Ilias Aris, Vassiliou Vassilis A, Vontas John, Tsagkarakou Anastasia
Hellenic Agricultural Organisation - "DEMETER", NAGREF - Institute of Olive Tree, Subtropical crops and Viticulture, Department of Viticulture, Floriculture, Vegetable crops and Plant Protection, Heraklion, Greece.
Agricultural Research Institute, Nicosia, Cyprus.
Pestic Biochem Physiol. 2017 Jan;135:9-14. doi: 10.1016/j.pestbp.2016.07.004. Epub 2016 Jul 21.
Avermectin and pyrethroid resistance mutations (the G314D and the G326E in the glutamate gated chloride channels, and the F1538I in the voltage gated sodium channel) have been reported in the spider mite Tetranychus urticae, one of the most devastating pests of protected and open field crops worldwide. We developed three TaqMan molecular diagnostic assays for monitoring the presence and frequency of these mutations in T. urticae field populations. The TaqMan assays were validated against known genotypes and subsequently used to monitor the frequency of the resistance mutations in eleven T. urticae populations from Greece and Cyprus, with variable history of avermectin and pyrethroids applications. The frequency of the F1538I pyrethroid resistance mutation largely varied among samples, with highest frequencies (75%-97%) detected in four populations derived from protected and open field crops from Crete and Peloponnesus, low frequencies in three populations (2.5%-11%) from Attiki, Cyprus and Crete and not detected in four populations from Crete, Peloponnesus and Cyprus. The frequency of the abamectin resistance mutations G314D and G326E also varied across populations (from 0 to 100%), showing fixation in two populations (>97.5% for the G314D and 100% for the G326E), originating from rose greenhouses from Greece, low frequencies in three populations (5%-12.5%) also originating from rose greenhouses (Crete, Peloponnesus and Cyprus) and not detected in six populations from protected and open field vegetable crops. The TaqMan diagnostics showed higher resolution in detecting specific alleles in low frequency, compared to massive quantitative sequencing approaches previously employed. They can be used, together with classical bioassays, to support evidence - based insecticide resistance management strategies.
阿维菌素和拟除虫菊酯抗性突变（谷氨酸门控氯离子通道中的G314D和G326E，以及电压门控钠通道中的F1538I）已在二斑叶螨中被报道，二斑叶螨是全球保护地和露地作物中最具破坏性的害虫之一。我们开发了三种TaqMan分子诊断检测方法，用于监测二斑叶螨田间种群中这些突变的存在情况和频率。这些TaqMan检测方法针对已知基因型进行了验证，随后用于监测来自希腊和塞浦路斯的11个二斑叶螨种群中抗性突变的频率，这些种群的阿维菌素和拟除虫菊酯应用历史各不相同。F1538I拟除虫菊酯抗性突变的频率在样本间差异很大，在来自克里特岛和伯罗奔尼撒半岛的保护地和露地作物的四个种群中检测到最高频率（75%-97%），在来自阿提卡、塞浦路斯和克里特岛的三个种群中频率较低（2.5%-11%），在来自克里特岛、伯罗奔尼撒半岛和塞浦路斯的四个种群中未检测到。阿维菌素抗性突变G314D和G326E的频率在不同种群中也有所不同（从0到100%），在两个种群中出现固定（G314D为>97.5%，G326E为100%），这两个种群来自希腊的玫瑰温室，在另外三个同样来自玫瑰温室（克里特岛、伯罗奔尼撒半岛和塞浦路斯）的种群中频率较低（5%-12.5%），在来自保护地和露地蔬菜作物的六个种群中未检测到。与先前采用的大规模定量测序方法相比，TaqMan诊断检测方法在检测低频特定等位基因时具有更高的分辨率。它们可以与经典生物测定法一起使用，以支持基于证据的杀虫剂抗性管理策略。
