应用微滴数字PCR同时检测瓜类种子携带果斑病菌和角斑病菌

细菌性果斑病和角斑病是葫芦科作物两大重要细菌病害,病原菌分别为西瓜嗜酸菌Acidovorax citrulli和丁香假单胞菌黄瓜致病变种Pseudomonas syringae pv.lachrymans。两种菌均可通过种子、种苗带菌进行远距离传播。种子检测是预防和控制这两种病害发生的首要环节。本研究应用微滴数字PCR技术(droplet digital PCR,ddPCR)建立了同时检测种子携带西瓜嗜酸菌和丁香假单胞菌的方法。结果显示:两种细菌菌悬液和DNA样品等浓度混合时,ddPCR能同时检测到两种靶标菌的最低混合菌悬液浓度和最低DNA浓度分别为10³ cfu/mL和10^-3 ng/μL,其检测灵敏度是平行测试的real-time PCR方法的10倍;对于非等浓度混合的菌悬液和DNA样品,两种靶标菌菌悬液按浓度比1∶1000(10³∶10⁶ cfu/mL)混合或其DNA浓度比为1∶10000(2.28×10^-3 ng/μL∶22.8 ng/μL)条件下,ddPCR可检测到低浓度的靶标菌,检测灵敏度同样是real-time PCR的10倍。此外,在人工接菌种子测试中,西瓜、甜瓜单粒种子平均带菌量10⁵~10⁶ cfu/粒时,ddPCR方法可检测到带菌率0.2%(n=500)的西瓜、甜瓜种子样品。将分别携带两种菌的种子按比例1∶10混合时ddPCR方法可以准确检出浓度相对低的靶标菌;而使用相同检测引物的real-time PCR检测方法则只能检出西瓜嗜酸菌和丁香假单胞菌带菌率分别为0.2%和2%(n=500)的甜瓜种子混合样品中的西瓜嗜酸菌,未能稳定检出丁香假单胞菌。综上所述,本研究基于ddPCR技术建立了可同时检测两种重要葫芦科种传细菌的方法,检测结果稳定可靠,丰富了当前种传病原细菌的检测技术体系。

Detection of Acidovorax citrulli and Pseudomonas syringae pv. lachrymans from cucurbit seeds by multiplex droplet digital PCR

Bacterial fruit blotch(BFB)and bacterial angular leaf spot are two major bacterial diseases of Cucurbitaceae crops,caused by Acidovorax citrulli(Ac)and Pseudomonas syringae pv.lachrymans(Psl),respectively.These two pathogens can achieve long-distance transmission through contaminated seeds or seedlings,therefore,seed detection plays the first key role in disease management.In this study,we adopted a new technique of droplet digital PCR(ddPCR)to establish a method for simultaneous detection of seeds carrying both Ac and Psl.The results showed that ddPCR could simultaneously detect Ac and Psl mixed in equal ratio,with the limits of 10³ cfu/mL of two bacterial suspensions and 10^-3 ng/μL of two DNA samples respectively.The detection sensitivity of ddPCR was 10 times higher than that of real-time PCR tested in parallel.For two bacterial suspensions and DNA samples mixed in unequal ratio,ddPCR can detected the low concentration of target bacteria at a ratio of 1∶1000(10³∶10⁶ cfu/mL)for bacterial suspension and 1∶10000(2.28×10^-3 ng/μL∶22.8 ng/μL)for DNA.Meanwhile,the detection sensitivity was 10 times higher than that of real-time PCR.In addition,when testing with artificially infested watermelon and melon seed(inoculated bacterial amount=10⁵-10⁶ cfu/seed),the seed sample with an infestation level of 0.2%(n=500)was detectable by using ddPCR.When the ratio of the two contaminated seeds was 1∶10,ddPCR still detected the target bacteria at low concentrations.However,using real-time PCR could only detect Ac in the melon seed at a level of 0.2%(n=500),and Psl was not consistently detected in replicates.In summary,this study established a seed detection method based on ddPCR that could simultaneously detect two important seed-transmitted pathogens in the Cucurbitaceae.The ddPCR-based method was stable and reliable,which enriches the current detection technology system for seed-borne pathogens.