Phytoplasma

Phytoplasma, formerly known as 'Mycoplasma-like organisms' or MLOs, are specialised bacteria that are obligate parasites of plant phloem tissue, and some insects. They were first discovered by scientists in 1967 in plants that were thought to be infected with viruses, but ultrathin sections of the plants phloem revealed the presence of mycoplasma like organisms.^[1] They can't 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 important crops, including coconuts and sugarcane, causing a wide variety of symptoms that ranges 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 replicate.

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Morphology

Being mollicutes, phytplasmas lack cell walls and instead are bound by a triple layered unit membrane.^[2] 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.^[3] Their shape is normally pleiomorphic or filamentous and normally have a diameter of less than 1 micrometer. Like other prokaryotes, DNA is free in the cytoplasm. They are believed to reproduce through Binary fission.^{[citation needed]}

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).^[4] This causes sepals to form where petals should. 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. ^[5]

Phytoplasma infected plants may also suffer from virescence - the development of green flowers due to the loss of pigment in the petal cells..^[6] Sometimes sterility of the flowers is also seen.

Many phytoplasma infected plants gain a bushy or witch's broom appearance due to changes in normal growth patterns caused by the infection. Most plants show apical dominance but phytoplasma infection can cause the proliferation of auxiliary (side) shoots and an increase in size of the internodes.^[6] Such symptoms are actually useful in the commercial production of poinsettia. The infection is necessary to produce more axillary shoots that enable to production of pionsettia plants that have more than one flower.^[7]

Phytoplasmas may cause many other symptoms that are induced because of the stress placed on the plant by infection rather than specific pathogencity of the phytoplasma. Photosynthesis, especially photosystem II, is inhibited in many phytoplasma infected plants.^[2] Phytoplasma infected plants often show yellowing which is caused by the breakdown of chlorophyll, whose biosynthesis is also inhibited.^[2]

Transmission

Movement between plants

The phytoplasmas are mainly spread by insects of the families Cicadellidea (leafhoppers) and Fulgoridea (planthoppers)^[6] 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 and this has been shown to interact with insect microfilament complexes and is believed to the determining factor is insect-phytoplasma interation.^[8]Phytoplasmas may overwinter in insect vectors or perrinial plants. Phytoplasmas can have varying affects on their insect hosts, examples of both reduced and increased fitness have been seen.^[9]

Phytoplasmas will be found in most of the major organs of an infected insect host once they are established. They will enter the insects body through the stylet and then move through the intestine and bein absorbed into the haemolymph.^[9] From here they proceeded to colonise the salivary glands, a process that can take up to three weeks.^[9] The time between phytoplasmas being taken up by the insect and the phytoplasmas reaching an infectious titre in the salivary gland is called the latency period.^[9]

Phytoplasmas can also be spread via vegetative propergation such as the grafting of a piece of infected plant onto a healthy plant.

Movement within plants

Phytoplasmas are able to move within the pholem from source to sink and they are able to pass through sieve tube elements, but spread more slowly than solutes, for this and other reasons movement by passive translocation is not supported.^[10]

Detection and Diagnosis

Before molecular techniques were developed the diagnosis of phytoplasma diseases was difficult due to the fact that 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.^[1] Another diagnostic technique used was to treat infected plants with antibiotics such as tetracycline to see if this cured the plant.

Molecular diagnostic techniques for the detection of phytoplasma began to emerge in the 1980s and included ELISA based methods. In the early 1990's 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.^[11]

There are also techniques that allow the assessement 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.^[10]

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.^[6]

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.^[12]

Work has also been carried out investigating the effectiveness of plantibodies targeted against phytoplasmas.^[13]

Tetracyclines are bacteriostatic to phytoplasmas, that is they inhibit their growth.^[14] However without continuous use of the antibiotic disease symptoms will reappear. Thus tetracycline is not a viable control agent in agriculture, but is used to protect ornamental coconut trees.^[15]

Genetics

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.^[16] In fact Bermuda grass white leaf phytoplasma has a genome size of just 530Kb, the smallest genome of any known living organism.^[17] Larger phytoplasma genomes are around 1350 Kb. Some phytoplasmas contain extrachromosomal DNA such as plasmids.^[18]

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.^[19] 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, rather than to code for tryptophan.^[20]

Phytoplasma genomes contain large numbers of transposon genes and insertion sequences. They also contain a unique family of repetative 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.^[21]

Taxonomy

Phytoplasmas are mollicutes and within this group belong to the monophyletic order Acholeplasmatales.^[6]The genus name Phytoplasma is yet to be formally recognised, and is currently at Candidatus status^[22] which is used for bacteria that can not be cultured.^[23] 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.^[6]. Phytoplasma taxonomic groups are based on differences in the fragment sizes produced by the restriction digest of the 16S rRNA gene sequence (Called RFLP).^[24] There is some disagreement over how many taxonomic groups the phytoplasmas fall into, recent work involving computer similuated restriction digests of the 16Sr gene suggest there maybe up to 28 groups^[25] where as 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.

