Publications

4523

Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’ (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp. clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.

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.

‘Candidatus Liberibacter solanacearum’ has recently been reported to be associated with vegetative disorders and economic losses in carrot and celery crops in Spain. The bacterium is a carrot seedborne pathogen and it is transmitted by psyllid vector species. From 2011 to 2014 seasonal and occasional surveys in carrot, celery and potato plots were performed. The sticky plant method was used to monitor the arthropods that visited the plants. The collected arthropods were classified into Aphididae and Cicadellidae, and the superfamily Psylloidea was identified to the species level. The superfamily Psylloidea represented 35.45% of the total arthropods captured on celery in Villena and 99.1% on carrot in Tenerife (Canary Islands). The maximum flight of psyllid species was in summer, both in mainland Spain and the Canary Islands, reaching a peak of 570 specimens in August in Villena and 6,063 in July in Tenerife. The main identified psyllid species were as follows: Bactericera trigonica Hodkinson, B. tremblayi Wagner and B. nigricornis Förster. B. trigonica represented more than 99% of the psyllids captured in the Canary Islands and 75% and 38% in 2011 and 2012 in Villena, respectively. In addition, Trioza urticae Linnaeus, Bactericera sp., Ctenarytaina sp., Cacopsylla sp., Trioza sp. and Psylla sp. were captured. ‘Ca. L. solanacearum’ targets were detected by squash real-time PCR in 19.5% of the psyllids belonging to the different Bactericera species. This paper reports at least three new psyllid species that carry the bacterium and can be considered as potential vectors.

Significance TM7 is one of the most enigmatic bacterial phyla among the uncultivated candidate phyla referred to as “microbial dark matter,” and it has potential pathogenic associations. We revealed molecular insights into its uncultivability and pathogenicity, as well its unique epibiotic and parasitic lifestyle phases. These novel discoveries shed significant light on the biological, ecological, and medical importance of TM7, as well as providing useful information for culturing other TM7 and currently uncultivable bacteria that may evade standard cultivation approaches.