Phytoplasma

Phytoplasma are specialised bacteria that are obligate parasites of plant phloem tissue and transmitting insects (vectors). They were first discovered by scientists in 1967 and were named mycoplasma-like organisms or MLOs. They cannot be cultured in vitro in cell-free media. They are characterised by their lack of a cell wall, a pleiomorphic or filamentous shape, normally with a diameter less than 1 micrometer, and their very small genomes.

Phytoplasmas are pathogens of agriculturally-important plants, including coconut, sugarcane, and sandalwood, causing a wide variety of symptoms that range from mild yellowing to death of infected plants. They are most prevalent in tropical and sub-tropical regions of the world. Phytoplasmas require a vector to be transmitted from plant to plant, and this normally takes the form of sap-sucking insects such as leaf hoppers, in which they are also able to survive and replicate.

History
There are references to diseases now known to be caused by phytoplasmas as far back as 1603 for Mulberry dwarf disease in Japan. Such diseases were originally thought to be caused by viruses, which, like phytoplasmas, require insect vectors, cannot be cultured, and have some symptom similarity. In 1967 phytoplasmas were discovered in ultrathin sections of plant phloem tissue and named mycoplasma-like organisms (MLOs), because they physically resembled mycoplasmas The organisms were renamed phytoplasmas in 1994, at the 10th congress of the International Organization of Mycoplasmology.

Morphology
Being mollicutes, phytoplasmas lack cell walls and instead are bound by a triple layered membrane. The cell membranes of all phytoplasmas studied so far usually contain a single immunodominant protein (of unknown function) that makes up the majority of the protein content of the cell membrane. The typical phytoplasma exhibits a pleiomorphic or filamentous shape and is less than 1 micrometer in diameter. As prokaryotes, phytoplasms' DNA is found throughout the cytoplasm, rather than being concentrated in a nucleus.

Symptoms
A common symptom caused by phytoplasma infection is phyllody, the production of leaf-like structures in place of flowers. Evidence suggests that the phytoplasma downregulates a gene involved in petal formation (AP3 and its orthologues) and genes involved in the maintenance of the apical meristem (Wus and CLV1). Other symptoms, such as the yellowing of leaves, are thought to be caused by the phytoplasma's presence in the phloem, affecting its function and changing the transport of carbohydrates.

Phytoplasma-infected plants may also suffer from virescence, the development of green flowers due to the loss of pigment in the petal cells. Photoplasma-harboring plants which are able to flower may nevertheless be sterile. A phytoplasma effector protein (SAP54) has been identified as inducing symptoms of virescence and phyllody when expressed in plants (discussed below).

Many plants infected by phytoplasma gain a bushy or "witches' broom" appearance due to changes in their normal growth patterns. Most plants show apical dominance, but phytoplasma infection can cause the proliferaztion of auxiliary (side) shoots and an increase in size of the internodes. Such symptoms are actually useful in the commercial production of poinsettia. The infection produces more axillary shoots, which enables production of poinsettia plants that have more than one flower.

Phytoplasmas may cause many other symptoms that are induced because of the stress placed on the plant by infection rather than specific pathogenicity of the phytoplasma. Photosynthesis, especially photosystem II, is inhibited in many phytoplasma infected plants. Phytoplasma infected plants often show yellowing which is caused by the breakdown of chlorophyll, whose biosynthesis is also inhibited.

Effector (virulence) proteins
Many plant pathogens produce virulence factors (or effectors) that modulate or interfere with normal host processes in a way that is beneficial to the pathogen. Aster Yellows phytoplasma strain Witch's Broom (often referred to as 'AY-WB') produces two effector proteins that have been characterized experimentally. SAP11 is a small protein (~10 kDa) that is released into plants upon infection with AY-WB. SAP11 selectively interacts with and destabilizes CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors in the model plant Arabidopsis. TCP transcription factors normally regulate plant development and control the expression of lipoxygenase (LOX) genes that are required for the biosynthesis of jasmonate. In AY-WB-infected Arabidopsis plants (and plants that express SAP11 transgenically), jasmonate levels are decreased due to the SAP11-mediated degradation of CIN-TCPs. The downregulation of jasmonate production is beneficial to the phytoplasma because jasmonate is involved in plant defence against herbivorous insects such as leafhoppers, and leafhoppers have been shown to lay more eggs on AY-WB-infected plants at least in part because of SAP11. For example, the leafhopper Macrosteles quadrilineatus lay 30% more eggs on plants that express SAP11 transgenically, and 60% more eggs on plants infected with AY-WB. Phytoplasmas cannot survive in the external environment and are dependent upon insects such as leafhoppers for transmission to new (healthy) plants. Thus, by interfering with jasmonate production, SAP11 'encourages' leafhoppers to lay more eggs on phytoplasma-infected plants, thereby ensuring that newly hatching leafhopper nymphs feed upon infected plants and become vectors for the bacteria.

AY-WB produces a second effector (called SAP54) that induces symptoms of virescence and phyllody in infected plants. The mechanism by which SAP54 converts flowers into leaf-like organs is presently being investigated by researchers at the John Innes Centre in Norwich, United Kingdom.

