Page 1 of 37 1 ‘Candidatus Phytoplasma asteris’ strains associated with oil palm lethal wilt in Colombia 2 3 Elizabeth Alvarez, Plant Pathology Program, International Center for Tropical Agriculture 4 (CIAT), Cali, Valle del Cauca, Colombia; Juan F. Mejía, CIAT and Department of 5 Agricultural Sciences and Technologies (DipSA), Alma Mater Studiorum, University of 6 Bologna, Italy; Nicoletta Contaldo and Samanta Paltrinieri, DipSA; Bojan Duduk, 7 Institute of Pesticides and Environmental Protection, Belgrade, Serbia; and Assunta 8 Bertaccini, DipSA. 9 10 Corresponding author: Elizabeth Alvarez 11 Email: E.ALVAREZ@CGIAR.ORG 12 13 GenBank accession numbers: JX681021, JX681022, JX681023, KF434318, KF434319, 14 KF434320 15 16 All authors have reviewed the manuscript and have approved its submission to the journal 17 Plant Disease. The manuscript is not being submitted elsewhere. 18 19 20 Alvarez et al. 1 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 2 of 37 21 22 Abstract 23 24 Alvarez, E., Mejía, J. F., Contaldo, N., Paltrinieri, S., Duduk, B., and Bertaccini, A. 25 ‘Candidatus Phytoplasma asteris’ strains associated with oil palm lethal wilt in 26 Colombia. Plant Dis. xx: xxx-xxx. 27 28 The distribution of lethal wilt, a severe disease of oil palm, is spreading throughout South 29 America. An incidence of about 30% was recorded in four commercial fields in Colombia. In 30 this study, phytoplasmas were detected in symptomatic oil palms by using specific primers, 31 based on 16S rDNA sequences, in nested polymerase chain reaction assays. The phytoplasmas 32 were then identified as ‘Candidatus Phytoplasma asteris’, ribosomal subgroup 16SrI-B, 33 through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing. 34 Cloning and sequencing of 16S rDNA from selected strains, together with phylogenetic 35 analysis, confirmed the classification. Moreover, collective RFLP characterization of the 36 groEL, amp, and rp genes, together with sequence data, distinguished the aster yellows strain 37 detected in Colombian oil-palm samples from other aster yellows phytoplasmas used as 38 reference strains, in particular from an aster yellows strain infecting corn in the same country. 39 40 ___________________________________________________________________________ 41 42 Production of oil palm (Elaeis guineensis Jacq.) is a recent and now a major agricultural 43 activity in Colombia. Because it constitutes a key alternative for generating employment, it is 44 considered strategic for the national economy. However, production has declined by 7.1% 45 since 2002. This drop occurred mainly in northeastern Colombia, where production decreased Alvarez et al. 2 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 3 of 37 46 by almost 10% (17). The cause of this decrease is a disease known as “lethal wilt of oil palm” 47 (“marchitez letal” in Spanish). Lethal wilt is present in Colombia in the Upía River area, in the 48 oil-palm plantations of Palmar del Oriente (July 1994), Palmas del Casanare (1999), Palmeras 49 Santana (2000), and Palmeras del Upía (2002) (44). By 2010, the disease had severely 50 infected oil palms in these areas, leading to their eradication on about 690 hectares with a total 51 of 97,619 plants (17). Symptoms of lethal wilt usually first appear as vascular discoloration 52 and leaf yellowing when the palm is mature (i.e., flowering and fruiting) at seven years old. 53 These symptoms are followed by leaf drying, wilt, and necrosis of infected tissues, and 54 eventual plant collapse (Fig. 1). Root necrosis often accompanies leaf discoloration. Internal 55 discoloration of trunk tissue may also occur but does not represent a distinctive symptom. 56 Lethal wilt is potentially destructive because it spreads rapidly and causes plant death within 4 57 to 6 months after symptoms first appear (43). 58 The pathogen was believed to be a phytoplasma, possibly related to that associated with 59 lethal yellowing disease, which infect other palms such as coconut (2). Phytoplasmas have 60 been associated with diseases in several hundred plant species (6). They are limited to the 61 plants’ phloem tissue, and to insect vectors that feed directly from phloem tissues. Vectors 62 include planthoppers and leafhoppers in the genera Macrosteles, Euscelis, Euscelidius, and 63 Scaphoideus, and Cacopsylla (52). 64 Phytoplasmas are associated not only with lethal yellowing in coconut palms in many 65 parts of the world, but also with diverse palm species. Worldwide this disease affects at least 66 30 species of palm, including Phoenix dactylifera (date palm), Veitchia merrilli (manila palm), 67 Caryota rumphiana (fishtail palm), Phoenix canariensis (Canary Island date palm), and Elaeis 68 guineensis (African oil palm) (39,40). The disease has killed millions of coconut palms (Cocos 69 nucifera) throughout the Caribbean, Florida, Mexico, and Central America (23,24,41,42). In 70 addition, a first report of phytoplasmas in symptomatic oil palms was confirmed by electron Alvarez et al. 3 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 4 of 37 71 microscopy in West New Britain, Papua New Guinea (50). The group 16SrIV lethal yellowing 72 phytoplasma has been shown to be vectored by Myndus crudus (American palm cixiid) and 73 possibly also by Cedusa species of derbid planthoppers (8). Phytoplasmas closely related to 74 the 16SrIV group have also been reported in date palm and other palm species in USA (22,23). 75 They were also recently detected in weeds such as Emilia fosbergii, Synedrella nodiflora, and 76 Vernonia cinerea (9,10). These weeds are all members of the Asteraceae, and were collected 77 in Jamaica near diseased coconut palms. 78 More recently, phytoplasmas from other 16S ribosomal groups have been associated with 79 symptoms in palms in other parts of the world. In Saudi Arabia, a 16SrI group was found 80 associated with the Al-Wijam disease of date palm (1). In North Sudan, a 16SrXIV group, 81 ‘Ca. P. cynodontis’, commonly associated with bermudagrass, was found in date palm 82 showing slow decline (11). Recently, a 16SrI phytoplasma was associated with coconut yellow 83 decline and oil palm in Malaysia (39,40). Similarly, the Weligama wilt disease of coconut in 84 Sri Lanka was associated with phytoplasmas belonging to the 16SrXI ‘Ca. P. oryzae’ group. 