Cabañas R, Abarca M L, Bragulat M R, Cabañes F J
Veterinary Mycology Group, Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Int J Food Microbiol. 2009 Jan 31;129(1):26-9. doi: 10.1016/j.ijfoodmicro.2008.10.025. Epub 2008 Nov 5.
Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples and is regarded as the major producer of the mycotoxin patulin. Imazalil is one of the fungicides used in Spain to control postharvest blue mold, but development of fungal resistance has been reported in P. digitatum and P. italicum. The most common used methods to detect antifungal susceptibility of fungal crop pathogens in vitro, are direct-plating isolates in media amended with various concentrations of fungicide and determining inhibition of growth and/or spore germination. These techniques are time- and labor-intensive and are not suitable if a large number of isolates has to be evaluated. On the other hand, the broth microdilution method M38-A is the reference method developed by the Clinical and Laboratory Standards Institute (CLSI) for antifungal susceptibility testing in some clinical fungi, but Penicillium spp. are not included. Due to the lack of a standard method, the aim of this work is to evaluate the suitability of an adaptation of the CLSI M38-A method to monitor P. expansum susceptibility to imazalil in comparison with other techniques. A total of 128 P. expansum strains have been studied (118 isolates from apples and pears, 5 from grapes and 5 reference strains). Imazalil has shown to be highly active in vitro against all the P. expansum isolates tested, as all the evaluated parameters were in the range reported for imazalil sensitive Penicillium spp. The mean minimum inhibitory concentration determined by broth microdilution method and by agar dilution method (48-72 h readings) was 0.0625 microg/ml and 0.11-0.12 microg/ml respectively. The mean concentration that inhibited the size of colonies (48-72 h) and spore germination by 50% was 0.05-0.06 and 0.04 microg/ml respectively. Our results highlight that the broth microdilution method CLSI M38-A is a good alternative to be used in screening the in vitro activity of imazalil against a large number of isolates.
扩展青霉是导致贮藏苹果发生青霉病的最重要病原菌之一，被认为是霉菌毒素棒曲霉素的主要产生菌。抑霉唑是西班牙用于控制采后青霉病的杀菌剂之一，但已有报道指在指状青霉和意大利青霉中出现了真菌抗性。体外检测真菌作物病原菌抗真菌药敏性最常用的方法是，将分离菌株直接接种于添加不同浓度杀菌剂的培养基中，并测定生长和/或孢子萌发的抑制情况。这些技术既耗时又费力，如果要评估大量分离菌株则并不适用。另一方面，肉汤微量稀释法M38 - A是临床和实验室标准协会（CLSI）针对某些临床真菌抗真菌药敏试验开发的参考方法，但不包括青霉属。由于缺乏标准方法，本研究的目的是评估对CLSI M38 - A方法进行改良后用于监测扩展青霉对抑霉唑药敏性的适用性，并与其他技术进行比较。共研究了128株扩展青霉菌株（118株来自苹果和梨的分离株、5株来自葡萄的分离株以及5株参考菌株）。抑霉唑在体外对所有测试的扩展青霉分离株均表现出高活性，因为所有评估参数均在报道的对抑霉唑敏感的青霉属菌株范围内。通过肉汤微量稀释法和琼脂稀释法（48 - 72小时读数）测定的平均最低抑菌浓度分别为0.0625微克/毫升和0.11 - 0.12微克/毫升。抑制菌落大小（48 - 72小时）和孢子萌发50%的平均浓度分别为0.05 - 0.06微克/毫升和0.04微克/毫升。我们的结果表明，肉汤微量稀释法CLSI M38 - A是用于筛选抑霉唑对大量分离株体外活性的良好替代方法。
