre, pour la première fois, de comparer dans une même étude la génotoxicité et la cytotoxicité de 160 mycotoxines sur quatre lignées cellulaires humaines issues des tissus cibles des mycotoxines (foie, colon, rein et cerveau).

Cultures of heat resistant molds (20) were isolated from spoiled canned tomato paste in order to estimate the pasteurization efficiency applied to commercially canned products. Ascospores of nine strains grown on malt extract agar for 30 days at 30°C, survived heating at 85°C for 20 min when initial numbers were near 10 5 /mL. Of these heat resistant strains were identified: two Byssochlamys nivea, three Byssochlamys fulva and four neosartorya fischeri strains. Ascospores of all cultures were more heat resistant in tomato juice than in phosphate buffer. Thermal death rate curves were nonlogarithmic but approached logarithmic death rates at higher temperatures. The thermal destruction time for I log 10 at 90°C was 1.5 min for a Byssochlamys nivea strain, 8.1 min for a Byssochlamys fulva strain and 4.4 to 6.6 min for Neosartorya fischeri strains.

Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and BYSSOCHLAMYS: Patulin is a common contaminant of ripe apples used for the production of apple juice concentrates and is also present in other fruits, vegetables and food products. Patulin has been reported to have mutagenic, carcinogenic and teratogenic properties. Nevertheless, these properties are still a matter of debate. In this report, we further investigated the genotoxicity of patulin in mammalian cells by two different approaches. Firstly, we evaluated the induction of micronuclei in cytokinesis-blocked human lymphocytes. This approach is important because available data concerning the genetic toxicity of patulin in human cells is sparse. Secondly, we chose an established model for patulin genotoxicity, i.e. the chromosomal aberration assay in V79 Chinese hamster cells, to clarify whether concomitant exposure to ascorbic acid with the mycotoxin modulates or not the clastogenicity of patulin. The results unequivocally show induction of DNA-damaged cells by patulin as assessed by both cytogenetic assays. In addition, an almost complete abolition of patulin (0.8 microM) clastogenicity was observed in the presence of 80 microM ascorbic acid (P < 0.05), showing that although a genetic risk is present, ascorbic acid could somehow partially modulate this problem., We are grateful to Dr L.Gonçalves for invaluable assistance with the statistical analysis. This study was supported in part by the FCT (Project PRAXIS/PSAU/C 67-96) and the Luso American Foundation for Development (FLAD).

WHEN seedlings of Phaseolus vulgaris var. Black Valentine were treated on one of the simple leaves with culture filtrates of P. thomii, P. oxalicum, or B. nivea (Arachniotus trisporus), subsequent growth of the plants was inhibited more or less selectively on the treated side1. As a result, the first, second, and often the third trifoliolate leaves were asymmetrical. In severe cases, both sides of the trifoliolate leaves were inhibited. ‘Asymmetrin’ has been proposed as a name for the growth inhibitor produced by these fungi2. Preliminary investigations showed that culture filtrates of the three fungi produced a red-purple colour when treated with ferric chloride. Because of their similar effects on bean plants and coloration with ferric chloride, the relationship of the inhibitors to each other and to the ferric chloride reactive compounds was investigated.

Beavan and Brown1 investigated the pectic enzymes of the fungus Byssochlamys fulva and were unable to detect pectin-esterase or polygalacturonase, although protopectinase was shown to be present by its disintegrating action on disks of potato tuber. They concluded that B. fulva elaborated a disaggregating enzyme which reduced the molecular size of pectin, without the production of galacturonic acid, on the lines suggested by Kertesz2.

Crozier formation in Byssochlamys nivea was studied by means of scanning electron microscopy (SEM) and light microscopic techniques. The continuous recording of crozier formation by time lapse cinematography combined with karyological analyses revealed an important deviation from the classical scheme of crozier formation. In B. nivea the divisions of the two nuclei during crozier formation are not simultaneous ("conjugate"). First, the nucleus in the crook region divides followed by septum formation, and then the nucleus in the basal region undergoes nuclear division followed by septum formation. The three-dimensional arrangement of crozier chains was analyzed by SEM. Subsequent croziers are turned against each other at an angle of 120?. There are two modes of crozier chain branching: formation of one crozier, each, from the penultimate cell and from the fused terminal cell, and simultaneous formation of the two croziers from the penultimate cell. Crozier formation is a prominent feature in the dikaryotic stage of the majority of higher Ascomycetes. The classical scheme is based on Claussen's observations of the development of Pyronema confluens Pers. ex Tul. and other Discomycetes (Claussen, 1912). Later work on other fungi, e.g., Saccobolus kerverni (Crouan) Boud. (Carroll, 1968), Pyronema domesticum (Sow. ex Gray) Sacc. (Hung and Wells, 1971) and Neurospora crassa Shear & Dodge (Raju, 1980) seemed to confirm the general validity of the pattern of crozier formation derived from studies of Pyronema confluens. In all cases, however, data were obtained from sections of fixed material since the croziers usually are hidden within the developing fruiting bodies. As a consequence, the time course and details of crozier formation had to be reconstructed from different croziers in various developmental stages. In all such investigations there was incomplete information as a result. In our paper, crozier formation is analyzed in a more primitive organism, Byssochlamys nivea Westling, than P. confluens and others. In this ascomycete, the croziers and, later on, the asci, are surrounded by very scanty, loose welts of hyphae. To suppress even this presumably primitive form of a peridium, a suitable nutrient medium which allowed normal ascus formation without any cover of hyphae was developed. Crozier formation could then be studied by timelapse cinematography of living material combined with other techniques to reveal the karyology. As a result we have traced a complete course of crozier chain formation obtained from individual crozier initials. Important deviations from the classical scheme of crozier development were found.

IN tests conducted in these laboratories, p-amino-benzoic acid and the n-butyl p-aminobenzoate have shown definite reduction in rate of growth of three representative moulds (species of Aspergillus, Penicillium and Byssochlamys) growing on Czapek-Dox agar medium, pH 4. Inhibition in the case of the free acid was very marked (at least 50 per cent) at a concentration of 5·6 millimolar (mm.) and a reduction could still be detected, in the case of Aspergillus, as low as 0·18 mm. The butyl ester proved more effective, greater than 70 per cent reduction being observed at 1·0 mm. and a marked effect persisting as low as 0·14 mm.—the lowest concentration tested. In cases of the intermediate and lower concentrations of the free acid, the colonies remained regular in outline and maintained a steady though reduced rate of increase; concentrations 9·8 and 5·6 mm. gave rise to very irregular and fluctuating growth. With the higher concentrations of both substances, particularly of the p-aminobenzoic acid, an orange-yellow pigmentation was observed both in the mycelial felt and in the surrounding medium1, 2.

Two earlier records exist of heat-resistant moulds causing spoilage in canned and bottled fruits: Byssochlamys fulva1,2, which produces ascospores capable of withstanding relatively high temperatures (94° C for 2 min), and a species of Penicillium the sclerotia of which are reported3 to survive exposure to 93.3° C for 9 min. We wish to recordl the isolation from canned strawberries of a fungus showing an even greater degree of heat resistance than these.