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.     

Effect of 2-deoxy-D-glucose on mycotoxins from apples inoculated with Penicillium expansum

Patulin concentration was not significantly different in Bramley and Cox's apples inoculated with Penicillium expansum, and treated with the biocontrol enhancer 2-deoxy-D-glucose (DOG) when compared to no DOG treatment, despite average numerical increases. Some additional small HPLC peaks were detected from some extracts, one of which corresponded to citrinin. This revised version was published online in August 2006 with corrections to the Cover Date.     

扩展青霉脂肪酶ep8与R182K叠加突变体的构建

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因进行体外定点突变,并将含突变基因的重组质粒pAO815-ep8-R182K在毕赤酵母(Pichiapastoris)GS115中进行表达。叠加突变体PEL-ep8-R182K表达产物与野生型PEL、PEL-ep8比较实验表明:叠加突变体表达蛋白PEL-ep8-R182K最适反应温度与野生型PEL、PEL-ep8一致,均为40℃;热稳定性与野生型相似,比PEL-ep8降低2.25℃。但是,在比活上,PEL-ep8-R182K与PEL-ep8、野生型PEL相比,其比酶活分别提高了14.03%和3.86%。     

Alkylresorcinols isolated from rye bran by supercritical fluid of carbon dioxide and suspended in a food-grade emulsion show activity against Penicillium expansum on apples

Apple cultivars are attacked by many fungal diseases, both on the tree and during storage. One of the most serious is blue mould, caused by Penicillium expansum. In this study, 5-(n)-alkylresorcinols (AR) were isolated from rye bran by the supercritical fluid of carbon dioxide and were used for the preparation of bioactive emulsions. These emulsions were applied to harvested fruit of five apple varieties to determine the levels of antifungal activity. A significant inhibition of disease symptoms was obtained after spraying some of the prepared AR emulsions on fruits that had been experimentally infected with Penicillium expansum. The most effective emulsions consisted of 0.025% (m/v) ARs, 0.1% (m/v) xanthan gum, 0.5% (m/v) Synperonic 91/6 or PDMs-copolymer, 0.2% (m/v) Tween 20, 1% (m/v) Trioleate, 2% (m/v) oleylalcohol, 2% (m/v) PEG 400, 5% (m/v) CaCl₂ or NaCl suspended in water.     

D92P点突变对扩展青霉碱性脂肪酶最适作用温度的改善

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因进行体外定点突变,并构建了含突变基因的重组质粒pPIC3．5K—lip-D92P。将该质粒在毕赤酵母GS115菌株中表达。与野生型表达产物PEL-GS相比较,突变体表达产物PELD92P—GS最适作用温度为45℃,比野生型提高了5℃;其热稳定性与野生型相当;突变体在40℃下的表达量为109U／mL,约为野生型的29％。结果分析表明,Pro替代Asp^92后,可能是由于Pro一级结构的特点,使酶结构更加稳定,在高温下更适于与底物结合。     

Activity of trans-2-hexenal against Penicillium expansum in 'Conference' pears

AIMS: To investigate the effects of trans-2-hexenal on blue mould disease, patulin content and fruit quality in 'Conference' pears. METHODS AND RESULTS: Fruits, wounded and inoculated with Penicillium expansum or non-inoculated, were exposed to trans-2-hexenal vapour treatment (12.5 microl l(-1)) at 20 degrees C. A greater reduction of decay was obtained by treatment application 24 or 48 h after inoculation, in contrast trans-2-hexenal application 2 h after inoculation was ineffective. Fruit storage temperature (-1 degrees C) after treatment did not affect the antifungal activity. Although 2-h exposure to trans-2-hexenal was effective in reducing blue mould, an exposure of at least 8 h was required to reduce fruit patulin content. Treatments did not affect fruit physical-chemical characteristics. After 6 days at 20 degrees C following exposure, trans-2-hexenal residue in treated fruits was less than the natural content of the compound in unripe fruits. CONCLUSIONS: trans-2-Hexenal treatment is effective in the reduction of blue mould infections and patulin content in Conference pears when applied 24-48 h after pathogen inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: trans-2-Hexenal could be a natural alternative to fungicides in the control of P. expansum infections. Further work is needed to study the methods and conditions avoiding the persistence of off-odours and off-flavours in pears after their exposure to trans-2-hexenal vapours.     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病（Peniclium expansum Link)的丝孢酵母（Trichosporon pullulans(Lindner.)Diddens and Lodder)。罗伦隐球酵母（Cryptococcus laurentii(Kuffer.)skin-ner)和粘红酵母（Rhodotorula glutinis(Fresen.)F.C.Harrison)。其中,抑病效果最好的T.pullulans是一种用于采后果实生物防治的新型拮抗菌,研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果。实验结果表明;酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C.laurentii的细胞悬浮液中加入0.18mol/L的CaCl2能显著提高其抑病能力。但增加CaCl2对T.pullulans和R.glutinis的抑病效果却没有明显作用。     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

由扩展青霉(Penicillium expansum)PF868产生脂肪酶催化油脂水解的研究

研究了由扩展青霉（Ｐｅｎｉｃｉｌｉｕｍｅｘｐａｎｓｕｍ）ＰＦ８６８产生脂肪酶催化水解三种油脂（橄榄油、豆油、鱼油）的影响因素与工艺条件,其中包括：水解时间、温度、ｐＨ、酶量、油水比及添加剂,并用气相色谱对产品脂肪酸进行了分析鉴定,初步分析其催化水解的脂肪酸的特异性     

