Morel Arry, Peeters Nemo, Vailleau Fabienne, Barberis Patrick, Jiang Gaofei, Berthomé Richard, Guidot Alice
LIPM, Université de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan, France.
Methods Mol Biol. 2018;1734:223-239. doi: 10.1007/978-1-4939-7604-1_18.
In this chapter, we describe different methods for phenotyping strains or mutants of the bacterial wilt agent, Ralstonia solanacearum, on four different host plants: Arabidopsis thaliana, tomato (Solanum lycopersicum), tobacco (Nicotiana benthamiana), or Medicago truncatula. Methods for preparation of high volume or low volume inocula are first described. Then, we describe the procedures for inoculation of plants by soil drenching, stem injection or leaf infiltration, and scoring of the wilting symptoms development. Two methods for measurement of bacterial multiplication in planta are also proposed: (1) counting the bacterial colonies upon serial dilution plating and (2) determining the bacterial concentration using a qPCR approach. In this chapter, we also describe a competitive index assay to compare the fitness of two strains coinoculated in the same plant. Lastly, specific protocols describe in vitro and hydroponic inoculation procedures to follow disease development and bacterial multiplication in both the roots and aerial parts of the plant.
在本章中，我们描述了在四种不同寄主植物上对青枯病菌（茄科劳尔氏菌）菌株或突变体进行表型分析的不同方法，这四种寄主植物分别是拟南芥、番茄（茄属番茄）、烟草（本氏烟草）或蒺藜苜蓿。首先介绍了制备大量或少量接种物的方法。然后，我们描述了通过土壤浇灌、茎部注射或叶片浸润接种植物的程序，以及对萎蔫症状发展的评分方法。还提出了两种测量植物体内细菌增殖的方法：（1）通过系列稀释平板计数细菌菌落，（2）使用qPCR方法测定细菌浓度。在本章中，我们还描述了一种竞争指数测定法，以比较在同一植物中共接种的两种菌株的适合度。最后，具体方案描述了体外和水培接种程序，以跟踪植物根部和地上部分的病害发展和细菌增殖情况。
