Phytoplasmas: bacteria that manipulate plants and insects

TAXONOMY: Superkingdom Prokaryota; Kingdom Monera; Domain Bacteria; Phylum Firmicutes (low-G+C, Gram-positive eubacteria); Class Mollicutes; Candidatus (Ca.) genus Phytoplasma. HOST RANGE: Ca. Phytoplasma comprises approximately 30 distinct clades based on 16S rRNA gene sequence analyses of approximately 200 phytoplasmas. Phytoplasmas are mostly dependent on insect transmission for their spread and survival. The phytoplasma life cycle involves replication in insects and plants. They infect the insect but are phloem-limited in plants. Members of Ca. Phytoplasma asteris (16SrI group phytoplasmas) are found in 80 monocot and dicot plant species in most parts of the world. Experimentally, they can be transmitted by approximately 30, frequently polyphagous insect species, to 200 diverse plant species. DISEASE SYMPTOMS: In plants, phytoplasmas induce symptoms that suggest interference with plant development. Typical symptoms include: witches' broom (clustering of branches) of developing tissues; phyllody (retrograde metamorphosis of the floral organs to the condition of leaves); virescence (green coloration of non-green flower parts); bolting (growth of elongated stalks); formation of bunchy fibrous secondary roots; reddening of leaves and stems; generalized yellowing, decline and stunting of plants; and phloem necrosis. Phytoplasmas can be pathogenic to some insect hosts, but generally do not negatively affect the fitness of their major insect vector(s). In fact, phytoplasmas can increase fecundity and survival of insect vectors, and may influence flight behaviour and plant host preference of their insect hosts. DISEASE CONTROL: The most common practices are the spraying of various insecticides to control insect vectors, and removal of symptomatic plants. Phytoplasma-resistant cultivars are not available for the vast majority of affected crops.     

Comparative Molecular Analyses of Phytoplasmas infecting Sophora japonica cv. golden and Robinia pseudoacacia

Phytoplasmas were detected in Sophora japonica cv. golden and Robinia pseudoacacia with diseased branches of witches'‐broom collected in Haidian district, Beijing, China. Phytoplasma cells were observed in phloem sieve elements of symptomatic S. japonica cv. golden by transmission electron microscopy. The presence of phytoplasmas was further confirmed by sequence determination of partial gene sequences of 16S rDNA, rp (ribosomal protein) and secY. Phylogenetic trees and virtual restriction fragment length polymorphism (RFLP) analyses indicated that the phytoplasmas causing S. japonica cv. golden witches'‐broom (SJGWB) and R. pseudoacacia witches'‐broom (RPWB) belong to the 16SrV (elm yellows) group, and they are most closely related to subgroup 16SrV‐B, rpV‐C and secYV‐C jujube witches'‐broom (JWB) phytoplasma. Comparative analyses indicated that the phytoplasma of RPWB was closer to the JWB and that R. pseudoacacia might serve as an alternative host plant of JWB phytoplasma.     

Dramatic transcriptional changes in an intracellular parasite enable host switching between plant and insect

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.     

Development of molecular markers and a diagnostic tool for investigation of coinfections by and interactions between potato purple top and potato witches'‐broom phytoplasmas in tomato

Columbia Basin potato purple top (PPT) phytoplasma and Alaska potato witches'‐broom (PWB) phytoplasma are two closely related but mutually distinct pathogenic bacteria that infect potato and other vegetable crops. Inhabiting phloem sieve elements and being transmitted by phloem‐feeding insect vectors, both pathogens are affiliated with ‘Candidatus Phytoplasma trifolii’ and are members of the clover proliferation phytoplasma group (16SrVI). The polyphagous nature and wide geographic distribution of their insect vectors make mixed infection inevitable. In this study, we experimentally constituted a simultaneous PPT and PWB phytoplasma infection in tomato (Solanum lycopersicum) and developed a sensitive diagnostic tool to investigate mixed infections by and in planta interactions of the two phytoplasmas. The distribution and relative abundance of the two co‐infecting phytoplasmas were monitored over a 45‐day post‐infection time course and for three serial passages in planta. Our results revealed that dual infections of the two phytoplasmas induce a new symptom unseen in infection by either phytoplasma alone. Our results also raised an interesting question as to whether the two phytoplasmas differ in ability of competitive dominance under co‐infection conditions. The molecular markers and the diagnostic tool devised in this study should be useful for further investigations of the interactions between the two closely related phytoplasmas in their hosts.     

