Co-evolution and symbiont replacement shaped the symbiosis between adelgids (Hemiptera: Adelgidae) and their bacterial symbionts

The Adelgidae (Insecta: Hemiptera), a small group of insects, are known as severe pests on various conifers of the northern hemisphere. Despite of this, little is known about their bacteriocyte‐associated endosymbionts, which are generally important for the biology and ecology of plant sap‐sucking insects. Here, we investigated the adelgid species complexes Adelges laricis/tardus, Adelges abietis/viridis and Adelges cooleyi/coweni, identified based on their coI and ef1alpha genes. Each of these insect groups harboured two phylogenetically different bacteriocyte‐associated symbionts belonging to the Betaproteobacteria and the Gammaproteobacteria, respectively, as inferred from phylogenetic analyses of 16S rRNA gene sequences and demonstrated by fluorescence in situ hybridization. The betaproteobacterial symbionts of all three adelgid complexes (‘Candidatus Vallotia tarda’, ‘Candidatus Vallotia virida’ and ‘Candidatus Vallotia cooleyia’) share a common ancestor and show a phylogeny congruent with that of their respective hosts. Similarly, there is evidence for co‐evolution between the gammaproteobacterial symbionts (‘Candidatus Profftia tarda’, ‘Candidatus Profftia virida’) and A. laricis/tardus and A. abietis/viridis. In contrast, the gammaproteobacterial symbiont of A. cooleyi/coweni (‘Candidatus Gillettellia cooleyia’) is different from that of the other two adelgids but shows a moderate relationship to the symbiont ‘Candidatus Ecksteinia adelgidicola’ of A. nordmannianae/piceae. All symbionts were present in all adelgid populations and life stages analysed, suggesting vertical transmission from mother to offspring. In sharp contrast to their sister group, the aphids, adelgids do not consistently contain a single obligate (primary) symbiont but have acquired phylogenetically different bacterial symbionts during their evolution, which included multiple infections and symbiont replacement.     

Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts

Endosymbioses are a major form of biological complexity affecting the ecological and evolutionary diversification of many eukaryotic groups. These associations are exemplified by nutritional symbioses of insects for which phylogenetic studies have demonstrated numerous cases of long-term codiversification between a bacterial and a host lineage. Some insects, including most leafhoppers (Insecta: Hemiptera: Cicadellidae), have more than one bacterial symbiont within specialized host cells, raising questions regarding the patterns of codiversification of these multiple partners and the evolutionary persistence of complex symbiotic systems. Previous studies reported the presence of two dominant symbiont types in a member of the leafhopper subfamily Cicadellinae (sharpshooters). In this study, 16S rRNA sequences were obtained and used to examine the occurrence and evolutionary relationships of the two dominant symbiont types across 29 leafhopper species. Candidatus Sulcia muelleri (Bacteroidetes) was detected in all leafhopper species examined, a finding that is consistent with a previous report of its ancient association with the Auchenorrhyncha (a grouping that includes leafhoppers, treehoppers, cicadas, planthoppers, and spittlebugs). Baumannia cicadellinicola (Proteobacteria), previously known from only five sharpshooter species, was found only in the sharpshooter tribes Cicadellini and Proconiini, as well as in the subfamily Phereurhininae. Mitochondrial and nuclear gene sequences were obtained and used to reconstruct host phylogenies. Analyses of host and symbiont data sets support a congruent evolutionary history between sharpshooters, Sulcia and Baumannia and thus provide the first strong evidence for long-term co-inheritance of multiple symbionts during the diversification of a eukaryotic host. Sulcia shows a fivefold lower rate of 16S rDNA sequence divergence than does Baumannia for the same host pairs. The term 'coprimary' symbiont is proposed for such cases.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

Coexistence of novel gammaproteobacterial and Arsenophonus symbionts in the scale insect Greenisca brachypodii (Hemiptera,Coccomorpha: Eriococcidae)

Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii, a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod‐shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae, whereas the rod‐shaped symbionts are close relatives of Arsenophonus symbionts in insects – to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (～90%), we propose the name ‘Candidatus Kotejella greeniscae’ for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic ‘symbiont ball’. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.     

Symbiosis in the green leafhopper,Cicadella viridis (Hemiptera,Cicadellidae). Association in statu nascendi?

The green leafhopper, Cicadella viridis lives in symbiotic association with microorganisms. The ultrastructural and molecular analyses have shown that in the body of the C. viridis two types of bacteriocyte endosymbionts are present. An amplification and sequencing of 16S rRNA genes revealed that large, pleomorphic bacteria display a high similarity (94–100%) to the endosymbiont ‘Candidatus Sulcia muelleri’ (phylum Bacteroidetes), whereas long, rod-shaped microorganisms are closely related to the γ-proteobacterial symbiont Sodalis (97–99% similarity). Both endosymbionts may be harbored in their own bacteriocytes as well as may co-reside in the same bacteriocytes. The ultrastructural observations have revealed that the Sodalis-like bacteria harboring the same bacteriocytes as bacterium Sulcia may invade the cells of the latter. Bacteria Sulcia and Sodalis-like endosymbionts are transovarially transmitted from one generation to the next. However, Sodalis-like endosymbionts do not invade the ovaries individually, but only inside Sulcia cells. Apart from bacteriocyte endosymbionts, in the body of C. viridis small, rod-shaped bacteria have been detected, and have been identified as being closely related to γ-proteobacterial microorganism Pectobacterium (98–99% similarity). The latter are present in the sheath cells of the bacteriomes containing bacterium Sulcia as well as in fat body cells.     