References

1. ^ ^{a b} Doi et al. (1967) Mycoplasma or PLT-group-like organisms found in the phloem elements of plants infected with mulberry dwarf, potato witches' broom, aster yellows or paulownia witches' broom. Annals of the Phytopathological Society of Japan 33 259-66
2. ^ ^{a b c} Bertamini et al (2003). Effects of Phytoplasm infection on pigments, chlorophyll protein complex and photosynthetic activities in field grown apples. Biologia Plantarum 47: 237-242
3. ^ Berg et al. Isolation of the gene encoding an immunodominant membrane proten of apple proliferation phytoplasma and expression and characterisation of the gene product. Microbiology 145:1937-1943
4. ^ Pracros et al. (2005) Tomato flower abnormalities induced by stolbar phytoplasma infection are associated with changes in expression of floral development genes. Molecular Plant Microbe Interactions 19 62-68
5. ^ Muast et al. (2003) Changes in carbohydrate metabolism in coconut palms infected with the lethal yellowing phytoplasma Phytopathology 93 976-981.
6. ^ ^{a b c d e f} Lee et al. (2000) Phytoplasmas: phytopathogenic mollicutes. Annual Review of Microbiology 54 221-255
7. ^ Lee et al. (1997) Phytoplasma induced free branching in commercial poinsettia cultivars. Nature Biotechnology 15:178-82
8. ^ Suzuki et al. (2006) Interactions between a membrane protein of a pathogen and insect microfilament complex determines insect vector specificity. Proceedings of the National Academy of Sciences 103: 4252-4257
9. ^ ^{a b c d} Chrsitensen et al. (2005) Phytoplasmas and their interactions with their hosts. Trends in Plant Sciences 10 526-535.
10. ^ ^{a b} Christensen et al. 2004. Distribution of phytoplasmas in infected plants as revealed by real time PCR and bioimaging. Molecular plant microbe interactions 17: 1175-1184
11. ^ Chen et al. (1992) Detection and identification of plant and insect mollicutes. In The Mycoplasmas, editor RF Whitcomb and JG Tully 5: 393-424
12. ^ Wang et al. (2007) Effective elimination of sweet potato little lead by cryotherapy of shoot tips. Plant Pathology online early edition.
13. ^ Chen, Y. D., and Chen, T. A. 1998. Expression of engineered antibodies in plants: A possible tool for spiroplasma and phytoplasma disease control. Phytopathology 88:1367-1371.
14. ^ Davies et al. (1968) Remission of aster yellows disease by antibiotics. Science 161: 793-794.
15. ^ Drug for Humans Checks Palm Trees Disease. New York Times, July 19 1983
16. ^ Dikinson, M. Molecular Plant Pathology (2003) BIOS Scientific Publishers
17. ^ Marcone et al. (1999) Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups. Phytopathology 89 805-810
18. ^ Nishigawa et al. (2003) Complete set of extrachromosomal DNAs from three pathogenic lines of onion yellows phytoplasma and use of PCR to differentiate each line. Journal of General Plant Pathology 69 194-198
19. ^ Bai et al. (2006) Living with genome instability: the adaption of phytoplasmas to diverse environments of their insect and plant hosts. Journal of Bacteriology 188 3682-3696
20. ^ Razin et al. (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiology Molecular Biology Review 62 1094-1156
21. ^ Jomantiene et al (2006). Clusters of diverse genes existing as multiple, sequence variable mosaics in a phytoplasma genomes. FEMS Microbiology letters 255: 59-65
22. ^ The IRPCM Phytoplasma/Spiroplasma Working Team - Phytoplasma taxonomy group: Candidatus Phytoplasma, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol. Microbiol., 2004, 54, 1243-1255.[1]
23. ^ Murry et al. Taxonomic Note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int. J. Syst. Bacteriol., 1995, 45, 186-187.
24. ^ Hodgetts et al. (2007) Toxonomic groupings based on the analysis on the 16s/23s spacer regions which shows greater variation than the normally used 16srRNA gene results in classification similar to that derived from 16s rRNA data but with more detailed subdivisions. Plant Pathology 56: 357-365
25. ^ Wei et al. (2007) Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. International journal of systematic and evolutionary microbiology 57:1855-1867
26. ^ Lee et al (2004) ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. International Journal of Systematic and Evolutionary Microbiology 54 1037-1048
27. ^ T Sawayanagi, N Horikoshi, T Kanehira, M Shinohara, A Bertaccini, MT Cousin, C Hiruki and S Namba (1999) International Journal of Systematic Bacteriology.49: 1275-1285
28. ^ Y. Arocha, B. Piñol, B. Picornell, R. Almeida, P. Jones (2006) First report of a 16SrII ('Candidatus Phytoplasma aurantifolia') group phytoplasma associated with a bunchy-top disease of papaya in Cuba. Plant Pathology 55 (6), 821–821.
29. ^ Jones et al. (2007) A stunting syndrome of Napier grass in Ethiopia is associated with a 16SrIII group phytoplasma. Plant Pathology 56 345
30. ^ Griffiths, H. M., Sinclair, W. A., Smart, C. M., and Davis, R. E. 1999. The phytoplasma associated with ash yellows and lilac witches’-broom: ‘Candidatus Phytoplasma fraxini.’ Int. J. Syst. Bacteriol. 49:1605-1614.