Plant Science

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.

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.

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.

Mung bean (Vigna radiata (L.) R. Wilczek) is an important edible legume grown in Asia, particularly in the Indian subcontinent, where it is used for human and animal consumption. In September 2013, 10% of a group of 90 mung bean breeding lines in experimental plots of S. V. Agricultural College, Tirupati, Andhra Pradesh, India, exhibited symptoms typical of a phytoplasma infection, including stunting, extensive proliferation of branches, reduction in leaf size, phyllody, and longitudinal splitting of green pods followed by germination of green seeds producing small plants. These symptoms have been associated with mung bean phyllody (1,3). While the severity of infection varied within each line, on average, 20% of symptomatic plants did not produce seeds at all. Leaf samples from two symptomatic plants and one asymptomatic plant were collected and DNA was extracted from leaves following a CTAB DNA extraction procedure (2). Direct PCR and nested PCR assay was performed using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (4). Both of the symptomatic samples produced 1.8 kb and 1.2 kb size amplicons after direct and nested PCR cycles, respectively. No amplicons were produced with DNA from the asymptomatic sample. Nested PCR products (1.2 kb) from the two symptomatic samples corresponding to the F2nR2 region of 16S rDNA were directly sequenced on an ABI 3730 XL automated sequencer at McLab sequencing services (McLab, CA). Both samples were 100% identical and the representative sequence was designated as APMBP and deposited in GenBank with the accession number KF811205. BLAST analysis revealed a 100% sequence identity with 16SrII group ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas that include sesame phyllody phytoplasmas isolated from sesame and tomato in India (KF429485 and JX104335, respectively). Subgroup identification was performed using the iPhyClassifier online tool (5). The samples were identified as 16SrII-D subgroup phytoplasma based on their 100% identity to the reference strain (GenBank Accession No. Y10097) and virtual RFLP profiles. Phylogenetic analysis based on the F2nR2 sequences with the representative sequences were placed the APMBP in a single distinct cluster with the 16SrII-D reference strain Y10097. Although occurrence of phyllody on mung bean in India was first reported in 1988 (3), the report was based on the appearance of symptoms. This pathogen was recently reported as associated with mung bean phyllody in Pakistan (1). To our knowledge, this is the first report of ‘Ca. P. aurantifolia’ strain infecting mung bean in India. Phytoplasmas belonging to subgroup 16SrII-D are known to have a wide host range, including chickpea, peanuts, sesame, and tomato, which are commonly grown in this region. Mung bean plants infected early failed to produce normal seeds indicating of the potential of this 16SrII-D phytoplasma to become a production constraint for mung bean and other host crops in the area. References: (1) K. P. Akhtar et al. Plant Pathol. 59:399, 2010. (2) J. J. Doyle and J. L. Doyle. Phytochem. Bull. 19:11, 1987. (3) P. Lakshmanan et al. Curr. Sci. 57:809, 1988. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.