“Candidatus Euplotechlamydia quinta,” a novel chlamydia-like bacterium hosted by the ciliate Euplotes octocarinatus (Ciliophora,Spirotrichea)

Our knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia-like bacteria have been described as symbionts of free-living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b-1 was performed, using metagenomic screening as well as classical full-cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia-like bacterium. Following the full-cycle rRNA approach, we obtained the full-length 16S rRNA gene sequence of this chlamydia-like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia-like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: “Candidatus Euplotechlamydia quinta.”     

Independent Origins of Vectored Plant Pathogenic Bacteria from Arthropod-Associated <Emphasis Type="Italic">Arsenophonus</Emphasis> Endosymbionts

The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sap-sucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood and Bayesian criteria, revealed that this bacterium is not a sister taxon to “Candidatus Phlomobacter fragariae,” a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropod-associated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, “Candidatus Arsenophonus phytopathogenicus” novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.     

Symbiosis and Insect Diversification: an Ancient Symbiont of Sap-Feeding Insects from the Bacterial Phylum Bacteroidetes

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 μm in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. “Candidatus Sulcia muelleri” is proposed as the name of the new symbiont.     

New bacterium symbiont in the bacteriome of the leafhopper Yamatotettix flavovittatus Matsumura

The leafhopper Yamatotettix flavovittatus Matsumura is the vector of the phytoplasma pathogen that causes white leaf disease in sugarcane crops. This study aimed to identify the bacterial symbionts associated with Y. flavovittatus by amplifying, cloning, and sequencing their 16S rRNA genes. Two types of bacteria were present; one is Candidatus Sulcia muelleri (Bacteroidetes), a well-known ancient primary symbiont found in a diverse range of insects in Auchenorrhyncha suborder. The other is a member of Gammaproteobacteria that differed from all other members deposited in the GenBank database and was therefore named Candidatus Yamatotia cicadellidicola. These bacteria were present throughout the leafhopper life cycle and were found in 100% of the natural host populations examined. Fluorescent in situ hybridization analysis revealed that these bacteria were co-localized in the same bacteriome and present in the full-grown oocyte. Our main finding suggests the Ca. Y. cicadellidicola is a new type of symbiont that co-occurs with Ca. S. muelleri in the leafhopper Y. flavovittatus (Deltocephalinae subfamily).     

Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 mum in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. "Candidatus Sulcia muelleri" is proposed as the name of the new symbiont.     

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.     

A cixiid survey for natural potential vectors of ‘Candidatus Phytoplasma phoenicium’ in Lebanon and preliminary transmission trials

Almond witches'‐broom (AlmWB) disease, associated with ‘Candidatus Phytoplasma phoenicium’, is an emerging threat with real risk of introduction in Euro‐Mediterranean Countries. Its rapid spread over large geographical areas suggests the presence of efficient insect vector(s). In the present work, a survey on cixiids was carried out in Lebanon in the years 2010–2013 in AlmWB‐infested almond and nectarine orchards. Insects were collected by means of different methods, identified with a stereo microscope, and analysed for phytoplasma identification through 16S rDNA PCR‐based amplification and nucleotide sequence analyses. Preliminary transmission trials were performed with the most abundant species. A list of the cixiid genera and species present in the studied area is given as well as some information about their biology. ‘Ca. Phytoplasma phoenicium’ strains were detected in the genera Cixius, Tachycixius, Eumecurus and Hyalesthes. Preliminary trials revealed that Tachycixius specimens were able to transmit the detected strains to healthy peach potted seedlings. Further studies are required to better clarify the taxonomic status and the bio‐ethology of collected planthoppers and deeply study their role as phytoplasma vectors.     

Cellular and potential molecular mechanisms underlying transovarial transmission of the obligate symbiont Sulcia in cicadas

Vertical transmission of symbionts in insects is critical to persistence of symbioses across host generations. The key time point and related cellular/molecular mechanisms underlying the transmission in most insects remain unclear. Here, we reveal that in the bacteriome–endosymbiont system of the cicada Meimuna mongolica, the obligate symbiont Candidatus Sulcia muelleri (hereafter Sulcia) proliferates and migrates to the ovaries mainly after the adult emergence of cicadas. Sulcia cells swell to approximately twice their previous size with the outer membrane changed to be more irregular during this process. Almost all the Sulcia genes involved in biosynthesis of essential amino acids, heat shock protein, energy metabolism, DNA replication and repair and protein export were highly expressed in all life stages of cicadas. Among which, genes involved in DNA replication and synthesis of leucine and arginine were upregulated in the newly emerged adults relative to fifth-instar nymphs. Signal transduction is the pronounced function exhibited in both Sulcia and the cicada bacteriomes in newly emerged adults. The results suggest host sensing of arginine and leucine integrate Sulcia's output of host-EAAs into mTORC1 signalling. This study highlights the importance of signalling pathways in regulating the host/symbiont interaction and symbiont transmission in sap-feeding auchenorrhynchous insects.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.     

