Food Technology Department, Lleida University, XaRTA-Postharvest, Agrotecnio Center. Av. Rovira Roure, 191, 25198 Lleida, Catalonia, Spain.
IRTA, Postharvest XaRTA, Av. Rovira Roure, 191, 25198 Lleida, Catalonia, Spain.
Int J Food Microbiol. 2014 Apr 3;175:45-52. doi: 10.1016/j.ijfoodmicro.2014.01.014. Epub 2014 Jan 31.
The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 10(3) cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2 populations on apple surfaces during large-scale experiments that could ensure efficient and successful treatments.
生防制剂的注册需要开发监测系统，以检测和量化环境中的制剂。成团泛菌 CPA-2 是防治柑橘和苹果采后病害的有效生防制剂。通过 Rodac 印迹平板评估了 CPA-2 在采后半商业试验中的监测，并用 SCAR 引物 PAGA1 和 PAGB1 进行常规 PCR 确认分离的菌落。从与 CPA-2 接触的不同表面、周围环境和操作人员穿着的工作服中采集样本。此外，还使用经典平板技术和实时定量 PCR(qPCR)确定了菌株 CPA-2 在苹果表面的种群动态。开发了一种使用 3'-小沟结合（MGB）探针的 qPCR 检测法，用于特异性检测和定量成团泛菌菌株 CPA-2。基于 CPA-2 的 SCAR 片段的核苷酸序列，设计了一组引物和 TaqMan MGB 探针。引物 SP2-F/SP2-R 和 TaqMan MGB 探针在苹果表面特异性检测到菌株 CPA-2，通过测试从 17 株成团泛菌、4 种相关泛菌属种和 21 株从整个和新鲜切割的水果和蔬菜中分离的其他属细菌菌株的纯化 DNA 进行了验证。检测水平约为 10(3)个细胞/反应，标准曲线在 5log 单位范围内呈线性。半商业试验结果表明，CPA-2 的影响较低。在 0°C 下储存的塑料盒中，P. agglomerans CPA-2 的最大持续时间不超过 5 天。通过 qPCR 和稀释平板获得的结果观察到 CPA-2 种群动态的显著差异。这些差异可能表明存在非存活细胞的未降解 DNA。总之，qPCR 是一种快速、特异性监测大规模试验中苹果表面 CPA-2 种群近期定殖的潜在新工具，可确保有效和成功的处理。
