Detection of Penicillium expansum by polymerase chain reaction

Penicillium expansum is a major causative agent of postharvest decay in a variety of fruits, including apples, peaches, nectarines, and cherries. It causes significant economic losses to the fruit industry and is also of potential public health significance, since it produces patulin, a mycotoxin known to cause harmful effects in animals. Rapid and specific detection of P. expansum is important for ensuring microbiological quality and safety of fruits and fruit juices. The traditional methods for identification of P. expansum are time-consuming and labor-intensive. In this study, we report a polymerase chain reaction utilizing primers based on the polygalacturonase gene of P. expansum. The PCR amplified a 404-bp DNA product from all the P. expansum isolates tested, but not in other common foodborne Penicillium species and Escherichia coli. Experiments to determine the sensitivity of the PCR indicated that it can detect the DNA equivalent from as low as 25 spores of P. expansum. The PCR could potentially be used as a rapid tool for screening fruits for the presence of P. expansum.     

Factors affecting patulin production by Penicillium expansum

Patulin, a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to human health because exposure can result in severe acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects. In this study, we investigated the effects of Penicillium expansum isolate, apple cultivar, storage temperature and time, and pH on the production of patulin. Patulin was analyzed by a previously developed micellar electrokinetic capillary electrophoresis method. P. expansum isolates originating from across Ontario produced widely differing levels of patulin, ranging from 0 to >6 mg/g by dry mycelial weight. The highest patulin levels were those for isolates displaying aggressive growth (characterized by rapidly increasing acidity) accompanied by profuse mycelial development. Distinct patterns in fungal growth rates and patulin production were evident among isolates grown in McIntosh, Empire, and Mutsu ciders. Extensive fungal growth and higher patulin levels (538 to 1,822 microg/ml on day 14) in apple ciders were associated with incubation at room temperature (25 degrees C), although potentially toxic patulin levels (75 to 396 microg/ml on day 24) were also found in refrigerated ciders (4 degrees C) inoculated with P. expansum.     

采后热处理对红富士苹果青霉病和灰霉病的控制

热空气处理能有效抑制灰葡萄孢(Botrytiscinerea)和扩展青霉(Penicilliumexpansum)的毒性。灰葡萄孢比扩展青霉的热敏感度高,Bcinerea孢子38℃热处理48h即能完全抑制孢子萌发。热处理72h的Bcinerea平板均无菌丝生长。Pexpansum的孢子活性和菌丝生长随热处理时间的延长而明显减缓。热处理后的霉菌孢子在苹果果实内生长缓慢,Bcinerea孢子在热处理72h后在苹果中的生长完全受抑制,腐烂发生率为零。Pexpansum热处理72h后才能显著抑制苹果的腐烂发生。苹果接种霉菌后再进行热处理(38℃,96h),无论是0℃还是20℃下放置,均未发现腐烂,大大减少贮期灰霉病、青霉病的发生。研究还发现,经过2个月冷藏后,与对照组相比,热处理过的红富士苹果果皮色泽更黄,糖酸比上升,呼吸强度较低,硬度保持较好,说明热处理能够正面影响苹果的贮藏品质。     

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.     

棒曲霉素的生物合成、调控机制及其控制技术研究进展

棒曲霉素(patulin)是一种真菌的次生代谢产物。它对人和动物具有急性和慢性毒性,是污染水果及其加工制品的最重要的真菌毒素之一。食品中的棒曲霉素污染是一个全球性的问题,受到世界各国的关注,100多个国家和地区对果汁等水果加工制品中棒曲霉素的最高含量做了限定。棒曲霉素主要由青霉属、曲霉属、拟青霉属和丝衣霉属中的部分真菌产生。其中,扩展青霉(Penicillium expansum)是生产上棒曲霉素最重要的产生菌,因此成为研究棒曲霉素生物合成和调控机制的模式材料。近年来,随着食品安全问题越来越被人们重视,水果及其加工制品的真菌毒素污染问题也受到越来越多的关注,相关领域的研究逐渐成为热点。本文主要阐述了棒曲霉素的生物合成途径、分子基础、内外源调控因子和分子调控机制以及控制技术等方面的最新研究进展,并展望了棒曲霉素相关研究未来的重点。     

Antifungal activity of TiO2 photocatalysis against Penicillium expansum in vitro and in fruit tests

