This document discusses the classification of phytoplasmas, which are wall-less bacteria that infect plants and are transmitted by insect vectors. It begins by describing early observations of phytoplasmas and their causal role in plant diseases. It then discusses various classification systems used over time, beginning with symptom-based classification and moving to methods based on serology, DNA hybridization, RFLP analysis of 16S rDNA, and sequencing of 16S rDNA and ribosomal protein genes. The latest classification system divides phytoplasmas into 18 major 16Sr groups and over 40 subgroups, providing an overview of the molecular tools that enabled increasingly precise classification of these obligate plant pathogens.

2. Phytoplasma : An Introduction and
Classification with Recent
Molecular Tools
by
SUJATA DANDALE

3. Phytoplasmas: historical
Pleomorphic cells observed in ultra-thin section
of leaves of mulberry infected with dwarf disease
(Doi et al.,1967)
These bodies disappear after tetracycline
treatment when seen in EM (Ishiie et al.,1967)
Called as MLOs (Mycoplasma Like Organisms)
 Causal agents of yellow diseases in plants
Trival name “Phytoplasma” adopted in 10th
congress of International Organization of
Mycoplasmology (1994)
Lee et al. (2000) Annu. Rev. Microbiol.

4.  Single celled, wall-less prokaryotes, resembling mycoplasmas
in morphology (Doi et al.,1967)
 Obligate parasites, can’t be grown in in-vitro cell free culture
(Lee et al., 1986)
 Transmitted by phloem feeding insects (leafhoppers, plant
hoppers, psyllids) and Cuscuta (Tsai et al., 1979)
 Sensitive to tetracycline but resistant to penicillin (Ishii et al.
1967)
 Descended from low G+C gram positive bacterium (Woese et al.,
1987)
Lee et al. (2000) Annu. Rev. Microbiol

5. Round to filamentous (Kirkpatrick,1982)
Size 200-800 nm
Phytos- plant + plasma- thing moulded (Greek)
In sieve elements of Plants
Cont..

6. Symptoms due to Phytoplasmas:
Virescence
Phyllody
Little leaf
Sterility of flowers
Witches’ broom
Slender shoots
Stunting, leaf curling
Generalized decline, Bunchy growth etc.
Virescence on horseradish
Lee et al. (2000) Ann. Rev. Microbiol.
Diseased control

7. lethal yellowingSesame phyllody Little leaf of brinjalCarrot yellow
Witches’ broom lime Palm wilt Brassica phyllody Grape vine yellow

8.  First reports of phytoplasma diseases:
 Clover phyllody (Merrett,1866)
 Peach yellow (Smith,1888)
 Aster yellow on China aster (1902)
First Etiology : Mulberry dwarf (1967)
Important plant diseases world wide:
 Aster yellows in Carrot & Onion
 Apple proliferation
 Coconut lethal yellowing
 Peach X disease
 Elm yellow
More than
300 plant
Diseases
in hundreds
of
plant genera
Lee et al. (2000) Ann. Rev. Microbiol.

9. Important diseases in India
Disease Host Area First Report of etiology
Little leaf Brinjal
Periwinkle
All India
Lukhnow
Varma et al. (1969)
Rao et al. (1983)
X disease Peach NE region Ahlawat & Chenulu (1979)
Bushy Stunt Brinjal New Delhi Mitra & Chakraborty (1988)
Phyllody Bottle gourd &
other gourd
Black pepper
Sesame
Banglore
Banglore
Karela
All India
Sastry & Singh (1981)
Bhat et al. (2006)
Sahambi (1970)
Witches’ broom Acid Lime
Winged bean
Sunhemp
MH, AP Ghosh et al. (1999)
Singh (1991)
Sharma et al. (1990)
Rubbery wood Citrus Darjeeling Ahlawat & Chenula (1985)
Root wilt Coconut Kerala Solomon et al. (1983)
Sandal spike Sandal Kerala,Kr Varma et al. (1969)
GSD Sugarcane All India Rishi et al. (1973)
White leaf Bermuda grass UP Rishi (1978) Rao et al. (2007)
Yellow dwarf Rice All India Raychaudhri et al. (1967)