Movement between plants
The phytoplasmas are mainly spread by insects of the families Cicadellidea (leafhoppers), Fulgoridea (planthoppers) and Psyllidae (jumping plant lice) , which feed on the phloem tissues of infected plants, picking up the phytoplasmas and transmitting them to the next plant they feed on. For this reason the host range of phytoplasmas is strongly dependent upon its insect vector. Phytoplasmas contain a major antigenic protein that makes up the majority of their cell surface proteins. This protein has been shown to interact with insect microfilament complexes and is believed to be the determining factor in insect-phytoplasma interaction. Phytoplasmas may overwinter in insect vectors or perennial plants. Phytoplasmas can have varying effects on their insect hosts; examples of both reduced and increased fitness have been seen.

Phytoplasmas enter the insect's body through the stylet, move through the intestine, and are then absorbed into the haemolymph. From here they proceed to colonise the salivary glands, a process that can take up to three weeks. Once established, phytoplasmas will be found in most major organs of an infected insect host. The time between being taken up by the insect and reaching an infectious titre in the salivary glands is called the latency period.

Phytoplasmas can also be spread via dodders cascutaceae or vegetative propagation such as the grafting of a piece of infected plant onto a healthy plant.

Movement within plants
Phytoplasmas are able to move within the phloem from source to sink, and they are able to pass through sieve tube elements. But since they spread more slowly than solutes, for this and other reasons, movement by passive translocation is not supported.

Detection and Diagnosis
Before molecular techniques were developed, the diagnosis of phytoplasma diseases was difficult because they could not be cultured. Thus classical diagnostic techniques, such as observation of symptoms, were used. Ultrathin sections of the phloem tissue from suspected phytoplasma infected plants would also be examined for their presence. Treating infected plants with antibiotics such as tetracycline to see if this cured the plant was another diagnostic technique employed.

Molecular diagnostic techniques for the detection of phytoplasma began to emerge in the 1980s and included ELISA based methods. In the early 1990s, PCR-based methods were developed that were far more sensitive than those that used ELISA, and RFLP analysis allowed the accurate identification of different strains and species of phytoplasma.

More recently, techniques have been developed that allow for assessment of the level of infection. Both QPCR and bioimaging have been shown to be effective methods of quantifying the titre of phytoplasmas within the plant.

Control
Phytoplasmas are normally controlled by the breeding and planting of disease resistance varieties of crops (believed to the most economically viable option) and by the control of the insect vector.

Tissue culture can be used to produce clones of phytoplasma infected plants that are healthy. The chances of gaining healthy plants in this manner can be enhanced by the use of cryotherapy, freezing the plant samples in liquid nitrogen, before using them for tissue culture.

Work has also been carried out investigating the effectiveness of plantibodies targeted against phytoplasmas.

Tetracyclines are bacteriostatic to phytoplasmas, that is they inhibit their growth. However, without continuous use of the antibiotic, disease symptoms will reappear. Thus, tetracycline is not a viable control agent in agriculture, but it is used to protect ornamental coconut trees.

Genetics
The genomes of three phytoplasmas have been sequenced: Aster Yellows Witches Broom, Onion Yellows (Ca. Phytoplasma asteris) and Ca. Phytoplasma australiense Phytoplasmas have very small genomes, which also have extremely low levels of the nucleotides G and C, sometimes as little as 23% which is thought to be the threshold for a viable genome. In fact Bermuda grass white leaf phytoplasma has a genome size of just 530Kb, one of the smallest known genomes of living organisms. Larger phytoplasma genomes are around 1350 Kb. The small genome size associated with phytoplasmas is due to their being the product of reductive evolution from Bacillus/Clostridium ancestors. They have lost 75% or more of their original genes, and this is why they can no longer survive outside of insects or plant phloem. Some phytoplasmas contain extrachromosomal DNA such as plasmids.

Despite their very small genomes, many predicted genes are present in multiple copies. Phytoplasmas lack many genes for standard metabolic functions and have no functioning homologous recombination pathways, but do have a sec transport pathway. Many phytoplasmas contain 2 rRNA operons. Unlike the rest of the Mollicutes, the triplet code of UGA is used as a stop codon in phytoplasmas.

Phytoplasma genomes contain large numbers of transposon genes and insertion sequences. They also contain a unique family of repetitive extragenic palindromes (REPs) called PhREPS whose role is unknown though it is theorised that the stem loop structures the PhREPS are capable of forming may play a role in transcription termination or genome stability.

Taxonomy
Phytoplasmas are mollicutes and within this group belong to the monophyletic order Acholeplasmatales. In 1992 the Subcommittee on the Taxonomy of Mollicutes proposed the use of the name Phytoplasma in place of the use of the term MLO (Mycoplasma-like organism) "for reference to the phytopathogenic mollicutes". In 2004 the genus name Phytoplasma was adopted and is currently at Candidatus status which is used for bacteria that can not be cultured. It's taxonomy is complicated by the fact that it can not be cultured and thus methods normally used for classification of prokaryotes are not possible. Phytoplasma taxonomic groups are based on differences in the fragment sizes produced by the restriction digest of the 16S rRNA gene sequence (Called RFLP) or by comparison of DNA sequences from the 16s/23s spacer regions. There is some disagreement over how many taxonomic groups the phytoplasmas fall into, recent work involving computer simulated restriction digests of the 16Sr gene suggest there may be up to 28 groups whereas other papers argue for less groups, but more sub-groups. Each group includes at least one Ca. Phytoplasma species, characterised by distinctive biological, phytopathological and genetic properties. The table below summaries some of the major taxonomic groups and the candidatus species that belong in them.