Lineage-specific decay of folate biosynthesis genes suggests ongoing host adaptation in phytoplasmas

Phytoplasmas are nonculturable cell wall-less, obligate intracellular pathogens of plants and insect vectors. In their descent from walled bacterial ancestors, phytoplasmas underwent massive genome reduction, resulting in some of the smallest cellular genomes known in nonsymbiotic bacteria. While requirements for in vitro culture of phytoplasmas remain unknown, two opposing reports have appeared concerning genes encoding the ability of phytoplasmas to synthesize folates de novo. One study found pseudogene homologs of folP and folK, obviating folate synthesis in "Candidatus Phytoplasma asteris"-related strain CPh, whereas, a separate study found intact genes encoding a complete folate biosynthesis pathway in "Ca. Phytoplasma asteris"-related strain OY. To resolve the apparent conflict, we hypothesized that evolutionary adaptation to the availability of folate and/or other metabolites in host cells is an ongoing process in the phytoplasma clade that is reflected in part by differences among phytoplasmas in the status of genes of the folate biosynthesis pathway. By studying folP and folK loci in 11 closely related phytoplasmas, we determined that these essential folate biosynthesis genes are intact in some phytoplasmas but are deteriorating in closely related strains. We suggest that the status of the folate biosynthesis pathway and the course of gene decay are lineage-specific, predicting the eventual, lineage-related loss of recognizable folP and folK homologs in phytoplasma genomes.     

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.     

Screening potential insect vectors in a museum biorepository reveals undiscovered diversity of plant pathogens in natural areas

Phytoplasmas (Mollicutes, Acholeplasmataceae), vector‐borne obligate bacterial plant parasites, infect nearly 1,000 plant species and unknown numbers of insects, mainly leafhoppers (Hemiptera, Deltocephalinae), which play a key role in transmission and epidemiology. Although the plant–phytoplasma–insect association has been evolving for >300 million years, nearly all known phytoplasmas have been discovered as a result of the damage inflicted by phytoplasma diseases on crops. Few efforts have been made to study phytoplasmas occurring in noneconomically important plants in natural habitats. In this study, a subsample of leafhopper specimens preserved in a large museum biorepository was analyzed to unveil potential new associations. PCR screening for phytoplasmas performed on 227 phloem‐feeding leafhoppers collected worldwide from natural habitats revealed the presence of 6 different previously unknown phytoplasma strains. This indicates that museum collections of herbivorous insects represent a rich and largely untapped resource for discovery of new plant pathogens, that natural areas worldwide harbor a diverse but largely undiscovered diversity of phytoplasmas and potential insect vectors, and that independent epidemiological cycles occur in such habitats, posing a potential threat of disease spillover into agricultural systems. Larger‐scale future investigations will contribute to a better understanding of phytoplasma genetic diversity, insect host range, and insect‐borne phytoplasma transmission and provide an early warning for the emergence of new phytoplasma diseases across global agroecosystems.     

The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.     

