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

Abstract

Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus 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 ‘Ca. 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.

Seaweed as a Natural Source against Phytopathogenic Bacteria

“…Besides their intervention in Erwinia transmission, insects are also responsible for the dispersion of the aggressive citrus disease, Huanglongbing disease (citrus greening), caused by the Gram-negative “ Candidatus Liberibacter”; this genus is responsible for the phloem vessels infections [ 118 , 119 , 120 ]. The term “ Candidatus ” refers to the impossibility to cultivate and grow this bacteria group in laboratory conditions [ 118 ], which is also difficult to detect as it is only possible through molecular methods such as PCR-based techniques [ 120 , 121 ]. There are three major species of this group mainly dispersed in Asia, Africa [ 122 , 123 ], and America [ 124 , 125 ] that can be transmitted by different vectors.…”

“…There are three major species of this group mainly dispersed in Asia, Africa [ 122 , 123 ], and America [ 124 , 125 ] that can be transmitted by different vectors. In Europe, the dispersion of these species is also a concern since there are already reports of the presence of vectors in the Atlantic Coast of Portugal and northwest of Spain [ 118 , 126 , 127 ].…”

A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects

“…All of them were positive by new protocol, with C q which ranged from 23.0 ± 2.9 to 33.2 ± 4.2 (data not shown). No amplification was detected in the 105 non-target plant and insect samples analyzed, including the strain BT-1 of L. crescens and those samples that gave undesired amplifications with other PCR protocols, as described in Morán et al 47 .…”

“…universal real-time PCR by Bertolini et al 46 has been removed as it produces false-positive results 36 . However, recent works have demonstrated that both real-time PCRs by Bertolini et al 46 and Li et al 44 , although with a good sensitivity, produced undesired amplifications in environmental samples due to their highly conserved nature of the 16S rRNA gene used as target 47 . Other universal real-time PCR protocols, such as Ananthakrishnan et al 48 , based on other genes, have been described but not validated according to EPPO requirements 49 .…”

A Synoptic Review of Plant Disease Epidemics and Outbreaks Published in 2021

“…In many cases, the detection of plant pathogenic bacteria is made from samples of different environmental origin: soils and plant debris, living plants, and insects. Diagnosis of HLB using standardised RT-PCR methods was attempted from samples taken from plants, psyllid vectors, and parasitoids of the psyllid (Morán et al, 2021), a total of 4915 samples. A total of 2783 samples were taken from asymptomatic plants collected in surveys made between 2009 and 2018 in Spain.…”