Identification of fruit tree phytoplasmas and their vectors in Bosnia and Herzegovina

Surveys were carried out in autumn 2004 and spring 2005 in the traditional areas dedicated to pome and stone fruit cultivation in Bosnia and Herzegovina to assess the presence, distribution and incidence of phytoplasma diseases in fruit trees. The occurrence of psyllid vectors was also considered. The detection of phytoplasmas in plant and insect samples and their identification were carried out by symptom observations in the field, double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA), nested polymerase chain reaction (nested-PCR) and restriction fragment length polymorphism (RFLP) analyses. Laboratory analyses showed the presence of phytoplasmas belonging to: (i) 16SrX group, subgroup A (' Candidatus Phytoplasma mali') in 23 out of 25 apple samples, in 4 groups out of 18 of Cacopsylla picta (synonym Cacopsylla costalis ) and in 2 groups out of 9 of Cacopsylla melanoneura ; (ii) 16SrX group, subgroup C (' Candidatus Phytoplasma pyri') in 11 out of 30 pears samples and in 2 groups out of 9 of Cacopsylla pyri ; (iii) 16SrX group, subgroup B (' Candidatus Phytoplasma prunorum') in 4 apricots, 2 peaches out of 42 stone fruit samples and in 1 group out of 14 of Cacopsylla pruni . The presence of different subtypes of Candidatus Phytoplasma mali, both in apple trees and in insects, was proven.     

Co-operational PCR coupled with dot blot hybridization for detection and 16SrX grouping of phytoplasmas

A protocol based on Co-operational PCR has been successfully applied to the detection of phytoplasmas. A triprimer reaction coupled with hybridization using general and specific probes permitted detection of ' Candidatus Phytoplasma mali', ' Ca . Phytoplasma prunorum' and ' Ca . Phytoplasma pyri', and their identification as members of 16S ribosomal quarantine group X. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR. The method was validated by testing field samples collected from Malus , Prunus and Pyrus spp. and Olea europaea and compared with seven phytoplasmas maintained in Catharanthus roseus .     

Characteristics of the spread of apple proliferation by its vector Cacopsylla picta

The distribution and natural phytoplasma infection of Cacopsylla picta were investigated during a long-term field survey between 2002 and 2009 in commercial and abandoned apple proliferation-infected orchards throughout Germany, northern Switzerland, and eastern France. Comparable population dynamics were described for the different sites whereas considerable variations in the absolute population densities were observed among the years. Individual polymerase chain reaction (PCR) testing revealed, for each year, a rather stable natural infection rate with ?Candidatus Phytoplasma mali? of ?10% for overwintered adults of C. picta. Both genders were equally highly infected although more females were caught. The overall male/female ratio was 1:1.5. No direct correlation was found between the infection status of the orchard and the infection rate of overwintered C. picta. No influence of agricultural practices was seen. However, a relationship between the incidence of the disease and the vector population density became evident on a regional scale. Successful transmission of ?Ca. P. mali? occurred each year with overwintered individuals as well as with new adults. The transmission efficiency varied among the years within 8 to 45% for overwintered adults and 2 to 20% for individuals of the new generation. The load of single C. picta with ?Ca. P. mali? was determined by quantitative real-time PCR. High phytoplasma titers were measured in overwintered adults already at their first appearance in the orchards after remigration from their overwintering hosts. Thus, the data indicate the transmission of the disease on a regional scale by remigrant adults of C. picta and at a local scale within the same season by emigrant adults which developed on infected plants.     

The insect vector Cacopsylla picta vertically transmits the bacterium ‘Candidatus Phytoplasma mali’ to its progeny

The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F₁ individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F₁ adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.     

Mapping the spread of apricot chlorotic leaf roll (ACLR) in southern France and implication of Cacopsylla pruni as a vector of European stone fruit yellows (ESFY) phytoplasmas

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc-Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY-infected apricot orchards, of which about 10 000 were used in PCR and nested-PCR assays with universal ribosomal and ESFY-specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa . Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni .     

Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

Incidence and distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in Spain: an approach to the epidemiology of the disease and the role of wild Prunus

‘Candidatus Phytoplasma prunorum’ is the causal agent of the European stone fruit yellows (ESFY) disease. This phytoplasma affects wild and cultivated species of Prunus to different degrees, depending on their susceptibility. ‘Candidatus Phytoplasma prunorum’ is present in the four regions of Spain surveyed in this study (Aragon, Catalonia, Extremadura and Valencia) with a variable incidence. Results showed that ‘Ca. Phytoplasma prunorum’ was detected in all of the cultivated Prunus species studied, except P. avium and P. dulcis, and was widespread in Spain. The most affected species was P. salicina, with symptoms including early bud break and blooming, leaf curling and yellowing, collapse, and a major decrease in production. In some plots in the Baix Llobregat area of Barcelona province (Catalonia), the incidence of ESFY on P. salicina was as high as 80%. The insect vector, Cacopsylla pruni, was present in all four of the regions studied, with the highest captures in yellow sticky traps in Catalonia on P. mahaleb and in Extremadura in peach orchards. In Baix Llobregat, large populations of C. pruni were present on infected P. mahaleb bushes, and with high infection rates. This was a key factor in the local pathogenic cycle that caused a major ESFY outbreak in the nearby P. salicina orchards. In the Ebro valley (Lleida and Aragon) and Valencia, the surveys showed very low incidences of the disease and low C. pruni populations.     

Identifying risk factors for European stone fruit yellows from a survey

ABSTRACT European stone fruit yellows (ESFY) is becoming a major economic problem for Prunus growers in Europe. The causal agent ("Candidatus Phytoplasma prunorum") and its vector (Cacopsylla pruni) have been identified, but the present knowledge of the risk factors for this disease relies, at best, on specific experiments. To assess the relative significance of several factors correlated with ESFY incidence in the field, an exhaustive survey was performed on apricot and Japanese plum orchards in the Crau plain (France). After a preliminary multivariate exploration of the data, we used a logistic regression model to analyze and predict the cumulative number of diseased trees on the basis of a set of quantitative (age, planting density, and area of the orchard) and categorical variables (species, cultivar, and rootstock). Because of the nature of the data, we used an overdispersed binomial model and we developed a parametric bootstrap procedure based on the beta-binomial distribution to obtain confidence intervals. Our results indicated that the age, species, and cultivar of the scion were the major factors explaining the observed number of diseased trees. The planting density and the rootstocks used in the zone under study were less significant, and the area of the orchard had no effect. The residuals of the model showed that some explanatory variables had not been taken into account, because part of the remaining variability could be explained by a grower effect. The spatial distribution of the residuals suggested that one of the reasons for this grower effect was the correlation between orchards closer than 100 m, possibly caused by the flight behavior of infectious vectors.     

Role of <Emphasis Type="Italic">Cacopsylla pyri</Emphasis> in the epidemiology of pear decline in Spain

The frequency of pear decline-positive insects and transmission of pear decline (PD) phytoplasma by Cacopsylla pyri in Spain has been studied. Psyllids were used for experiments on phytoplasma transmission both to healthy Pyrus communis trees and to an artificial feeding medium. Over a period of 1 year, about 100 psyllids were collected monthly from pear trees, cv. Williams, using the beating tray method, and tested for the presence of PD phytoplasma. Results indicate that the frequency of PD positive psyllids changes through the year and that C. pyri transmits the pear decline associated disease agent. Phytoplasma transmission was also effective under laboratory conditions using a feeding medium. The relationship between PD positive Cacopsylla pyri, Pear decline phytoplasma transmission and the sex of the vector was also evaluated. Although the percentage of PD positive psyllids was similar in both genders, PD phytoplasma transmission by females was significantly higher than by males. Since the sex ratio (male/female) was less than 1:1 for most of the year, these results should be taken into consideration for controlling Pear decline in Mediterranean climates.     