85 Phytoplasmas from two phylogenetic groups, 16SrXI and 16SrXIII, Mexican periwinkle 86 virescence, were associated with Kalimantan wilt in Indonesia (51). 87 In South America, symptoms similar to those observed in Colombia were also described 88 from oil palms in Brazil suffering from a disease known as “fatal yellowing” (7,38). Although 89 the cause of this disease is still unknown, the symptoms and distribution of the problem in 90 both Colombian and Brazilian plantations suggest that infectious agents are involved (49). A 91 preliminary study also detected phytoplasmas in symptomatic plants in commercial crops of 92 the susceptible oil-palm hybrid (Elaeis guineensis × Elaeis oleifera) (2) in Colombia. 93 Phytoplasma identification and classification rely on 16S ribosomal gene analysis to 94 identify ‘Candidatus Phytoplasma’ species and distinguish between the 16Sr groups and 95 subgroups (6). In particular, ‘Candidatus Phytoplasma asteris’ (‘Ca. P. asteris’) is classified in Alvarez et al. 4 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 5 of 37 96 the 16SrI group, in which at least 18 subgroups are recognized (30). Finer differentiation can 97 also be obtained by studying polymorphisms on other genes (35,37) in order to monitor the 98 spread of specific phytoplasma strains. 99 The large survey carried out in this work allowed us to verify phytoplasma presence in diverse 100 tissues from diseased oil palms collected in four areas of Colombia affected by “marchitez 101 letal”. The detected aster yellows strains were then characterized on four phytoplasma genes 102 with a multilocus typing technique that allowed comparison and distinction of the strains 103 infecting diseased oil palm from reference strains (5) and from a strain infecting corn in 104 Colombia. 105 Materials and Methods 106 Plant samples. Samples from 44 symptomatic and seven asymptomatic oil palms were 107 collected between 2003 and 2011 from four plantations in two sites: Villanueva (Department 108 of Casanare) and Barranca de Upía (Department of Meta), Colombia. The four plantations 109 belonged to (A) Palmar del Oriente S.A. (located at 4°30′15″ N and 72°56′20″ W), (B) Palmas 110 del Casanare S.A. (4°35′58.33″ N and 72°50′58.74″ W), (C) Palmeras Santana Ltda. 111 (4°32′24.18″ N and 72°52′51.38″ W), and (D) Palmeras del Upía Ltda. (4°26′8.13″ N and 112 72°56′29.39″ W). 113 Samples were taken from each symptomatic and asymptomatic plant by collecting entire 114 meristems and about 50 to 100 g from each of three tissue types: chlorotic leaves, spears, and 115 inflorescences. Three 10 × 10 cm segments were also excised from the base of the trunk, 116 together with ten 25-cm-long root segments from the root ball of each palm at 50 cm from the 117 collar. From 44 symptomatic trees 85 samples from different tissues were tested; about half of 118 these samples were collected from palms with severe symptoms (see below). Comparable 119 tissues from seven asymptomatic plants were collected at the same time from all four Alvarez et al. 5 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 6 of 37 120 plantations surveyed (three plants from plantation A, two from B, and one plant each from 121 plantations C and D). A total of 44 samples were tested as negative controls. 122 A symptom severity scale was used to rate each symptomatic plant, where 1 represented a 123 dead inflorescence and fruit rot; 2, chlorosis or necrosis of the oldest leaves; 3, leaf chlorosis 124 in the upper canopy; and 4, a dead spear leaf and apical meristem rot. Plants receiving a score 125 of 1 or 2 were characterized as having mild symptoms, 3 as having moderate symptoms, and 126 4 as having severe symptoms. The ability of detecting phytoplasmas from infected tissues was 127 then compared between plants with mild symptoms and those exhibiting severe symptoms. 128 Detecting and identifying phytoplasmas. DNA was extracted from 0.4 to 1.0 g of tissues 129 from each plant sample as according to previously described protocols (19,44). Tissue samples 130 were frozen and ground in liquid nitrogen using a sterilized mortar and pestle. After the final 131 ethanol precipitation, nucleic acid extracts were resuspended in 30 to 50 µL of Tris-EDTA 132 buffer (10 mM Tris-HCl, pH 8.0; and 1 mM EDTA, pH 8.0) and stored at -20°C. 133 The phytoplasma universal primer pair P1/P7 (13,46) was used to amplify DNA from the 134 16Sr region and the beginning of the 23S rDNA genes, including the internal spacer region. 135 Nested-PCR assays were performed on amplicons diluted at 1:29 with sterilized high- 136 performance-liquid-chromatography-grade water, using primers R16F2n/R2 (20). Each PCR 137 reaction was carried out in 0.5-ml tubes in 25-µl reactions, using final concentrations of 20 ng 138 of DNA, 1X buffer, 0.05 U/µL Taq polymerase (Sigma-Aldrich Co., St. Louis, MO, USA), 139 0.2 mM dNTPs (Invitrogen Life Technologies, Carlsbad, CA, USA), and 0.4 µM of each 140 primer. 141 Positive controls employed for the molecular analyses included DNA from phytoplasma 142 reference strains that represented different ribosomal 16S rDNA subgroups. These strains had 143 been either maintained in periwinkle [Catharanthus roseus (L.) G. Don.] or were extracted 144 from the original host plant, as for maize bushy stunt from Colombia (Table 1). Samples Alvarez et al. 6 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 7 of 37 145 devoid of DNA template and from asymptomatic oil palms were added as negative controls 146 for the PCR reactions. 147 Direct and nested-PCR assays were carried out in a PTC-100 thermal cycler with a heated 148 lid (MJ Research, Inc., Waltham, MA), using the following thermal profile: 30 s (90 s for the 149 first cycle) of denaturation at 94°C, annealing for 50 s at 55°C, and extension of the primer for 150 80 s (10 min in the final cycle) at 72°C. For primer pair R16F2n/R16R2, amplifying about 151 1,200 bp within the 16S rDNA region in nested-PCR assays, the annealing temperature was 152 50°C. The PCR products were visualized in a 1.5% agarose gel, stained with 0.75 µg/ml ® 153 ethidium bromide, and analyzed in a Stratagene Eagle Eye II video system (La Jolla, CA). 