Phytoplasma Group Research Articles

Multilocus sequence analysis of a ‘Candidatus Phytoplasma australasia’-related strain associated with peanut little leaf disease in India

Severe little leaf and excessive shoot proliferation symptoms typical of the peanut little leaf (PnLL) disease were observed in different peanut (Arachis hypogaea) fields, variety K-6, at Kadiriand Gooty regions, of Andhra Pradesh, India, during the monsoon season of 2018–19. Disease incidence was 2–3%. The phytoplasma etiology of the recorded disease was confirmed by amplifying ~1.25 kb DNA products of 16S rRNA gene from symptomatic plants using nested polymerase chain reaction with phytoplasma specific primers, P1/P7 and R16F2n/R16R2, respectively. A further confirmation of the phytoplasma etiology was obtained by amplification of 840 bp, 1094 bp and 465 bp DNA fragments using phytoplasma specific primers targeting secA, tuf and SAP11 genes, respectively. Sequence and phylogenetic analyses of the amplified 16S rRNA, secA, tuf and SAP11 genes revealed that the detected phytoplasma is a member of the peanut witches’-broom phytoplasma group or 16SrII group (‘Candidatus Phytoplasma australasia’). Also, on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified 16S rRNA gene, the detected phytoplasma was assigned to subgroup D (16SrII-D). Parthenium hysterophorus plants showing witches’ broom and Cleome viscosa plants with little leaf symptoms, both collected in the mentioned peanut fields were also infected by similar strain of phytoplasma which proved to be identical with each of the molecular marker employed to the peanut-infecting agent in India. This is the first report on the association of ‘Ca. P. australasia’ (16SrII-D subgroup phytoplasma) with PnLL disease in India.

First report on the association of ‘Candidatus Phytoplasma asteris’ with lethal wilt disease of coconut (Cocos nucifera L.) in India

Lethal wilt disease of coconut, reported as a disease of unknown etiology as early as 2007, was for the first time found consistently in association with a phytoplasma in diseased coconut trees in Thanjavur, Thiruvarur and Pudukkottai districts in Tamil Nadu on the southeastern coast of India. Nested PCR with phytoplasma-specific universal primers and in silico restriction analysis of 16S rDNA sequences revealed the association of ‘Candidatus Phytoplasma asteris’ (16SrI-B) group phytoplasma, which was confirmed by sequence analysis of translation elongation factor (tuf) and ribosomal protein (rp) genes. Because this disease kills affected palms within 5 months, recommendations are given to arrest the spread of the pathogen to other coconut-growing regions in the country.

Diversity of the ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma fraxini’ isolates that infect urban trees in Bogotá, Colombia

Phytoplasmas have been associated with a disease that affects trees of at least 11 species from different botanic families in Bogotá, Colombia. 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' are the major groups of phytoplasma in the area of Bogotá. In this study, the genetic diversity within 'Ca. P. asteris' and 'Ca. P. fraxini' was studied in five urban tree species: Croton species (Euphorbiaceae), Fraxinus uhdei (Oleaceae), Magnolia grandiflora (Magnoliaceae), Populus nigra (Salicaceae) and Quercus humboldtii (Fagaceae). Analyses of the 16S rRNA gene using nested PCR, RFLP and sequencing showed that phytoplasmas of 'Ca. P. asteris' could be assigned to: subgroup 16SrI-B; a new subgroup named 16SrI-AF, with a restriction pattern similar to that of 16SrI-B; and a new subgroup named 16SrI-AG, with a restriction pattern similar to that of 16SrI-K and 16SrI-AH with a restriction pattern similar to that of 16SrI-AC. 'Ca. P. fraxini' isolates belonged to a new subgroup named 16SrVII-G, with a restriction pattern similar to that of 16SrVII-A. To complement the identification of the phytoplasma strains, we amplified nonribosomal genes such as leuS and secA. Unexpectedly, it was observed that in 16 trees in which 16S rRNA gene analysis showed the presence of 'Ca. P. fraxini' only, the leuS or secA primers amplified sequences exclusively affiliated to 'Ca. P. asteris. In those plants, sequences belonging to 'Ca. P. fraxini' leuS or secA genes were not amplified. The present work contributes to the identification of novel strains of both species in Colombia, and supports previous suggestions that phytoplasmas in South America are highly variable.

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification.