The antifungal activity of TiO2 photocatalytic reaction in the form of TiO2 powder and TiO2 coated on a plastic film against Penicillium expansum was investigated in vitro and in fruit tests. The mixture of P. expansum conidial suspension and TiO2 powder was added to potato dextrose agar (PDA) plates for vitro test. The TiO2 photocatalytic reaction reduced conidial germination of the fungal pathogen. It was found that the ability of the TiO2 photocatalytic reaction to suppress P. expansum growth correlated to the amount of TiO2 added. Lower numbers of viable colonies of P. expansum were observed with increasing amount of TiO2. Regardless of the kind of selected fruit inoculated with P. expansum, both TiO2 powder and TiO2-coated film exhibited antifungal activity to control fruit rot. Development of Penicillium rot in apple was significantly (P = 0.05) retarded by the TiO2 photocatalytic reaction. Similarly the TiO2 photocatalytic reaction was the only treatment where no tomato fruit rot was noticeable after 1 week of storage. TiO2-coated film also decreased brown lesions and Penicillium rot infection in lemons. The mean severity fruit rot scores (browning and softening flesh) were 3.2 and 1.9 for uncoated and TiO2-coated film, respectively. Our findings suggest that "TiO2 photocatalytic reaction" shows antifungal activity against P. expansum which may have potential for postharvest disease control.     

鼠尾藻多酚提取纯化及其抗果蔬病原菌活性研究

为了获得具有抗菌活性的天然海洋生物活性物质,利用褐藻门马尾藻属的鼠尾藻(Sargassum thunbeergii Kuntze)乙醇提取物,通过对Botrytis cinerea和Penicillium expansum的抑制率来评价其抗菌活性。结果表明,鼠尾藻提取物有很强的抗果蔬病原菌活性,抑制Botrytis cinerea和Penicillium expansum生长的最小抑菌浓度(MIC)分别为16mg/mL和12mg/mL。采用10mg/mL多酚处理的草莓,其发病率和病斑直径分别比对照低28.4%和47.6%。     

Fungal profiles of Spanish country-cured hams

The fungal population of Spanish country-cured hams was analyzed. For 160 surface samples from 40 hams yeast counts between 10⁴ and 3 × 10⁵ cells per g and filamentous fungi from 5 × 10² to 3 × 10⁴ colony-forming units per g were found. The yeasts isolated belonged to the species Debaryomyces marama and were capable of growth at 16% of NaCl. The molds isolated belonged to the genera Eurotium, Penicillium and Trichoderma. 70% of the isolates were identified as Eurotium repens the species most frequently isolated. Five species of Penicillium: Penicillium expansum, P. cyclopium, P. viridicatum P. brevicompactum and P. simplicissimum, represented about 25% of the isolates.     

Ecophysiological characterization of Penicillium expansum population in lleida (Spain)

Penicillium expansum, a patulin producer fungus, is the most important fungus causing decay in cold stored both apples and pears. This can lead to patulin contaminated by-products. The aim of this assay was to evaluate the phenotypical and physiological variability in the population of P. expansum that cause fruit spoilage in post-harvest stages in Lleida (Spain). In total, 101 isolates of P. expansum from the 2004 and the 2005 seasons were obtained from decayed fruits. Significant differences were found in the observations from both seasons. Variability of the isolates in each season seemed to be partially explained by differences in growth in media, patulin accumulation and resistance to fungicides. Patulin production was detected in almost 100% of the isolates. Variability existing in P. expansum population could not be totally explained, but the above mentioned variables explained up to 74% of the diversity in some cases. The results obtained point to the existence of different populations of P. expansum in each season and may explain the differences in fungicide resistance observed between both seasons. The capacity to colonize apple flesh and some variables involved in fruit colonization were not a source of variation neither in each season nor when both seasons were compared. As storage rooms are cleaned and disinfected each season, this suggests that each season, the populations in storage rooms develop only from strains capable to colonize apple flesh. This may lead to rapid sporulation and spreading of spores.     

Evaluation of several culture media for production of patulin by Penicillium species

The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.     

Synergistic effects of combining biocontrol agents with silicon against postharvest diseases of jujube fruit

The synergistic effects of biocontrol yeasts Cryptococcus laurentii and Rhodotorula glutinis combined with silicon (Si) against Alternaria alternata and Penicillium expansum molds were investigated in jujube fruit (Chinese date, Zizyphus jujuba) stored at 20 and 0 degrees C, respectively. Combinations of C. laurentii and R. glutinis at 5 x 10(7) cells/ml with 2% Si was most effective in controlling the diseases caused by A. alternata and P. expansum on jujube fruit stored at 20 degrees C. When fruits were stored at 0 degrees C, combining C. laurentii and R. glutinis with Si was as effective against P. expansum as was Si or the yeasts applied alone and was more effective in controlling A. alternata. Si may have a fungistatic effect by directly inhibiting pathogen growth, but it did not greatly influence the growth of the antagonists.     