154 The 98 amplicons obtained with the R16F2n/R16R2 primers (1.2 kb) were then digested 155 with restriction enzymes, Tru1I and HhaI (Fermentas, Vilnius, Lithuania), following the 156 manufacturer’s instructions. Separation of bands generated from restriction digestion was 157 performed in 6.7% polyacrylamide gels. The DNA was then stained and visualized as 158 described above. 159 Direct sequencing in both directions [using primers P1/F1 (12) as forward primers and P7 160 as reverse primer] was performed on the P1/P7 amplicons after purification with a QIAquick 161 PCR Purification Kit (QIAGEN, Valencia, CA). The sequences were assembled using 162 Sequencher 4.1 software. They were then compared with selected nucleotide sequences in the 163 GenBank database using BLAST (version BLASTN 2.2.18) (National Center for 164 Biotechnology Information, Bethesda, MD). 165 Sequence alignments were performed using ClustalX and BioEdit (21,48). Before 166 constructing phylogenetic trees all sequences were trimmed to contain only 16S rDNA (1,245 167 bp). Phylogenetic analyses were carried out on 16S rDNA sequences from oil palm and from 168 several ‘Candidatus’ phytoplasmas strains using Acholeplasma laidlawii as the outgroup. 169 GenBank accession numbers and other sources of 16S rRNA gene sequences used in Alvarez et al. 7 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 8 of 37 170 phylogenetic analyses are given in Table 1. Phylogenetic trees were constructed with 171 maximum parsimony (MP) analysis, using the Close-Neighbor-Interchange algorithm, with 172 the initial tree created by random addition for 10 replications of neighbor-joining (NJ) method, 173 using MEGA version 5 (47). For all methods, all default values (gaps excluded) were 174 performed with 1,000 replications for bootstrap analysis to estimate stability and support for 175 the clades. 176 Strain characterization on groEL, rp, and amp genes. These gene regions were chosen 177 because they were useful in distinguishing among phytoplasma strains in several studies 178 (28,35,37). Amplification was carried out on 44 positive oil-palm samples obtained from 179 previous phytoplasma identification on 16S rDNA gene. 180 The samples were employed for amplification in nested-PCR with groEL primers 181 AYgroelF/AYampR, followed by AYgroelF/AYgroelR amplicons, diluted at 1:30 as 182 described in published protocols (36,37). The negative and positive controls were as described 183 above. RFLP analyses were carried out on amplicons using AluI and Tru1I restriction enzymes 184 (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. Restriction 185 fragments were separated as described above. Direct sequencing and sequence assembly were 186 performed on the AYgroelF/AYgroelR amplicon from sample OP47. A phylogenetic tree was 187 produced, using available reference strains (Table 1), as described above. 188 Previous studies indicated that the part of the ribosomal operon that includes the complete 189 l22 and s3 genes can be used as a phylogenetic marker, as it has fine resolving power for 190 differentiating distinct phytoplasma strains in 16S rDNA subgroups (35). The 44 oil-palm 191 samples were employed for direct amplification with the rpF1/rpR1 primer pair (33), using the 192 reaction mix and the negative and positive controls as described above. Thirty-eight PCR 193 cycles were conducted under the following conditions: 1 min (2 min for the first cycle) for 194 denaturation step at 94°C, 2 min for annealing at 55°C, and 3 min (10 min for the last cycle) Alvarez et al. 8 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 9 of 37 195 for primer extension at 72°C. RFLP analyses of obtained amplicons with Tru1I, Hpy8I, TaaI, 196 and AluI were then performed. The rpF1/rpR1 fragment of OP47 samples was also sequenced 197 as described above and a search for SNPs presence in comparison with reference strains was 198 also carried out using Mega version 5 (47). 199 The amp gene codes for a surface membrane protein that was recently reported as being 200 involved in insect to phytoplasma transmission. It is therefore also suitable for phytoplasma 201 strain differentiation (4,28). Direct PCR assays with Amp-N1/C1 primers, which amplify 702 202 bp of the amp gene, were carried out according to reported procedures (29). The 44 oil-palm 203 samples tested and the negative and positive controls were all as described above. RFLP 204 profiles generated with TruI and Tsp509I were compared with those of the reference strains 205 (Table 1). Direct sequencing and sequence assembly were performed on the amplicon from 206 sample OP47. A phylogenetic tree was produced using available reference strains (Table 1) as 207 described above. The full sequence of the amp gene was also analyzed with translated 208 nucleotide query, using BLASTP (version BLASTP 2.2.18) (National Center for 209 Biotechnology Information, Bethesda, MD) (Table 2). 210 211 Results 212 Detecting and identifying phytoplasmas. Nested-PCR assays amplified 1.2-kb DNA 213 fragments of the 16S rDNA in samples from the various tissues tested at different percentages. 214 The assays detected phytoplasmas in samples from all 44 symptomatic oil-palm plants from 215 the four plantations surveyed. All samples collected from the seven asymptomatic plants, 216 together with the template without nucleic acid, were negative according to nested PCR. 217 Symptoms were evaluated and compared with phytoplasma detection percentages in the 218 diverse oil-palm tissues showing symptoms at different stages in two localities (A and B) (Fig. 219 2). Leaves or spears showed 86% to 100% incidence of phytoplasma detection in samples Alvarez et al. 9 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 10 of 37 220 collected from plants with either mild (scoring 1 or 2) or severe (scoring 3 or 4) symptoms. 