Agronomy and Crop Science

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.

Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated CandidatusLiberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorinacitri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.

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.

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.

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.

‘Candidatus Phytoplasma prunorum,’ which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to ‘Ca. P. pyri,’ which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, ‘Ca. P. pyri’ is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, ‘Ca. P. prunorum’ is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and ‘Ca. P. pyri’ is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of ‘Ca. P. prunorum,’ was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km2 area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR. References: (1) C. L. Blomquist et al. Plant Dis. 86:759, 2002. (2) J. L. Danet et al. Microbiology 157:438, 2011. (3) M. Garcia-Chapa et al. J. Phytopathol. 151:584, 2003. (4) E. Seemüller et al. Int. J. Syst. Evol. Microbiol. 54:1217, 2004.

Corn reddening (CR) or maize redness is a severe disease of corn (Zea mays L.) associated with ‘Candidatus Phytoplasma solani’ or stolbur phytoplasma (16SrXII-A). In Serbia, CR is continually present at a low frequency, while two outbreaks occurred in the late 1950s and 1990s. Its etiology was molecularly determined in 2006 (1). The first severe outbreak in Bulgaria was observed in Kneja in 1992, and in 2010 typical CR symptoms (leaf reddening, premature drying, and shriveled grains) were observed from Byala Slatina to Pleven. Although the number of CR affected plants was highly variable in different fields, the disease incidence in most cases was 30 to 50%, with an estimated yield reduction of about 20%. Leaf samples from four symptomatic corn plants were collected from Kneja, northwestern Bulgaria, in mid-August 2013. Extraction of DNA was performed from the main leaf midrib tissues using the CTAB method. Separate PCRs were carried out for amplification of the phytoplasma 16S rDNA and tuf genes using the phytoplasma specific primers P1/P7 and TufAyf/r, respectively. DNA from asymptomatic corn plants and reactions without template DNA were employed as negative controls, while DNA from periwinkle tissue infected with ‘Ca. P. asteris’ was used as a positive control. Amplicons of the expected sizes (1.7 and 0.9 kbp, respectively) were produced with DNA from three out of four symptomatic corn samples, while no amplification was observed with DNA from one symptomatic corn sample (probably because samples were collected during a drought period) nor the DNA from asymptomatic plants and negative control. RFLP analyses performed on 16S rDNA and tuf gene amplicons using Tru1I and HpaII restriction enzymes, respectively, revealed the presence of ‘Ca. P. solani’ (16SrXII-A, tuf type b) in all three positive samples (3). Both amplicons of a selected representative sample 241/13 were directly sequenced by a commercial service and the obtained sequences were deposited in NCBI GenBank under the accession number KF907506 for the16S rDNA (1,684 bp) and KF907507 for the tuf gene (896 bp). The 16S rDNA sequence of phytoplasma detected in Bulgarian corn shared a complete sequence identity with ‘Ca. P. solani’ strain from Serbian corn (JQ730750.1) and >99.7% sequence identity with the reference strain STOL (AF248959), while the tuf gene nucleotide sequence shared complete sequence identity with several ‘Ca. P. solani’ (e.g., Serbian strain 284/09, FO393427), thus confirming, with both genes, the affiliation of phytoplasmas in Bulgarian corn to ‘Ca. P. solani’. This study adds new information on CR prevalence, previously reported in neighboring countries. Further studies will investigate the roles of Hyalesthes obsoletus and Reptalus panzeri, both polyphagous Cixiidae reported as CR vectors (2,4) in disease transmission in Bulgaria. References: (1) B. Duduk and A. Bertaccini. Plant Dis. 90:1313, 2006. (2) J. Jović et al. Eur. J. Plant Pathol. 118:85, 2007. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) N. Mori et al. Bull. Insectol. 66:245, 2013.