Publications

4526

‘Candidatus Phytoplasma solani’ is the causal agent of the Bois noir (BN), affecting grapevine worldwide. The complex epidemiology of BN, which involves multiple ‘Ca. P. solani’ host plants and insect vectors, as well as the occurrence of recovery (loss of symptoms on grapevine canopy), makes disease investigations and containment in vineyards difficult. To achieve early detection of ‘Ca. P. solani’, a droplet digital PCR (ddPCR)-based approach and quantitative (q)PCR assay were compared, testing specific primers based on the elongation factor Tu (tuf) gene using SYBR Green chemistry. The regression curve analysis of the ddPCR assay showed good linearity. Compared with the qPCR method, the sensitivity of ddPCR improved about 10-fold. The analysis of grapevine roots spiked with serial dilutions of ‘Ca P. solani’. PCR tuf fragments showed that qPCR was inhibited, while ddPCR was not affected. Testing 66 grapevine samples from 50 grapevine plants, the ddPCR provided superior diagnostic performance compared to qPCR in roots of symptomatic plants (75% detected by ddPCR, 41.6% by qPCR), roots of recovered plants (58.8% detected by ddPCR, 25% by qPCR), and asymptomatic leaf tissues from recovered plants (75% detected by ddPCR, 25% by qPCR). The ddPCR analysis allowed us to detect ‘Ca. P. solani’ on 40% of leaf samples from recovered plants and 20% of roots from asymptomatic plants. No differences among ddPCR and qPCR were found in detecting phytoplasma on symptomatic leaf samples. The ddPCR assay allowed the absolute quantification of ‘Ca. P. solani’ in complex matrices, such as roots, and when low titer of phytoplasma is present.

This is a general protocol for the vegetative propagation of citrus material infected with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (HLB), using an aeroponic cloning apparatus. The following procedure details a workflow using an EZ-Clone Pro Low Cloning System from Hydrobuilder.com. This system comes in varying cell-sizes (number of clones). We have found the most versatile to be the 16- or 32-cell size based on ease of preparation, keeping algae growth to a minimum, and maintenance. Cloning versus seed sowing has been useful in producing infected material for experimental assays requiring cloned, mature citrus trees as opposed to genetically variable plants from seeds. Where seed germination can take up to 12+ months to achieve plants with stem diameters ≥ 10 mm and 5+ years for flowering, infected branches from flowering trees can be directly propagated using this protocol within a few months and retain their maturity. Clone viability rate differs by citrus cultivar and general health of the cutting used, with citron and CLas negative material having the highest success rate. Infected citron cuttings have had a 95% survival rate, regardless of the diameter of the cutting. The cutting diameter ranged from 2mm-12mm and was limited to 12mm maximum, which is the size of the collar on the EZ-Clone Pro Low cloner.

‘ Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, and so far is being mainly restricted to Europe. Here, we report draft genome sequences for ‘ Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of P. persica in Germany, and LNS1 is the causal agent of leptonecrosis disease of P. salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases assembled into 28 and 16 contigs, with an average GC content of 21.1 and 20.7%, respectively. Analysis of average nucleotide identity (ANI) values between genomes further supported the delineation of ‘ Ca. P. prunorum’ as a species distinct from ‘ Ca. P. mali’, in agreement with previous phylogenetic data. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

‘ Candidatus Phytoplasma pyri’, the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of ‘ Ca. P. pyri’ using a strain from Chile, with its genomic features analyzed in comparison with the closely related ‘ Ca. Phytoplasma’ species ‘ Ca. P. mali’ and ‘ Ca. P. prunorum’. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of ‘ Ca. P. mali’ was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from 10 Chilean and 10 Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy, collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of ‘ Ca. P. pyri’.