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

柑橘黄单胞菌亚种

• 批准号: BB/S018891/1
• 负责人: Mark Enright
• 金额: $ 44.67万
• 依托单位: Manchester Metropolitan University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS018891%2F1
• 关键词: Xanthomonas; citri; subsp.; citrus; canker

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.

圣保罗州是迄今为止世界上最大的甜橙子生产国，也是欧盟和美国的最大出口国。仅2016年全球浓缩果汁市场就价值895. 6亿美元，预计到2025年将上升至1178. 9亿美元。这一至关重要的行业雇用了大约20万人，因此对圣保罗州和巴西国家产生了巨大的积极社会和经济影响。橙子生产业受到亚洲柑橘溃疡病（ACC）的威胁，一种柑橘属植物的毁灭性且无法治愈的疾病，只能通过昂贵的移除和破坏受感染的树木以及经常使用对环境有害的铜来预防已经出现耐药性并正在蔓延的杀菌剂。2009年之前实施的ACC根除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使用情况在果园中进行不同程度的疾病发生。这导致了整个州的地方病和越来越多地使用对环境有毒的铜杀菌剂，威胁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。柑桔属citri（X.柑橘），这会导致过早落果、茎枯病和落叶。我们目前对圣保罗引起ACC的细菌菌株的多样性知之甚少，因为很少有已发表的研究，其中大多数使用了不适合进行强有力的流行病学或进化分析的歧视性方法。基因组DNA测序是检测细菌病原体和追踪抗生素耐药性和毒力的传播和进化的金标准方法，但这仅应用于少数已发表的X.在本项目中，我们将研究X. citri的遗传多样性和流行病学。从圣保罗的受感染植物中提取的柑橘。我们将在2018/19年夏季疾病发病率最高的时候从全州48个地点采集植物样本，并将其与历史分离株和其他国家的分离株进行比较。本文对X.从ACC水平高、中、低的地区取样的柑橘将产生关于ACC如何传播的重要信息，是否存在超毒力菌株，与相同和其他物种的菌株的DNA交换频率，以及铜抗性问题的规模和性质。我们还将分离和表征X。柑橘特有的病毒称为噬菌体。这些被提议作为人类健康中抗生素的替代品，并且它们目前用于食品工业，例如用于控制奶酪生产中的李斯特菌污染。在美国，它们也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，测试它们杀死X的能力。柑橘分离物在实验室研究中的作用，以及它们在温室研究中保护免受ACC的能力。通过噬菌体基因组测序，我们已经确定了产生有可能破坏X的酶的基因。以生物膜形式存在的柑橘群落。这些生物膜由细菌产生的多糖“粘液”保持在一起，帮助它在环境中生存，并且是导致疾病的关键因素。我们将确定并产生这些噬菌体多糖解聚酶的库，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酶，这可能会提高它们在田间的有效性。这项研究将为圣保罗ACC疾病提供重要的新信息，以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虫剂替代品，用于2021年可能的现场试验。

项目成果

Core-Genome Multilocus Sequence Typing for Epidemiological and Evolutionary Analyses of Phytopathogenic Xanthomonas citri.

• DOI: 10.1128/aem.02101-22
• 发表时间: 2023-05-31
• 期刊: APPLIED AND ENVIRONMENTAL MICROBIOLOGY
• 影响因子: 4.4
• 作者: Ragupathy, R.;Jolley, K. A.;Zamuner, C.;Jones, J. B.;Redfern, J.;Behlau, F.;Ferreira, H.;Enright, M. C.
• 通讯作者: Enright, M. C.

Core genome MLST for epidemiological and evolutionary analyses of phytopathogenic Xanthomonas citri

用于植物病原柑橘黄单胞菌流行病学和进化分析的核心基因组 MLST

• DOI: 10.1101/2022.12.20.521341
• 发表时间: 2022
• 作者: Ragupathy R