Landi Lucia, Murolo Sergio, Romanazzi Gianfranco
Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Via Brecce Bianche, I60131 Ancona, Italy.
Biology (Basel). 2025 Sep 11;14(9):1251. doi: 10.3390/biology14091251.
' Phytoplasma solani' is the causal agent of the Bois noir (BN), affecting grapevine worldwide. The complex epidemiology of BN, which involves multiple '. P. solani' host plants and insect vectors, as well as the occurrence of recovery (loss of symptoms on grapevine canopy), makes disease investigations and containment in vineyards difficult. To achieve early detection of '. P. solani', a droplet digital PCR (ddPCR)-based approach and quantitative (q)PCR assay were compared, testing specific primers based on the elongation factor Tu () gene using SYBR Green chemistry. The regression curve analysis of the ddPCR assay showed good linearity. Compared with the qPCR method, the sensitivity of ddPCR improved about 10-fold. The analysis of grapevine roots spiked with serial dilutions of ' P. solani'. PCR fragments showed that qPCR was inhibited, while ddPCR was not affected. Testing 66 grapevine samples from 50 grapevine plants, the ddPCR provided superior diagnostic performance compared to qPCR in roots of symptomatic plants (75% detected by ddPCR, 41.6% by qPCR), roots of recovered plants (58.8% detected by ddPCR, 25% by qPCR), and asymptomatic leaf tissues from recovered plants (75% detected by ddPCR, 25% by qPCR). The ddPCR analysis allowed us to detect '. P. solani' on 40% of leaf samples from recovered plants and 20% of roots from asymptomatic plants. No differences among ddPCR and qPCR were found in detecting phytoplasma on symptomatic leaf samples. The ddPCR assay allowed the absolute quantification of '. P. solani' in complex matrices, such as roots, and when low titer of phytoplasma is present.
“茄植原体”是黑痘病（BN）的病原体，影响着全球的葡萄树。黑痘病复杂的流行病学涉及多种“茄植原体”寄主植物和昆虫媒介，以及恢复现象（葡萄树冠症状消失）的发生，这使得葡萄园中的病害调查和防治变得困难。为了实现对“茄植原体”的早期检测，比较了基于液滴数字PCR（ddPCR）的方法和定量（q）PCR检测，使用SYBR Green化学法检测基于延伸因子Tu（）基因的特异性引物。ddPCR检测的回归曲线分析显示出良好的线性。与qPCR方法相比，ddPCR的灵敏度提高了约10倍。对用“茄植原体”系列稀释液加标的葡萄树根进行分析，PCR片段显示qPCR受到抑制，而ddPCR不受影响。对来自50株葡萄树的66个葡萄树样本进行检测，在有症状植物的根（ddPCR检测到75%，qPCR检测到41.6%）、恢复植物的根（ddPCR检测到58.8%，qPCR检测到25%）以及恢复植物的无症状叶片组织（ddPCR检测到75%，qPCR检测到25%）中，ddPCR的诊断性能优于qPCR。ddPCR分析使我们能够在40%的恢复植物叶片样本和20%的无症状植物根中检测到“茄植原体”。在检测有症状叶片样本中的植原体时，未发现ddPCR和qPCR之间存在差异。ddPCR检测能够对复杂基质（如根）中存在低滴度植原体时的“茄植原体”进行绝对定量。
