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：近年来巴西甘蔗产业的首次重大变革，是机械收割取代了焚烧甘蔗秆的人工砍收作业。当前，第二次产业变革正随着种植材料机械化技术的普及而推进，其核心依托的是预萌芽种蔗（葡萄牙语缩写MPB）这类全新种植方法。为保障这类创新种植技术的推广成效，使用健康的MPB种植材料至关重要，尤其是针对由Leifsonia xyli subsp. xyli（缩写Lxx）引发的甘蔗宿根矮化病。该病害被认为是甘蔗最具危害性的病害，且病原菌唯一的跨区域传播途径为种植带菌繁殖材料。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。在第一组检测实验中，本研究在原实验条件及本研究自行优化的适配条件下，测试了4组引物对（Cxx ITSf#5/Cxx ITSr#5、Cxx1/Cxx2、C2F/C2R、LX23SF/LX23SR）对携带不同浓度Lxx的成熟甘蔗植株的鉴定效果，实验材料为成熟甘蔗植株。第二组检测实验中，研究选取表现最优的2组引物及对应反应条件，对源自Lxx带菌材料的45日龄和60日龄MPB进行检测。本研究数据显示，在优化后的反应条件下，引物对CXX ITSF #5/CxxITSr #5的检测准确性最高，可在源自10^9至10^6 CFU/mL带菌繁殖材料的全部52份45日龄MPB中检出Lxx。