Root (wilt) disease of coconut palms in South Asia – an overview

Root (wilt) disease (RWD) caused by phytoplasma is one of the most devasting diseases of coconut palms. The major symptoms of the disease in leaves are wilting and drooping and flaccidity; ribbing, paling/yellowing and necrosis of leaflets are typical symptoms of foliar diseases. Unopened pale yellow leaflets of spindle leaves are more susceptible to leaf rot disease, which is caused by Exerohilum rostratum and Colletotrichum gloeosporioides. RWD is caused by phytoplasmas, the cell wall-less prokaryotes that are bounded by a “unit” membrane. In ultrathin sections, they appear as a complex multi-branched, beaded, filamentous or spheroidal pleomorphic bodies. The disease was transmitted by plant hoppers (Proutista moesta) and lace wing bug (Stephanitis typica). Phytoplasmas are generally present in the phloem sieve tubes and in the salivary glands of these insect vectors. Phytoplasmas cannot be cultured in vitro, and hence it is very difficult to identify them. Using polymerase chain reaction technique, group-specific primers have been applied to detect mixed-phytoplasma infections in a single host. RWD, is a non-lethal, debilitating disease, and hence an integrated approach for the management of this disease in coconut palms has been discussed in this study.     

An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp?Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp?Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp?Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts

Phytoplasmas ("Candidatus Phytoplasma," class Mollicutes) cause disease in hundreds of economically important plants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches' broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organized into large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes for specialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and therefore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of approximately 250 kb located between the lplA and glnQ genes are syntenic between the two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be further along in the reductive evolution process than OY-M. The AY-WB genome is approximately 154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M.     

Phytoplasmas and their interactions with hosts

Phytoplasmas are bacteria without cell walls and are responsible for plant diseases that have large economic impacts. Knowledge of their biology is limited because they are uncultivable and experimentally inaccessible in their hosts. It is a mystery how these bacteria use the sugar-rich phloem sap in which they live and how they interact with the host. This makes it difficult to develop means to control them. Recently, the full genomes of two phytoplasmas have been sequenced, allowing new insights into their requirements. Phytoplasmas contain a minimal genome and lack genes coding for ATP synthases and sugar uptake and use, making them dependent on their host. This dependency can be exploited to elucidate the particular physiology of the phloem.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

Presence of two glycolytic gene clusters in a severe pathogenic line of Candidatus Phytoplasma asteris

Phytoplasmas are plant-pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY-M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30-kb region including the glycolytic genes was tandemly duplicated in the genome of OY-W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop-codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full-length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6-phosphofructokinase is known as a rate-limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY-W and OY-M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY-W was higher than that of OY-M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.     

Characterization of Phytoplasmas Related to 'Candidatus Phytoplasma asteris' and Peanut WB Group Associated With Sweet Cherry Diseases in Iran

Symptoms suggestive of phytoplasma diseases were observed in infected sweet cherry trees growing in the central regions of Iran. Phytoplasmas were detected in symptomatic trees by the nested polymerase chain reaction (nested PCR) using phytoplasma universal primer pairs (P1/Tint, PA2F/R, R16F2/R2 and NPA2F/R). Restriction fragment length polymorphism analyses of 485 bp DNA fragments amplified in nested PCR revealed that different phytoplamas were associated with infected trees. Sequence analyses of phytoplasma 16S rRNA gene and 16S-23S intergenic spacer region indicated that the phytoplasmas related to ' Ca. Phytoplasma asteris ' and peanut WB group infect sweet cherry trees in these regions. This is the first report of the presence of phytoplasmas related to ' Ca. Phytoplasma asteris' and peanut WB group in sweet cherry trees.     

Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.     

Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to “Candidatus Phytoplasma asteris” (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.     

Shotgun proteomic analysis of mulberry dwarf phytoplasma

Background  

Mulberry dwarf (MD), which is caused by phytoplasma, is one of the most serious infectious diseases of mulberry. Phytoplasmas have been associated with diseases in several hundred plant species. The inability to culture phytoplasmas in vitro has hindered their characterization at the molecular level. Though the complete genomes of two phytoplasmas have been published, little information has been obtained about the proteome of phytoplasma. Therefore, the proteomic information of phytoplasmas would be useful to elucidate the functional mechanisms of phytoplasma in many biological processes.