Pujol Marta, Badosa Esther, Manceau Charles, Montesinos Emilio
Institute of Food and Agricultural Technology-CIDSAV-CeRTA, University of Girona, Campus Montilivi, 17071 Girona, Spain.
Appl Environ Microbiol. 2006 Apr;72(4):2421-7. doi: 10.1128/AEM.72.4.2421-2427.2006.
The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10(2) cells g (fresh weight)(-1) and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10(7) to 10(8) cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.
在温室和田间试验中，通过可培养细胞计数和实时荧光定量PCR监测荧光假单胞菌EPS62e在苹果花和叶片上的定殖情况。实时荧光定量PCR为检测菌株EPS62e提供了一种特异性定量方法。检测水平约为10(2)个细胞/g（鲜重）(-1)，标准曲线在5个对数范围内呈线性。EPS62e能积极定殖于花朵，每朵花中的细胞数量达到10(7)至10(8)个。在苹果花中，实时荧光定量PCR和平板计数获得的菌量水平之间未观察到显著差异，这表明不存在活的但不可培养（VBNC）细胞和残留的未降解DNA。相反，在苹果叶片上，EPS62e的可培养菌量随时间减少，实时荧光定量PCR和平板计数之间观察到显著差异。这些差异表明细胞死亡后存在VBNC细胞或未降解DNA。因此，EPS62e在花朵定殖期间处于最佳条件，但在叶片上受到胁迫且存活不佳。得出的结论是，为监测这种生物防治剂，有必要联合使用可培养细胞计数和实时荧光定量PCR。
