Agronomy and Crop Science

The bacterium ‘Candidatus Liberibacter solanacearum’ is associated with zebra chip disease (ZC), a threat to potato production in North America and New Zealand. It is vectored by potato psyllids. Previous studies observed that ‘Ca. L. solanacearum’ infection causes potato tubers to undergo ZC-symptom-associated shifts in physiology, such as increased levels of amino acids, sugars, and phenolics. However, little is known about how ‘Ca. L. solanacearum’ infections caused by psyllid vector feeding may affect metabolism in potato foliage and stems. This study compared metabolism in potato plants fed upon by ‘Ca. L. solanacearum’-positive psyllids with potato plants not exposed to psyllids. Foliar levels of asparagine, aspartic acid, glutamine, fructose, glucose, sucrose, a ferulic acid derivative, and quinic acid were lower in ‘Ca. L. solanacearum’-inoculated than noninfected plants. However, foliar levels of proline, serine, four phenolic compounds, and most terpenoids were greater in ‘Ca. L. solanacearum’-inoculated than noninfected plants. Upper stem levels of asparagine and aspartic acid, upper and lower stem levels of ellagitannins and most monoterpenoids, and lower stem level of sesquiterpenoids were greater in ‘Ca. L. solanacearum’-inoculated than noninfected plants. These results suggest that many defense-related terpenoid compounds might increase in plants which had psyllids inoculate ‘Ca. L. solanacearum’. This could impact progression and spread of ZC.

Juncus articulatus L. (Juncaceae) is a species of rush occurring in Eurasia, Canada, and the United States. In 2013, symptoms of “witches'-brooms,” similar to those associated with phytoplasma infection in other plants, were observed on jointleaf rush plants in Lower Silesia (southwest Poland), with some pests feeding on them. Livia junci (Liviinae, Hemiptera) is a small plant-feeding sap-sucking insect that affects monocotyledonous plants. To confirm the presence of phytoplasma in 15 examined plants, total DNA was extracted from 100 mg of inflorescence and leaf samples collected in July 2013 in Bogatynia, Poland, from six symptomatic and six asymptomatic plants using a DNeasy Plant Mini Kit (Qiagen, Syngen Biotech, Wrocław, Poland) according to the manufacturer's protocol. Additionally, three leaf samples from asymptomatic rush plants, collected from a location where the disease was not observed (Wrocław, Poland), as well as water blank samples were included as negative controls. Moreover, thirty-two insects were collected from symptomatic plants and preserved in ethanol (75%). DNA from L. junci specimens (the imago and the last larva stage) was extracted using DNeasy Blood and Tissue Kit (Qiagen, Syngen Biotech). Extracted nucleic acids were used as templates for PCR employing a) phytoplasma universal rRNA primer pairs P1/P7 followed by R16F2n/R16R2 (1), b) primers rp1-rp2 followed by rp3-rp4, allowing amplification of fragments of ribosomal protein rpl22 and rps3 genes (3), and c) primers AYsecYF1/AYsecYR1 (2) for amplification of the secY gene. The phytoplasma was detected in all tested insects as well as in all six symptomatic and four out of six asymptomatic plant samples (10 out of 12 plant samples from Bogatynia were positive). No amplification products were detected in negative control samples from Wrocław or in water blanks. The fact that we detected the pathogen in some asymptomatic plants indicated that a low concentration may have been present prior to the development of disease symptoms. Amplicons representing three genetic loci were sequenced in an AbiPrism 3100 Genetic Analyzer apparatus (Applied Biosystems, USA), at the Maria Skłodowska Memorial Cancer Center and Institute of Oncology, Warsaw, Poland. To avoid sequencing errors, all DNA samples were sequenced twice in both directions. The obtained sequences were nearly identical, and representative sequences of 16S rDNA fragments (Accession Nos. KF774297, KF774298, and KF774299), secY gene (KJ394481, KJ394482, and KJ394483) and ribosomal protein gene (KJ394484, KJ394485, and KJ394486), isolated from two plants and one insect, were deposited in GenBank. BLAST analysis of the sequenced 16S rDNA fragments revealed that tested strains shared more than 99% sequence identity with the sequences of phytoplasmas from the aster yellows group (e.g., KJ556903, KJ494330, and KJ491100). The same analysis performed for ribosomal proteins and secY genes confirmed the highest identity (99%) of analyzed sequences with those of ‘Candidatus Phytoplasma asteris’ (HM626105 and KC354611, respectively). The impact of the detected phytoplasma in the regional ecosystem and the role of L. junci as a possible vector of this pathogen are being assessed. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 48:1153, 1998. (2) I.-M. Lee et al. Mol. Cell. Probes 20:87, 2006. (3) H. Nakamura et al. Plant Dis. 80:302, 1996.

