Abstract：
\'Candidatus Liberibacter asiaticus\', the putative causal agent of citrus greening disease is transmitted by the Asian citrus psyllid, Diaphorina citri in a propagative, circulative, and persistent manner. Unfortunately, \'Ca. L. asiaticus\' is not yet available in pure culture to carry out Koch\'s postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (w/v) sucrose and 0.1% ascorbic acid (w/v) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (ten nymphs/seedling) for an inoculation period of two weeks. A transmission rate of up to 80% was recorded at six months post-inoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch\'s postulates if a pure culture of \'Ca. L. asiaticus\' becomes available.
摘要：
\'亚洲自由念珠菌\'，柑橘绿化病的推定病原体是由亚洲柑橘木虱传播的，在一个传播者中，循环,和坚持不懈的态度。不幸的是,\'Ca.在纯培养中还没有L.asiaticus\'来执行Koch的假设并确认其病因。当纯粹的文化可用时，测试其在昆虫媒介和植物宿主中的感染性的方法将是至关重要的。在这里，我们描述了一种基于使用从受感染的柑橘植物中提取的韧皮部汁液和局部饲喂柑橘若虫的传播试验。通过离心收集韧皮部汁液，用含有20％（w/v）蔗糖和0.1％抗坏血酸（w/v）作为抗氧化剂的0.1M磷酸盐缓冲液pH7.4稀释，并通过将液滴放在口器上传递给第三至第五龄若虫。若虫将探针展开，并获得含有细菌病原体的韧皮部汁液。然后将若虫置于柑橘幼苗(10个若虫/幼苗)上两周的接种期。在接种后6个月记录到高达80%的传播率。该方法可以是测试细菌病原体在各种处理后的可传播性以降低细菌的生存力或阻断其传播的有力工具。此外,如果纯培养Ca，这可能是实现科赫假设的有效检测方法。L.asiaticus\'变得可用。