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrIB subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64oC for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/μl, namely approximately 53 copies/μl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

Molecular characterization of group 16SrI and 16SrIII phytoplasmas associated with loofah witches’ broom disease in Sinaloa, Mexico

Loofah [Luffa acutangula (L.) Roxb.] is grown in some regions of Asia, Africa and the Americas. This vegetable is well known for its high nutritional and medicinal value. Loofah plants showing typical symptoms of phytoplasma infection, such as witches’ broom, fruit deformation, flower proliferation and phyllody were observed in a commercial field in Sinaloa, Mexico, in January 2015. Nineteen symptomatic loofah plants and seven asymptomatic weeds (Helianthus annuus, Sarcostemma sp., Chenopodium sp., Amaranthus palmeri, Rumex sp., Ambrosia sp., and Raphanus sp.) growing nearby were collected and analyzed by phytoplasma-specific nested PCR. Phytoplasmas were detected in 79% of symptomatic loofah plants and in two of the seven weeds. Conventional and virtual restriction fragment length polymorphism (RFLP) analyses of the 16S rRNA-encoding gene sequences indicated the presence of strains of the 16SrIII and 16SrI phytoplasma groups in loofah, which occurred as both single and mixed infections. The phylogenetic analysis confirmed four new phytoplasma strains associated with loofah diseases, two ‘Candidatus Phytoplasma asteris’-related strains (16SrI-AG and 16SrI-AH), and two ‘Candidatus Phytoplasma pruni’-related strains (16SrIII-AA, and 16SrIII-AB). Furthermore, phytoplasmas belonging to groups 16SrI and 16SrIII also were identified in the weeds H. annuus and A. palmeri, respectively; 16SrIII-AC represents a new phytoplasma subgroup described in A. palmeri in Sinaloa. To our knowledge, this is the first report of loofah disease associated with phytoplasmas belonging to the 16SrI and 16SrIII groups in Mexico.

First report of a subgroup 16SrII-D phytoplasma associated with Opuntia cylindrica fasciated disease in Oman.

Cacti are evergreen perennial succulent plants that are used as ornamental and hedge plants. The fruits and leaves are also used as forage in some areas (Dewir, 2016). Cactus species are susceptible to several pathogens, including phytoplasma. In March 2020, three cactus plants (Opuntia cylindrica) out of ten (30% incidence) exhibited phytoplasma symptoms, including stunted growth, fasciation in stems and cladodes, color changes of the tips of cladodes to purple, and having clusters of highly proliferating cladodes. The plants were located in the Botanic Garden at Sultan Qaboos University, Muscat, Oman (N:23º59'14"; E:58º16'34"). PCR assays were carried out on the DNA samples extracted from young cladodes of three each of symptomatic and asymptomatic plants using phytoplasma-universal 16S rRNA primers, P1/P7 in direct PCR followed by R16F2n/R16R2, P4/P7 in the nested PCR. Distilled water (DW) and Alfalfa witches' broom phytoplasma (AlfWB) were used as negative and positive controls in each assay, respectively. In addition, amplification of the partial translocase protein A (secA) gene in the symptomatic cactus samples was done using SecA-II-F1/SecA-II-R1 (targeting 2140 bp) followed by SecA-II-F1/SecAR4 (targeting 1510 bp) (Al-Subhi et al., 2018). All the symptomatic plants and the positive control were positive for both genes (16S and secA), but no amplification was observed from the asymptomatic samples and DW. Sequence analysis and similarity searches against BLASTn revealed that the phytoplasma 16S rRNA (MT327813) shared 100% sequence identity with that of 'Candidatus Phytoplasma aurantifolia' isolate CB04 (MT555412) from India. The secA gene sequence (MT331815) analysis showed 100% identity with Cicer arietinum phyllody (KX358585). The associated phytoplasma was designated as cactus fasciated phytoplasma (CFP). Phylogenetic trees based on CFP 16Sr rRNA,secA genes, and a combined phylogenetic tree showed clustering of the CFP with the 16SrII-D subgroup phytoplasmas. The association of the aster yellows and peanut witches'-broom phytoplasma groups with other cactus species has already been reported from Lebanon, Mexico, China, Italy and Egypt (Dewir, 2016). The 16SrII phytoplasma in association with O. cylindrica showing fasciated stem has been reported from Egypt (Omar et al., 2014). A series of diverse plant species in association with 16SrII-D phytoplasma has been reported from Oman (Al-Subhi et al., 2018). However, this is the first report of a cactus phytoplasma disease in Oman belonging to the 16SrII-D subgroup phytoplasmas. Some fasciated cactus species are attractive and therefore cultivated as new ornamental plants and transported around the world, which may pose a new threat to other economically important crops.

