FIRST REPORT OF A 16Sr IV GROUP PHYTOPLASMA ASSOCIATED WITH LETHAL YELLOWING IN AGAVE TEQUILANA

Jimsonweed (Datura stramonium), family Solanaceae, a common weed in spring crops in Greece, includes two botanical varieties (var. stramonium and var. tatula). During a field trial at the Aristotle University Farm (40o32N’ 22o59’E, 6 m asl), in which four accessions of D. stramonium were tested for growth rate and alkaloid content, phytoplasma-like symptoms were observed. Initially, the affected plants showed interveinal chlorosis of the upper leaves, stunting and flower malformation, whereas at maturity they did not form normal fruits and developed leaf necrosis. To investigate the possibility of a phytoplasma infection, DNA was extracted from leaf samples of symptomatic and apparently healthy plants of both varieties according to Psifidi et al. (2010). A nested PCR was performed using two universal primer sets specific to the phytoplasma 16S rRNA gene: P1/P7 (Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). The expected ca. 1,200 bp product, amplified esclusively from symptomatic plants of both varieties, was cloned and sequenced. BLAST analysis revealed 99% similarity with sequence AF248959 of stolbur phytoplasma (16SrXII group, Ca. Phytoplasma solani). Sequences from both varieties were identical, indicating infection by the same phytoplasma strain and were deposited in EMBL-EBI (accession Nos HE598778 and HE598779). Diseased plants of var. tatula and stramonium showed 49-69% and 38% reduction of the above-ground fresh weight, respectively, compared to healthy plants, hence making the phytoplasma an important pathogen of jimsonweed, which constitutes a reservoir plant in the field. This is, to our knowledge, the first report of a Ca. Phytoplasma solani-related disease in jimsonweed in Greece.

Land use changes and transnational migration

Cabbage (Brassica oleracea var. capitata) is one of the most im- portant vegetable crops in Greece grown on an area of 7,006 ha with a production of 188,000 tons (FAOSTAT, 2009). During a survey conducted on winter 2009 in northern Greece for pathogens infecting plant species of the Brassicaceae family, two cabbage plants showing phytoplasma-like symptoms were collect- ed near the village of Vasilika-Thessaloniki (40o28’N 23o08’E). These plants exhibited reduction of leaf size (little leaf), opening of the head, proliferation and generalized stunting. In order to in- vestigate the possibility of a phytoplasma infection, DNA was ex- tracted from leaf samples of two symptomatic and two apparently healthy plants according to the in house protocol developed by Psifidi et al. (2010). A nested PCR was done using two universal primer sets specific to the phytoplasma 16S rRNA gene: P1/P7 (Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). The expected ca. 1200 bp amplicon was obtained from both symptomatic cabbage plants but not from the symp- tomless ones. One of the two amplicons was purified and directly sequenced. BLAST comparisons of the partial 16S rRNA se- quence revealed 99% identity with the homologous sequence from Ca. Phytoplasma asteris reference strain (M30790). The ob- tained sequence was deposited in EMBL-EBI database (accession No. HE601634). In silico RFLP analysis (Zhao et al., 2009) classi- fied the isolate in the 16SrI-B subgroup. Ca. Phytoplama asteris is an important pathogen of many herbaceous and woody plants; it has a worldwide distribution and has been previously associated with various diseases of several Brassicaceae plant species. How- ever this is, to our knowledge, the first record of a cabbage dis- ease associated with Ca. Phytoplasma asteris in Greece.

Five samples from neem trees exhibiting witches broom (NeWB) symptoms were collected from the Raichur district, Karnataka State, India. The identity of the phytoplasma associated with all five neem samples was confirmed through PCR using phytoplasma 16Sr RNA gene specific universal primers. The amplified products were cloned, sequenced and nucleotide (nt) sequence comparisons were made with published phytoplasmas 16S rRNA gene nt sequences available at NCBI database. The 16Sr RNA gene nt sequence of NeWB phytoplasma had 99 to 99.8% identity with ‘Candidatus Phytoplasma’ group (16SrVI) isolates reported from different parts of the world. This was supported by the close clustering of NeWB phytoplasma in the current study with members of clover proliferation group-16SrVI in the phylogenetic analysis. The virtual RFLP pattern generated for the phytoplasma from neem was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI and subgroup D (Brinjal little leaf-16VI-D, NCBI Ac.No.: X83431). The analysis further confirmed that the phytoplasma associated with NeWB disease of neem belongs to 16Sr group VI and subgroup 16Sr IV-D. This is the first report of ‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group associated with witches broom disease of Azadirachta indica from India.