甘肃河西走廊产区酿酒葡萄产毒霉菌的分离与鉴定

目的：了解甘肃河西走廊酿酒葡萄产区污染霉菌的主要类群,分析酿酒葡萄潜在的霉菌毒素污染。方法：采集河西走廊不同地区的酿酒葡萄,进行霉菌的分离纯化和鉴定,并通过高效液相色谱法测定霉菌代谢产物。结果：鉴定出4 株污染酿酒葡萄的优势菌,分别为青霉属的黄绿青霉（Penicillium decumbens）和扩展青霉（Penicillium expansum）,曲霉属的杂色曲霉（Aspergullus versicolor）和烟曲霉（Aspergillus fumigatus）;扩展青霉能够产生展青霉毒素。结论：根据污染酿酒葡萄霉菌的情况,可确定河西走廊酿酒葡萄产区存在展青霉毒素污染的风险。     

茶树精油对扩展青霉线粒体功能的影响

为了揭示茶树精油(tea tree oil,TTO)对果蔬采后病原真菌扩展青霉(Penicillium expansum)的抑菌作用机制,以TTO处理的P.expansum孢子、菌丝和线粒体为研究对象,测定活性氧(reactive oxygen species,ROS)积累及6种线粒体功能相关酶类的活力变化,并采用扫描...     

Molecular Identification of Spoilage Fungi Isolated from Fruit and Vegetables and Their Control with Chitosan

This study investigated fungal spoilage of fruits and vegetables in Saudi Arabia. Activities of cell wall degrading enzymes in fungal isolates were measured. We evaluated the fungicidal activities of chitosan against spoilage fungi. Thirty-three fungal species belonged to 21 fungal genera were collected during this study. ITS sequencing results showed that Alternaria tenuissima, Fusarium chlamydosporum, Penicillium sumatrens, Rhizopus stolonifera, and Trichoderma harzianum were the most common species. The diversity of cell wall degrading enzyme produced by the spoilage fungi revealed that xylanase was the most widely distributed enzyme in fungi followed by amylase and cellulase. Some strains of Macrophomina phaseolina, Trichoderma harzianum, Trichoderma asperellum, Aspergillus niger and Alternaria consortialis showed the highest enzymes activities among tested fungi. Four different concentrations of chitosan (20, 30, 50, and 100 mg/L) were investigated against 22 fungal species. All investigated concentrations of chitosan showed highly significant reduction in the average of radial growth of all tested fungi.     

Mould and yeast flora in fresh berries, grapes and citrus fruits

Fresh fruits are prone to fungal contamination in the field, during harvest, transport, marketing, and with the consumer. It is important to identify fungal contaminants in fresh fruits because some moulds can grow and produce mycotoxins on these commodities while certain yeasts and moulds can cause infections or allergies. In this study, 251 fresh fruit samples including several varieties of grapes, strawberries, blueberries, raspberries, blackberries, and various citrus fruits were surface-disinfected, incubated at room temperature for up to 14 days without supplemental media, and subsequently examined for mould and yeast growth. The level of contamination (percent of contaminated items/sample) varied depending on the type of fruit. All raspberry and blackberry samples were contaminated at levels ranging from 33% to 100%, whereas 95% of the blueberry samples supported mould growth at levels between 10% and 100% of the tested berries, and 97% of strawberry samples showed fungal growth on 33-100% of tested berries. The most common moulds isolated from these commodities were Botrytis cinerea, Rhizopus (in strawberries), Alternaria, Penicillium, Cladosporium and Fusarium followed by yeasts, Trichoderma and Aureobasidium. Thirty-five percent of the grape samples tested were contaminated and supported fungal growth; the levels of contamination ranged from 9% to 80%. The most common fungi spoiling grapes were Alternaria, B. cinerea and Cladosporium. Eighty-three percent of the citrus fruit samples showed fungal growth at levels ranging from 25% to 100% of tested fruits. The most common fungi in citrus fruits were Alternaria, Cladosporium, Penicillium, Fusarium and yeasts. Less common were Trichoderma, Geotrichum and Rhizopus.     

Quantitative analysis of mycoflora on commercial domestic fruits in Japan

A comprehensive and quantitative analysis of the mycoflora on the surface of commercial fruit was performed. Nine kinds of fruits grown in Japan were tested. Overall fungal counts on the fruits ranged from 3.1 to 6.5 log CFU/g. The mean percentages of the total yeast counts were higher than those of molds in samples of apples, Japanese pears, and strawberries, ranging from 58.5 to 67.0%, and were lower than those of molds in samples of the other six fruits, ranging from 9.8 to 48.3%. Cladosporium was the most frequent fungus and was found in samples of all nine types of fruits, followed by Penicillium found in eight types of fruits. The fungi with the highest total counts in samples of the various fruits were Acremonium in cantaloupe melons (47.6% of the total fungal count), Aspergillus in grapes (32.2%), Aureobasidium in apples (21.3%), blueberries (63.6%), and peaches (33.6%), Cladosporium in strawberries (38.4%), Cryptococcus in Japanese pears (37.6%), Penicillium in mandarins (22.3%), and Sporobolomyces in lemons (26.9%). These results demonstrated that the mycoflora on the surfaces of these fruits mainly consists of common pre- and postharvest inhabitants of the plants or in the environment; fungi that produce mycotoxins or cause market diseases were not prominent in the mycoflora of healthy fruits. These findings suggest fruits should be handled carefully with consideration given to fungal contaminants, including nonpathogenic fungi, to control the quality of fruits and processed fruit products.     

