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

英国的油菜（OSR）作物受到两种主要真菌病害的威胁：由芸苔核盘菌（Pyrenopeziza brassicae）（谱系1）引起的光叶斑病（LLS）和由Plenodomus lingam / Plenodomus biglobosus引起的茎点霉（Phoma）。在英国，茎点霉和LLS各自造成超过1亿英镑的作物损失，尽管使用了昂贵的杀真菌剂（主要是脱甲基抑制剂（DMIs））。这些病原体的新变种和抗杀真菌剂菌株最近已从其他地理区域报道，并可能威胁英国OSR作物，如果他们出现在那里。第一个风险与杀真菌剂敏感性降低有关，其中在包括英国在内的欧洲P. brassicae种群中发现了杀真菌剂抗性。CYP 51基因的改变介导了CYP敏感性降低，包括编码区（G460 S或S508 T）的点突变和上游控制（启动子）区的插入。来自爱尔兰的具有G460 S + S508 T基因型的真菌分离株具有大大降低的耐药性。因此，需要开展工作以监测英国芸苔类枯落物种群的杀真菌剂敏感性状态的变化并筛选G460 S + S508 T基因型。最近发现了一个来自P. brassicae的额外风险，该风险是导致美国太平洋西北部褪绿叶斑病爆发的谱系2变异体。在那里，谱系2似乎是最近引入的，经历了快速的侵入性传播。这种破坏性的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和谱系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，以监测目前存在于英国病原体种群的谱系。对于茎点霉，对UK OSR的一个威胁来自抗杀真菌剂的P. lingam /P. biglobosus。直到2015年，体外测试显示这两种病原体对DMIs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P. lingam种群具有耐药性。这种耐药性与CYP 51启动子区的插入片段有关。在英国的P. lingam和P. biglobosus病原体种群中是否也出现了耐药性需要调查。另一个茎点霉的威胁与最近在欧洲发现的P. biglobosus 'canadensis'亚支有关。在此之前，只有P. biglobosus 'brassicae'亚支在欧洲被报道。越来越多的证据表明，P. biglobosus亚进化枝变体对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，需要在英国OSR作物中存在的分支的病原体种群结构的数据。 在拟议的项目中，我们将与我们的合作伙伴巴斯夫公司合作，巴斯夫公司将从显示茎点霉（1月23日）和LLS（3月23日）症状的田间试验中收集OSR叶片。至少有10项试验将从英国和可能的其他一些欧盟地点抽样。将样品送至RRes进行真菌分离，目的是收集50-100株芸苔假单胞菌、P. lingam和P. biglobosus分离株。这些分离株将保藏在RRes和OREGIN培养物保藏中心。提取分离物DNA，并通过谱系特异性扩增对芸苔假单胞菌菌株谱系进行分型。将使用P. lingam / P. biglobosus的种特异性DNA引物对Plenodomus分离株进行分型。对于大裂叶假单胞菌分离株，将对内部转录间隔区（ITS）进行测序，以确定亚进化枝（亚组）身份。将在体外筛选所有三个种属的分离株对杀真菌剂的敏感性，包括DMI（丙硫康唑-地硫，甲呋唑）和QoI（吡唑醚菌酯），对于最不敏感的分离株，将对CYP 51进行分子检查，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.