Production of patulin and citrinin by <Emphasis Type="Italic">Penicillium expansum</Emphasis> from British Columbia (Canada) apples

Twenty-four isolates of Penicillium expansum Link from British Columbia (Canada) apples were cultured in yeast-extract sucrose (YES) at 25°C for 28 days to investigate production of patulin and citrinin. These isolates proved to be potent producers of citrinin, patulin, or in most cases, both mycotoxins. In every isolate, citrinin, patulin, or both compounds were produced at levels as high as 565 μg/mL (mean 269 μg/mL) and 100 μg/mL (mean 31 μg/mL), respectively. Of the 24 isolates, 4 produced citrinin only, and 2 produced patulin only. Overall, 83% of the isolates formed patulin and 91% formed citrinin. YES broth proved to be an effective medium for patulin and citrinin production. Other workers have noted that production of these mycotoxins in culture often presages production in fruits, so these results might help Canadian fruit processors evaluate and minimize mycotoxin levels in their products.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

Mycelium of fungi isolated from mouldy foods inhibits Staphylococcus aureus including MRSA – A rationale for the re-introduction of mycotherapy?

Fungal mycelium capable of producing antibacterial agents was isolated from samples of apple, beetroot, lemon and orange; the mycelium of all isolates produced penicillin, while the apple and beetroot samples also produced the antibacterial mycotoxin patulin. The known penicillin-producing fungi were shown to produce penicillin, but not patulin. The mycelial discs of all of fruit and vegetable isolates, as well as the two known penicillin producing fungi, inhibited Staphylococcus aureus, and mycelium of all isolates inhibited MRSA, in contrast, only one of the two known penicillin-producers did so. The results are discussed in relation to the possibility of using the mycelium of Penicillium species in mycotherapy.     

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.     

Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation

Aspergillus flavus, A. niger, Penicillium expansum and Rhizopus stolonifer were the most frequently isolated fungi from healthy apple fruits. Alternaria alternata was the most common organism of rotten apple fruits, followed by A. niger, A. flavus, P. expansum and R. stolonifer. The prevalent type of decay, brown rot lesion, is caused by R. stolonifer followed by A. flavus, A. niger, A. alternata and P. expansum. Sodium hypochlorite had good curative properties against fruit rots. The main natural mycotoxins produced in rotten apple were patulin and aflatoxins. The optimum temperature for patulin production by P. expansum was 15 °C after 15 days. Complete inhibition of patulin formation was attained using 0.2% lemon oil and > 90% inhibition using 0.05% lemon and 0.2% orange oils. Also significant inhibition (> 90%) of aflatoxin production was observed with 0.2% lemon oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Patulin accumulation in apples by Penicillium expansum during postharvest stages

AIMS: The aim of this study was to assess the opportunities of Penicillium expansum to develop and produce patulin in apples during cold storage and in the steps prior to processing of apple products. METHODS AND RESULTS: Two lots of apples var. Golden with different ripeness degree were used. Half of each lot was fungicide treated. Apples were inoculated with P. expansum and stored at 1 degrees C for 6 weeks. The extent of lesions and patulin accumulation both at the end of cold storage and after 3 days at 20 degrees C were assessed. Short storage at 20 degrees C aimed to simulate the transport and storage steps at room temperature before processing. Lesion size significantly increased during the storage at 20 degrees C. An interaction between fungicide treatment and ripeness degree was found; efficiency of fungicide treatment was higher for ripe apples. Although lesions were evident after cold storage, no patulin was detected. Patulin was detected only when fruits were further stored at 20 degrees C. Neither ripeness degree nor fungicide treatment affected patulin accumulation. CONCLUSIONS: Cold storage periods of 6 weeks do not lead to patulin accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: Shortening preprocessing times at warm temperatures would result into a reduction in patulin content at initial steps of fruits entering the processing plants.     

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose‐responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175‐ and five‐fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe‐21 from Israel and Pe‐T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch‐Pe‐T01 strains to 15%–25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is‐Pe‐21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.     

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.     

