Association of ‘Candidatus Liberibacter solanacearum’ with a Vegetative Disorder of Celery in Spain and Development of a Real-Time PCR Method for Its Detection

Abstract

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

Transmission of ‘Candidatus Liberibacter solanacearum’ by Bactericera trigonica Hodkinson to vegetable hosts

“…Used in the following experiments, individuals from this colony were regularly tested by real-time PCR (see below) to confirm the presence of CaLso. The CaLso haplotype found in the colony was the haplotype E, which was determined according to Teresani et al (2014).…”

“…The detection of CaLso was performed with a complete real-time PCR Kit CaLso/100 (Plant Print Diagnòstics) according to the manufacturer's instruction and based on the protocol described by Teresani et al (2014). The assays were conducted with the StepOne Plus (Applied Biosystems) machine, and the data acquisition and analyses were performed with the thermal cycler's software.…”

“…CaLso is transmitted by Bactericera cockerelli Sulc in potato and tomato (Secor et al, 2009) and by Trioza apicalis Förster in carrot in northern Europe (Nissinen et al, 2014). Bactericera trigonica Hodkinson is associated with vegetative disorders of carrot and celery in Spain (Alfaro-Fernández et al, 2012;Teresani et al, 2014), and Antolínez et al (2017aAntolínez et al ( , 2017b described the feeding behaviour of B. trigonica on carrot plants and reported that the transmission of CaLso was not affected by the vector gender. Moreover, Munyaneza et al (2016) assessed the ability of the potato psyllid B. cockerelli to transmit CaLso from potato to carrot, and Antolínez et al (2017b) the risk of transmission of CaLso from Apiaceae to potato by B. trigonica.…”

Haplotypes of “Candidatus Liberibacter europaeus” also separate by geography and host species

“…L. solanacearum" (Lso) is a Liberibacter species vectored by a number of species of Triozid psyllids and causes considerable damage in both Solanaceae and Apiaceae crops in North America and western Europe respectively (Munyaneza, 2013). Five haplotypes have now been described within this bacterial species through SNPs on the 16S, 16S-23S intergenic spacer region and 50S genes (Nelson, Fisher & Munyaneza, 2011;Teresani et al, 2014), as well as describing differences in geographic and plant/insect host ranges. Haplotyping has a number of important disease management aspects, for example as a means of aiding in determining the source of incursions (Nelson et al, 2014;Thomas et al, 2011), or apparent differences in host responses (Rashed et al, 2014).…”

“…Similarly, one of the haplotypes of Lso is recorded to produce symptoms on celery very similar to those associated with "Ca. Phytoplasma" (Teresani et al, 2014).…”