Comparison of the activity of antifungal hexapeptides and the fungicides thiabendazole and imazalil against postharvest fungal pathogens

In this study, we evaluated the activity of short antimicrobial peptides against different fungal isolates that cause postharvest decay of fresh fruits. The previously identified hexapeptides PAF19, PAF26 and LfcinB4-9 inhibited the in vitro growth of isolates from Penicillium digitatum and P. italicum, and from Alternaria and Geotrichum genera, being no active against Rhizopus, Mucor and Aspergillus. The results extend our previous observations on the specific and distinct activity profiles of this class of antifungal peptides. In addition, peptide activities were compared with that of two fungicides used for citrus fruit preservation, thiabendazole (TBZ) and imazalil (IMZ). We observed a lack of correlation between peptide and fungicide sensitivity among different species. Importantly, P. digitatum and P. italicum isolates resistant to fungicides were susceptible to peptides and our data suggest that common multiple drug resistance mechanisms are not active against this class of peptides. The in vitro peptide inhibition was correlated with a retard of the decay caused by Penicillium on citrus fruits, and this effect was comparable for both fungicide-resistant and -sensitive isolates. Comparison of PAF26 and TBZ in vitro minimum inhibitory concentration (MIC) values and their in vivo effect on citrus decay indicated that PAF26 performed in vivo better than TBZ.     

Activity of Electrolyzed Oxidizing Water Against Penicilium expansum in Suspension and on Wounded Apples

ABSTRACT: Spores of Penicillium expansum , the primary organism responsible for the occurrence of patulin in apple juice, were exposed to electrolyzed oxidizing (EO) water in an aqueous suspension and on wounded apples. Full-strength and 50% EO water decreased viable spore populations by greater than 4 and 2 log units, respectively. Although EO water did not prevent lesion formation on fruit previously inoculated with P. expansum , cross-contamination of wounded apples from decayed fruit or by direct addition of spores to a simulated dump tank was substantially reduced. EO water, therefore, has potential as an alternative to chlorine disinfectants for controlling infection of apples by P. expansum during handling and processing operations.     

Inoculum size and intraspecific interactions affects Penicillium expansum growth and patulin accumulation in apples

Penicillium expansum is the most important fungal species causing spoilage in cold stored apples. P. expansum produces patulin, a toxic secondary metabolite. The aim of this work was to study the changes in growth parameters and patulin accumulation that may occur when two different isolates of P. expansum germinate and develop in the same wound. The effect of the inoculum size was also studied. For that purpose, apples were point inoculated with two different isolates of P. expansum at two different conidia concentrations. Both isolates were also inoculated together. Apples were cold stored at 1 degrees C or incubated at 20 degrees C until lesion diameter reached 2.5 cm. Patulin from decayed tissue was analysed at the end of cold storage/incubation. It seems that intraspecific interactions occur. The interactions resulted in slower growth in which patulin content was lower. A competition for resources followed by either a "combat-like" interaction may be involved in this. Higher inoculum size led to shorter lag phase, which reflects the importance of the hygiene in packinghouses facilities. The inoculum size also affected growth rate and patulin accumulation, which suggests that interactions between individuals of the same strain may occur. Competition for resources and a combat-like interaction may take place. In vitro studies should be carried out in order to assess the patterns of the interactions observed.     

杨梅贮藏期真菌的分离鉴定及基于ITS序列的聚类分析

目的 分离并鉴定杨梅贮藏期真菌。方法 通过稀释涂布平板分离法以及划线纯化, 从贮藏期杨梅果实中分离、纯化真菌菌种, 并结合传统形态学与ITS (internal transcribed spacer)全序列分析对其进行准确鉴定。结果 最终鉴定出共14株真菌, 其中1株毛霉(Mucor racemosus)、1株白地霉(Geotrichum candidum)、 1株腐皮壳属霉菌(Diaporthe sp.)、1株好食链孢霉(Neurospora sitophila)、3株出芽短梗霉(Aureobasidium pullulans)、4株酵母菌(Pichia kudriavzevii, Pichia terricola, Hanseniaspora opuntiae, Saccharomycopsis crataegensis)以及3株青霉(Penicillium sp., Penicillium expansum, Penicillium herquei)。从进化关系上看有3类显示较近的亲缘关系, 第1类为青霉属, 第2类为出芽短梗霉, 第3类为毕赤酵母。结论 结合传统形态学与ITS全序列分析可对杨梅贮藏期真菌进行更为准确的鉴定。