Mycotoxin production and evolutionary relationships among species of Aspergillus section Clavati

Aspergillus clavatus is a commonly encountered fungus in the environment, producing a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins. A. clavatus belongs to Aspergillus section Clavati together with six other species, all of which possess clavate-shaped vesicles. Patulin production was analysed by thin layer chromatography and high performance liquid chromatography, while a primer pair developed for the detection of an iso-epoxydon dehydrogenase gene involved in the biosynthesis of patulin in penicillia was used to detect the ability of patulin production in the isolates examined. A good correlation was observed between patulin producing properties, and the presence of an iso-epoxydon dehydrogenase gene fragment among the isolates tested. A. longivesica was found for the first time to produce patulin. Ribotoxin production was also examined using a PCR-based approach. Ribotoxins were detected for the first time in an A. pallidus and a Hemicarpenteles acanthosporus isolate. A phylogenetic analysis of intergenic transcribed spacer sequence data indicated that most isolates belong to two main clades that have also been identified earlier based on 26 S rDNA sequence data. A. pallidus isolates clustered together with A. clavatus strains. Although A. clavatus isolates produced highly homogeneous random amplified polymorphic DNA profiles, phylogenetic analysis of these data let us cluster A. clavatus isolates into distinct clades. Correlations were not observed between either patulin or ribotoxin production, and the taxonomic position of the isolates tested, indicating that patulin and ribotoxin producing abilities were lost several times during evolution of Aspergillus section Clavati. Although patulin was earlier found to inhibit mycovirus replication, one of the mycovirus carrying isolates also produced patulin, and both carried the iso-epoxydon dehydrogenase gene. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on Apple Canker Disease. The Necrotic Toxins Produced by Valsa ceratosperma

Several metabolites responsible for the toxic manifestations of Valsa ceratosperma (Toda et Fries) Maire, a phytopathogenic fungus of the Japanese apple canker, have been isolated from its culture filtrate after growth on apple branch extract. Chemical and spectrometric studies revealed the products to be degradation products of phlorizin which is a dominant component distributed in leaves, stems, fruits and roots of apple. The toxic substances were identified as 3-(p-hydroxyphenyl) propionic acid, phloroglucinol, p-hydroxyacetophenone, p-hydroxybenzoic acid and protocatechuic acid. All of these compounds except p-hydroxyacetophenone were detected in the lesions of apple trees infected by V. ceratosperma. The fungus cultivated in a medium containing added phlorizin also produced the five toxic substances mentioned above. These results suggest that phlorizin is involved in the specific relationship between the host and the pathogen, indicating that the degradation products of phlorizin play important roles in the production of symptoms of infected apple trees.     

Low occurrence of patulin‐ and citrinin‐producing species isolated from grapes

Aims: To assess the ability of fungi isolated from grapes to produce patulin and citrinin. Methods and Results: A total of 446 Aspergillus isolates belonging to 20 species and 101 Penicillium isolates were inoculated in Czapek yeast extract agar and yeast extract sucrose agar and incubated for 7 days at 25°C. Extracts were analysed for patulin and citrinin by thin‐layer chromatography. None of the isolates of Aspergillus spp. produced either patulin or citrinin. Patulin was produced by three isolates of Penicillium expansum and two of Penicillium griseofulvum. Citrinin was produced by five isolates of P. expansum, two of Penicillium citrinum and one of Penicillium verrucosum. Conclusions: Our results show that the Aspergillus and Penicillium species commonly isolated from grapes are not a source of the mycotoxins, patulin and citrinin. Significance and Impact of the Study: The possibility of co‐occurrence of patulin and citrinin with ochratoxin A in grapes and grape products remain low, owing to the low frequency of isolation of potentially producing species.     