Genetic diversity of phytoplasma strains infecting chrysanthemum varieties in India and their possible natural reservoirs

Symptoms typical of phytoplasma infection such as phyllody, virescence, witches’ broom and yellowing were observed in 12 varieties of Chrysanthemum morifolium in floral nurseries and experimental fields at New Delhi, Karnataka, Maharashtra and Andhra Pradesh, India, during surveys made from 2015 to 2017. Disease incidence ranged from 15 to 30%. Phytoplasma presence was confirmed in all symptomatic chrysanthemum varieties by molecular identification assays. Sequence comparison, phylogenetic and in silico RFLP analyses of 16S rDNA sequences allowed the identification of the chrysanthemum infecting phytoplasma strains into different ribosomal groups and subgroups, namely 16SrI, 16SrII-D, 16SrVI-D and 16SrXIV. Detection of phytoplasma strains of 16SrII-D subgroup were also confirmed in symptomatic Chenopodium album and Parthenium hysterophorus plants grown in and around the surveyed chrysanthemum fields at New Delhi, whereas 16SrVI-D phytoplasma strains were detected in symptomatic Cannabis sativa weed and leafhopper Hishimonus phycitis individuals collected from the symptomatic chrysanthemum fields at New Delhi. This is the first report on the presence of 16SrVI and 16SrXIV groups of phytoplasmas in chrysanthemum plants. Studies on genetic diversity of phytoplasmas infecting the major chrysanthemum varieties in India and their epidemiological aspects had previously not been reported. The detection and identification of phytoplasmas in different chrysanthemum varieties could contribute to increase the awareness among farmers in the management of these diseases.

Haplaxius crudus (Hemiptera: Cixiidae) Transmits the Lethal Yellowing Phytoplasmas, 16SrIV, to Pritchardia pacifica Seem. & H.Wendl (Arecaceae) in Yucatan, Mexico.

Lethal yellowing (LY) affects several palm species in the Americas. It is caused by 16SrIV group phytoplasmas. In Florida (USA), LY was shown to be transmitted by the planthopper Haplaxius crudus ( Van Duzee ) (Hemiptera, Cixiidae) to different palm species, including Pritchardia pacifica Seem . & H. Wendl . (Arecaceae) in insect-proof cage experiments in the 1980s, a result that had never been reproduced later. LY has destroyed many coconut plantations as well as other palm species in the Caribbean and Mexico. In order to evaluate if H. crudus is a vector of LY phytoplasmas in Mexico, experiments were carried out in Yucatan (Mexico). Several H. crudus from palms infected by LY in the field were introduced into cages containing young P. pacifica palms. These insects were able to transmit 16SrIV group phytoplasmas to P. pacifica palms. According to DNA sequences comparative analysis, virtual restriction fragment length polymorphism, and phylogenetic analysis, the phytoplasmas detected in these infected P. pacifica were of subgroups A and D. All of ten P. pacifica palms infected with the subgroup D phytoplasmas developed symptoms of LY and died, whereas only one of two palms infected with subgroup A developed LY symptoms and died. This is the first time, more than 30years later, that the role of H. crudus as a vector of LY is confirmed.

Survey of Incidence and Nested PCR Detection of Sugarcane White Leaf in Different Varieties.

Sugarcane white leaf (SCWL) is a devastating sugarcane (Saccharum officinarum) disease caused by a 16SrXI group phytoplasma, which is extremely harmful to sugarcane production. To determine the occurrence of SCWL in different varieties in 2018, we conducted a field survey and performed nested PCR detection of SCWL phytoplasma in cane-planting areas of Mangweng and Hepai in Gengma, Yunnan province, which are the areas most severely affected by SCWL in China. The results of the field survey showed that the symptomatic incidence of SCWL differed among varieties. The mean symptomatic incidence of SCWL on variety Yuetang60 was the highest (73.50%), and it was the lowest on Liucheng05-136 (13.67%). Using nested PCR, the SCWL phytoplasma was detected in symptomatic plants of all varieties more than 90% of the time; the SCWL phytoplasma was detected in 91 and 97% of symptomatic plants of Yingyu91-59 and Liucheng05-136 varieties, respectively. The SCWL phytoplasma was detected by PCR in 82% of the asymptomatic plant samples. The results of this study showed that field survey based on white leaf symptoms did not accurately reflect the actual occurrence of the SCWL phytoplasma.