First report of Almond witches’ broom phytoplasma1 (‘Candidatus Phytoplasma phoenicium’) causing a severe disease on nectarine and peach trees in Lebanon

Symptoms of shoot proliferation characteristic of phytoplasma diseases were observed on nectarine (Prunus persica var. nucipersica) and peach (P. persica) trees in the Sarada plain, south of Lebanon. The presence of phytoplasmas in the two orchards visited was confirmed by nested polymerase chain reaction using universal primers. The amplified DNA fragments were cloned and sequenced. Blast analysis of over 1000 nucleotides demonstrated the presence of ‘Candidatus Phytoplasma phoenicium’ which is considered to be the causal agent of Almond witches’ broom. This phytoplasma which belongs to the pigeon pea witches’ broom group (16SrIX) can be devastating since Almond witches’ broom has killed thousands of almond trees in Lebanon and Iran. Previous reports indicated that Almond witches’ broom may be transmitted by grafting to peach and nectarine under experimental conditions. This is the first report of a natural and epidemic spread of ‘Ca. Phytoplasma phoenicium’ in peach and nectarine. Farmers in the region were advised to eradicate the infected trees immediately. Further studies on the epidemiology of ‘Ca. Phytoplasma phoenicium’ and its vector(s) are recommended in order to develop successful eradication or disease management programmes.     

Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species

Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.     

Differences in Virulence and Genomic Features of Strains of 'Candidatus Phytoplasma mali', the Apple Proliferation Agent

ABSTRACT Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with 'Candidatus Phytoplasma mali' were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52 degrees C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in 'Ca. P. mali'. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.     

Role of wild Prunus species in the epidemiology of European stone fruit yellows

Several uncultivated trees of the species Prunus spinosa , P. cerasifera and P. domestica , sampled both adjacent to European stone fruit yellows (ESFY)-infected orchards and in isolation from cultivated stone fruit plants, were found to be infected by ESFY phytoplasma. These species were also colonized by Cacopsylla pruni , vector of the ESFY agent. In contrast, uncultivated species of Prunus avium , P. cerasus and P. mahaleb hosted neither the pathogen nor the vector. Insect- and graft-transmission trials of ESFY phytoplasma conducted under controlled conditions confirmed the data obtained in the field. The role played by the wild Prunus species is discussed and appears to be fundamental in the epidemic cycle of the disease.     

Transmission Characteristics of the European Stone Fruit Yellows Phytoplasma and its Vector Cacopsylla Pruni

A study was carried out on the transmission parameters of the European stone fruit yellows phytoplasma by the vector Cacopsylla pruni. In the greenhouse, using groups of psyllids, the minimum acquisition period was 2–4 days, the minimum latent period 2–3 weeks and the minimum inoculation period 1–2 days. The vectors retained infectivity until their death. Under natural conditions retention of infectivity in C. pruni lasts through the winter and the following spring, when the overwintering insects reach the stone fruit trees, they are already infected and infective. The research shows that the vector C. pruni transmits the European stone fruit yellows phytoplasma in a persistent manner.     

Composition,abundance and phytoplasma infection in the hawthorn psyllid fauna of northwestern Italy

Hawthorn (Crataegus monogyna) is one of the natural hosts of Cacopsylla melanoneura, the acknowledged vector of ‘Candidatus Phytoplasma mali’, the causal agent of Apple Proliferation disease, a serious and growing problem for apple production in Europe, particularly in northern Italy. Wild plants could be important sources of both insects and phytoplasmas, but their role in the epidemiology of phytoplasma diseases and their insect vectors has never been thoroughly examined. Cacopsylla melanoneura’s primary host is hawthorn, a plant closely related to apple which often grows wild near orchards. Other psyllid species feed on hawthorn, but no data are available on their possible role as phytoplasma vectors. We investigated the hawthorn’s psyllid fauna in northwestern Italy using yellow sticky traps, beat trays, and molecular analyses from 2003–2005, to study the relationship between hawthorn, the phytoplasma and the insect vector. Population dynamics were monitored, and insects and hawthorn samples were analysed by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and DNA sequencing for the presence of phytoplasmas. Cacopsylla melanoneura was the dominant psyllid species, followed by C. peregrina, C. affinis and C. crataegi. PCR and RFLP analyses revealed the presence of different fruit tree phytoplasmas in hawthorn plants, and in all four psyllid species.     

"Candidatus Phlomobacter fragariae" Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China)

ABSTRACT Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.     

Distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in European fruit‐growing areas: a review

European stone fruit yellows (ESFY) is an EU‐listed I/AII disease affecting Prunus spp. caused by ‘Candidatus Phytoplasma prunorum’. This paper reports the results from a systematic literature review approach that sought to determine the geographic distribution of ‘Ca. Phytoplasma prunorum’ in European fruit‐growing areas. Evidence for the presence of the phytoplasma was found for 15 of the 27 EU countries. It is prevalent in the most important stone fruit production areas of Central and Southern Europe, where it causes substantial impact in apricots (Prunus armeniaca), Japanese plums (P. salicina) and peaches (P. persica). In Northern European areas where these hosts are not produced, it is occasionally found on tolerant species (P. domestica). However, because surveys of the disease status of tolerant hosts are not performed, it remains unclear whether the pathogen is absent in Northern Europe or survives in tolerant cultivated or wild hosts. No reports of ESFY were found from the southernmost part of Europe: Portugal, Spain (Andalucia, Castile–La Mancha), Italy (Sicily, Puglia), Greece (Crete), Cyprus and Malta. This may be explained by the absence of the favoured wild hosts of the vector. Moreover, it remains unclear if the vector finds suitable conditions for aestivation and overwintering in these regions.     

Association of 'Candidatus Phytoplasma australiense' with green petal and lethal yellows diseases in strawberry

The identity of phytoplasmas detected in strawberry plants with green petal (SGP) and lethal yellows (SLY) diseases was determined by RFLP analysis of the 16S rRNA gene and adjacent spacer region (SR). RFLP and sequence comparisons indicated that the phytoplasmas associated with SGP and SLY were indistinguishable and were most closely related to ' Candidatus Phytoplasma australiense', the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. This taxon lies within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify only members of the AY strain cluster, amplified a DNA product from the SGP and SLY phytoplasmas. Primers deduced from the 16S rRNA/SR of P. australiense that amplify only members of this taxon amplified rDNA sequences from the SGP and SLY phytoplasmas. Primers that selectively amplify members of the faba bean phyllody (FBP) phytoplasma group, the most commonly occurring phytoplasma group in Australia, did not amplify rDNA from the SGP and SLY phytoplasmas.     

Untersuchungen zur Europäischen Steinobstvergilbung (ESFY) in Deutschland

From 2003 to 2007 surveys have been conducted in different stone fruit growing regions in southwest Germany to detect European stone fruit yellows (ESFY) disease in Germany. Samplings have been done regularly in selected reference orchards in the regions Neuwieder Becken, Rheinhessen, Vorderpfalz and Südpfalz in summer on trees showing ESFY typical symptoms as well as on branches of trees with unspecific symptoms. All samples have been analysed by PCR for infection with Candidatus Phytoplasma prunorum. The phytoplasma could be detected in all investigated regions on the cultivated Prunus species P. armeniaca, P. persica and P. domestica. No infection was found in wild Prunus species. The main spread of the disease appeared on apricot while peach and European plum were less affected. A good correlation between symptoms and molecular detection of the pathogen could be shown for the typical symptoms in summer and winter for apricot as well as for peach. During regular psyllid captures in the reference orchards the population dynamics of Cacopsylla pruni could be described in southwest Germany for several years. By PCR-testing all collected insects individually a yearly natural infection rate of about 1–2% of all individuals of C. pruni could be calculated.     

Genetic variation in Candidatus Phytoplasma australiense

This study examined whether genes that are less conserved than the 16S rRNA gene can distinguish Candidatus Phytoplasma australiense strains that are identical based on their 16S rRNA genes, with a view to providing insight into their origins and distribution, and any patterns of association with particular plant hosts. Sequence analysis of the tuf gene and rp operon showed that Ca . P. australiense strains could be differentiated into four subgroups, named 16SrXII-B ( tuf -Australia I; rp -A), 16SrXII-B ( tuf -New Zealand I; rp -B), 16SrXII-B ( tuf -New Zealand II) and 16SrXII-B ( rp -C). Strawberry lethal yellows 1, strawberry green petal, Australian grapevine yellows, pumpkin yellow leaf curl and cottonbush witches' broom phytoplasmas were designated members of the 16SrXII-B ( tuf -Australia I; rp -A) subgroup. The strawberry lethal yellows 2 and cottonbush reduced yellow leaves phytoplasmas were assigned to the 16SrXII ( tuf -New Zealand II; rp -B) subgroup. No relationship was observed between these phytoplasma subgroups and collection date, location or host plant. However, the study revealed evolutionary divergence in the 16SrXII group.