221 Tissues from roots and trunks resulted in only 10% to 60% incidence of phytoplasma 222 detection, regardless of symptom severity. RFLP analysis of the 1.2-kb 16S rDNA amplicons 223 indicated that a phytoplasma belonging to subgroup 16SrI-B (‘Ca. P. asteris’) was present in 224 all symptomatic oil palms. RFLP patterns from the positive samples were indistinguishable 225 from each other and from phytoplasma reference strains belonging to subgroup 16SrI-B (Fig. 226 3A). Phytoplasma strain OP47, obtained from a palm hybrid growing in a Palmar del Oriente 227 field, was then selected for further molecular characterization. The 1,491-bp 16S rDNA 228 sequence was deposited in GenBank under accession number JX681021 (Table 1), and 229 showed 99% with a number of strains in group 16SrI, ‘Ca. P. asteris’. The sequence of strain 230 OP47 was then employed for phylogenetic analysis and 20 equally parsimonious trees were 231 constructed, using 27 additional strains of aster yellows (AY) phytoplasmas from different 232 crops (Table 1). Results confirmed its placement in the 16SrI group (Fig. 4A). 233 Strain characterization on groEL, rp, and amp genes. The expected length (about 1.4 kb) 234 of the amplicons of the partial groEL gene was amplified from 21 of the 44 oil-palm samples 235 tested. They showed identical RFLP profiles after Tru1I and AluI digestion. This profile was 236 identical to the one observed in maize bushy stunt (MBS) strain from Colombia, thus 237 differentiating aster yellows phytoplasmas in oil palm from other AY strains and assigning 238 them to the groELI RFLP subgroup V (Fig. 3B). The groEL sequence from OP47 (1,397 bp) 239 was deposited in GenBank under accession number JX681023. The phylogenetic tree 240 confirmed the differentiation of phytoplasmas from oil palm and maize from Colombia (Fig. 241 4B). 242 PCR assays with the rpF1/rpR1 primer pair amplified the expected fragment length of 243 about 1,200 bp from 18 oil-palm samples. RFLP analyses with four restriction enzymes 244 produced restriction profiles that were identical to each other and allowed clear differentiation Alvarez et al. 1 0 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 11 of 37 245 of the two oil-palm phytoplasma strains from all the other AY strains, including maize from 246 Colombia (Fig. 5). The rpF1/rpR1 sequence from OP47 (1,168 bp) was deposited in GenBank 247 under accession number KF434318. The SNPs comparison confirmed the differentiation of 248 phytoplasmas from oil palm and maize from Colombia in the restriction site Hpy8I (Table 2). 249 However the further differentiation observed after RFLP analyses with AluI and TaaI that 250 allow distinguishing oil palm aster yellows phytoplasma from maize as well as from all other 251 reference strains employed was not retrieved in SNPs comparison, presumably due to the 252 position of the sequenced fragment outside of the rp gene. 253 Amplification of the amp gene was obtained for 22 samples. Restriction analysis showed 254 RFLP profiles of all strains from oil palms to be identical to each other and to the one from 255 maize (data not shown). Sequencing and alignment for oil-palm strain OP47 provided a 702- 256 bp sequence. It was deposited in GenBank under accession number JX681022. This sequence 257 encodes 233 amino acids, and its predicted translation showed no significant similarities to 258 any predicted amino acid sequence of aster yellows phytoplasmas available in GenBank 259 (Table 3). The phylogenetic tree confirmed the differentiation of phytoplasmas from oil palm 260 and maize in Colombia from other strains tested (data not shown). 261 262 Discussion 263 The results of this study confirmed the association of a phytoplasma strain related to ‘Ca. 264 P. asteris’ with oil palms severely affected by a lethal wilt in Colombia. The work carried out 265 expanded knowledge of this oil palm disease since a large geographic area was surveyed and 266 a range of samples from different parts of the plants and from different stages of the disease 267 were examined. Considering the sampling sites from which plants were tested and the 268 presence of phytoplasmas in at least one of the samples from each symptomatic plant, the 269 association of the disease with aster yellows phytoplasmas is clear. Alvarez et al. 1 1 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 12 of 37 270 The 16S rDNA is a valuable classification tool, but it is not always able to discriminate 271 phytoplasma strains. The fine-scale molecular characterization of the phytoplasma from oil 272 palm indicates that it can be differentiated from all other phytoplasmas in the same 273 ribosomal subgroup enclosing those infecting corn in Colombia. Multilocus sequence 274 analysis on amp, groEL and rp genes indicated that they could be useful molecular markers 275 to follow the Colombian oil palm epidemic. 276 Only some of the diverse types of samples tested from symptomatic oil palms were negative in 277 PCR assays. This result may be explained by uneven phytoplasma distribution in woody hosts, 278 as recently described (3,16). The amplification of other genes allowed finer characterization of 279 the phytoplasma strain infecting oil palms in Colombia, and indicated that it can be 280 differentiated from all the other phytoplasma strains in the AY group, including a MBS strain 281 from Colombia (15). To our knowledge, this is the first study in which the phytoplasma 282 previously associated with lethal wilt of oil palm in Colombia (2) was classified and its 283 molecular identity characterized. The phytoplasma was assigned to the 16SrI-B AY group, 284 which was clearly differentiated from the other reference phytoplasma strains. 285 The association of more than one group of phytoplasmas with a specific set of disease 286 symptoms at different locations is not unusual. Napier grass stunt disease in Kenya was shown 287 to be associated with a phytoplasma from group 16SrXI (27), while in Ethiopia a phytoplasma 288 from group 16SrIII was found associated with the same symptoms in Napier grass (26). These 289 findings suggest that such phytoplasmas are being transmitted among plant species at these 290 locations, although vectors have yet to be identified. 