Using experimental evolution to create phage therapy agents to target the Horse Chestnut bleeding canker pathogen, Pseudomonas syringae pv. aesculi

英国约47万棵具有重要生态和社会意义的欧洲栗树正受到一种溃烂病疫情的困扰。该病是由细菌病原体紫丁香假单胞菌引起的。Aesculi(PAE)，它侵染树干和树枝上的韧皮部和木质部维管，当树木分泌物从受感染的地方流出时，导致‘出血’(Green等人。2009年植物病理学58,731-744)；它最终可能导致树木死亡，从而可能引发另一场“荷兰榆树”灾难。噬菌体在治疗细菌疾病方面具有巨大的潜力，因为它具有很大的多样性，并且能够在实验室中进化出多种基因类型作为多基因混合物使用。噬菌体疗法也可以被用作预防性应用，作为感染的进一步屏障。我们建议对噬菌体进行采样：分析多样性；与宿主一起进化噬菌体以创造新的基因类型；分析噬菌体在治疗树木方面的效果。目的1：采样和多样性为了采集环境中的PAE噬菌体，将从健康和患病的树木组织和周围土壤中分离出噬菌体，并将其以琼脂平板半固体覆盖的方式接种到PAE草坪上：裂解噬菌体将根据其斑块形态进行鉴定。平板计数将决定感染PAE的裂解噬菌体的丰度。将使用显微镜和分子技术对裂解噬菌体进行表征和比较。目标2：实验进化(两个阶段)阶段1-进行大筛选以评估分离的噬菌体的共同进化潜力。每个独特的噬菌体分离物(IF&GT；50，分离物的代表性亚样将被使用)的三个重复群体和PAE将通过分批培养(每个群体的1%将被转移到新鲜的微型世界中，每48小时被转移到新鲜的微型世界中)进行12次转移。每隔2次转移群体样本在-80℃20%甘油中冷冻，噬菌体样本用氯仿处理后分离并保存在4℃。每4次转移，我们将使用标准的时移协议来测量共同进化(Brockhurst 2003 EcolLett 6：975-979)。细菌-噬菌体共同进化的特点是随着时间的推移，传染性和耐药性范围不断扩大。因此，如果在复制种群中，并且在多个时间步骤中，噬菌体感染性和细菌抗药性随时间呈正斜率，则将定义显著的协同进化。显示最大协同进化的5个噬菌体将被选择进行进一步的实验。阶段2-进行两种类型的长期选择实验：[1)协同进化，选择增加的噬菌体宿主范围(即，可以感染多种细菌基因型的噬菌体)，将进行如上所述的50次转移的实验，将使用标准的时移实验来评估共同进化的进展；[2)在固定的细菌基因型上传代，选择高宿主利用率，将通过分批培养进行实验，在每次转移时，只将噬菌体从以前的微观世界转移到新的微观世界，细菌被从实验室种群中生长的祖先基因所取代。在50次转移后，将通过执行一步生长曲线实验来评估进化噬菌体系对选择的响应，以确定关键的生活史参数(即吸附速度、裂解时间、破裂大小)。目的3：树上噬菌体疗法的疗效试验将在案例合作伙伴BTE提供的受控环境生长室中生长的树上进行。不同滴度的单一和混合噬菌体接种剂将使用不同的应用模式(喷雾和高压注射)应用于受感染和预感染的树木，以确定噬菌体作为预防和治疗的效率。

A bleeding canker disease epidemic is afflicting the circa 470,000 ecologically and socially important European Horse Chestnut (Aesculus hippocastanum) trees in Britain. The disease is caused by the bacterial pathogen, Pseudomonas syringae pv. aesculi (Pae), which infects phloem and xylem vessels in the trunk and branches, causing 'bleeding' as tree exudates flow from infected sites (Green et al. 2009 Plant Pathology 58, 731-744); it can eventually lead to tree death thus posing the potential for another 'Dutch Elm' disaster. Bacteriophages have enormous potential for treating bacterial disease because of the large diversity and the ability to evolve multiple genotypes in the laboratory to use as multi-genotype mixtures. Phage therapies could also be used as a prophylactic application to act as a further barrier to infection. We propose to sample phages to: analyse diversity; evolve phage with its host to create new genotypes; analyse the efficacy of phage in treating trees. Objective 1: Sampling and diversity To sample environmental Pae phages, phages will be isolated from healthy and diseased tree tissue and from the surrounding soil and plated onto Pae lawns in semi-solid overlays on agar plates: lytic phages will be identified by their plaque morphology. Plate counts will determine the abundance of Pae-infecting lytic phages. Lytic phages will be characterised and compared using microscopy and molecular techniques. Objective 2: Experimental evolution (two stages) Stage 1 - Perform a large screen to assess the coevolutionary potential of isolated phages. Three replicate populations of each unique phage isolate (if >50 a representative subsample of isolates will be used) and Pae will be propagated in microcosms (30ml glass universals containing 6ml of KB liquid medium) by batch culture (1% of each population will be transferred to a fresh microcosm every 48 hours) for 12 transfers. Every 2nd transfer population samples will be frozen at -80C in 20% glycerol, and phage samples will be isolated by treatment with chloroform and stored at 4C. Every 4th transfer we will measure coevolution using a standard time-shift protocol (Brockhurst 2003 Ecol Lett 6:975-979). Bacteria-phage coevolution is characterised by the evolution of increasingly broad infectivity and resistance ranges through time. Therefore, significant coevolution will be defined if in replicate populations and for multiple timesteps there is a positive slope of phage infectivity and bacterial resistance with time. The 5 phages that display the greatest coevolution will be selected for further experiments. Stage 2 - Perform two types of long-term selection experiment: [1] coevolution, which selects for increased phage host range (i.e. phage that can infect a wide range of bacterial genotypes), experiments will be performed as described above for 50 transfers, standard time shift experiments will be used to assess the progress of coevolution; [2] passaging on a fixed bacterial genotype, which selects for high rates of host exploitation, experiments will be performed by batch culture whereby at each transfer only the phage is transferred from the previous to the new microcosm, and the bacteria are replaced by the ancestral genotype grown up from the laboratory stock. After 50 transfers the response to selection will be assessed in evolved phage lines by performing one-step growth curve experiments to determine key life history parameters (i.e. adsorbtion rate, lysis time, burst size). Objective 3: Therapy trials The efficacy of phage therapies in trees will be done using trees grown in controlled environment growth rooms supplied by the CASE partner, BTE. Different titers of single and mixed phage inocula will be applied to infected and pre-infected trees using different modes of application (spray and high pressure injection) to determine the efficiency of phage as a prophylactic as well as a therapy.