新型水稻黄单胞菌噬菌体资源的发掘与功能鉴定

Exploration and functional identification of novel Xanthomonas oryzae pv. oryzae(Xoo)phages

由水稻黄单胞菌（Xanthomonas oryzae pv.oryzae,简称Xoo）引起的白叶枯病是水稻种植过程中毁灭性的细菌病害,对我国经济和食品安全造成巨大威胁。施用抗生素和化学生物防治手段的防控效果并不稳定,且易污染环境,还存在食品安全问题。为了应对该病害不可预知的爆发,利用噬菌体防控水稻白叶枯病可以作为一种备选方案,以减少化学杀菌剂和抗生素的使用。本研究利用中国不同水稻产区的9株水稻黄单胞菌和模式菌株PXO99A为靶标,从32份土壤样品中分离到了15个高效的Xoo噬菌体单株,说明黄单胞菌噬菌体广泛存在于中国各地的土壤中。选取其中Xoo_sp8和Xoo_sp9,电镜观察确定其具有二十面体的头部和细长的尾部,为典型的有尾噬菌体。宿主谱检测分析发现Xoo_sp8和Xoo_sp9都可以感染除PXO99A以外的9株不同生理小种的水稻黄单胞菌,且在培养基条件下能有效抑制其生长。测定其基因组序列后,根据末端酶大亚基（large terminase subunit,terL）相似性建立其与已报道的不同细菌噬菌体间的系统发育树,发现这两株噬菌体都与已报道的Xoo噬菌体亲缘关系很远,为新型的Xoo噬菌体。本研究分离发掘了15个对Xoo高效感染的噬菌体,为利用噬菌体防控水稻白叶枯病相关杀菌剂产品开发提供了宝贵的种质资源,同时也提出了土壤环境是分离Xoo噬菌体的重要来源的观点。

Bacterial leaf blight（BLB）caused by Xanthomonas oryzae pv.oryzae（Xoo）is a destructive bacterial disease in rice plantation,which produces tremendous threats on economy and food security in China.Phage therapy is an alternative approach to reduce the use of chemical agent and antibiotics in case the disease may break out unpredictably.In this study,nine wildtype Xoo strains isolated from different rice-growing areas of China and the type strain PXO99 A were used as targets to detect the Xoo phages in thirty-two soil samples,fifteen Xoo phages were isolated.This means that Xoo phages are widespread in soil.Xoo_sp8 and Xoo_sp9 were chosen to detect their morphology under electron microscope.They had isometric heads and long tails,belonging to Siphoviridae.Host range detection analysis showed that both Xoo_sp8 and Xoo_sp9 could infect nine wildtype strains of Xoo except PXO99 A,and could inhibit their growth effectively under the condition of medium.We sequenced the whole genome of Xoo_sp8 and Xoo_sp9,then built the evolution tree between these two Xoo phages and bacteriophages of other bacteria based on the similarity of the large terminase subunit（terL）.The two phages were found to be distantly related to the reported Xoo phages,and are new Xoo phages.Fifteen phage materials with high efficiency for Xoo treatment were obtained in this work,which provids valuable materials for the development of related fungicides for the control of BLB.We also foundthat the soil environment is a potential natural warehouse for the isolation of phages against plant bacterial disease.