ITS序列结合培养特征鉴定梨树腐烂病菌

对采自中国4个省份的9个梨树腐烂病菌分离株和7个苹果树腐烂病菌分离株的ITS序列进行了测定和分析,并结合GenBank的有性型Valsa ceratosperma、V.ambiens和V.mali的ITS序列构建了系统发育树。结果表明梨和苹果树的各分离株在ITS核苷酸序列上分化较小(p-distance=1.55%),均在V.ceratosperma聚类组,但二者又分别处于两个独立小分支。其与V.ambiens和V.mali处在不同的聚类组中,且亲缘关系较远,表明供试梨树腐烂病菌并非V.ambiens。培养性状和生物学特点的研究结果还发现,梨树腐烂病菌各分离株无论在菌落颜色、产孢特点、还是37℃高温的生长情况都和苹果腐烂病菌有一定差别。前者菌落始在PDA终为乳白色,而后者菌落初为白色后期变褐色;在20%ABA上,前者形成的产孢体较大而数量较少,在37℃高温下能正常生长,后者则形成的产孢体较小而数量较多,在37℃高温下不能正常生长。并未发现二者在子实体上有稳定明显的差异。因而表明梨树腐烂病菌应为V.ceratosperma,但可用培养性状和生物学特点进行区分其和苹果树腐烂病菌。     

苹果盘二孢的分离培养研究

苹果盘二孢Marssonina coronaria引起的褐斑病是造成我国苹果树早期落叶的主要原因,由于该病菌分离培养困难,阻碍了对其生物学特性的研究,进而影响了对其防治机理和流行规律的研究。本研究应用4种培养基质,探索了3种方法对苹果盘二孢的分离效果。结果表明,3种方法均可分离到病原菌,但组织块分离法和分生孢子团分离法成功率仅有10%左右,而单孢分离法污染少,成功率高达到90%以上,明显优于其他两种方法。不同培养基上菌落形态、大小和产孢情况差异也很大,培养1个月(25℃)后PDA上菌落黑褐色隆起,表面蚯蚓粪状,无气生菌丝,无子实体和基内菌丝;10%V8培养基上菌落中央隆起,黑褐色,表面生少量气生菌丝,边缘放射状,基内菌丝深褐色,有子实体;苹果叶片葡萄糖琼脂培养基(LDA)上菌落平坦,黄褐色,表面生茂密的金黄色气生菌丝,基内菌丝深褐色,有子实体;苹果叶片煎汁葡萄糖琼脂培养基(LEDA)上菌落有明显的不规则隆起,黄褐色至黑褐色,表面生少许气生菌丝,菌落生长缓慢,无基内菌丝,分生孢子盘菌落表面生,菌落直径仅2mm左右,而在其他培养基上的菌落直径可达6-8mm,说明培养基质、分离方法均对苹果盘二孢的分离培养和生长发育有明显的影响。     

A monoclonal antibody to ochratoxin A

The production of patulin by P. expansum Link ex Gray growing in apple juice was determined over a range of pH. Patulin was found to be produced in large quantities over a narrow range of pH from 3-2 to 3–8. The biomass produced increased with increasing pH. The changes in pH due to growth of the organism were found to be small.     

Influence of temperature and water activity on growth and patulin production by Byssochlamys nivea in apple juice

The influence of temperature and water activity (aw) on growth and patulin production by Byssochlamys nivea in apple syrups was determined over a 44-day incubation period. The minimum aw at which the mold was capable of growing was 0.915 and 0.886 at 21 and 30 degrees C, respectively. Growth at 37 degrees C was observed at 0.871 aw. Minimum aw values for patulin production were 0.978, 0.968, and 0.959 at 21, 30 and 37 degrees C, respectively.     

Influence of temperature and water activity on growth and patulin production by Byssochlamys nivea in apple juice.

The influence of temperature and water activity (aw) on growth and patulin production by Byssochlamys nivea in apple syrups was determined over a 44-day incubation period. The minimum aw at which the mold was capable of growing was 0.915 and 0.886 at 21 and 30 degrees C, respectively. Growth at 37 degrees C was observed at 0.871 aw. Minimum aw values for patulin production were 0.978, 0.968, and 0.959 at 21, 30 and 37 degrees C, respectively.     