291 The epidemiological and etiological significance of the ability of phytoplasmas to move 292 among plant species and into coconut and oil palm is unclear. Some phytoplasmas are known 293 to be associated with ‘dead-end’ hosts, that is, plants to which the vector can transmit 294 pathogens, but from which it cannot acquire them (52). For example, grapevine is a ‘dead-end’ Alvarez et al. 1 2 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 13 of 37 295 host for the stolbur phytoplasma, although this phytoplasma is associated with “bois noir” in 296 grapevine. However, phytoplasmas are also known to have variable genomes and ‘potential 297 mobile units’ of DNA within their genomes (25). Spreading into ‘dead-end’ hosts is a first step 298 towards these phytoplasmas eventually becoming adapted to these new hosts. Future studies in 299 comparative genomics on more phytoplasma sequences and identification of insect vectors 300 will be key to determining how these organisms are evolving and adapting to old and new 301 plant and insect hosts. 302 To our knowledge, this is the first time where a multigenic characterization of conserved 303 genes other than 16S rDNA distinguished an AY strain in a specific host plant. The close 304 association of this pathogen with oil palm lethal wilt disease was also confirmed since all 305 symptomatic plants tested positive for the phytoplasma. In spite of the limitations of the data 306 set in number of samples, sampling scheme, number of strains used for molecular 307 characterization, and number of asymptomatic samples, the results of this survey provide 308 important information and tools that can be employed to further study the disease. The 309 epidemiology and insect vector identity can be defined for planning disease management 310 strategies and contains further epidemics. 311 312 Acknowledgements 313 The authors gratefully thank the company Palmar del Oriente S.A., particularly Fernando 314 Rodríguez and Rubén Darío Bedoya, for financial and sampling support. They also thank 315 Palmas del Casanare, Palmeras Santana, and Palmeras del Upía for sampling and technical 316 support. 317 318 319 Alvarez et al. 1 3 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 14 of 37 320 Literature Cited 321 1. Alhudaib, K., Arocha, Y., Wilson, M., and Jones, P. 2008. First report of a 16SrI, 322 ‘Candidatus Phytoplasma asteris’ group phytoplasma associated with a date palm disease 323 in Saudi Arabia. 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Pages 959-962 in: Quarantine Pests Alvarez et al. 1 5 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 16 of 37 367 for Europe. 2nd edn. Eds I.M. Smith, D.G. McNamara, P.R. Scott and K.M. Harris. 368 Wallingford, UK: CAB International. 369 17. FEDEPALMA. 2010. Es possible controlar la marchitez letal si se aplican las 370 recommendaciones de Cenipalma. El Palmicultor 458:12-15. 371 18. Galetto, L., Fletcher, J., Bosco, D., Turina, M., Wayadande, A., and Marzachì, C. 2008. 372 Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma 373 asteris’, chrysanthemum yellows isolate. Can. J. Microbiol. 54:341-351. 374 19. Gilbertson, L., and Dellaporta, S. L. 1983. Molecular extraction DNA protocols. Pages 375 395–397 in: Molecular biology of plants. C. S. Harbor, ed. Cold Spring Harbor 376 Laboratory, New York. 377 20. Gundersen-Rindal, D. E. and Lee, I-M. 1996. Ultrasensitive detection of phytoplasmas by 378 nested-PCR assays using two universal primer pairs. Phytopath. Mediterr. 35:144-151. 379 21. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and 380 analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98. 381 22. Harrison, N. A., Helmick, E. E., and Elliot, M. L. 2008. Lethal yellowing-type diseases of 382 palms associated with phytoplasmas newly identified in Florida, USA. Ann. Appl. Biol. 383 153:85-89. 384 23. Harrison, N. A., Helmick, E. E., and Elliott, M. L. 2009. First report of a phytoplasma- 385 associated lethal decline disease of Sabal palmetto in Florida, USA. Plant Pathol. 58:789. 386 24. Harrison, N. A., Myrie, W., Jones, P., Carpio, M. L., Castillo, M., Doyle, M. M., and 387 Oropeza, C. 2002. 16S rRNA interoperon sequence heterogeneity distinguishes strain 388 populations of palm lethal yellowing phytoplasma in the Caribbean region. Ann. Appl. 389 Biol. 141:183-193 Alvarez et al. 1 6 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 17 of 37 390 25. Hogenhout, S. A., Oshima, K., Ammar, E. D., Kakizawa, S., Kingdom, H. N., and Namba, 391 S. 2008. Phytoplasmas: bacteria that manipulate plants and insects. Mol. Plant Pathol. 392 9:403-423. 393 26. Jones, P., Arocha, Y., Sheriff, T., Proud, J., Abebe, G., and Hanson, J. 2007. A stunting 394 syndrome of Napier grass in Ethiopia is associated with a 16SrIII group phytoplasma. 395 Plant Pathol. 56:345. 396 27. Jones, P., Devonshire, B. J., Holman, T. J., and Ajanga, S. 2004. Napier grass stunt: a new 397 disease associated with a 16SrXI group phytoplasma in Kenya. Plant Pathol. 53:519. 398 28. Kakizawa, S., Oshima, K., Jung, H.-Y., Suzuki, S., Nishigawa, H., Arashida, R., Miyata, 399 S., Ugaki, M., Kishino, H., and Namba, S. 2006. Positive selection acting on a surface 400 membrane protein of the plant-pathogenic phytoplasmas. J. Bacteriol. 188:3424-3428. 401 29. Kakizawa, S., Oshima, K., Nishigawa, H., Jung, H.-Y., Wei, W., Suzuki, S., Tanaka, M., 402 Miyata, S., Ugaki, M., and Namba, S. 2004. Secretion of immunodominant membrane 403 protein from onion yellows phytoplasma through the Sec protein translocation system in 404 Escherichia coli. Microbiology 150:135-142. 405 30. Lee, I-M., Gundersen-Rindal, D. E., Davis, R. E., Bottner, K. D., Marcone, C., and 406 Seemüller, E. 2004. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon 407 associated with aster yellows and related diseases. Int. J. Syst. Evol. Microbiol. 54:1037- 408 1048. 409 31. Lee, I-M., Zhao, Y., and Bottner, K. D. 2005. Novel insertion sequence-like elements in 410 phytoplasma strains of the aster yellows group are putative new members of the IS3 411 family. FEMS Microbiol. Letters 242:353-360. 412 32. Lee, I-M., Zhao, Y., and Bottner, K. D. 2006. SecY gene sequence analysis for finer 413 differentiation of diverse strains in the aster yellows phytoplasma group. Mol. Cell. Probes 414 20:87-91. Alvarez et al. 1 7 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 18 of 37 415 33. Lim, P. O., and Sears, B. B. 1992. Evolutionary relationships of a plant pathogenic 416 mycoplasma-like organism and Acholeplasma laidlawii deduced from two ribosomal 417 protein gene sequences. J. Bacteriol. 174:2606–2611. 418 34. Marcone, C., Lee, I-M., Davis, R. E., Ragozzino, A., and Seemüller, E. 2000. 419 Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA 420 and tuf gene sequences. Int. J. Syst. Evol. Microbiol. 