The Challenge of Environmental Samples for PCR Detection of Phytopathogenic Bacteria: A Case Study of Citrus Huanglongbing Disease

• Main Authors: Félix Morán, Silvia Barbé, Saskia Bastin, Inmaculada Navarro, Edson Bertolini, María M. López, Estrella Hernández-Suárez, Alberto Urbaneja, Alejandro Tena, Felipe Siverio, Ester Marco-Noales
• Format: Article
• Language: English
• Published: MDPI AG 2021-12-01
• Series: Agronomy
• Subjects: undesired amplification; 16S; real-time PCR; sequence identity; greening; HLB
• Online Access: https://www.mdpi.com/2073-4395/11/1/10

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘<i>Candidatus</i> Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘<i>Ca.</i> Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.