Candidates for symbiotic control of sugarcane white leaf disease

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) is the most important vector of a phytoplasma pathogen causing sugarcane white leaf (SCWL) disease. The purpose of this study was to evaluate candidate bacterial symbionts for possible use as vehicles in the control of the disease. 16S rRNA bacterial genes were amplified from whole bodies of M. hiroglyphicus leafhoppers and analyzed by cloning and sequencing. Two dominant groups were found: one belonged to the Betaproteobacteria that did not closely match any sequences in the database and was named bacterium associated with M. hiroglyphicus (BAMH). Another one found to be abundant in this leafhopper is "Candidatus Sulcia muelleri" in the order Bacteroidetes, which was previously reported in the insect members of the Auchenorrhyncha. Most M. hiroglyphicus leafhoppers carry both BAMH and "Ca. Sulcia muelleri." Fluorescent in situ hybridization showed that BAMH and "Ca. Sulcia muelleri" colocalized in the same bacteriomes. BAMH was present in the midgut and ovaries of the leafhopper and was found in all developmental stages, including eggs, nymphs, and adults. Because BAMH appears to be specific for the SCWL vector, we evaluated it as a candidate for symbiotic control of sugarcane white leaf disease.     

A novel subspecies of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter africanus’ found on native <Emphasis Type="Italic">Teclea gerrardii</Emphasis> (Family: Rutaceae) from South Africa

The phloem limited bacterium ‘Candidatus Liberibacter africanus’ is associated with citrus greening disease in South Africa. This bacterium has been identified solely from commercial citrus in Africa and the Mascarene islands, and its origin may lie within an indigenous rutaceous host from Africa. Recently, in determining whether alternative hosts of Laf exist amongst the indigenous rutaceous hosts of its triozid vector, Trioza erytreae, three novel subspecies of Laf were identified i.e. ‘Candidatus Liberibacter africanus subsp. clausenae’, ‘Candidatus Liberibacter africanus subsp. vepridis’ and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ in addition to the formerly identified ‘Candidatus Liberibacter africanus subsp. capensis’. The current study expands upon the range of indigenous rutaceous tree species tested for liberibacters closely related to Laf and its subspecies. A collection of 121 samples of Teclea and Oricia species were sampled from Oribi Gorge and Umtamvunu nature reserves in KwaZulu Natal. Total DNA was extracted and the presence of liberibacters from these samples determined using a generic liberibacter TaqMan real-time PCR assay. Liberibacters from positive samples were further characterised through amplification and sequencing of the 16S rRNA, outer-membrane protein (omp) and 50S ribosomal protein L10 (rplJ) genes. A single Teclea gerrardii specimen tested positive for a liberibacter and, through phylogenetic analyses of the three genes sequenced, was shown to be unique, albeit closely related to ‘Ca. L. africanus’ and ‘Ca. L. africanus subsp. zanthoxyli’. We propose that this newly identified liberibacter be named ‘Candidatus Liberibacter africanus subsp. tecleae’.     

Bacteriome-associated endosymbionts of the green rice leafhopper <Emphasis Type="Italic">Nephotettix cincticeps</Emphasis> (Hemiptera: Cicadellidae)

The green rice leafhopper Nephotettix cincticeps (Uhler) is a commonly distributed pest of rice in East Asia. Early histological studies describe the presence of two bacteriome-associated symbionts and a rickettsial microorganism in N. cincticeps, but their microbiological affiliations have been elusive. We identified these bacterial symbionts using modern microbiological techniques. Cloning and sequencing of the 16S ribosomal RNA gene from dissected bacteriomes yielded two major and a minor bacterial sequences: a major sequence was placed in the Bacteroidetes clade of Sulcia muelleri, an ancient symbiont lineage associated with diverse hemipteran insects; another major sequence was allied to a β-proteobacterial sequence from a leafhopper Matsumuratettix hiroglyphicus; the minor sequence fell in the α-proteobacterial genus Rickettsia. In situ hybridization and transmission electron microscopy showed that the Sulcia symbiont and the β-proteobacterial symbiont are harbored within different types of bacteriocytes that constitute the outer and inner regions of the bacteriome, respectively. Oral administration of tetracycline to nymphal N. cincticeps resulted in retarded growth, high mortality rates, and failure in adult emergence, suggesting important biological roles of the symbionts for the host insect. The designation Candidatus Nasuia deltocephalinicola is proposed for the β-proteobacterial symbiont clade associated with N. cincticeps and allied leafhoppers of the subfamily Deltocephalinae.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.