50:1703-1713. 421 35. Martini, M., Lee, I-M., Bottner, K. D., Zhao, Y., Botti, S., Bertaccini, A., Harrison, N., 422 Carraro, L., Marcone, C., Khan, A. J., and Osler, R. 2007. Ribosomal protein gene-based 423 phylogeny for differentiation and classification of phytoplasmas. Int. J. Syst. Evolut. 424 Microbiol. 57:2037-2051. 425 36. Mitrović, J., Contaldo, N., Paltrinieri, S., Mejía, J. F., Mori, N., Bertaccini, A., and Duduk, 426 B. 2011a. The use of groEL gene in characterisation of aster yellows phytoplasmas in field 427 collected samples. Bull. Insectol. 64 (Suppl.):S17-S18. 428 37. Mitrović, J., Kakizawa, S., Duduk, B., Oshima, K., Namba, S., and Bertaccini, A. 2011b. 429 The groEL gene as an additional marker for finer differentiation of ‘Candidatus 430 Phytoplasma asteris’-related strains. Ann. Appl. Biol. 159:41-48. 431 38. Montano, H. G., Brioso, P. S. T., and Pimentel, J. P. 2007. List of phytoplasma hosts in 432 Brazil. Bull. Insectol. 60:129-130. 433 39. Nejat, N., Sijam, K., Abdullah, S., Vadamalai, G., and Dickinson, M. 2009. Phytoplasmas 434 associated with disease of coconut in Malaysia: phylogenetic groups and host plant 435 species. Plant Pathol. 58:1152-1160. 436 40. Nejat, N., Vadamalai, G., Davis, R. E., Harrison, N. A., Sijam, K., Dickinson, M., 437 Abdullah, S. N., and Zhao, Y. 2012. ‘Candidatus Phytoplasma malaysianum’, a novel 438 taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus 439 roseus). Int. J. Syst. Evol. Microbiol. DOI: 10.1099/ijs.0.041467-0. Alvarez et al. 1 8 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 19 of 37 440 41. Ntushelo, K., Harrison, N. A., and Elliott, M. L. 2012. Comparison of the ribosomal RNA 441 operon from Texas Phoenix decline and lethal yellowing phytoplasmas. Eur. J. Plant 442 Pathol. 133:779-782. 443 42. Oropeza, C., Cordova, I., Chumba, A., Narváez, M., Sáenz, L., Ashburner, R., and 444 Harrison, N. 2011. Phytoplasma distribution in coconut palms affected by lethal yellowing 445 disease. Ann. Appl. Biol. 159:109-117. 446 43. Pérez, A. P., and Cayón, G. 2010. Metabolism of carbohydrates in oil palms (Elaeis 447 guineensis Jacq.) affected by lethal wilt. Agron. Colomb. 28:181-187. 448 44. Prince, J. P., Davis, R. E., Wolf, T. K., Lee, I-M., Mogen, B., Dally, E., Bertaccini, A., 449 Credi, R., and Barba, M. 1993. Molecular detection of diverse mycoplasma-like organisms 450 (MLOs) associated with grapevine yellows and their classification with aster yellows, X- 451 disease, and elm yellows MLOs. Phytopathology 83:1130-1137. 452 45. Schneider, B., Ahrens, U., Kirkpatrick, B. C., and Seemüller, E. 1993. Classification of 453 plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR- 454 amplified 16S rDNA. J. Gen. Microbiol. 139:519-527. 455 46. Schneider, B., Seemüller, E., Smart, C. D., and Kirkpatrick, B. C. 1995. Phylogenetic 456 classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. Pages 369- 457 380 in: Molecular and Diagnostic Procedures in Mycoplasmology. S. Razin and J. G. 458 Tully, eds. Academic Press, New York, NY. 459 47. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. 460 MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, 461 evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731-2739. 462 48. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. 1997. 463 The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment 464 aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882. Alvarez et al. 1 9 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 20 of 37 465 49. Tovar, J. P., and Torres, E. 2004. Estudio epidemiológico de la enfermedad marchitez letal 466 de la palma de aceite en plantaciones de Villanueva, Casanare–Cenipalma. Palmas 25:210- 467 219. 468 50. Turner, P. D. 1981. Oil palm diseases and disorders. Oxford University Press, Oxford, UK. 469 51. Warokka, J. 2005. Studies on the etiology and epidemiology of kalimantan wilt disease of 470 coconut in Indonesia. Dissertation. University of Nottingham, Nottingham, UK. 471 52. Weintraub, P. G., and Beanland, L. 2006. Insect vectors of phytoplasmas. Ann. Rev. 472 Entomol. 51:91-111. 473 Alvarez et al. 2 0 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 21 of 37 474 Table 1. Aster yellows-related reference phytoplasma (‘Candidatus Phytoplasma asteris’) strains employed for 16S rDNA, groEL, 475 ribosomal protein l22, and s3 characterization b Phytoplasma associated disease Geographic GenBank accession numbers RFLP classification a c d e (acronym) origin 16S rDNA groEL rp 16SrI groELI rpI References New Jersey aster yellows (NJ-AY) NJ, USA HM590622 AB599703 - A I A (31,35,37) Plantago virescence (PVM) Germany AY265216 AB599706 AY264867 A I A (5,35,37) Carrot yellows (ca2006/1) Serbia EU215424 AB599708 EU215428 A I A (14,37) Grey dogwood stunt (GD1) NY, USA DQ112021 AB599694 AY264864 A II M (32,37) Periwinkle virescence (NA) Italy HM590621 AB599702 - B III - (5,37) Primula green yellows (PrG) UK HM590623 AB599696 - B III - (5,37) Oilseed rape virescence (RV) France HM590625 AB599698 - B III - (5,37) Carrot yellows (ca2006/9) Serbia EU215426 AB599709 EU215430 B III B (14,37) Primrose virescence (PRIVA) Germany AY265210 AB599705 - B(L) III B (35,45) Aster yellows (AV2192) Germany AY180957 AB599687 AY183708 B(L) III B (34,35,37) Aster yellows (AVUT) Germany AY265209 AB599686 AY264855 B(M) III B (34,35,37) Aster yellows (AY-J) France HM590616 AB599689 - B IV - (37) Alvarez et al. 2 1 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 22 of 37 Carrot yellows (ca2006/5rrnA) Serbia EU215425/ AB599711 EU215429 B(?) IV - (14,37) Carrot yellows (ca2006/5rrnB) Serbia GQ175789 Maize bushy stunt (MBS Col) Colombia HQ530152 AB599712 KF434319 B V - (15,37,Unpublished) Maize bushy stunt (MBS) Mexico AY265208 – AY264858 B – L (30) Oil palm lethal wilt (OP47) Colombia JX681021 JX681023 KF434318 B V - This work Leontodon yellows (LEO) Italy HM590620 AB599701 - C VI - (5,37) Carrot yellows (CA) Italy HM448473 AB599690 - C VI - (5,37) Clover phyllody (KVE) France AY265217 – AY264861 C – C (30,35) Clover phyllody (KVF) France HQ530150 AB599695 - C VII - (34,37) Potato purple top (PPT) France HQ530151 AB599704 - C VII - (34,37) Paulownia witches’ broom (PaWB) Taiwan AY265206 AB124810 AY264857 D – D (28,30) Blueberry stunt (BBS3) MI, USA AY265213 – AY264863 