Phytoplasma detection and identification: from 16S ribosomal gene ...

Phytoplasma detection and identification: 

from 16S ribosomal gene to multiple gene 

identification 

Assunta Bertaccini ‐ Samanta Paltrinieri ‐ Nicoletta 

Contaldo Jana Franova 

Mogens Nicolaisen 

Bojan Duduk 

DiSTA Plant Pathology ‐ Alma Mater Studiorum University of Bologna, Italy 

Biology Centre of the ASCR, Institute of Plant Molecular Biology, Biology, 

Department of Plant Virology, Czech 

Republic 

Department of Agroecology, Agroecology, 

Aarhus University, Slagelse, Slagelse, 

Denmark 

Institute of Pesticides and Environmental protection, protection, 

Belgrade, Belgrade, 

Serbia

● plant pathogenic mycoplasmas that 

infect over 700 plant species 

● Induce yellowing, stunting, witches’ 

broom, and phyllody 

● Non yet culturable and classified in 

the class Mollicutes 

● Cell‐wall‐less, pleomorphic, 0.1~0.8 

µm in diameter 

● Inhabit phloem and emolimph and 

are transmitted by insects and…

New ‘Candidatus Candidatus species’ species must have 16S 

rDNA homology lower of 97.5% compared to 

those already described 

Today 34 species officially described as 

‘Candidatus’…

‘Candidatus Phytoplasma’ species Ribosomal group Associated diseases 

‘Ca. Phytoplasma asteris’ Aster Yellows group (16SrI) Aster yellows 

‘Ca. Phytoplasma japonicum’ Aster Yellows group (16SrI) Japanese Hydrangea phyllody 

‘Ca. Phytoplasma aurantifolia’ Peanut Witches' Broom group (16SrII) Acid lime witches’ broom 

‘Ca. Phytoplasma pruni’ X‐disease (16SrIII) Peach X disease 

‘Ca. Phytoplasma castaneae’ Coconut Lethal Yellowing group (16SrIV) Korean chestnut witches' broom 

'Ca. Phytoplasma pini' Coconut Lethal Yellowing group (16SrIV) 

‘Ca. Phytoplasma ziziphi’ Elm Yellows group (16SrV) Ziziphus jujube witches' broom 

‘Ca. Phytoplasma ulmi’ Elm Yellows group (16SrV) Elm yellows 

‘Ca. Phytoplasma trifolii’ Clover Proliferation group (16SrVI) Clover proliferation 

‘Ca. Phytoplasma fraxini’ Ash Yellows group (16SrVII) Ash yellows 

‘Ca. Phytoplasma phoenicium’ Pigeon Pea Witches' Broom group (16SrIX) Almond lethal disease 

‘Ca. Phytoplasma mali’ Apple Proliferation group (16SrX) Apple proliferation 

‘Ca. Phytoplasma pyri’ Apple Proliferation group (16SrX) Pear decline 

‘Ca. Phytoplasma prunorum’ Apple Proliferation group (16SrX) European stone fruit yellows 

‘Ca. Phytoplasma spartii’ Apple Proliferation group (16SrX) Spartium witches’ broom 

‘Ca. Phytoplasma rhamni’ Apple Proliferation group (16SrX) Buckthorn witches’ broom 

‘Ca. Phytoplasma allocasuarinae Apple Proliferation group (16SrX) Allocasuarina yellows 

‘Ca. Phytoplasma oryzae’ Rice Yellow Dwarf group (16SrXI) Rice yellow dwarf 

‘Ca. Phytoplasma australiense’ Stolbur group (16SrXII) Australian grapevine yellows 

‘Ca. Phytoplasma cynodontis’ BGWL group (16SrXIV) Bermuda grass white leaf 

‘Ca. Phytoplasma brasiliense’ ‘Ca. P. brasiliense’ group (16SrXV) Brazilian hibiscus witches' broom 

'Ca. Phytoplasma graminis' 'Ca. P. graminis' group (16SrXVI) Sugarcane yellow leaf from Cuba 

'Ca. Phytoplasma caricae' 'Ca. P. caricae' group (16SrXVII) Papaya bunchy top from Cuba 