Evaluating the effect of tuf and secA gene sequence length for discrimination of phytoplasmas

Phytoplasmas are a major liming factor to the growth and production of many plant species worldwide. Most studies have used the conserved 16S rRNA gene for phytoplasma identification and classification. The efficiency of the tuf and secA genes length in the characterization of phytoplasmas was evaluated by designing universal primers to amplify the long and short tuf (740 bp and 414 bp) and secA (1232 bp and 536 bp) genes from the phytoplasma 16SrII-A and -D sub-groups, which are closely related phytoplasma strains. The neighbour joining (NJ) and the maximum likelihood (ML) methods were used to test the accuracy and stability of the phylogenetic trees. The NJ and ML phylogenetic trees from the long and short DNA sequences of the 16S rDNA, tuf, and secA genes produced two major clades (WB and AS). The NJ and ML phylogenetic trees showed high similarity in their differentiation between and within the 16S phytoplasma groups. This study indicates that the long DNA sequences of tuf and secA genes produced phylogenetic trees with higher resolution and separated very closely related phytoplasma groups. The accurate characterization of phytoplasmas is important for epidemiological studies and in disease management.

Genetic diversity of phytoplasma strains inducing phyllody, flat stem and witches' broom symptoms in Manilkara zapota in India.

During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches' broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25kb and ~ 1.3kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by 16S rRNA gene sequence comparison analysis. Virtual RFLP analysis of 16S rRNA gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.

A survey of causal agents associated with sugarcane yellowing in Northeast Brazil

The etiological agents of the yellow leaf syndrome (YLS) are the Sugarcane yellow leaf virus (ScYLV), and phytoplasmas of different groups. Due to a lack of knowledge on the occurrence of YLS and the importance of sugarcane to Brazil, the aims of this study were to assess the occurrence and distribution of the pathogens associated with YLS in sugarcane cultivation areas in three states of Northeast Brazil. For this purpose, 101 sugarcane samples with symptoms of YLS were collected in 2018 crop cycle. ScYLV and a new phytoplasma were detected by RT-PCR and nested PCR, respectively. The identity of virus- and phytoplasma-positive samples was confirmed by Sanger sequencing, and these sequences were used in subsequent phylogenetic studies. Both pathogens were present in all sampled regions. ScYLV and the phytoplasma were detected in 40.4% and 62.2% of the samples, respectively. Mixed infections were found in 33.5% of the samples. Phylogenetic analysis showed that the ScYLV isolates identified were most closely related to ScYLV genotypes REU, PER, HAW, CHN2 and BRA. The phytoplasma 16S rDNA gene nucleotide sequences shared 99.0% identity with sequences of the 16SrVII phytoplasmas from Argentina and Brazil. This is the first report of the presence of a member of the 16SrVII phytoplasma group in association with sugarcane leaf yellowing in the world. These findings are important to raise awareness among sugarcane producers on the impact of pathogens causing YLS in Northeast Brazil.

Natural phytoplasma infections on fruit, vegetable and weed plants at the same agroecosystem and their molecular properties

The phytoplasma associated diseases are an emerging threat to fruit and vegetable crops leading severe yield losses worldwide. Pear (Pyrus communis L.) trees, with symptoms of severe reddening, dwarfing and shoot proliferation were observed in pear orchards of Malatya province of Turkey. Tomato (Solanum lycopersicum L.) plants grown nearby the symptomatic pear orchard displaying leaf rolling, severe flower sterility and purple leaves were observed at the same agroecosystem. To verify the presence and diversity of phytoplasmas, symptomatic pears and tomatoes were sampled and weeds nearby the symptomatic plants were collected. Total plant DNA was purified from midrib of collected leaves using a commercial kit. The DNA samples were analyzed by nested polymerase chain reaction (PCR) using universal primer pairs to amplify 16S rDNA fragments. The phytoplasmas detected in collected samples were differed according to the host. Here we detected and characterized ‘Candidatus Phytoplasma pyri’ belonging to apple proliferation group (subgroup 16SrX-C) from a pear tree, ‘Candidatus Phytoplasma trifolii’ belonging to clover proliferation group (subgroup 16SrVI-A) from a weed (Amaranthus retroflexus) and ‘Candidatus Phytoplasma solani’ belonging to the stolbur phytoplasma group (subgroup 16SrXII-A) from a tomato plant. Direct sequencing of PCR products verified the phytoplasmal nature of the infections. The occurrence of ‘Ca. P. trifolii’ on A. retroflexus is the first report for the world. The irregular presence of the phytoplasmas in fruit and vegetable crops and weeds indicates continuous spread of the phytoplasmas threatening the new crops and new horizons.