E – E (30) Aster yellows apricot (A-AY) Spain AY265211 AB599699 AY264866 F VIII N (30,37) Strawberry multiplier (STRAWB2) FL, USA U96616 – U96617 K – J (30) Ipomea obscura witches’ broom (IOWB) Taiwan AY265205 – AY264859 N – F (30,34) Populus decline (PopD) Serbia HM590626 AB599710 - P IX - (37) Alvarez et al. 2 2 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 23 of 37 a 476 Strains in bold were used as references for PCR-RFLP analysis. b 477 (?) refers to a strain with interoperon heterogeneity that is tentatively classified in this subgroup; – refers to a sequence not available in the 478 GenBank. c 479 Different letter represent diverse RFLP subgroups in the 16S rDNA gene of aster yellows strains d 480 Different Roman number represent diverse RFLP groups in the groEL gene e 481 Different letter represent diverse RFLP groups in the rp gene 482 483 484 485 486 487 488 489 490 491 Alvarez et al. 2 3 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 24 of 37 492 Table 2. Differential SNP positions in ribosomal protein s3 sequences, of fifteen 'Candidatus Phytoplasma asteris' strains compared with OP47 493 (Oil palm lethal wilt). Single nucleotide positions in ribosomal protein s3 Strain 18 124* 139 252 277* 278* 285* 444* 486 571 667* 673 OP47 C C A T A A C T C C C A MBS Col . A . . . . . . . . . . MBS T . . . G . . . . . . . PVM T . G C G C T A T G T G ca2006/1 T . G C G C T A T G T G GD1 T . G C G C T A . G T G ca2006/9 T . . . G C . . T G . G AV2192 T . . . G C . . T G . G AVUT T . . . G C . . T G . G ca2006/5 T . . . G . . . . . . G KVE T . G C G C A A T G . G PaWB T . . . G C . . T G . G Alvarez et al. 2 4 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 25 of 37 BBS3 T . G C G T . A T G . G A-AY T . G C G C . A T G T G STRAWB2 T . G C G T . A T G . G IOWB T . . C G C . . T T . G 494 *SNPs making differential restriction sites for RFLP differentiation: 124 and 278 (Hpy8I), 277 and 285 (Tru1I), 444 (AluI), 667 (TaaI). Dots 495 represent nucleotides identical to the OP47 consensus sequence. 496 Alvarez et al. 2 5 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 26 of 37 497 Table 3. Aster yellows-related reference phytoplasma strains employed for amp characterization, and their homology percentages. GenBank % nucleotide (nt) and amino Phytoplasma associated disease Geographic 16SrI RFLP accession acid (aa) identity Reference a (acronym) origin classification number nt aa Oil palm lethal wilt (OP47) Colombia JX681022 B 100 100 This work Maize bushy stunt (MBS Col) Colombia KF434320 B 100 100 Unpublished Paulownia witches’ broom (PaWB) Taiwan AB124810 D 95.3 89.5 (28) Onion yellows (OYW) Japan AB124806 B 98.6 95.7 (28) Periwinkle leaf yellowing (PLY) Taiwan GQ845122 – 98.3 94.8 Unpublished Chrysanthemum yellows phytoplasma (CY) Italy DQ787852 B 95.3 89.5 (18) Onion yellows (OY-M) Japan AB124807 B 97.6 95.2 (28) Onion yellows (OY-NIM) Japan AB124808 B 96.3 95.2 (28) Mulberry dwarf (MD) AB124809 – 91.6 93.8 (28) Rape virescence (RV) France AF244540 B 97.6 84.2 (4) Bermudagrass white leaf (AYBG) Thailand AB124811 B 97.9 95.2 (28) Iceland poppy yellows (IPY) Japan AB242234 B 98.3 95.2 (28) Alvarez et al. 2 6 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 27 of 37 Eggplant dwarf (ED) Japan AB242231 B 98.4 94.8 (28) Sumac witches' broom (SWB) Japan AB242236 – 90.6 95.3 (28) Porcelain vine witches’ broom (PvWB) Korea AB242237 – 92.3 80.9 (28) Lettuce yellows (LeY) Japan AB242233 B 98.1 83.7 (28) Marguerite yellows (MarY) Japan AB242235 B 98.7 94.4 (28) Tomato yellows (TY) Japan AB242232 B 98.4 96.1 (28) a 498 – means “not described as a ribosomal group”. 499 In bold oil palm phytoplasma strain used for similarity comparison Alvarez et al. 2 7 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 28 of 37 500 Figure legends 501 502 Fig. 1. Two diseased oil palms in Colombia, one with mild symptoms (severity score of 2) (A) 503 and the other with severe symptoms (severity score of 4) (B) of lethal wilt. 504 505 Fig. 2. Oil palm wilt symptoms observed in relation to percentage of phytoplasma detection in 506 samples from diverse host tissues in localities A and B. Severity scale: mild symptoms with a 507 score of 1 or 2; severe symptoms, 3 or 4. Vertical bars represent standard errors of the means. 508 Two-way analysis of variance for phytoplasma detection related to symptomatic tissues tested 509 indicated significant differences between tissue types sampled (F = 57.81 at P < 0.001) and no 510 significant differences between phytoplasma detection and symptom severity or geographic 511 locality (F = 4.90 at P =0.0624 and F = 6.45 at P = 0.0387, respectively). 512 513 Fig. 3. Restriction fragment length polymorphism patterns of oil-palm phytoplasma strains 514 OP45 and OP47 compared with those of several reference strains from periwinkle. (A) 515 Phytoplasma 16S rDNA amplified in nested-PCR with R16F2n/R16R2 primers and digested 516 with Tru1I and HhaI restriction enzymes. (B) Phytoplasma groEL gene amplified in nested- 517 PCR with AYgroelF/R primers and digested with Tru1I and AluI restriction enzymes. Strains 518 employed were maize bushy stunt Colombia (MBS Col); periwinkle virescence (NA); aster 519 yellows (AY-J); primula green yellows (PrG); grey dogwood stunt (GD1); carrot yellows 520 (CA); clover phyllody-France (KVF); aster yellows from apricot (A-AY); clover phyllody- 521 England (KVE); New Jersey aster yellows (NJ-AY); primrose virescence (PRIVA); plantago 522 virescence (PVM); leontodon yellows (LEO); oilseed rape virescence (RV); potato purple top 523 (PPT); aster yellows Germany (AVUT) Markers: phiX174, phiX174 HaeIII digested; and 524 pBR322, pBR322 HaeI digested. Alvarez et al. 2 8 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 29 of 37 525 Fig. 4. Phylogenetic trees constructed by maximum parsimony analysis of (A) 16S rDNA 526 gene sequences and (B) groEL gene sequence from selected phytoplasma strains. Strains 527 employed were paulownia witches’ broom (PaWB); Ipomea obscura witches’ broom (IOWB); 528 oil-palm lethal wilt (OP47); maize bushy stunt (MBS); primula green yellows (PrG); maize 529 bushy stunt Colombia (MBS Col); periwinkle virescence (NA); aster yellows apricot (A-AY); 530 blueberry stunt (BBS3); strawberry multiplier (STRAWB2); carrot yellows (ca2006/5rrnA; 531 ca2006/5rrnB; ca2006/5); carrot yellows (ca2006/9); ‘Ca. P. asteris’ strain OY-M 532 (NC_005303); aster yellows (AY2192); aster yellows (AVUT); oilseed rape virescence (RV); 533 primrose virescence (PRIVA); aster yellows (AY-J); Populus decline (PopD); grey dogwood 534 stunt (GD1); plantago virescence (PVM); New Jersey aster yellows (NJ-AY); carrot yellows 535 (ca2006/1); carrot yellows (CA); clover phyllody England (KVE); clover phyllody France 536 (KVF); leontodon yellows (LEO); potato purple top (PPT);. ‘Ca. P. australiense’ (NC 537 010544); ‘Ca. P. mali’ strain AT (NC 011047) and Acholeplasma laidlawii PG-8A 538 (CP000896). Numbers on the branches are bootstrap values obtained for 1,000 replicates (only 539 values above 60% are shown). 