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.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病(Penicilium expansum Link)的丝孢酵母(Trichosporon pullulans (Lindner.) Diddens and Lodder)、罗伦隐球酵母(Cryptococcus laurentii (Kuffer.) Skinner)和粘红酵母(Rhodotorula glutinis (Fresen.) F. C. Harrison).其中,抑病效果最好的T. pullulans 是一种用于采后果实生物防治的新型拮抗菌.研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果.实验结果表明:酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C. laurentii的细胞悬浮液中加入0.18 mol/L 的CaCl2能显著提高其抑病能力,但增加CaCl2 对T. pullulans 和R. glutinis 的抑病效果却没有明显作用.     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases,the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique.The recombinant plasmid containing mutant E83 V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS 115.Comparison experiments of the mutant PEL-E83 V-GS and the wild-type PEL-GS showed that the optimum temperature (45℃) of the mutant was 5℃ higher than that of the wild type.The thermostability of the mutant was similar to that of the wild type.The enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80% that of the wild type in the same conditions.Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.     

假单胞菌YL11对扩展青霉的抑制作用及其机理初探

【背景】苹果青霉病是由扩展青霉引起的一种重要的果实采后病害,影响果实品质导致苹果腐烂从而造成经济损失。【目的】研究假单胞菌YL11对扩展青霉的抑制作用和苹果采后青霉病的防治效果,并对抑菌机理进行初步探讨。【方法】以扩展青霉为供试菌株,研究不同浓度的假单胞菌YL11无菌发酵液对扩展青霉菌落直径、孢子萌发率、菌丝体干重、苹果损伤接种病斑直径扩展的影响,利用对电导率、核酸及蛋白释放量、AKP含量、SDH活性、ATP酶活性和ATP含量的影响对抑菌机理进行探究。【结果】假单胞菌YL11无菌发酵液能有效抑制扩展青霉生长,抑菌圈直径为22.33±0.27 mm,抑菌效价为71.67 mm/mL;能有效抑制孢子萌发,100%无菌发酵液对孢子萌发抑制率达到80.2%;对扩展青霉的生物量也有一定抑制作用,体积分数为100%时,菌丝体干重为4.7mg/mL,抑制率达到39.74%;无菌发酵液处理能有效抑制苹果青霉病病斑的扩展,3d时对病斑扩展的抑制率最大,达到47.1%;无菌发酵液处理均能引起电导率升高、胞内核酸和蛋白释放量增大、胞外AKP含量升高、SDH活性降低、ATP酶活性和ATP含量均降低,且随着发酵液浓度的增加效果越明显。【结论】假单胞菌YL11能显著抑制扩展青霉的生长,破坏细胞膜结构、降低能量代谢酶活性,从而扰乱扩展青霉的正常生长,对苹果青霉病有较好的生防效果,具有潜在的开发价值。     

一株扩展青霉拮抗菌的分离、鉴定及抑菌活性物质

【目的】筛选有效抑制扩展青霉(Penicillium expansum)的拮抗菌,并鉴定其所产抑菌物质的主要种类及相对含量。【方法】从苹果表面分离到拮抗扩展青霉的菌株BA-16,经形态学、生理生化及16S r RNA基因序列分析对该菌进行鉴定;根据已知脂肽类抗生素合成相关基因序列设计3对特异性引物对菌株BA-16进行检测,对PCR产物克隆、测序和BLAST分析,采用酸沉淀法从菌株发酵液中制备出抑菌物质粗提液,对活性粗提物进行HPLC和MALDI-TOF-MS分析。【结果】经鉴定,BA-16被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),所得PCR产物经测序和BLAST分析,证实BA-16带有sfp和fen B基因。HPLC和MS结果显示菌株发酵液中含有Fengycin和Surfactin两种脂肽类产物,Fengycin是拮抗扩展青霉的主要因素。【结论】本研究对于苹果采后青霉病的生物防治具有良好的应用开发前景。     

E83V对扩展青霉脂肪酶最适作用温度的影响

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因作了体外定点突变,获得了最适作用温度有所提高的突变体。含突变基因的重组质粒pPIC3．5K-lip-E83V在Pichia pastoris GS115中表达。对突变体表达产物PEL-E83V-GS与野生型表达产物PEL-GS作了比较：前者最适作用温度为45℃,比野生型提高了5℃;其热稳定性基本不变;突变体在37℃下的表达量为188U／mL,约为野生型的80％。最适作用温度的提高可能是由于83位亲水性的Glu用疏水性的Val取代,增加了脂肪酶表面的疏水作用,使其在高温下更适于与底物的结合。     

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.     

Antifungal activity of crude extract produced by Bacillus sp. associated with entomopathogenic nematode from media formulated by six nitrogen sources with fructose against Penicillium expansum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different nitrogen sources in combination with fructose on the production of antifungal crude extract by Bacillus sp. against Penicillium expansum. The yield of crude extract and antifungal activity against the test fungi differed significantly when the nitrogen sources in the fermentation media were changed. The highest yield was recorded for beef extract plus fructose (921?mg/L). The antifungal activity was higher in yeast extract plus fructose [P. expansum (46.5?±?2.12?mm)], followed by beef extract. High performance liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation medium, the antimicrobial production was substantially reduced almost eight times. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Yeast extract and fructose as nitrogen and carbon sources in the fermentation medium produced maximum antimicrobial activity.