During 2008-13 surveys, Eucalyptus camaldulensis little leaf (ELL) disease was observed in Firuzabad, Shiraz and Darab (Fars province, Iran), consisting of proliferation of auxiliary buds, formation of brooms on branches and trunk, yellowing, reddening and reduction of the leaf size, tree decline and death within 3-4 years from the onset of symptoms. Total DNA extracted from 15 ELL-affected and five symptomless plants was tested by direct and nested PCR using the phytoplasma primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). Amplicons of ca. 1.25 kbp were obtained from 9 of 15 symptomatic but not from all symptomless and six symptomatic trees, probably due to the low titres and unevenly distribution of phytoplasma or the presence of PCR inhibitors in the latter. Nine R16F2n/R16R2 amplicons from infected samples (three from each sampled area) were directly sequenced in both directions, one of which from Firuzabad ELL (FELL) was deposited in GenBank (Accession No. KT992689). BLAST search showed that the FELL phytoplasma had 100% sequence identity with four 16SrI ('Candidatus Phytoplasma asteris') group-related phytoplasmas including mulberry dwarf, aster yellows, onion yellows, sasa witches' broom and epilobium phyllody phytoplasmas (AB693124, FJ824597, AP006628, AB293421, AY101386). Computer-simulated restriction analysis using iPhyClassifier revealed that the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment of FELL is identical (similarity coefficient 1.00) to onion yellows phytoplasma (NC_005303) representative of subgroup B of the 16SrI group. Phylogenetic analysis revealed that FELL phytoplasma clustered with 16SrI- B members closer to a phytoplasma associated with Eucalyptus yellows and witches' broom (AY685054). To our knowledge, this is the first report of the association of a 16SrI phytoplasma with ELL in Iran

Previous studies have shown that an immunodominant membrane protein is a major portion of the total cellular membrane proteins in most phytoplasmas. Genes encoding immunodominant membrane proteins have been identified and sequenced from several taxonomic groups of phytoplasmas and have been classified into three distinct types: type 1 immunodominant membrane protein Imp, type 2 immunodominant membrane protein IdpA, and type 3 immunodominant membrane protein Amp. No amino acid similarity was revealed among different type of immunodominant membrane proteins. The sequence identity of immunodominant membrane protein genes between phytoplamas was also evidenced to be lower than those of their upstream or downstream genes or non-coding regions. In this study, cloning and sequencing of these three types immunodominant membrane protein genes in Taiwan were performed for three groups of phytoplasmas including the pear decline phytoplasma (PDTWII phytoplasma) and peanut witches'-broom (PnWB) phytoplasma in 16SrII group which have imp genes, poinsettia branch-inducing (PoiBI) phytoplasma in 16SrIII group which has idpA gene, and periwinkle leaf yellowing (PLY) phytoplasma and paulownia witches'-broom (PaWB-Taiwan) phytoplasma in 16SrI group which have amp genes. Sequence analysis of these cloned immunodominant membrane protein gene fragments revealed that the sequence identities were very low, especially among the sequences of hydrophilic domains. Each phytoplasmal immunodominant membrane protein gene has their own distinct sequence even the phytoplasmas belong to the same group. These distinct immunodominant membrane protein gene sequences can be used for the differentiation of groups or subgroups of various phytoplasmas. Specific PCR primer pairs ampf5/Amp1R and Amp1F/ampr4 were designed based on distinct regions of PLY phytoplasma and PaWB-Taiwan phytoplasma amp gene sequences for their identification and classification. RT-PCR analysis showed that the imp, amp genes of PLY phytoplasma and the imp gene of PnWB phytoplasma were transcribed in PLY phytoplasma and PnWB phytoplasma.

Epazote (Chenopodium ambrosioides L.), a plant native to South America, is a serious invasive weed in China. In May 2011, epazotes exhibiting small leaves and fasciation indicative of phyto- plasma infection were found in a pepper field in Qijiang County (China), with an incidence lower than 3%. Stem and leaf samples were collected from symptomatic and symptomless plants, total DNA was extracted and nested PCR performed using primers P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by primers R16F2n/R16R2 (Lee et al., 1998). Fragments of 1.2 kb corresponding to 16S rDNA were amplified exclusively from DNA extracts from symptomatic plants. Purified PCR products were cloned in pGEM-T Easy Vector (Promega, USA), sequenced (GenBank accession No. JN836942) and analyzed using iPhyClas- sifier online tool. The sequence shared 99.4% identity with the 16S rDNA sequence of the ‘Candidatus Phytoplasma asteris’ refer- ence strain (M30790) that belongs to 16SrI-B subgroup. The vir- tual RFLP pattern of the epazote phytoplasma 16S rDNA gene se- quence showed maximum similarity with the reference pattern of the 16SrI-B subgroup (NC005303), with a similarity coefficient of 0.98. A phylogenetic tree based on the 16S rDNA phytoplasma se- quences showed that the sequences clustered with members of the 16SrI-B subgroup. Taken together, the epazote phytoplasma was classified in subgroup 16SrI-B. Phytoplasmas identified as 16SrI-B strains have previously been identified in Morus alba, Amaranthus hypochondriacus and Sasa fortunei in China. To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma associat- ed with diseased epazote in our country. As a widely distributed weed in southwest China, phytoplasma-infected epazote may be a potential source of inoculum for crops.