540 541 Fig. 5. Restriction fragment length polymorphism (RFLP) patterns of oil-palm phytoplasma 542 strains OP45 and OP47 compared with several reference strains from periwinkle that were 543 amplified with primers rpF1/rpR1 and digested with restriction enzymes TruI (A); TaaI (B); 544 Hpy8I (C), and AluI (D). Strains employed were maize bushy stunt Colombia (MBS Col); 545 periwinkle virescence (NA); aster yellows (AY-J); primula green yellows (PrG); grey 546 dogwood stunt (GD1); carrot yellows (CA); clover phyllody-France (KVF); aster yellows 547 from apricot (A-AY); clover phyllody-England (KVE); New Jersey aster yellows (NJ-AY); 548 primrose virescence (PRIVA); plantago virescence (PVM); leontodon yellows (LEO); oilseed Alvarez et al. 2 9 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 30 of 37 549 rape virescence (RV); potato purple top (PPT). Markers: phiX174, phiX174 HaeIII digested; 550 and pBR322, pBR322 HaeI digested. Alvarez et al. 3 0 Plant Disease Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 31 of 37 Fig. 1. Two diseased oil palms in Colombia, one with mild symptoms (severity score of 2) (A) and the other with severe symptoms (severity score of 4) (B) of lethal wilt. 253x159mm (150 x 150 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 32 of 37 Fig. 2. Oil palm wilt symptoms observed in relation to percentage of phytoplasma detection in samples from diverse host tissues in localities A and B. Severity scale: mild symptoms with a score of 1 or 2; severe symptoms, 3 or 4. Vertical bars represent standard errors of the means. Two-way analysis of variance for phytoplasma detection related to symptomatic tissues tested indicated significant differences between tissue types sampled (F = 57.81 at P < 0.001) and no significant differences between phytoplasma detection and symptom severity or geographic locality (F = 4.90 at P =0.0624 and F = 6.45 at P = 0.0387, respectively). 427x239mm (96 x 96 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 33 of 37 Fig. 3. Restriction fragment length polymorphism patterns of oil-palm phytoplasma strains OP45 and OP47 compared with those of several reference strains from periwinkle. (A) Phytoplasma 16S rDNA amplified in nested-PCR with R16F2n/R16R2 primers and digested with Tru1I and HhaI restriction enzymes. (B) Phytoplasma groEL gene amplified in nested-PCR with AYgroelF/R primers and digested with Tru1I and AluI restriction enzymes. Strains employed were maize bushy stunt Colombia (MBS Col); periwinkle virescence (NA); aster yellows (AY-J); primula green yellows (PrG); grey dogwood stunt (GD1); carrot yellows (CA); clover phyllody-France (KVF); aster yellows from apricot (A-AY); clover phyllody-England (KVE); New Jersey aster yellows (NJ-AY); primrose virescence (PRIVA); plantago virescence (PVM); leontodon yellows (LEO); oilseed rape virescence (RV); potato purple top (PPT); aster yellows Germany (AVUT) Markers: phiX174, phiX174 HaeIII digested; and pBR322, pBR322 HaeI digested. 440x256mm (150 x 150 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 34 of 37 218x278mm (96 x 96 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 35 of 37 Fig. 4. Phylogenetic trees constructed by maximum parsimony analysis of (A) 16S rDNA gene sequences and (B) groEL gene sequence from selected phytoplasma strains. Strains employed were paulownia witches’ broom (PaWB); Ipomea obscura witches’ broom (IOWB); oil-palm lethal wilt (OP47); maize bushy stunt (MBS); primula green yellows (PrG); maize bushy stunt Colombia (MBS Col); periwinkle virescence (NA); aster yellows apricot (A-AY); blueberry stunt (BBS3); strawberry multiplier (STRAWB2); carrot yellows (ca2006/5rrnA; ca2006/5rrnB; ca2006/5); carrot yellows (ca2006/9); ‘Ca. P. asteris’ strain OY-M (NC_005303); aster yellows (AY2192); aster yellows (AVUT); oilseed rape virescence (RV); primrose virescence (PRIVA); aster yellows (AY-J); Populus decline (PopD); grey dogwood stunt (GD1); plantago virescence (PVM); New Jersey aster yellows (NJ-AY); carrot yellows (ca2006/1); carrot yellows (CA); clover phyllody England (KVE); clover phyllody France (KVF); leontodon yellows (LEO); potato purple top (PPT);. ‘Ca. P. australiense’ (NC 010544); ‘Ca. P. mali’ strain AT (NC 011047) and Acholeplasma laidlawii PG-8A (CP000896). Numbers on the branches are bootstrap values obtained for 1,000 replicates (only values above 60% are shown). Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 36 of 37 241x288mm (96 x 96 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ. Page 37 of 37 Fig. 5. Restriction fragment length polymorphism (RFLP) patterns of oil-palm phytoplasma strains OP45 and OP47 compared with several reference strains from periwinkle that were amplified with primers rpF1/rpR1 and digested with restriction enzymes TruI (A); TaaI (B); Hpy8I (C), and AluI (D). Strains employed were maize bushy stunt Colombia (MBS Col); periwinkle virescence (NA); aster yellows (AY-J); primula green yellows (PrG); grey dogwood stunt (GD1); carrot yellows (CA); clover phyllody-France (KVF); aster yellows from apricot (A-AY); clover phyllody-England (KVE); New Jersey aster yellows (NJ-AY); primrose virescence (PRIVA); plantago virescence (PVM); leontodon yellows (LEO); oilseed rape virescence (RV); potato purple top (PPT). Markers: phiX174, phiX174 HaeIII digested; and pBR322, pBR322 HaeI digested. 380x232mm (150 x 150 DPI) Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-12-12-1182-RE • posted 10/10/2013 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.