'Ca. Phytoplasma fragariae' ribosomal group not designated Strawberry yellows ‐ Lithuania 

'Ca . Phytoplasma americanum' 'Ca . P. americanum' (16SrXVIII) Potato purple top wilt ‐ USA 

'Ca . Phytoplasma omanense' 'Ca. P. omanense' (16SrXIX) Cassia witches' broom ‐ Oman 

'Ca . Phytoplasma lycopersici' ribosomal group not designated Lycopersicum esculentum ‐ Bolivia 

'Ca . Phytoplasma rubi' Rubus stunt (16SrV‐E) Rubus sp.‐ Italy 

'Ca . Phytoplasma costaricanum' Soybean stunt (16SrXXXI‐A) Soybean stunt 

'Ca . Phytoplasma tamaricis' Salt cedar trees witches' broom (16SrXXX) Salt cedar trees witches' broom ‐ China 

'Ca . Phytoplasma sudamericanum' Passion fruit witches' broom (16SrVI‐I) Passion fruit witches' broom 

'Ca . Phytoplasma convolvuli' Bindweed yellows (16SrXII‐H) Bindweed yellows 

'Ca . Phytoplasma malaysianum' Periwinkle virescence Virescence and phyllody of periwinkle 

'Ca . Phytoplasma balanitae' Balanites witches' broom Balanites phyllody

‘Candidatus Phytoplasma’ 

species 

Ribosomal group Associated diseases 

[‘Ca. Phytoplasma palmae’] Coconut Lethal Yellowing group (16SrIV) Coconut lethal yellowing (Mexico) 

[‘Ca. Phytoplasma 

Coconut Lethal Yellowing group (16SrIV) Coconut lethal yellowing 

cocostanzaniae’] 

(Tanzania) 

[‘Ca. Phytoplasma cocosnigeriae’] Coconut Lethal Yellowing group (16SrIV) Coconut lethal yellowing (Nigeria) 

[‘Ca. Phytoplasma vitis’] Elm Yellows group (16SrV) Flavescence dorée 

[‘Ca. Phytoplasma luffae’] Loofah Witches' Broom group (16SrVIII) Loofah witches'‐broom 

[‘Ca. Phytoplasma solani’] Stolbur group (16SrXII) Stolbur and Bois Noir 

Today 8 species NOT officially described as 

‘Candidatus’…

16S ribosomal groups 

Aster yellows: AY 16SrI 

Peanut witches’ witches broom: broom: 

PnWB 16SrII 

Peach X disease: disease: 

CX 16SrIII 

Coconut letal yellowing: LY 16SrIV 

Elm yellows: EY 16SrV 

Clover proliferation: CP 16SrVI 

Ash yellows: ASHY 16SrVII 

Loofah witches’ witches broom: broom: 

LWB 16SrVIII 

Pigeon pea witches’ witches broom: broom: 

PPWB 16SrIX 

Apple proliferation: proliferation: 

AP 16SrX 

Rice yellow dwarf: RYD 16SrXI 

Stolbur: STOL 16SrXII 

Mexican periwinkle virescence: MPV 16SrXIII 

Bermudagrass white leaf: BGWL 16SrXIV 

Hibiscus witches’ witches broom: broom: 

HiWB 16SrXV 

Sugarcane yellow leaf: leaf: 

SCYL 16SrXVI 

Papaya bunchy top: PAY 16SrXVII 

American potato purple top wilt: wilt: 

APPTW 16SrXVIII 

Cassia witches’ witches broom: broom: 

CaWB 16SrXIX…… 

16SrXIX…….. 

..