Zebra chip (ZC) is a disease of potato, putatively caused by the vectorborne bacterium ‘Candidatus Liberibacter solanacearum’. Although ZC has been a major concern due its significant negative impact on both potato yield and quality, its effect on seed potato sprouting has been the subject of recent evaluations. The present study was conducted to determine whether variation in emergence is affected by the infection duration of ‘Ca. L. solanacearum’-infected seed potato prior to harvest. Furthermore, changes in pathogen detectability and titer levels in late-season-infected plants also were evaluated during and after cold storage. The rate of ZC-affected seed potato emergence following cold storage was not affected by the time of infection in the field, and the majority of ZC-infected tubers failed to sprout. Time to “seedling” emergence also was significantly longer in seed potato from plants infected ≥2 weeks before harvest. The small percentage of plants that emerged from ZC-affected seed potato produced stunted, nonvigorous plants that often died after a few weeks. The rate of successful ‘Ca. L. solanacearum’ detection increased during cold storage, suggesting a continued ‘Ca. L. solanacearum’–tuber interaction postharvest. After tubers were removed from cold storage and held at room temperature, ‘Ca. L. solanacearum’ titer started to increase. Although none of the tubers from plants infected 1 week before harvest exhibited any disease symptoms or tested positive for ‘Ca. L. solanacearum’ at harvest, up to 38% of these tubers tested positive following placement at room temperature after cold storage. Results of this study suggest that the role of seedborne ZC in disease epidemiology is likely to be insignificant. Furthermore, the findings of this study emphasized the importance of continued control measures until at least a week before harvest, and highlighted the need for improved methods of ‘Ca. L. solanacearum’ detection at harvest, especially in tubers infected late in the season.

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jović et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.

In March of 2014, carrot plants (Daucus carota L. var. Mascot) exhibiting symptoms of yellowing, purpling, and curling of leaves, proliferation of shoots, formation of hairy secondary roots, general stunting, and plant decline were observed in commercial fields in the Gharb region of Morocco. The symptoms resembled those caused by phytoplasmas, Spiroplasma citri, or ‘Candidatus Liberibacter solanacearum’ infection (1,2,3). About 30% of the plants in each field were symptomatic and plants were infested with unidentified psyllid nymphs; some psyllids are known vectors of ‘Ca. L. solanacearum.’ A total of 10 symptomatic and 2 asymptomatic plants were collected from three fields. Total DNA was extracted from petiole and root tissues of each of the carrots, using the CTAB buffer extraction method (3). The DNA samples were tested for phytoplasmas and spiroplasmas by PCR (3) but neither pathogen was detected in the samples. The DNA extracts were tested for ‘Ca. L. solanacearum’ by PCR using specific primer pairs OA2/OI2c, Lso adkF/R, and CL514F/R, to amplify a partial fragment of the 16S rDNA, the adenylate kinase gene, and rpIJ/rpIL50S rDNA ribosomal protein genes, respectively (1,2,5). DNA samples from all 10 symptomatic carrots yielded specific bands; 1,168 bp for the 16S rDNA fragment, 770 bp for the adk fragment, and 669 bp for rpIJ/rpIL, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in asymptomatic plants. DNA amplicons of three plant samples (one plant/field) for each primer pair were directly sequenced (Macrogen Inc., Amsterdam). Sequencing results identified two distinct products for the OA2/OI2c primer pair (GenBank Accession Nos. KJ740159 and KJ740160), and BLAST analysis of the 16S rDNA amplicons showed 99 and 100% identity to ‘Ca. L. solanacearum’ (KF737346 and HQ454302, respectively). Two different sequences of the adk amplicon were obtained (KJ740162 and KJ740163), both of which were 98% identical to ‘Ca. L. solanacearum’ (CP002371). Sequencing results also identified two distinct products for the CL514F/R primer pair (KJ754506 and KJ754507), and BLAST analysis of the 50S rDNA ribosomal protein showed 99 and 100% identity to ‘Ca. L. solanacearum’ (KF357912 and HQ454321, respectively). The differences in our 16S and 50S rDNA sequences identified the presence of both ‘Ca. L. solanacearum’ haplotypes D and E (4). To our knowledge, this is the first report of the occurrence of ‘Ca. L. solanacearum’ in Morocco and Africa, suggesting a wider distribution of the bacterium in carrot crops in the Mediterranean region, including North Africa. ‘Ca. L. solanacearum’ has caused economic damages to carrot and celery crops in the Canary Islands and mainland Spain, France, Sweden, Norway, and Finland (3). This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in the United States, Mexico, Central America, and New Zealand (1,2,5). Given the economic impact of ‘Ca. L. solanacearum’ on numerous important crops worldwide, it is imperative that preventive measures be taken to limit its spread. References: (1) L. W. Liefting et al. Plant Dis. 93:208, 2009. (2) J. E. Munyaneza et al. Plant Dis. 93:552, 2009. (3) J. E. Munyaneza et al. J. Plant Pathol. 93:697, 2011. (4) W. R. Nelson et al. Eur. J. Plant Pathol. 135:633, 2013. (5) A. Ravindran et al. Plant Dis. 95:1542, 2011.