Correction to: Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.

Occurrence of ‘Candidatus Phytoplasma omanense’-related strains and other phytoplasmas in Sophora alopecuroides plants showing dwarfing and yellowing

Sophora alopecuroides dwarfing and yellowing symptoms were observed at the edge of several Damaneh and Fereydun Shahr (Isfahan province, Iran) fields. Polymerase chain reaction using primers P1/P7 and nested PCR using the same primers followed by R16mF2/R16mR2 and R16F2n/R16R2 primer pairs amplified the 16S ribosomal RNA-encoding gene in all the symptomatic plants tested, but not in the asymptomatic ones. RFLP analysis of R16F2n/R16R2 amplicons with AluI, HaeIII, HhaI, HpaI, HpaII, RsaI, MseI and TaqI restriction enzymes showed profiles referable to 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX phytoplasma ribosomal groups indicating also the presence of mixed infection in several samples. The identity percentage and the phylogeny obtained after direct sequencing of selected 16S rRNA-encoding gene sequences (R16F2n/R2 amplicons) from symptomatic S. alopecuroides confirmed the presence of phytoplasma strains related to ‘Candidatus Phytoplasma omanense’ (group 16SrXXIX), ‘Ca. P. australasia’ (group 16SrII), and ‘Ca. P. trifolii’ (16SrVI). Moreover, in a few samples/amplicons, 16SrXII-A, 16SrIX-H, 16SrVI-F and 16SrXXIX-A phytoplasma subgroups were identified by real and virtual RFLP analyses. The other phytoplasmas detected were classified in subgroups 16SrII-D and 16SrVI-A. This is the first report of the presence of 16SrII and 16SrXXIX phytoplasma groups and of mixed infection of phytoplasma strains belonging to groups 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX in S. alopecuroides worldwide.

Detection of 16SrVI and 16SrIX phytoplasma groups in pot marigold and tickseed plants in northeastern Iran.

Pot marigold and tickseed are ornamental plants with many medicinal and cosmetic uses and for landscape, respectively. During a survey in 2018, phyllody symptoms were observed in high percentages in these plants in some regions of the Razavi Khorasan province (northeastern Iran). Total DNA was extracted from symptomatic and asymptomatic plants and polymerase chain reaction was carried on using universal phytoplasma primer pairs P1/P7 and nested primer pairs R16F2n/R16R2. The nested amplification of 1200-bp fragments confirmed the presence of phytoplasmas only in the symptomatic plants. BLAST search, phylogenetic analysis, and virtual RFLP patterns of cloned amplicons allowed to classify the pot marigold phyllody phytoplasma in the 16SrVI-A subgroup while tickseed phyllody was enclosed in the 16SrIX-I subgroup. This is the first report of the association of a 16SrVI phytoplasma with pot marigold phyllody in Iran and of the phytoplasma presence in tickseed.

A timetree for phytoplasmas (Mollicutes) with new insights on patterns of evolution and diversification

The first comprehensive timetree is presented for phytoplasmas, a diverse group of obligate intracellular bacteria restricted to phloem sieve elements of vascular plants and tissues of their hemipteran insect vectors. Maximum likelihood-based phylogenetic analysis of DNA sequence data from the 16S rRNA and methionine aminopeptidase (map) genes yielded well resolved estimates of phylogenetic relationships among major phytoplasma lineages, 16Sr groups and known strains of phytoplasmas. Age estimates for divergences among two major lineages of Mollicutes based on a previous comprehensive bacterial timetree were used to calibrate an initial 16S timetree. A separate timetree was estimated based on the more rapidly-evolving map gene, with an internal calibration based on a recent divergence within two related 16Sr phytoplasma subgroups in group 16SrV thought to have been driven by the introduction of the North American leafhopper vector Scaphoideus titanus Ball into Europe during the early part of the 20th century. Combining the resulting divergence time estimates into a final 16S timetree suggests that evolutionary rates have remained relatively constant overall through the evolution of phytoplasmas and that the origin of this lineage, at ~641 million years ago (Ma), preceded the origin of land plants and hemipteran insects. Nevertheless, the crown group of phytoplasmas is estimated to have begun diversifying ~316 Ma, roughly coinciding with the origin of seed plants and Hemiptera. Some phytoplasma groups apparently associated with particular plant families or insect vector lineages generally arose more recently than their respective hosts and vectors, suggesting that vector-mediated host shifts have been an important mechanism in the evolutionary diversification of phytoplasmas. Further progress in understanding macroevolutionary patterns in phytoplasmas is hindered by large gaps in knowledge of the identity of competent vectors and lack of data on phytoplasma associations with non-economically important plants.

Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

Characterization of 16SrII group phytoplasma associated with sesame phyllody disease in different cropping seasons

A survey was conducted in the sesame fields of Karnataka, India during April–June 2010 (summer) and in Telangana State, Maharashtra and Karnataka during July–September 2010 (Kharif). In summer, disease incidence of 18.57–31.6% was recorded, whereas, in Kharif, incidence of 44.66–68%, 13–47.14% and 32.14% was recorded in Telangana State, Karnataka and Maharashtra respectively. Three symptomatic and two non-symptomatic samples from summer crop and sixteen symptomatic and two non symptomatic samples from Kharif season fields were collected. The disease etiology was confirmed using nested polymerase chain reaction (PCR) with phytoplasma universal primers P1/P7 followed by R16F2n/R16R2 which amplified ~ 1.8 and ~ 1.25 kb in the 16SrDNA gene. The BLAST and phylogenetic analysis of 16S rDNA gene further revealed that phytoplasma associated with phyllody disease in both the season were belonged to peanut witches’ broom group (16SrII). Further, iPhyClassifier analysis of 1.25 kb product of 16S rDNA sequence classified the isolates into 16SrII-A and 16SrII-D subgroups. All summer crop isolates were classified under 16SrII-D subgroup whereas; Kharif season isolates were grouped under 16SrII-A and 16SrII-D group. Phylogenetic analysis revealed the distinct subgroup-wise as well as season-wise clustering pattern where subgroup 16SrII-A and 16SrII-D clustered separately and summer and Kharif season isolates clustered separately. Our study reports that the sesame phyllody disease in Kharif season is caused by the taxonomically distinct strains (16SrII-A and 16SrII-D) whereas, in the summer crop, it is associated with 16SrII-D subgroup alone.

Detection, characterization and evolutionary aspects of S54LP of SP (SAP54 Like Protein of Sesame Phyllody): a phytoplasma effector molecule associated with phyllody development in sesame (Sesamum indicum L.).

SAP54, an effector protein secreted by phytoplasmas has been reported to induce phyllody. S54LP of SP (SAP54 Like Protein of Sesame Phyllody), a SAP54 ortholog from phyllody and witches' broom affected sesame (Sesamum indicum L.) was amplified, cloned and sequenced. Comparative sequence and phylogenetic analysis of diverse phytoplasma strains was carried out to delineate the evolution of S54LP of SP. The degree of polymorphism across SAP54 orthologs and the evolutionary forces acting on this effector protein were ascertained. Site-specific selection across SAP54 orthologs was estimated using Fixed Effects Likelihood (FEL) approach. Nonsynonymous substitutions were detected in the SAP54 orthologs' sequences from phytoplasmas belonging to same (sub) group. Phylogenetic analysis based on S54LP of SP grouped phytoplasmas belonging to same 16SrDNA (sub) groups into different clusters. Analysis of selection forces acting on SAP54 orthologs from nine different phytoplasma (sub)groups, affecting plant species belonging to twelve different families across ten countries showed the orthologs to be under purifying (negative) selection. One amino acid residue was found to be under pervasive diversifying (positive) selection and a total of three amino acid sites were found to be under pervasive purifying (negative) selection. The location of these amino acids in the signal peptide and mature protein was studied with an aim to understand their role in protein-protein interaction. Asparagine residues (at positions 68 and 84) were found to be under pervasive purifying selection suggesting their functional importance in the effector protein. Our study suggests lack of coevolution between SAP54 and 16SrDNA. Signal peptide appears to evolve at a rate slightly higher than the mature protein. Overall, SAP54 and its orthologs are evolving under purifying selection confirming their functional importance in phytoplasma virulence.