Potato (Solanum tuberosum), one of the most important crops worldwide, is susceptible to a high number of viruses and phytoplasmas. During the summer of 2013 symptoms similar to those caused by phytoplasmas, such as dwarfing, bright red discoloration, and rolling of the top leaves were observed in potato crops of cvs. Spunta and Jelly in Kastoria (40° 31'0 .12 "N21° 16'0 .12" E), Northern Greece. The affected plants were scattered in the field and their incidence ranged among the plots between 10% and 20%. In order to investigate the etiology of the aforementioned symptoms, leaves from 10 symptomatic and 10 asymptomatic plants of each variety were collected and subjected to DNA extraction. The extracts were tested using a generic nested PCR that targets the highly conserved 16S rRNA gene of phytoplasmas. More specifically, the universal primer set P1/P7 (Schneider et al., 1995) was used in the first round of PCR, whereas R16F2n/R16R2 primers (Gundersen and Lee, 1996) were applied in the nested step. The expected PCR products of ca. 1200 bp were amplified from all samples coming from symptomatic plants but not from the asymptomatic ones. One amplicon from each variety was further sequenced in both directions and their BLAST analysis revealed 98% similarity with the sequences of several “Candidatus Phytoplasma solani” (16SrXII-A stolbur group) strains (e.g. Bulgarian strain 241/13, KF907506). The two Greek sequences were 99% similar between them and were deposited in the EMBL-EBI under the accession Nos HG531809 and HG792592. “Ca. Phytoplasma solani” is endemic in Greece and was first detected in Datura stramonium plants in the area of Thessaloniki (Macedonia, Northern Greece). However, this is to our knowledge the first report of a potato disease caused by a “Ca. Phytoplasma solani” related strain in our country.

Almond witches’ broom associated with ‘ Candidatus Phytoplasma phoenicium’ is an economically important disease of almond in Iran and Lebanon. During surveys of almond witches’ broom in 2012–2015, an apricot yellows disease was observed in Fars Province of Iran. The characteristic symptoms of the disease were leaf yellowing, inward leaf curl, scorch of leaf margins, shortened internodes, production of rosettes at the tips of the branches, and decline, stunting, and death of affected trees. Healthy bitter almond and apricot seedlings, grafted with shoots from symptomatic trees, exhibited phytoplasma-type symptoms. A 16S rDNA fragment of 1,250 bp was amplified by nested-PCR from affected trees and grafted seedlings. Nucleotide sequence identity, presence of species-specific signature sequences, and phylogenetic analysis of 16S rDNA allowed the assignation of the phytoplasma strains identified to the ‘ Ca . P. phoenicium’. In vitro and in silico RFLP analyses of the amplified fragment allowed affiliation of the apricot yellows phytoplasma to a molecular variant in the subgroup 16SrIX-B. Within the population strains identified in this and previous studies, 16 genetic lineages were determined within 16S rDNA nucleotide sequences by the combination of 19 single nucleotide polymorphisms. The apricot yellows phytoplasma strains belong to a unique genetic lineage distinguished by the presence of three lineage-specific SNPs. This first report of ‘ Ca . P. phoenicium’ in association with apricot yellows in Iran opens new perspectives on the epidemiology of almond witches’ broom, suggesting possible adaptation of the phytoplasma to other fruit tree species.

A new disease named as periwinkle leaf yellowing (PLY) was first observed in a flower production farm in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Sequence analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group) which cause diseases in many horticultural and vegetable crops worldwide, and can be delineated into 10 subgroups. Six cultivated plants including periwinkle plant, chrysanthemum, cosmos, torenia, Persian violet, goosegrass and cucumber, were determined to be the host plants of PLY phytolasma. Monthly PCR detection indicated that PLY phytoplasma was detected in host plants from June to October in 2007, and from July to October in 2007. However, it can be detected earlier since April in 2009. To further clarify the phylogenetic relationship of strain PLY among 16SrI phytoplasmas, six phylogenetic trees were constructed in this study. Beside the phylogenetic trees based on the independent analysis of 16S rRNA, rplV-rpsC, secY and tuf gene sequences, two other trees based on the analysis of the comprising gene sequence of 16S rRNA, rplV-rpsC and secY, and the comprising gene sequence of rplV-rpsC and secY were also constructed. The results indicate that the strain PLY was closely related to 16SrI-B and 16SrI-D subgroup, and could be a new subgroup based on the phylogenetic tree constructed by rplV-rpsC gene sequences. In addition, the phylogenetic analysis of the comprised gene sequences showed similar tree topology when compared with sequence analysis of the rplV/ rpsC gene or of the secY gene alone. The main difference is that the branch lengths were elongated in the comprised gene tree. To further confirm the subgroup affiliation of PLY phytoplasma, the 16S rRNA gene sequences of 10 closely related phytoplasma strains were digested in silico, and the similarity coefficients were then calculated. The results also support the conclusion that PLY phytoplasma might belongs to a new 16SrI subgroup. The putative restriction site analysis can also distinguish PLY phytoplasma from other close related phytoplasma strains in phylogenetic analysis.