Full genome sequence 

available for 4 strains 

2 ‘Ca Ca. . P. asteris’ asteris 

1 ‘Ca Ca. . P. australiense’ 

australiense 

1 ‘Ca Ca. . P. mali’ 

mali

Multiple locus analyses 

Elongation factor tuf tuf all 

all 

Ribosomal protein S3 S3 all* 

Nitroreductase gene 16SrX 

Rib. Rib Rib. . recycling factor + aminoacid kinase 16SrI‐A/B 16SrI 16SrI‐A/B A/B 

Amp/Imp Amp Amp/Imp Imp genes all* 

Translocation genes SecA all 

all 

Translocation genes SecY all* 

GroEL genes all*

QBOL‐developed 

QBOL developed tuf marker

Phytoplasma strains from micropropagated 

collection 

16SrI 16SrII 16SrIII 16SrV 16SrVI 16SrVII 16SrX 16SrXI 16SrXII 16SrXV 

RFLP on 3% agarose gel

Insect phytoplasma vectors 

‘Ca. Ca. P. asteris’ asteris (Europe Europe) 16SrI‐B 16SrI B Macrosteles 

Macrosteles quadripuctulatus, 

quadripuctulatus, 

Euscelis spp., spp., 

Euscelidius spp. spp 

Mulberry dwarf (Korea Korea) 16SrI‐D 16SrI Hishimonoides sellatiformis 

Various diseases (Australia) 16SrII‐D 16SrII Orosius argentatus 

Peach X disease (USA) 16SrIII‐A 16SrIII A Paraphlepsius irroratus, irroratus 

spp. spp 

‘Ca Ca. . P. ulmi’ ulmi (USA) 16SrV‐A 16SrV A Scaphoideus caphoideus luteolus (USA) 

Grapevine flavescence dorée dor 16SrV‐C/ 16SrV C/–D Scaphoideus titanus 

‘Ca Ca. . P. mali’ mali 

16SrX‐A 16SrX A Cacopsylla costalis, costalis, 

C. 

‘Ca Ca. . P. prunorum’ prunorum 

16SrX‐B 16SrX C. pruni 

‘Ca Ca. . P. pyri’ pyri 

16SrX‐C 16SrX C C. 

Grapevine bois noir 16SrXII‐A 16SrXII A Hyalestes 

Suger beet stolbur 16SrXII‐A 16SrXII A Pentastiridius 

, Colladonus 

melanoneura, 

melanoneura, 

Fieberiella florii 

C. pyri, pyri, 

C. pyricola e C. pyritsuga 

Hyalestes obsoletus, obsoletus, 

Reptalus spp. spp 

Pentastiridius beierii

Identification of phytoplasma strains associated 

with epidemic diseases: flavescence dorée

RFLP with TaqI Taq on 16Sr DNA amplicons

FD‐C 

Liguria (I) 

Tuscany (I) 

Serbia 

FD‐C 

Serbia 

Serbia 

Serbia 

Tuscany (I) 

FD‐D 

France 

Spain 

Emilia (I) 

Veneto (I) 

FD‐70 FD 70 France 

France 

Emilia (I)

GroELI gene ‐ ‘Ca. P. asteris’ (16SrI) 

group 

270 bp 1610 bp 701 bp 392 bp 

groES groEL amp nadE 

AYgroelF AYgroelR 

(22 ‐ 40) (1399 ‐ 1418)

Phytoplasma strains from 

micropropagated collection

Strain differentiation 

below the 16Sr 

subgroup level

Nested PCR primers 

270bp 1610bp 701bp 392bp 

groES groEL amp nadE 

AYgroelF AYgroelR 

(22‐40) (1399 ‐ 1418) 

AYgroesF 

(96‐115) 

AYampR 

(46‐65)

Samples Lettuce 

yellows 

(Imperia) 

Onion 

phyllody 

and 

virescence 

Sensitivity 

Onion 

phyllody 

and 

virescence 

Cabbage 

4 

phyllody 

Cabbage 

1 

phyllody 

Periwinkle 

virescence 

(Ferrara) 

Periwinkle 

virescence 

(Torino) 

Periwinkle 

virescence 

(Saint Louis) 

USA 

Year of 

extraction 

1992 1994 1994 1994 1994 1998 1998 1994 

16SrDNA group I‐B I‐B I‐B I‐B I‐B I‐B I‐B I‐B 

groELI group III III IV I XI III III X 

Direct 

PCR 

Nested 

PCR 

+ ‐ ‐ ‐ ‐ ‐+ ‐ ‐ ‐ 

+ + + + + + + +

Field‐collected samples 

Phytoplasma disease Geographical location 

Number of tested 

samples (year) 

