Plant Science

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.

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.

Potatoes are a major crop in the Columbia Basin of Oregon and Washington, representing an annual farm gate value of almost $750 million. Zebra chip disease (ZC), a new and economically important disease of potato, was first reported in Oregon and Washington in 2011 (1). The disease is caused by the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso, also referred to as ‘Ca. L. psyllaurous’), which is vectored by the potato psyllid (Bactericera cockerelli Sulc) (1,2). Identifying alternative hosts for Lso may facilitate management of ZC disease, which has increased potato production costs in the region. The perennial weed, bittersweet nightshade (Solanum dulcamara L.), is a year-round host of the potato psyllid (3) and is also a suspected host of Lso. However, little is known about the role of this weed in ZC epidemiology. Naturally occurring bittersweet nightshade plants (n = 21) were sampled at six different locations near Hermiston, Oregon, between May and October in 2012. These plants exhibited several symptoms associated with Lso, ranging from asymptomatic to slight purpling, chlorosis, or scorching of the foliage. However, S. dulcamara exhibits similar symptoms under a variety of environmental conditions (drought stress, etc.); therefore, it was difficult to identify potentially infected plants based solely on symptomology. Leaf and stem tissue (n = 21) was analyzed with high-fidelity PCR using species-specific primers for the 16S rDNA gene, CLipoF, and OI2c (2,4). Approximately 27.3% of the plants tested positive for Lso using these primers, including plants from the following locations on 16 April, 16 May, and 24 May, respectively: Hat Rock, OR (45°55.033′ N, 119°10.495′ W), Irrigon, OR (45°54.560′ N, 119°24.857′ W), and Stanfield, OR (45°46.971′ N, 119°13.203′ W). Three plants were selected for further PCR analysis with primers for the outer membrane protein gene, 1482f and 2086r (1). Amplicons obtained with both sets of PCR primers were directly sequenced. A BLAST analysis showed that the 16S rDNA gene sequence (993 to 1,000 bp) shared 99 to 100% identity with several Lso accessions, including JN848751.1 (from Washington) and JN848753.1 (from Oregon). Likewise, the outer membrane protein gene sequence (600 to 601 bp) shared 99 to 100% identity with ‘Ca. L. solanacearum’ accession KC768330.1 (from Honduras). All six sequences were deposited in GenBank (Accession Nos. KJ854199 to KJ854204). According to these findings, bittersweet nightshade may be an important annual source of Lso in the region, particularly since it serves as a host for the potato psyllid. Potato psyllids were also detected at two of the locations with infected S. dulcamara: Irrigon, OR, and Stanfield, OR. A subsample of the psyllids collected in 2012 were analyzed with PCR and Lso was detected in a sample from Stanfield, OR (5). Identifying perennial hosts of Lso promotes a better understanding of both ZC disease epidemiology and management. To our knowledge, this is the first report of Lso causing natural infections in S. dulcamara in the United States. References: (1) J. M. Crosslin et al. Plant Dis. 96:452, 2012. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) A. F. Murphy et al. Am. J. Pot. Res. 90:294, 2013. (4) G. A. Secor et al. Plant Dis. 93:574, 2009. (5) K. D. Swisher et al. Am. J. Pot. Res. 90:570, 2013.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with ‘Candidatus Liberibacter solanacearum’ and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect ‘Ca. L. solanacearum’. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of ‘Ca. Liberibacter’ were observed using electron microscopy in celery plant tissues. A fifth haplotype of ‘Ca. L. solanacearum’, named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

A survey carried out in Georgian vineyards, located in the Khaketi region, in September 2013, showed the presence of vines of the cultivar Chardonnay with typical grapevine yellows (GY) symptoms including leaf discoloration and curling, berry shriveling, and irregular maturation of wood. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing shoot proliferation and leaf yellowing were found, suggesting the involvement of phytoplasmas in the disease etiology. Total DNA was extracted by a CTAB method from leaf veins of 18 symptomatic and two asymptomatic grapevines, and from four symptomatic and two asymptomatic bindweeds, and analyzed by PCR assays. Moreover, DNA extracted from ‘Candidatus Phytoplasma asteris’ strain SAY (group 16SrI), ‘Ca. P. solani’ strain STOL (group 16SrXII), and ‘Ca. P. ulmi’ strain EY1 (group 16SrV) were used as positive controls. DNA extracted from healthy periwinkle and a reaction mixture without template were employed as negative controls. Nested PCRs targeting the 16S rDNA, carried out using the primer pairs P1/P7 followed by R16F2n/R16R2 (1), produced a band of the expected size (1,250 nt) in all the symptomatic grapevine and bindweed plants, and in the positive controls. No amplification was observed with DNA from asymptomatic plants nor the negative controls. PCR products were sequenced by a commercial sequencing service (Primm, Milan, Italy). The 16S rDNA nucleotide sequences of phytoplasmas identified in all grapevines and in two bindweed samples shared >99.5% sequence identity with ‘Ca. P. solani’ reference strain STOL (GenBank Accession No. AF248959), and carried identical STOL-unique signature sequence and distinguishing sequence blocks (3). Moreover, nucleotide sequences of phytoplasmas identified in the other two bindweed samples shared >99.6% sequence identity with ‘Ca. P. convolvuli’ reference strain BY-S57/11 (JN833705) (2). RFLP and phylogenetic analyses confirmed the affiliation of the phytoplasma strains identified in grapevine and bindweed plants in Georgia to the species ‘Ca. P. solani’ (subgroup 16SrXII-A) and ‘Ca. P. convolvuli’ (subgroup 16SrXII-H). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank website with accession nos. KF996535 and KF996536 (‘Ca. P. solani’ from grapevine and bindweed, respectively), and KF996537 (‘Ca. P. convolvuli’). Future studies will focus on investigating the spread and impact of ‘Ca. P. solani’-associated bois noir (BN) in Georgia. In particular, the identification of ‘Ca. P. solani’ in bindweeds suggested the presence of the insect Hyalesthes obsoletus, a polyphagous cixiidae responsible for BN phytoplasma transmission in vineyards in Europe. Accurate surveys and molecular analyses will be performed for identifying the insect vector(s) of the BN associated phytoplasma strains in Georgia. Additional studies will be performed to study the spread and impact of ‘Ca. P. convolvuli,’ identified only in Italy, Germany, Serbia, and Bosnia and Herzegovina (2), throughout the Caucasian countries. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) M. Martini et al. Int. J. Syst. Evol. Microbiol. 62:2910, 2013. (3) F. Quaglino et al. Int. J. Syst. Evol. Microbiol. 63:2879, 2013.

Abstract In two of Iran's central provinces, several herbaceous plants showing phytoplasma disease symptoms were collected to detect 'Canididatus Phytoplasma asteris'-related phytoplasmas. Confirmation of an association of phytoplasmas with diseased plants was done using polymerase chain reaction (PCR) assays having the phytoplasma universal primer pairs P1/P7 followed by R16F2n/ R16R2 in nested PCR. Then, for detection of 'Ca. P. asteris', DNA samples were subjected to amplification of rp and tuf genes using specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy, respectively. Restriction fragment length polymorphism or RFLP analyses of rp gene fragments using Tsp509I restriction enzyme as well as sequence analyses indicated that 'Ca. P. asteris'-related phytoplasmas associated with carrot, niger seed and scallion plants in these regions, belong to the rpI-L subgroup. This research is the first report of carrot, niger seed, and scallion infection with phytoplasmas belonging to the rpI-L subgroup.