10. “After a time the growth of and accumulation of
specimens or phenomena forces people to try to
classify” - Pirie (1995)
Hurdles to definite description and classification:
Obligately parasitic habit
Structural fragility
Presence in low numbers in infected plants
Intimate association with host tissues
Firraro et al. (2005) J. Pl. Pathol

11. Based on biological properties
(1970s)
Based on serological properties
(1980s)
Based on molecular properties
(1990s onward)
Working Team on
Phytoplasmas of International
Research Program of
Comparative Mycoplasmology
(IRPCM)
International Committee on Systematic Bacteriology (ICSB)-
Subcommittee on the Taxonomy of Mollicutes
Development of classification systems in Phytoplasmas
Bergeys’ manual of systematic bacteriology: Vol. III , class- Mollicutes

12.  First attempts:
 Symptomatology
 Host Range (eg. Aster yellow, Clover phyllody)
 Transmission by insect vectors (vector species)
Groups based on Symptoms
Kirkpatrick et al. (1992)
 Decline agents
 Proliferation agents
 Virescence agents
Kirkpatrick et al. (1992)
 Aster yellows
 Stolbur
 Witches’ broom
 Decline
 Phyllody
Grunewald et al. (1977)

13. Chyokowski & Sinha (1989)
Phytoplasmas
Mutually exclusive
floral
symptoms
Reduced flower size
and colour with other
symptoms
On experimental host Periwinkle
Catharanthus roseus
Classification based on symptoms is not reliable

14.  Serology mostly used for detection/identification
 Phytoplasma enriched extract used for production of :
 Monoclonal antibodies (Chen et al., 1988)
 Polyclonal antibodies (Kirkpatrick et al., 1992)
 Monoclonal antibodies suited for differentiation of closely
related strains (Lee et al., 1993a )
Limitations:
 Difficult to obtain pure phytoplasmas
 Low concentration
 Non-specific reaction
Lee et al. (1993a)

15. Molecular Era
Year Work Scientists
1987 Improvement in phytoplasma extraction from
infected hosts
Kirkpatrick et al.
1989 First estimate of phytoplasma DNA composition -
Reported low G+C value: 23-26 mol%
Kollar et al.
1989 Plant pathogenic MLOs different from
Mycoplasmas by 16S rDNA sequence analysis
Lims & Sears
1991 Suggested primer pair for 16S rRNA gene
amplification for wider array of MLO identification
Deng & Hiruki
1992 Cloned DNA fragments as probes in dot blot
hybridization to identify phytoplasma
Lee et al.
1992 Suggested RFLP analysis of amplified 16S rDNA Ahrens & Seemuller
1993 by pulse field gel electrophoresis showed genome
size of phytoplasma
Neimark et al.
1993 Used oligo-nuceotide primers that amplify 16S
DNA of AY-MLO Cluster
Davis & Lee

16. Table Cont..
Year Work Scientists
1994 Cloned DNA fragments used in southern
hybridization to identify phytoplasmas from hosts
Schneider et al.
1994 combined RFLP (16S rDNA) & ribosomal protein
gene sequence for classification
Gundersen et al.
1997 characterization & classification by using enzymes
and sequence analysis
Schneider et al.
1998 Based on RFLP of 16S rRNA gene & rp gene
classified phytoplasmas to 14 groups
Lee et al.
2004,2006 Expanded phytoplasma classification to 18 16Sr
groups
Lee et al.
2005 Gave description of 21 Candidatus phytoplasma sp. Firrao et al.
2007 Expanded RFLP based 16Sr classification through
in silico analysis
Wei w. et al.

17. Classification based on DNA hybridization assays
SNo. Strain Cluster Ref.
1 Little leaf disease of
periwinkle-MLO
Davis et al.
(1990)
2 Ash yellows-MLO Davis et al.
(1991)
3 Clover proliferation-MLO Lee et al.
(1992)
4 Aster yellows-MLO Lee et al.
(1992)
5 Canadian peach X disease ,
Western X disease, Clover
yellow edge
Lee et al.
(1992)
6 Italian periwinkle virescence Davis et al.
(1992)
•Cloned DNA
fragments from
known phytoplasma
were used for
hybridization
• Each Strain Cluster
consists of strains
with extensive
sequence homology
Lee & Davis et al. (1992)

