Targeting symptomless pathogen growth to control fungal plant diseases

无症状病原菌在寄主组织中的传播是疾病发生发展的关键阶段，包括大多数植物病害。更好地了解这种无症状的传播对于制定控制这类疾病的有效措施至关重要，无论是通过宿主抗性还是使用杀菌剂喷雾。油菜茎溃疡病是包括英国在内的世界油菜最重要的病害。当病原体最初感染叶片时，它随后沿着叶柄（茎）无症状地生长，到达植物茎，在那里发生流行病的破坏性阶段。最近的证据表明，田间（定量）对病原菌的抗性在该病无症状阶段起作用，在感染无症状阶段用杀菌剂处理作物对有效控制该病至关重要。洛桑研究所最近发展的表达水母绿色荧光蛋白（GFP）基因的病原体菌株和定量聚合酶链反应（qPCR）方法来量化无症状组织中病原体的生物量，为研究这种疾病的无症状阶段提供了一个独特的机会。此外，这些方法可用于最近由INRA （Rennes, France）生产的宿主材料，该材料提供了包含有助于定量抗性的基因的染色体区域的更高遗传分辨率。杜邦公司对作物育种和杀菌剂都很感兴趣，该公司支持这项工作作为IPA申请，旨在回答两个问题。1. 油菜抗性是否限制了瘤茎溃疡病病菌沿叶柄向下并进入植株茎部的无症状生长，是田间抗病的关键成分？2. 目前的杀菌剂是否只有在病原体引起症状前使用才对病原体（黄斑钩球绦虫）有效？这将涉及四个任务。任务1将通过比较受控环境（CE）实验（GFP, qPCR）中获得的对病原菌在茎中无症状传播的抗性结果与田间实验（qPCR，收获前取样评估茎溃疡病严重程度）的数据来解决问题1。任务2将通过比较受控环境（CE）实验（GFP, qPCR）中获得的对植物茎中病原体无症状传播的抗性结果与田间实验数据来解决问题1。因此，应该有可能确定数量抗性的主要成分是在叶柄向下生长还是在茎组织定植期间发生的。任务3将利用与现有和新的遗传作图信息相关的田间和受控环境实验结果，研究植物茎秆和植物茎部对病原菌无症状生长的抗性遗传控制。任务4将通过检查杀菌剂和遗传抗性对叶片和植物茎中无症状病原体生长的相互作用来解决问题2。它将涉及抗性和易感品系的CE （GFP）实验。将根据接种（由任务2的结果决定）的不同时间施用杀菌剂，并观察对若干遗传上不同的寄主系无症状生长的影响。CE试验将辅以一系列杀菌剂时间的田间试验。这些实验结果将用于鉴定和表征对黄斑霉的定量抗性，以便在抗性育种计划中容易地利用它。它们还将使杀菌剂应用的时机得到优化。

Symptomless pathogen spread in host tissues is a crucial stage in the development of diseases, including most plant diseases. Better understanding of this symptomless spread is essential to devise effective measures for control of such diseases, whether it be through host resistance or application of fungicide sprays. Phoma stem canker is the most important disease of oilseed rape in the world, including the UK. Whilst the pathogen initially infects the leaves, it then grows symptomlessly down their petioles (stalks) to reach plant stems, where the damaging phase of epidemics occurs. Recent evidence suggests that field (quantitative) resistance to the pathogen operates during the symptomless phase of the disease and that treatment of crops with fungicides when infections are symptomless is crucial to effective disease control. The recent development at Rothamsted of pathogen strains expressing the jellyfish green fluorescent protein (GFP) gene and quantitative polymerase chain reaction (qPCR) methods to quantify the biomass of the pathogen in symptomless tissues provides a unique opportunity to investigate the symptomless phase of this disease. Furthermore, these methods can be used on host material recently produced by INRA (Rennes, France) that provides greater genetic resolution of the chromosomal regions containing genes contributing to quantitative resistance. This work, supported as an IPA application by DuPont, who have interests in both crop breeding and fungicides, will aim to answer two questions. 1. Is oilseed rape resistance restricting symptomless growth of the phoma stem canker pathogen down the leaf stalk and into the plant stem the key component of field resistance to the disease? 2. Are current fungicides effective against the pathogen (Leptosphaeria maculans) only if applied before the pathogen causes stem symptoms? This will involve four tasks. Task 1 will address question 1 by comparing results obtained in controlled environment (CE) experiments (GFP, qPCR) on resistance to symptomless spread of the pathogen in leaf stalks with data from field experiments (qPCR, stem canker severity assessed by sampling stems before harvest). Task 2 will address question 1 by comparing results obtained in controlled environment (CE) experiments (GFP, qPCR) on resistance to symptomless spread of the pathogen in plant stems with data from field experiments. Thus it should be possible to determine whether the main component of quantitative resistance occurs during growth down the leaf stalk or during colonisation of stem tissues. Task 3 will investigate the genetic control of resistance to symptomless growth of the pathogen in leaf stalks and plant stems, exploiting results of field and controlled environment experiments in relation to existing and new genetic mapping information. Task 4 will address question 2 by examining interactions between fungicide and genetic resistance effects on symptomless pathogen growth in leaf stalks and plant stems. It will involve CE experiments (GFP) with resistant and susceptible lines. Fungicide applications will be made at different times in relation to inoculation (determined by results of task 2) and the effects on symptomless growth in a number of genetically different host lines observed. CE experiments will be complemented by field experiments with a range of fungicide timings. Results of these experiments will be used to identify and characterise the quantitative resistance to L.maculans, so that it can be easily exploited in resistance breeding programmes. They will also enable timing of fungicide applications to be optimised.