Xanthomonas citri subsp. citri citrus canker in São Paulo. Molecular epidemiology and biocontrol potential of bacteriophage and their depolymerases.

<s:1>圣保罗州是目前世界上最大的甜橙生产国，也是对欧盟和美国最大的甜橙出口国。2016年全球浓缩果汁市场价值为895.6亿美元，预计到2025年将增至1178.9亿美元。这个至关重要的行业雇佣了大约20万人，因此对<s:1>圣保罗州和巴西国家产生了巨大的积极的社会和经济影响。柑橘生产工业受到亚洲柑橘溃疡病（ACC）的威胁，这是柑橘植物的一种毁灭性和无法治愈的疾病，只有通过昂贵的清除和摧毁受感染的树木和频繁使用破坏环境的铜杀菌剂才能预防，这种杀菌剂已经出现并正在蔓延。直至2009年的根除ACC政策已被风险缓解系统所取代，该系统允许根据水果的最终用途在果园中不同程度地发生疾病。这导致了整个州的地方病，并越来越多地使用对环境有害的铜杀菌剂，威胁到土壤和水道的生物多样性。ACC是由细菌感染黄单胞菌引起的。citri无性系种群。柑橘（X. citri），导致果实过早掉落，茎枯死和落叶。目前，我们对<s:1>圣保罗市引起ACC的细菌菌株的多样性知之甚少，因为发表的研究很少，而且大多数研究使用的方法不适合进行强有力的流行病学或进化分析。基因组DNA测序是检查细菌病原体和追踪抗菌素耐药性和毒力传播和进化的金标准方法，但这只应用于少数已发表的柑橘X. citri研究，其中只包括来自<s:1>圣保罗的一种分离物。在本项目中，我们将利用基因组测序技术研究圣保罗受感染植株的柑橘X. citri的遗传多样性和流行病学。我们将在2018/19年夏季（疾病发病率最高）对全州48个地点的植物进行取样，并将其与历史分离株和来自其他国家的分离株进行比较。从ACC高、中、低水平地区取样的柑橘柑橘的研究，将提供关于ACC如何传播、是否存在高毒菌株、与相同菌株和其他物种的菌株交换DNA的频率，以及铜抗性问题的规模和性质的重要信息。我们也将分离和表征柑橘X.特异性病毒称为噬菌体。这些被提议作为人类健康抗生素的替代品，它们目前被用于食品工业，例如控制奶酪生产中的李斯特菌污染。在美国，它们也被用于控制番茄和辣椒作物的感染。我们将收集约100个噬菌体，我们将在实验室研究中测试它们杀死柑橘X.分离物的能力，以及在温室研究中测试它们对ACC的保护能力。通过噬菌体基因组测序，我们已经确定了产生酶的基因，这些酶有可能破坏叶片上作为生物膜存在的柑橘X.群落。这些生物膜是由细菌产生的多糖“黏液”结合在一起的，这种黏液帮助细菌在环境中生存，也是导致疾病的关键因素。我们将鉴定和生产这些噬菌体多糖解聚合酶的曲目，并在实验室和温室研究中测试它们的抗生物膜活性。我们还将生产具有叶结合基序的修饰解聚合酶，以提高其在田间的有效性。该研究将为<s:1>圣保罗地区ACC病害提供重要的新信息，指导未来的作物管理和疾病预防策略。它还将生产潜在的可生物降解和环境可持续的铜杀菌剂替代品，可能在2021年进行现场试验。

São Paulo state is by far the largest sweet orange producer in the world and the biggest exporter to the EU and USA. The 2016 global juice concentrate market alone was valued at US$ 89.56bn and is forecast to rise to US$ 117.89bn by 2025. This crucially important industry employs approximately 200 000 people and therefore has enormously positive social and economic impacts on São Paulo state and the country of Brazil.The orange production industry is threatened by Asiatic Citrus Canker (ACC), a devastating and untreatable disease of citrus plants that is only preventable by the costly removal and destruction of infected trees and frequent application of environmentally damaging copper biocides to which resistance has emerged and is spreading. An ACC eradication policy, in place until 2009, has been replaced with a risk mitigation system that allows for differing levels of disease incidence in orchards depending on the fruits final usage. This has led to endemic disease throughout the state and increasing use of environmentally toxic copper biocides that threaten soil and water course biodiversity.ACC is caused by bacterial infection Xanthomonas. citri subsp. citri (X. citri), which results in premature fruit drop, stem dieback and defoliation. We currently know little about the diversity of strains of bacteria causing ACC in São Paulo as there have been few published studies and most of these have used poorly discriminatory methods that are unsuitable for robust epidemiological or evolutionary analyses. Genomic DNA sequencing is the gold-standard method for examining bacterial pathogens and tracing transmission and evolution of antimicrobial resistance and virulence but this has only been applied in a small number of published studies of X. citri that include just one isolate from São Paulo.In this project we will examine the genetic diversity and epidemiology of X. citri from infected plants in Sao Paulo using genome sequencing. We will sample plants from 48 sites throughout the state in the summer of 2018/19 when disease incidence is highest and compare these to historical isolates and to those from other countries. This study of X. citri sampled from regions with high, medium and low levels of ACC will yield important information on how ACC has spread, whether hyper-virulent strains are present, frequency of DNA exchange with strains of the same and other species, and the scale and nature of the problem of copper resistance. We will also isolate and characterize X. citri - specific viruses known as phage. These are proposed as an alternative to antibiotics in human health and they are currently used in the food industry e.g. for controlling Listeria contamination in cheese production. They are also used in the control of infections in tomato and pepper crops in the USA. We will assemble a collection of ~100 phage that we will test for their ability to kill X. citri isolates in laboratory studies and also their ability to protect from ACC in greenhouse studies. Through phage genome sequencing we have identified genes that produce enzymes with the potential to disrupt X. citri communities on leaves that exist as biofilms. These biofilms are held together by a polysaccharide 'slime' produced by the bacteria that helps it survive in the environment and is a key factor in causing disease. We will identify and produce a repertoire of these phage polysaccharide depolymerase enzymes and test their anti-biofilm activity in the laboratory and in greenhouse studies. We will also produce modified depolymerases with leaf-binding motifs that may promote their effectiveness in the field.This study will give important new information on ACC disease in São Paulo to guide future crop-management and disease prevention strategies. It will also produce potential biodegradable and environmentally sustainable alternatives to copper biocides for possible field trials in 2021.