18. DNA fragments were cloned only from limited numbers of
Phytoplasmas
Difficulty in obtaining desired concentration of phytoplasma
strains from infected hosts
Standardized DNA probes for general detection & identification
were not available
PCR based Molecular
tools became more
popular
Lee & Davis et al. (1992)
Classification based on DNA hybridization assays:
Difficulties

19. Classification systems based on recent molecular tools
Tools for classification:
Most conserved Less conserved regions
16S rRNA ( primers- P1/P7,
R16F2n/R16R2)
 23S rRNA
 Rp gene operon (rpl22,rps3)
(primers-rpF1, rpR1)
 16S-23S spacer
Tuf elongation factor
Gene Phytoplasma Phytoplasma &
Acholeplasma
16S rRNA 88-99% 87.0-88.5%
Ribosomal protein 60-79% 50-57%
16S rRNA Spacer 23S rRNA
1.5 Kb 0.3 Kb 0.4 Kb
Lee et al. (1998, 2000)
Sequence similarity:

20. DNA extracted from 52 isolates and digested with BclI
30 PCR amplification Cycles (primers- fD1, rp1)
Amplification product (1500bp) digested with BclI
and then with AlulI, RsaI, EcoRI
Amplified DNA cloned and sequenced
Schneider et al. (1992) J. Gen. Microbiol.
Seven Groups determined

21. OAY
I(AAY)
II(ACLR)
III(ASHY)
IV(EY)
V(AT)
VI(WX)
VII(SCWL)
a b c d e f g h i j
Fig: AluI restriction map of 16S rDNA depicting the seven
(I-VII) Restriction profile
Schneider et al. (1992)

22. 16S rRNA gene sequence homology:
52 isolates from 4 symptom groups (Aster yellows, Clover phyllody,
Periwinkle virescence and stolbur) were divided to 7 groups
Isolates from
Same group
Different gp
Schneider et al. (1992)
V IV II III I VI
Fig: AluI Restriction profile Phytoplasma 16S rDNA
Inference
97.8 to 99.5%
89.6 to 92%

23. Total nucleic acid extracted from 40 strains
35 PCR amplification cycles with primer pair R16F2/R2
PCR product digested separately with 15 Restriction enzymes
Gel electrophoresis
Similarity coefficient (F)= 2Nxy
Nx+Ny
Nei & Li (1979)
Lee et al. (1993) Phytopathol.
F > 0.9 for members of same group

24. The reference RFLP pattern obtained with 15 restriction enzymes
Lee et al. (1993)
Group MseI AluI HpaI HpaII Sau3A Rsa
I
Hinf
I
TaqI HhaI Hae
III
KpnI DraI EcoR
I
Tha
I
EcoR
II
I-A 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
I-B 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1
I-C 2 2 1 1 1 1 1 1 1 2 1 1 1 1 1
I-D 3 1 1 1 1 1 1 1 2 1 1 1 1 1 1
I-E 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1
II 4 3 1 3 2 2 2 2 3 2 2 1 1 1 1
III-A 5 4 1 4 3 2 2 1 1 3 2 1 1 2 2
III-B 6 4 1 5 3 2 2 1 1 3 2 1 1 2 2
IV 7 5 1 4 3 3 3 1 1 3 2 1 1 2 2
V 8 6 1 4 3 4 2 1 3 4 2 1 1 2 3
VI 9 7 1 4 3 5 2 1 4 4 2 1 1 2 3
VII 10 8 1 4 3 5 2 3 5 3 2 1 1 2 3
VIII 8 4 1 4 3 5 2 1 4 4 2 1 1 1 3
IX 11 9 1 4 3 6 4 4 6 5 2 2 1 1 3
Numbers - distinct RFLP types

25. Key enzymes for Group and subgroup classification:
MseI & AluI – sufficient
to classify in 16Sr-I
MseI, AluI, HpaII &
HhaI – Further
differentiation
RFLP from 2
Strains (BBSI, HyphI)
16SrI-B, 16SrI-E
(newly classified)
Lee et al. (1993)
AB B C

