Lin, H.

In January 2011, tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers, and reduced-size fruits, symptoms similar to those exhibited by plants infected by ‘Candidatus Liberibacter solanacearum’ (2), were observed in approximately 5% of tomato plants in greenhouses in Jocotitlan in the State of Mexico, Mexico. Occasional plant recovery was also observed. Tomato plants in this facility were previously shown to be infected by Mexican papita viroid (MPVd), Pepino mosaic virus (PepMV), and aster yellows phytoplasma. Eight symptomatic leaf samples (designated MX11-01 to MX11-08) were collected and screened against selected tomato viruses and pospiviroids by reverse transcription (RT)-PCR using purified plant RNA or for ‘Ca. L. solanacearum’ by PCR using purified plant DNA. As expected, both PepMV and MPVd were detected in these samples. However, two ‘Ca. L. solanacearum’-specific PCR products (1,168 and 669 bp) were also amplified in two samples (MX11-02 and MX11-05) using primers OA2 (2) and OI2c (1) or CL514F/CL514R (3), respectively. Each ‘Ca. L. solanacearum’-specific PCR product was gel purified with Geneclean (Q-Biogene, Carlsbad, CA) and cloned into pCR2.1 using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced (Functional Biosciences, Madison, WI). Sequences of 16S rRNA (1,168 bp) in both isolates (GenBank Accession Nos. JF811596 and JF811597) were identical. However, the 669-bp 50S rRNA sequences in these two isolates (GenBank Accession Nos. JF811598 and JF811599) contained two single nucleotide polymorphism (SNP) mutations. BLASTn searches showed that both 16S rRNA and 50S gene sequences in MX11-05 were identical to the ‘Ca. L. solanacearum’ previously identified on potato in Chihuahua (GenBank Accession Nos. FJ829811 and FJ829812) and Saltillo (GenBank Accession Nos. FJ498806 or FJ498807) in eastern Mexico. These ‘Ca. L. solanacearum’ isolates were recently classified as the “b” haplotype (4). Alignment analysis of the ‘Ca. L. solanacearum' 16S rRNA sequences also revealed the conserved SNP mutations (g.212T > G and g.581T > C) in MX11-02 and MX11-05 as previously identified for other “b” haplotype isolates (4). ‘Ca. L. solanacearum’ was first identified in greenhouse tomatoes in 2008 in New Zealand (2). It has also been identified in greenhouse and field tomatoes in the United States. ‘Ca. L. solanacearum’ was previously reported to infect field tomatoes in Sinaloa, Mexico (3), which was recently considered as the “a” haplotype (4). To our knowledge, this is the first report of ‘Ca. L. solanacearum’ naturally infecting tomatoes in Jocotitlan in the State of Mexico, Mexico. The greenhouse tomato ‘Ca. L. solanacearum’ may be transmitted from infected solanaceous plants by potato psyllids (Bactericera cockerelli), which were observed in this facility. References: (1) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009 (4) W. R. Nelson et al. Eur. J. Plant Pathol. 130:5, 2011.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.