Selection of indigenous isolates of entomopathogenic soil fungus Metarhizium anisopliae under laboratory conditions

Eight native isolates of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) Sorokin were obtained by monitoring soils cultivated in a conventional manner. These isolates were compared in three areas: (a) conidial germination, (b) radial growth and sporulation and (c) ability of conidia to infect Tenebrio molitor larvae. All bioassays were carried out at constant temperatures of 10, 15, and 20 °C. Conidia of individual isolates demonstrated differences in germination after a 24-h long incubation at all evaluated temperatures. At 20 °C, the germination ranged from 67 to 100 % and at 15 °C from 5.33 to 46.67 %. At 10 °C, no germination was observed after 24 h; nevertheless, it was 8.67–44.67 % after 48 h. In terms of radial growth, the culture diameters and the associated production of spores of all isolates increased with increasing temperature. At 10 °C, sporulation was observed in three isolates while all remaining cultures appeared sterile. Three weeks post-inoculation, conidia of all assessed isolates caused 100 % cumulative mortality of treated larvae of T. molitor at 15 and 20 °C with the exception of isolate 110108 that induced 81.33 % mortality at 15 °C. At 10 °C, larval cumulative mortality ranged from 6.67 to 85.33 % depending on the isolate. Isolates 110108 and 110111 showed significantly slower outset and a much lower rate of infection at all temperatures compared to other tested isolates of M. anisopliae. The bioassays were carried out with the purpose to sort and select indigenous isolates of M. anisopliae useful as biocontrol agents in their original habitat.     

Identification of Colletotrichum Species Causing Bitter Rot of Apple and Pear in Croatia

Symptoms of bitter rot were observed on apple and pear fruits in the field and in storage in Croatia between 2009 and 2011. Fifteen Colletotrichum isolates from apple and two from pear were collected and identified by sequencing of the ITS1 and ITS2 regions of the ribosomal DNA. Phylogenetic analysis revealed that ten isolates from apple and two isolates from pear could be identified as Colletotrichum fioriniae, five isolates from apple were clustered in Colletotrichum clavatum, while one isolate was in the Colletotrichum acutatum A7 group. All isolates caused typical bitter rot symptoms when inoculated on apple and pear fruits.     

Effect of Light, Temperature and Substrate during Spore Formation on the Germinability of Conidia of Colletotrichum falcatum

This paper presents results on the effect of light, temperature and substrate during spore formation on the germinability of conidia in Colletotrichum falcatum. Light seems to have no effect on the germination of conidia unless the cultures were exposed to a high intensity of light during sporulation, in which case the spores showed a reduced germination and an increased appressoria formation. Conidia produced at temperatures higher than the optimum showed better germination and less appressoria formation than the spores produced at the temperature optimum for the growth and sporulation of the fungus. A similar increase in germination was also observed in conidia obtained from inoculated sugarcane leaves as compared to those produced on culture media. The light type virulent isolates of C. falcatum showed greater sensitivity to all these treatments than the dark type weakly pathogenic isolates.     

Virulence and the Production of Endo-1,4-β-glucanase by Isolates of Alternaria alternata Involved in the Moldy-core Disease of Apples

Alternaria alternata, is the predominant fungal pathogen responsible for moldy‐core in apple cultivars of the Red Delicious group. Here we report on the association between virulence of natural isolates of A. alternata, their production of endo‐1,4‐β‐glucanase (EG) and moldy‐core development in apple fruits. Based on decay development following wound inoculations of mature fruits, three of 150 isolates, collected in three orchards in northern Israel and representing low, moderate and high virulence, were selected and designated Rm44, Er30 and Sh42, respectively. All three isolates secreted EG when grown on enzyme‐inducing medium (EIM) containing commercial cellulose or apple cell walls and this production was related to their degree of virulence. Polyacrylamide gel electrophoresis (PAGE) revealed quantitative differences between the three isolates, relative to their virulence. When fungal extracts were run in native gels, a single band with a molecular mass of 23 kDa showing EG activity was produced by the high‐ (Sh42) and the medium‐virulence (Er30) isolate but not by the low‐virulence (Rm44) isolate. A commercial cellulase preparation (containing endo‐ and exo‐1,4‐β‐glucanase) placed on pricked fruit led to the formation of symptoms similar to those developing on A. alternata‐inoculated fruits within 2–4 days. Inoculation of bloom clusters at full bloom with the highly virulent isolate (Sh42) of A. alternata resulted in a significantly higher infection in fruits (58%) than in those inoculated with the low‐virulence isolate (Rm44) (30%). Our results suggest that the moldy‐core symptoms caused by A. alternata in apple, could be related to the ability of the fungus to produce EG in developing lesions.