26. Resulted nine major 16Sr groups and 14 subgroups based on F
Comparison of 16Sr groups with previous strain clusters
Lee et al. (1993)-16Sr groups Lee & Davis (1992)-Strain Clusters
16SrI (5 subgroups) AY-MLO
16SrII Peach X disease -MLO
16SrV EY-MLO
16SrVI CP-MLO
16SrVII Ash Y-MLO
Lee et al. (1993)
16SrI: 5 subgroups (largest)
16SrIII: 2 subgroups
Inference

27. Nested PCR of 34 representative phytoplasma strains (16SrRNA)
RFLP with 17 restriction enzymes and similarity coefficient
Based on 16S rRNA
similarity
coefficient
--14 groups
-- 41 subgroups
By combined RFLP
16S rDNA &
rp gene sequence
--46 subgroups
Similarity coefficient between distinct Groups < 90%
Lee et al.(1998) IJSB

28. 16S DNA
Lee et al. (1998)

29. 16Sr RNA groups based on phylogeny
Lee et al. (1998)
Fig: RFLP analysis Fig: Phylogenetic Tree

30. Finer subgroup classification
Strain 16S r-rp subgp. Strain 16S r-rp subgp
Tomato big bud BB 16SrI-A (rp-A) Maize bushy stunt MBS 16SrI-B (rp-L)
New Jersey AY NJAY 16SrI-A (rp-A) Clover phyllody CPh 16SrI-C (rp-C)
Periwinkle little leaf CNI 16SrI-A (rp-A) Strawberry green petal SGP 16SrI-C (rp-C)
Oklahoma AY OKAYI 16SrI-A (rp-A) Annulus phyllody RPh 16SrI-C (rp-C)
Maryland aster yellow AYI 16SrI-B (rp-B) Paulownia WB PaWB 16SrI-D (rp-D)
Dwarf aster yellow DAY 16SrI-B(rp-B) Blueberry stunt BBSI 16SrI-E (rp-E)
Hydrangea phyllody HyPH 16SrI-B (rp-K) Grey dogwood WB GDI 16SrI (rp-M)
Ipomoea WB IOB 16SrI-B (rp-F)
Fig: RFLP of rp gene Lee et al. (1998)

31. Approach based on
16S r Groups: 16S rRNA gene sequence
Subgroups: 16S r RNA gene & rp gene cluster
16S rRNA sequence homologies
88-94% : Two distinct 16S rRNA Groups
95-98%: Two subgroups with in a Group
Grouping consistent with strain clusters (identified based on DNA-
DNA homology and serological data)
Lee et al. (1998)
Inference
So valid classification system

32. Case Study 4. Candidatus phytoplasma approach
Taxonomic Notes Reference
Major part of gene to be sequenced (1000bp from 16S rRNA)
for taxonomy
Murray et al.
(1990)
Candidatus (L. Candidatus, a candidate to indicate that
assignment is provisional) and must include:
 Sequence (16S rRNA)
 Identification of morphotype with probes from
characteristic sequence
Murray &
Schleifer (1994)
Organisms with less than 97% sequence homology of 16S
rRNA will not have more than 60-70% reassociation
Stackebrandt &
Goebel (1994)
Int. J. Syst. Bacteriol. (1994)

33. Candidatus phytoplasma description
Character Description Refrence
Morphology Single unit membrane, pleomorphic Doi et al. (1967)
Habitat Phloem sieve, gut, haemolymph of
sapsucking insects
Tsai et al. (1979)
Antibiotic
sensitivity
Tetracycline Ishii et al. (1967)
DNA base
composition
G+C : 23-29% Kollar & Seemuller
(1989)
Chromosomal Size 530-1350 bp Neimark & Kirkpatrick
(1993)
Codon usage UGA- stop codon, not for tryptophan Lims & Sears (1991)
Sterol in
membrane
Non sterol requiring Lim et al. (1992)
Ribosomal RNA Two rRNA operons & a spacer 16s
-23s rRNA genes
Kuske & Kirkpatrick
(1992)
Phytoplasma/Spiroplasma Working Team IRPCM (2000) IJSEM

34. Rules:
1. Single, unique 16S rRNA gene sequence (>1200bp) from the
‘reference strain’
2. A strain- novel Ca. Phytoplasma sp if sequence <97.5%
similarity to previously defined Ca. Phytoplasma
3. Even if >97.5% similarity but ecologically separated population:
 Transmitted by different vectors
 Have different natural plant host/ different response on same host
 Significant molecular diversity ( DNA probe hybridization,
serology, PCR assay)
Phytoplasma/Spiroplasma working team
IRPCM (2000)

