Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with boscalid.

In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing 50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype, specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine replacing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam.

Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depigmented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation between lack of pigmentation and increasing aflatoxin production was also observed.

黄曲霉是一种常见且普遍存在的真菌，​​能够在收获前和收获后的环境中定殖于几种农产品。该物种由于其合成黄曲霉毒素B1的能力而代表了一种非常有害的植物病原体，黄曲霉毒素B1负责人类原发性肝细胞癌，并被国际癌症研究机构归为I组（人类致癌物）。已经提出了几种方法来控制田间和储存条件下黄曲霉的发展以及相关的黄曲霉毒素的生产。琥珀酸脱氢酶抑制剂（SDHI）杀菌剂Boscalid已被证明可在体外控制黄曲霉的生长和黄曲霉毒素的污染并在野外实验中。但是，该化合物被归类为触发真菌抗药性的中高风险杀真菌剂，实际上，抗药性菌株可能发生在经过Boscalid处理的农作物上。

在本文中，我们选择了在含有50 mg / L的Boscalid琼脂培养基上生长的抗Boscalid的实验室A. flavus菌株。为了研究造成耐药表型的分子机制，设计了特定的引物对来扩增整个SdhB，SdhC和SdhD基因。通过氨基酸序列分析，两个点突变，酪氨酸在更换组氨酸密码子的249 SDHB（H249Y）和精氨酸取代甘氨酸以91位密码子SDHC（G91R），已被识别。评估了SDHI的Boscalid和isopyrazam对菌丝生长和分生孢子萌发的影响。两种耐药基因型均表现出对Boscalid的高耐药性（MIC和EC50> 1000 mg / L）。在Boscalid和异丙基吡嗪之间发现了正的交叉电阻。

两种杀真菌剂的特定亚致死剂量（0.5 mg / L的Boscalid杀虫剂和0.01 mg / L的异丙吡嗪）均会干扰与菌落色素沉着相关的机制。尤其是，真菌菌落出现了色素沉着，缺乏未经修饰的杀菌剂培养基上显示的典型的黄曲霉绿色。还观察到缺乏色素沉着和黄曲霉毒素产量增加之间的严格相关性。