Grouping 

TruI AluI groELI 16SrI 

Papaver virescence Forlì (Italy) 1 (2009) 1 1 III ‐B 

Oil seed rape phyllody Padova (Italy) 6 (2009) 1 1 III ‐B 

Onion phyllody and 

virescence 

Potenza (Italy) 4 (1994) 

1 

2 

1 

2 

III 

IV 

‐B 

‐B 

Cabbage phyllody Ravenna (Italy) 6 (1994) 

1 

7 

1 

1 

III 

XI 

‐B 

Lettuce yellows Florence (Italy) 1 (2000) 1 1 III ‐B 

Lettuce yellows Imperia (Italy) 5 (1992) 1 1 III ‐B 

Oil palm ‘machite lethal’ Colombia 1 (2004) 2 8 V ‐B 

Maize bushy stunt Palmira (Colombia) 2 (2007) 2 8 V ‐B 

Periwinkle virescence Belgrade (Serbia) 2 (2010) 4 4 VI ‐C 

Periwinkle virescence Torino (Italy) 1 (1998) 1 1 III ‐B 

Periwinkle virescence Saint Louis, USA 1 (1994) 1 9 X ‐B 

Periwinkle virescence Ferrara (Italy) 1 (1998) 1 1 III ‐B 

Carrot proliferation Begeč (Serbia) 3 (2006) 

Populus decline Belgrade (Serbia) 1 (2009) 6 7 IX ‐P 

Grindelia virescence Ravenna (Italy) 1 (2008) 2 2 IV ‐B 

1 

2 

3 

1 

2 

3 

III 

IV 

I 

‐B 

? 

‐A

ML variability in 

apple proliferation 

‘Ca. P. mali’ 

strains (16SrX‐A) 

Support

Serbia/Hungary 

Italy 

Strain 

acronyms 

Primers 

F1/B6 

HpaII FauI 

Group 

Primers 

AP13/AP10 

RcaI HincII 

Group 

Primers 

rpAP15f/rpAP15r 

AluI 

Group 

H-1 - - nd A A AT2 A rpX-A 

H-2 A+B A PI+PII - - nd A rpX-A 

H-3 B A PII B B AP A rpX-A 

H-4 A+B A PI+PII B A AT1 A rpX-A 

H-5 A+B A PI+PII B B AP A rpX-A 

H-6 A A PI B B AP A rpX-A 

RS-135 A A PI B B AP A rpX-A 

RS-151 A A PI B B AP A rpX-A 

I-VE11 B A PII - - nd C rpX-C 

I-VE12 A A PI - - nd A rpX-A 

I-VE14 A A PI A A AT2 A rpX-A 

I-VE16 B A PII B A AT1 C rpX-C 

I-VE22 B A PII - - nd D rpX-D 

I-VE27 B A PII B A AT1 D rpX-D 






I-TN1 A A PI A A AT2 A rpX-A 

I-TN2 A A PI - - nd A rpX-A 

I-TN3 B A PII - B nd B rpX-B 

AP-15 A A PI B B AP A rpX-A 

AT-1 A A PI B A AT1 B rpX-B 

AT-2 A A PI A A AT2 A rpX-A

Apple proliferation in the Czech Republic

East Bohemia 1 
























16S‐23S profile 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I 

P‐I+P‐I 

P‐I+P‐II 

P‐I+P‐II 

P‐I 

P‐I+ P‐II 

P‐I+P‐II 

AP‐15 

AT‐2 

AT‐2 

AT‐2 

AT‐2 

AT‐2 

AP‐15 

AT‐2 

AT‐2 

AT‐2 

AP‐15 

AP‐15 

AP‐15 

AT‐1+AT‐2+AP‐15 

AP‐15 

AT‐1 

AP‐15 

AP‐15 

AT‐2 

AT‐1 

AT‐2 

AP‐15 

AP‐15 

AT‐2 

AP subtype 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp X‐A 

rp subgroup

http://ipwgnet.org 

http:// 

Collection of in micropropagated 

periwinkle and other species of 

worldwide 

phytoplasma strains 

ipwgnet.org Plant pathology ‐ Alma Mater Studiorum University of Bologna Italy

Phytoplasma detection and identification: from 16S ribosomal gene ...

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