35. 4. No rank of subspecies
5. Description of new species submitted to IJSEM
6. Reference strain should be made available to scientists
7. Abbreviation of Candidatus is Ca.
Reference sequence alignment available from TreeBase
(accession no. S1048-1788)
Phytoplasma/Spiroplasma working
team, IRPCM (2000)

36. 16S rRNA groups and Ca. Phytoplamas
(IRPCM Phytoplasma/spiroplasma Working Team, 2004)
Phylogenetic Group Candidatus Phytoplasma sp. Reference
Aster yellows (16SrI) Ca. Phytoplasma asteris Lee et al. (2004a)
Peanut witches’-broom
(16SrII)
Ca. Phytoplasma aurantifolia Zreik et al. (1995)
X-disease(16SrIII) Ca. Phytoplasma pruni
Coconut lethal yellowing
(16SrIV)
Ca. Phytoplasma palmae
Ca. Phytoplasma cocostanzaniae
Ca. Phytoplasma castaneae
Elm yellows (16SrV) Ca. Phytoplasma ziziphi
Ca. Phytoplasma vitis
Ca. Phytoplasma ulmi
Jung et al. (2003a)
Lee et al. (2004b)
Clover proliferation
(16SrVI)
Ca. Phytoplasma trifolii Hiruki & Wang (2004)
Ash yellows(16SrVII) Ca. Phytoplasma fraxini Griffith et al. (1999)
Firraro et al. (2005) J. Plant pathol.

37. Table cont…
Phylogenetic Group Candidatus Phytoplasma sp Reference
Loofah witches’-broom
(16SrVIII)
Ca. Phytoplasma luffae
Pigeon pea witches’-broom
(16SrIX)
Ca. Phytoplasma phoenicum Verdin et al. (2003)
Apple proliferation (16SrX) Ca. Phytoplasma mali
Ca. Phytoplasma pyri
Ca. Phytoplasma prunorum
Seemuller & Schneider
(2004)
Rice yellow dwarf (16SrXI) Ca. Phytoplasma oryzae Jung et al. (2003b)
Stolbur (16SrXII) Ca. Phytoplasma australiense
Ca. Phytoplasma japonicum
Davis et al. (1997)
Sawayanagi et al. (1999)
BGWL (16SrXIV) Ca. Phytoplasma cynodontis Marcone et al. (2004)
Hibiscus witches’- broom (XV) Ca. Phytoplasma brabilience
Mexican periwinkle virescence
(16SrXIII)
No name suggested
Firraro et al. (2005)Firraro et al. (2005) J. Plant pathol.

38. Phytoplasma 16S rRNA gene sequences retrieved (NCBI)
Aligned and trimmed to 1.25 Kb (F2nR2) fragment bounded
by two Conserved Nucleotide blocks
In-silico restriction enzyme digestion (17 enzymes)
Virtual gel plotting (3.0% agrose)
Comparison of RFLP pattern and Similarity coefficient
Calculation (51 patterns)
Wei et al. (2007) IJSEM

39. Wei et al. (2007)

40. New 16Sr Groups
Arabic no - subclades
Roman no - Groups
Wei et al. (2007)

41. Wei et al. (2007)Fig: In silico RFLP

42.  New 16Sr groups based on 90% threshold of similarity
Each strain in new groups F< 0.85 with other Group strains
A total of 28 groups and more than 100 subgroups given
New groups contains 3 previously defined Ca. Phytoplasma Sp.
and 7 Potential sp. to be described
Provided feasible method for future extension of classifation
Wei et al. (2007)

43. Conclusion
Phytoplasma are important plant pathogens causing
economic losses in number of crop plants and tree species
RFLP analysis of PCR-amplified 16S rRNA gene with
restriction enzymes remains a valuable tool for studying
phytoplasma diversity and classification
Till now the most accepted and stable classification is to
describe phytoplasmas in ‘Candidatus phytoplasma species’
rank which combines both molecular (16S rRNA gene
sequence) as well as biological, phytopathological properties.