Detection of Leifsonia xyli subsp. xyli in presprouted seedcane

ABSTRACT The first great change in recent years in the Brazilian sugarcane industry occurred when mechanical harvest replaced manual cuttings of burned canes. The second transformation has started with mechanization of planting materials thanks to new methodology such as presprouted seedcane (MPB in Portuguese). To ensure the success of such innovation, the use of healthy MPB is fundamental, especially against ratoon stunt, caused by Leifsonia xyli subsp. xyli (Lxx). This is considered the most important disease of sugarcane and the only means of introducing it to new areas is by the planting of contaminated propagative materials. Therefore, the aim of this study was to develop a diagnostic assay for early detection of Lxx in MPB. In the first assay, the efficacy of four primer pairs (Cxx ITSf#5/Cxx ITSr#5, Cxx1/Cxx2, C2F/C2R, LX23SF/LX23SR) was examined to identify adult plants contaminated with various titers of Lxx, under original conditions, as well as adapted conditions proposed in this study, our own modifications. Thus, adult sugarcane was used. In the second assay, the two best primers/conditions were tested in 45- and 60-day-old MPB derived from Lxx-contaminated materials. Data of the present study showed that the primer pair CXX ITSF # 5/CxxITSr # 5, under our modification conditions, was the most accurate since Lxx was detected in all 52 MPB aged 45 days from propagative materials contaminated with 109 to 106 CFU/mL.

摘要 巴西甘蔗产业近年的首次重大变革，是机械收割取代了人工砍伐并焚烧蔗秆的传统作业模式。第二次变革则随着种植材料机械化的推进而启动，依托预芽种蔗（葡萄牙语简称MPB）这类新型栽培技术得以落地。为保障此类创新的产业化应用，使用健康的MPB是核心前提，尤其需要防控由Leifsonia xyli subsp. xyli（以下简称Lxx）引发的宿根矮化病。该病害被公认为甘蔗最具危害性的病害，其唯一的跨区域传播途径为使用携带病菌的繁殖材料进行种植。因此，本研究旨在开发一种可在MPB中早期检测Lxx的诊断检测方法。在第一项检测试验中，我们分别在原始实验条件与本研究自主优化的自定义条件下，测试了4对引物（Cxx ITSf#5/Cxx ITSr#5、Cxx1/Cxx2、C2F/C2R、LX23SF/LX23SR）对携带不同浓度Lxx的成年甘蔗植株的检测效能，试验所用材料为成年甘蔗植株。第二项试验中，我们将筛选得到的最优两对引物及对应检测条件，应用于源自Lxx污染繁殖材料的45日龄与60日龄MPB样本的检测。本研究数据表明，经本研究优化条件适配的CXX ITSF#5/CxxITSr#5引物对检测精度最高，在来自10⁹至10⁶ CFU/mL（菌落形成单位每毫升）污染繁殖材料的52份45日龄MPB样本中，均成功检测到Lxx。