ARABLE: Monitoring for emerging threats to UK OSR crops posed by novel variants and fungicide resistant strains of fungal pathogens

英国油菜（OSR）作物主要受到两种真菌病害的威胁：由Pyrenopeziza brassicae（1系）引起的轻叶斑病（LLS）和由Plenodomus lingam / Plenodomus biglobosus引起的Phoma。在英国，尽管使用了昂贵的杀菌剂（主要是去甲基化抑制剂（DMIs）），但Phoma和LLS各自造成了100亿英镑的作物损失。这些病原体的新变种和抗杀菌剂菌株最近已经在其他地理区域报道，如果它们出现在那里，可能会威胁到英国OSR作物。第一个风险与杀菌剂敏感性下降有关，在包括英国在内的欧洲油菜种群中发现了DMI抗性。DMI敏感性的降低是由CYP51基因的改变介导的，包括编码区（G460S或S508T）的点突变和上游控制区（启动子）的插入。来自爱尔兰的g4600s +S508T基因型真菌分离株对DMI的敏感性大大降低。因此，需要对英国油菜种群的杀菌剂敏感性变化进行监测，并对g4600s +S508T基因型进行筛选。来自芸苔属油菜的另一个风险最近确定了导致美国太平洋西北地区发生的叶斑病暴发的谱系2变异。该谱系2似乎是最近引进的，经历了快速的侵入性传播。这种破坏性变异现在威胁到邻近的加拿大OSR生产，并可能影响欧洲OSR生产，特别是考虑到谱系1和2接触的未知影响以及美国太平洋西北部生产的芸苔覆盖作物种子的使用增加。因此，需要进行研究以监测目前在英国病原体种群中存在的谱系。对于Phoma来说，对英国OSR的一个威胁来自于抗杀菌剂P. lingam / P. biglobosus。直到2015年，体外测试显示这两种病原体都对dmi敏感。然而，最近的研究表明，在澳大利亚和捷克共和国，15-24%的lingam种群对DMI具有抗性。这种抗性与CYP51启动子区域的插入物有关。是否DMI抗性也出现在P. lingam和P. biglobosus中，英国的病原体种群需要调查。进一步的phoa威胁与最近在欧洲发现的P. biglobosus“加拿大”亚分支有关。在此之前，欧洲只报道过P. biglobosus ‘brassicae’亚支系。越来越多的证据表明，大叶假单品亚支变体对OSR健康构成不同的风险，这可能需要不同的疾病管理策略。因此，需要有关英国OSR作物中亚支系病原体种群结构的数据。在拟议的项目中，我们将与合作伙伴巴斯夫合作，巴斯夫将从田间试验中收集显示Phoma（1月23日）和LLS（3月23日）症状的OSR叶片。至少有10个试验将在英国和其他一些欧盟国家进行。样品将被送到RRes进行真菌分离，目的是收集P. brassicae， P. lingam和P. biglobosus各50-100株。这些分离株将被保存到RRes和OREGIN培养集合中。分离的DNA将被提取，并通过谱系特异性扩增进行菌株谱系分型。Plenodomus分离株将使用针对lingam / P. biglobosus的物种特异性DNA引物进行分型。对于大叶假单胞菌，将对其内部转录间隔区（ITS）进行测序，以确定亚支（亚群）的身份。这三种菌株将在体外筛选对杀菌剂的敏感性，包括DMI （prothioconazole-desthio, mefentrifluconazole）和QoI (pyraclostrobin)，对于DMI最不敏感的菌株，将对CYP51进行分子检测，以探索可能的耐药机制。

Oilseed rape (OSR) crops in the UK are threatened by two main fungal diseases: light leaf spot (LLS) caused by Pyrenopeziza brassicae (lineage 1) and Phoma caused by Plenodomus lingam / Plenodomus biglobosus. In the UK, Phoma and LLS each cause >£100M in crop losses despite costly fungicide applications (primarily DeMethylation Inhibitors (DMIs)). New variants and fungicide resistant strains of these pathogens have been reported recently from other geographic regions and may threaten UK OSR crops should they emerge there. The first P. brassicae risk relates to decreased fungicide sensitivity, where DMI resistance was found in European P. brassicae populations including the UK. Decreased DMI sensitivity is mediated by alterations in the CYP51 gene, including point mutations in the coding region (G460S or S508T) and inserts in the upstream control (promoter) region. Fungal isolates from Ireland with a G460S+S508T genotype had a much reduced DMI sensitivity. Therefore, work is required to monitor for changes in the fungicide sensitivity status of UK P. brassicae populations and screen for the G460S+S508T genotype. An additional risk from P. brassicae has recently identified a lineage 2 variant responsible for chlorotic leaf spot outbreaks in the US Pacific northwest. There lineage 2 appears to be a recent introduction that has undergone rapid invasive spread. This damaging variant now threatens adjacent Canadian OSR production and could affect European OSR production, particularly given the unknown implications of lineages 1 and 2 coming into contact and increasing use of brassica cover crop seed produced in the US Pacific northwest. Research is thus required to monitor the lineages currently present in UK pathogen populations. For Phoma, one threat to UK OSR comes from fungicide resistant P. lingam / P. biglobosus. Until 2015, in vitro testing had shown both pathogens to be sensitive to DMIs. However, recent work has shown DMI resistance in 15-24% of the P. lingam population in Australia and the Czech Republic. Such resistance is associated with inserts in the CYP51 promoter region. Whether DMI resistance has also emerged in P. lingam, and also P. biglobosus, pathogen populations in the UK requires investigation. A further Phoma threat relates to the recent discovery in Europe of the P. biglobosus 'canadensis' subclade. Prior to this only the P. biglobosus 'brassicae' subclade had been reported in Europe. There is increasing evidence that the P. biglobosus subclade variants pose different risks to OSR health, that may require distinct disease management strategies. Data is thus required on the pathogen population structure of subclades present in the UK OSR crops. In the proposed project, we will work with our collaborator, BASF who will collect OSR leaves from field trials showing Phoma (Jan 23) and LLS (Mar 23) symptoms. At least ten trials will be sampled from the UK and potentially some additional EU locations. Samples will be sent to RRes for fungal isolation, with the aim of collecting 50-100 isolates each of P. brassicae, P. lingam, and P. biglobosus . These isolates will be deposited into the RRes and OREGIN culture collections. Isolate DNA will be extracted, and P. brassicae strains lineage typed via lineage-specific amplification. Plenodomus isolates will be typed using species-specific DNA primers specific for P. lingam / P. biglobosus. For P. biglobosus isolates, the internal transcribed spacer (ITS) region will be sequenced to determine subclade (subgroup) identities. Isolates of all three species will be screened in vitro for sensitivity to fungicides, including DMIs (prothioconazole-desthio, mefentrifluconazole) and a QoI (pyraclostrobin), and for the least DMI sensitive isolates, CYP51 will be examined molecularly to explore possible resistance mechanisms.