Phytoplasmas are wall-less prokaryotes associated with diseases in numerous plant species worldwide. In nature they are transmitted by phloem-sucking insects. Yellowing, decline, witches’ broom, leaf curl, floral virescence and phyllody are the most conspicuous symptoms associated with phytoplasmas, although infections are sometimes asymptomatic. Since phytoplasmas cannot be cultured in vitro, molecular techniques are needed for their diagnosis and characterization. The titer of phytoplasma cells in the phloem of infected plants may vary according to the season and the plant species, and it is often very low in woody hosts. Different DNA extraction procedures have therefore been tried out to obtain phytoplasma DNA at a concentration and purity high enough for effective diagnosis. DNA/DNA hybridization methods were reported in the nineties to be appropriate for the detection of phytoplasmas, but at present PCR is considered the most suitable. Universal and group-specific primers have been designed on the rRNA operon of the phytoplasma genome and on plasmid sequences. RFLP analysis of the obtained amplicons has classified these pathogens into major 16Sr RNA groups. Group-specific primers have also been designed on other genomic sequences. PCR is a very sensitive technique, but due to the low titre of phytoplasmas a further increase in sensitivity may be required for accurate diagnosis. This is routinely obtained with a second round of PCR (nested PCR). The drawback of nested PCR is that there is a greater chance of obtaining false positives due to contamination. Many authors have therefore developed protocols based on hybridization (PCR/dot blot) or serological approaches (PCR/ELISA) to increase the sensitivity and specificity of the direct PCR, reducing the risks due to nested PCR. Real time PCR protocols may also improve the sensitivity and specificity of the direct PCR assay.

Argentinian catharanthus little leaf phytoplasma (ACLL) was found naturally infecting Daucus carota L. (carrot), Catharanthus roseus (LG Don) (periwinkle) and wild Matricaria chamomilla L. (chamomile). All infected plants showed symptoms of yellowing, little leaf and internodes shortening. Flower scape distortion, malformations of umbels and roots were observed in infected carrots and witches´ broom in chamomile. Only periwinkles showed virescence and phyllody. On the basis of RFLP analyses of partial 16S rDNA, ribosomal protein rpl22 and tuf (EF-Tu) genes, ACLL phytoplasma was classified in group 16SrI (‘Ca. Phytoplasma asteris’), new subgroup 16SrI-S (rr-rp) tuf-H. All ACLL phytoplasma strains have 16S rRNA interoperon sequence heterogeneity as shown by PCR-RFLP and nucleotide sequence analyses. Phylogenetic analysis using 16S rDNA sequences clustered ACLL phytoplasma strains into a new phylogenetic lineage within group 16SrI.

Lethal yellowing (LY) is a devastating disease, affecting more than 35 palm species. One of them, Pritchardia pacifica, was found to be very susceptible in Florida and it is affected by a LY-type syndrome in Yucatan, Mexico. In this study, plants of P. pacifica were exposed naturally to feral insects in an area affected by LY in Mexico. All the plants exposed died showing symptoms of leaf decay with death occurring within approximately four months after first symptom. Real-time (polymerase chain reaction) PCR analysis of DNA extracts for phytoplasma detection and in silico sequence analysis of nested-PCR amplicons showed an association with 16SrIV-D phytoplasmas in plants studied. Time-course changes were studied for the detection of phytoplasmas, stomatal conductance and leaf temperature by thermography. One month before appearance of the first symptom, changes in all the parameters were observed: positive detection of phytoplasma DNA, a reduction in stomatal conductance that is complete and irreversible, and the appearance of a peak in leaf temperature. These results provide insights into a better understanding of this disease in P. pacifica. Moreover, from a practical point of view, the changes described could be useful for developing methods for early detection of the disease. Key words: Lethal yellowing-type disease, phytoplasmas, Pritchardia pacifica, stomatal conductance, thermography.

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia.