Bertaccini 2018

Chapter 1
Phytoplasmas: An Update
Assunta Bertaccini and Ing-Ming Lee
Abstract A summary of the research carried out on phytoplasma-associated dis-

eases 50 years after their discovery is presented. The great majority of this research
was devoted to classification and differentiation of these prokaryotes by molecular
and bioinformatic tools applied to specific phytoplasma genes. The availability of a
robust classification system has greatly facilitated phytoplasma identification lead-
ing to an increased knowledge of plant diseases worldwide. Phytoplasma biology
study still needs to be improved to allow better management solutions to reduce the
impact of these diseases in both agricultural and natural environments.
Keywords Taxonomy · Biology · Insect vector · Plant disease · Epidemiology
1.1 Introduction
Many yellows-type diseases, including aster yellows and paulownia witches’

broom, were believed to be caused by viruses until 1967 when a group of Japanese
scientists observed microorganisms resembling animal mycoplasmas in the phloem
sieve tube elements of diseased plants by electron microscopy (Doi et al. 1967).
These pleomorphic cell wall lacking bacteria were then named mycoplasma-like
organisms (MLOs) (Fig. 1.1). They possess a unique lifestyle that allows them to
live across plant and insect kingdoms. In the following decades, their detection was
primarily based on electron microscopy images of diseased phloem tissue and bio-
logical properties, such as unique disease symptoms, specific insect vector, and
plant host range. In subsequent years, the development of serological and molecular
A. Bertaccini (*)
Department of Agricultural and Food Sciences, Alma Mater Studiorum – University of
Bologna, Bologna, Italy
e-mail: [email protected]
I-M. Lee
Molecular Plant Pathology Laboratory, United States Department of Agriculture, Agriculture
Research Service, Beltsville, MD, USA
© Springer Nature Singapore Pte Ltd. 2018 1

G. P. Rao et al. (eds.), Phytoplasmas: Plant Pathogenic Bacteria - I,
https://doi.org/10.1007/978-981-13-0119-3_1
2 A. Bertaccini and I-M. Lee
Fig. 1.1 On the right electron microscopy picture of cross section of sieve tubes with phytoplas-
mas (X 6,000), and on the left symptoms associated with phytoplasma presence in aster
(Callistephus chinensis)
tools such as monoclonal antibodies and cloned DNA probes greatly improved their
detection.
DNA-specific amplification and sequencing provided evidence that MLOs con-
stitute a large monophyletic group within the class Mollicutes, and the trivial name
of “phytoplasma” was adopted followed by designation of the ‘Candidatus
Phytoplasma’ genus (IRPCM 2004). Phytoplasmas have variable sizes and shapes
(Fig. 1.1) and survive and multiply in the isotonic environments provided by plant
phloem and insect hemolymph. The full genome sequence has been completed for
two strains of aster yellows (‘Candidatus Phytoplasma asteris’), two strains of ‘Ca.
P. australiense’, and a strain of ‘Ca. P. mali’ (Oshima et al. 2004; Bai et al. 2006;
Kube et al. 2008; Tran-Nguyen et al. 2008; Andersen et al. 2013). A number of
draft genome sequences are also reported (Saccardo et al. 2012; Mitrović et al.
2014; Zamorano and Fiore 2016). Phytoplasmas possess one of the smallest
genomes among living organisms (Marcone et al. 1999) yet code complex meta-
bolic pathways that allow them to interact with both their plant and insect hosts
(Hogenhout et al. 2008). Phytoplasmas are quite often associated with severe and
rapidly spreading plant diseases, they are also able to increase the metabolic activ-
ity of their hosts, modify insect fitness, increase plant shoot production, and change
flower shape and color (Bertaccini et al. 2014); in other cases they are associated
with severe decline and death of the infected plants. It is however not uncommon to
detect phytoplasmas in asymptomatic plants, and this leaves open questions related
to their pathogenicity and biology as relevant characteristics that must be provided
together with phytoplasma identification when describing a new phytoplasma-
associated disease.
1 Phytoplasmas: An Update 3
1.2 Biological and Molecular Basis for Classification
Fifty years after their discovery, the role of phytoplasmas as plant pathogens is still
only based on indirect biological proof, such as electron microscopy observation,
specific DNA amplification, and symptom elimination after tetracycline treatments
(Ishiie et al. 1967). Insect and dodder transmission are the main tools available to
confirm phytoplasma association with plant disease. Plants infected by phytoplas-
mas very often exhibit symptoms indicative of profound unbalance of growth regu-
lators. Symptoms include virescence and phyllody of flowers, sterility, loss of apical
dominance generating the proliferation of axillary buds with witches’ broom forma-
tion (Fig. 1.1), abnormal internode elongation, and generalized stunting. The char-
acteristic symptomatology is very useful for preliminary indication of the possible
phytoplasma involvement in a disease. Some phytoplasmas also confer desirable
features such as for poinsettia, for which the presence of a specific strain allowed it
to be grown as an ornamental pot plant (Bertaccini et al. 1996; Lee et al. 1997).
Serological diagnostic techniques for detection of phytoplasmas began to emerge
in the 1980s when polyclonal and monoclonal antisera were produced and tested
(Lee et al. 1993b; Chen et al. 1993, 1994; Saeed et al. 1994) in plant tissues as well
as in leafhopper vectors or potential vectors using immunofluorescence (Lherminier
et al. 1990) and immunosorbent electron microscopy (Sinha 1979; Sinha and
Benhamou 1983), dot blot, or ELISA (Boudon-Padieu et al. 1989). In other
approaches, tissue blotting with direct or indirect antigen detection has been used
for specific phytoplasma detection (Lin and Chen 1985). In more recent years, anti-
bodies have been prepared to partial sequences of the major immunodominant pro-
teins of some phytoplasmas (Berg et al. 1999; Blomquist et al. 2001; Hong et al.
2001; Mergenthaler et al. 2001; Kakizawa et al. 2001; Barbara et al. 2002; Wei et al.
2004; Arashida et al. 2008; Siampour et al. 2012). Starting from the 1990s, the
application of molecular probes (Kirkpatrick et al. 1987; Lee and Davis 1988; Lee
et al. 1992; Bertaccini et al. 1993), PCR (Ahrens and Seemüller 1992; Namba et al.
1993; Lee et al. 1993a; Schneider et al. 1993), and nested-PCR (Lee et al. 1994,
1995) together with restriction fragment length polymorphism (RFLP) analyses or
sequencing allowed the broad detection of phytoplasmas (Lee et al. 1998a). The
introduction of quantitative PCR assays (qPCR) has shown these assays to be sensi-
tive with reduced risk of contamination making this technique a reliable alternative
to nested PCR assays in routine testing (Bianco et al. 2004; Torres et al. 2005;
Crosslin et al. 2006; Angelini et al. 2007; Baric et al. 2006; Hren et al. 2007;
Hodgetts et al. 2009; Aldaghi et al. 2009; Berger et al. 2009; Pelletier et al. 2009;
Nejat et al. 2010; Manimekalai et al. 2011; Monti et al. 2013; Córdova et al. 2014;
Ikten et al. 2016; Satta et al. 2017; Linck et al. 2017). Microarray (Nicolaisen and
Bertaccini 2007; Lenz et al. 2015), deep amplicon sequencing (Nicolaisen et al.
2011), and LAMP (Tomlinson et al. 2010; Bekele et al. 2011; Obura et al. 2011;
Sugawara et al. 2012; Kogovšek et al. 2015; Vu et al. 2016) are other techniques
used for phytoplasma detection, but not yet fully exploited and not always adequate
due to lack of specificity or sensitivity.
1.3 16S Ribosomal DNA-Based Classification
The development of a robust and quite exhaustive classification system based on

16S ribosomal gene sequence (Lee et al. 1998a) was followed by the multilocus
typing on other genes, which are usually different according to the diverse
‘Candidatus Phytoplasma’ species (Bertaccini 2015). A consensus for naming
novel phytoplasmas was reached in 2004 (IRPCM) for which “a ‘Candidatus (Ca.)
Phytoplasma’ species description should refer to a single, unique 16S rRNA gene
sequence (>1200 bp),” and “a strain can be recognized as a novel ‘Ca. Phytoplasma’
species if its 16S rRNA gene sequence has <97.5% similarity to that of any previ-
ously described ‘Ca. Phytoplasma’ species.” Phytoplasma taxonomy still relies on
these rules due to the lack of phenotypic characteristics of these bacteria that are
needed to classify them in formal genus and species. Some phytoplasma strains
which may warrant designation of a new taxon, but fail to meet the requirement of
sharing <97.5% sequence similarity with existing ‘Ca. Phytoplasma’, can be dif-
ferentiated and classified using additional unique biological properties such as anti-
body specificity, host range, and vector transmission specificity (Seemüller and
Schneider 2004).
Phytoplasma 16S rRNA genes are highly conserved; however, they possess suf-
ficient diversity to be used for their classification. Phytoplasmas are classified either
into a ‘Candidatus Phytoplasma’ species based on percent sequence identity
(IRPCM 2004) or into ribosomal groups and subgroups based on the presence of
restriction sites (Lee et al. 1998a; Wei et al. 2007; Zhao et al. 2009b). The number
of ‘Candidatus Phytoplasma’ species and ribosomal groups and subgroups is con-
tinually growing due to increased awareness of the importance of these pathogens
in agriculture and environment worldwide (Bertaccini et al. 2014; Maejima et al.
2014). A formal description remains to be published for phytoplasmas associated
with some well-known diseases such as grapevine “flavescence dorée” and some of
the lethal yellowing associated agents for which still the grouping and subgrouping
are the only official taxonomy (Table 1.1). A number of new ‘Candidatus’ species
were proposed in the last few years indicating the great biodiversity in these micro-
organisms intensified by the frequent report of interoperon heterogeneity and/or
mixed phytoplasma infection in both insects and plants (Schneider and Seemüller
1994; Lee et al. 1995, 1998b; Ho et al. 2001). Very recently two other ‘Candidatus’
species were officially proposed ‘Ca. P. wodyetiae’ and ‘Ca. P. noviguineense’
(Naderali et al. 2017; Miyazaki et al. 2018). There are thousands of 16S rRNA gene
sequences of phytoplasmas deposited in the public databases as well as sequences
to other conserved genomic regions used as supplemental tools for finer phyto-
plasma differentiation (Duduk and Bertaccini 2011). A barcode screening system
was also developed in agreement with the 16S rDNA classification for fast phyto-
plasma detection and identification (Makarova et al. 2012). The rp, tuf, and secY
genes show more variation than the 16S rRNA gene (Gundersen et al. 1996;
Schneider et al. 1997; Cimerman et al. 2009; Marcone et al. 2000; Lee et al. 2006b,
2010, 2012; Danet et al. 2007; Arnaud et al. 2007; Martini et al. 2002, 2007;
Table 1.1 Phytoplasma classification based on RFLP analyses and/or sequencing of 16S rDNA
GenBank
16Sr accession
grouping Strain (acronym) ‘Candidatus’ sp. number References
16SrI – aster yellows
I-A Aster yellows witches’ NC_007716 Bai et al. (2006)
broom (AY-WB)
I-B Aster yellows (MAY) ‘Ca. P. asteris’ M30790 Lee et al. (2004a)
I-C Clover phyllody (CPh) AF222065 Lee et al. (2004a)
I-D Paulownia witches’ AY265206 Lee et al. (2004a)
broom (PaWB)
I-E Blueberry stunt (BBS3) AY265213 Lee et al. 2004a
I-F Aster yellows apricot AY265211 Lee et al. (2004a)
Spain (A-AY)
I-I Strawberry witches’ U96614 Jomantiene et al.
broom (STRAWB1) (1998)
I-K Strawberry witches’ U96616 Jomantiene et al.
broom (STRAWB2) (1998)
I-L Aster yellows (AV2192) AY180957 Lee et al. (2003)
I-M Aster yellows (AVUT) AY265209 Lee et al. (2004a)
I-N Aster yellows (IoWB) AY265205 Lee et al. (2004a)
I-O Soybean purple stem AF268405 Lee et al. (2002)
(SPS)
I-P Aster yellows from AF503568 Šeruga et al.
Populus (PopAY) (2003)
I-Q Cherry little leaf (ChLL) AY034089 Valiunas et al.
(2005)
I-R Strawberry phylloid fruit AY102275 Jomantiene et al.
(StrawbPhF) (2002)
I-S Pepper little leaf (PeLL) DQ092321 Santos-Cervantes
et al. (2008)
I-T Tomato little leaf (ToLL) DQ375238 Santos-Cervantes
et al. (2008)
I-U Mexican potato purple FJ914650 Santos-Cervantes
top (JAL8) et al. (2010)
I-V Mexican potato purple FJ914642 Santos-Cervantes
top (SON18) et al. (2010)
I-W Peach rosette-like HQ450211 Arocha-Rosete
disease (PRU0382) et al. (2011)
I-X Papaya bunchy top JF781308 Acosta et al.
(BTS) (2013)
I-Y Tomato “brote grande” ‘Ca. P. lycopersici’ EF199549 Arocha et al.
(2007)
I-Z Papaya bunchy top JF781311 Acosta-Pérez
(BTS) et al. (2017)
(continued)
Table 1.1 (continued)

GenBank
16Sr accession
16SrII – peanut witches’ broom
II-A Peanut witches’ broom L33765 Gundersen et al.
(PnWB) (1994)
II-B Lime witches’ broom ‘Ca. P. U15442 Zreik et al.
(WBDL) aurantifolia’ (1995)
II-C Faba bean phyllody X83432 Seemüller et al.
(FBP) (1998)
II-D Papaya mosaic (PpM) ‘Ca. P. Y10096 White et al.
australasia’ (1998)
II-E Picris echioides phyllody Y16393 Seemüller et al.
(PEY) (1998)
II-F Cotton phyllody (CoP) EF186827 Martini et al.
(2007)
II-G Cactus witches’ broom EU099568 Cai et al. (2008)
(CWB)
II-J Cactus witches’ broom EU099552 Cai et al. (2008)
(CWB)
II-H Cactus witches’ broom EU099569 Cai et al. (2008)
(CWB)
II-K Cactus witches’ broom EU099572 Cai et al. (2008)
(CWB)
II-I Cactus witches’ broom EU099551 Cai et al. (2008)
(CWB)
II-L Cactus witches’ broom EU099546 Cai et al. (2008)
(CWB)
II-M Potato purple top FJ914643 Yadav et al.
(2014)
II-N Papaya BTSp JF781309 Acosta et al.
(2013)
II-O Tabebuia witches’ broom EF647744 Mafia et al.
(2007)
II-P Cuban papaya DQ286948 Perez-López
phytoplasma et al. (2016)
II-Q Papaya bunchy top JF78131 Perez-López
(TSpHav02-IIA) et al. (2016)
II-R Echinopsis yellow patch DQ535900 Perez-López
et al. (2016)
II-S Amaranthus FJ357164 Perez-López
hypochondriacus strain et al. (2016)
52A
II-T Tomatillo witches’ U125185 Perez-López
broom et al. (2016)
II-U Papaya little leaf KP057205 Yang et al.
(2016)
(continued)

GenBank
16Sr accession
16SrIII – X-disease
III-A Peach X-disease ‘Ca. P. pruni’ JQ044393 Davis et al.
(PX11CT1) (2013)
III-B Clover yellow edge AF173558 Davis et al.
(CYE) (2013)
III-C Pecan bunch (PB) GU004371 Davis et al.
(2013)
III-D Goldenrod yellows GU004372 Davis et al.
(GR1) (2013)
III-E Spiraea stunt (SP1) AF190228 Davis et al.
(2013)
III-F Milkweed yellows AF510724 Davis et al.
(MW1) (2013)
III-G Walnut witches’ broom AF190226/ Davis et al.
(WWB) AF190227 (2013)
III-J Chayote witches’ broom AF147706 Montano et al.
(ChWBIII) (2000)
III-K Strawberry leafy fruit AF274876 Jomantiene et al.
(SLF) (1998)
III-H Poinsettia branch- AF190223 Davis et al.
inducing (PoiBI) (2013)
III-I Virginia grapevine AF060875 Davis et al. 1998
yellows (VGYIII)
III-L Cassava frog skin EU346761 Alvarez et al.
disease (CFSD) (2009)
III-M Potato purple top FJ226074 Davis et al.
(MT117) (2013)
III-N Potato purple top GU004365 Davis et al.
(AKpot6) (2013)
III-O Dandelion virescence AF370120 Jomantiene et al.
(DanVir) (2002)
III-P Dandelion virescence AF370119/ Jomantiene et al.
(DanVir) AF370120 (2002)
III-Q Black raspberry witches’ AF302841 Davis et al.
broom (BRWB7) (2001)
III-R Cirsium white leaf AF373105 Zhao et al.
(CirWL) (2009b)
III-S Western peach X-disease L04682 Zhao et al.
(WX) (2009b)
III-T Sweet and sour cherry FJ231728 Valiunas et al.
(ChD) (2009)
III-U Cirsium white leaf AF373105/ Jomantiene et al.
(CWL) AF373106 (2002)
(continued)

GenBank
16Sr accession
III-V Passion fruit GU292082 Davis et al.
phytoplasma (2012)
(PassWB-Br4)
III-W Heterothalamus little KC412029 Galdeano et al.
leaf (HetLL) (2013)
III-X Conyza witches’ broom KC412026 Galdeano et al.
(2013)
III-Y Cranberry false blossom KF62652 Lee et al. (2014)
III-Z Broccoli stunt strain JX626327 Perez-López
BSP-21 et al. (2016)
16SrIV – coconut lethal yellowing
IV-A Coconut lethal yellowing AF498307 Harrison et al.
(LYJ-C8) (2002)
IV-B Yucatan coconut lethal U18753 Harrison et al.
decline (LDY) (1994)
IV-C Tanzanian coconut lethal X80117 Harrison et al.
decline (LDT) (1994)
IV-D Texas phoenix decline AF434969 Harrison et al.
(TPD) (2008)
IV-E Coconut lethal yellowing DQ631639 Martinez et al.
(LYDR-B5) (2008)
IV-F Washingtonia robusta EU241512 Harrison et al.
decline (2008)
16SrV – elm yellows
V-A Elm yellows (EY) ‘Ca. P. ulmi’ AY197655 Lee et al. (2004b)
V-B Jujube witches’ broom ‘Ca. P. ziziphi’ AB052876 Jung et al.
(JWB-G1) (2003a)
V-C “Flavescence dorée” X76560 Martini et al.
(FD-C) (1999)
V-D “Flavescence dorée” AJ548787 Martini et al.
(FD-D) 1999
V-E Rubus stunt (RuS) ‘Ca. P. rubi’ AY197648 Malembic-Maher
et al. (2011)
V-F Balanite witches’ broom ‘Ca. P. balanitae’ AB689678 Win et al. (2013)
(BltWB)
V-G Korean jujube witches’ AB052879 Jung et al.
broom (2003a)
V-H Bischofia polycarpa KJ452547 Lai et al. (2014)
witches’ broom
V-I Blackberry witches’ KR233473 Fránová et al.
broom (2016)
(continued)

GenBank
16Sr accession
16SrVI – clover proliferation
VI-A Clover proliferation (CP) ‘Ca. P. trifolii’ AY390261 Hiruki and Wang
(2004)
VI-B Strawberry multiplier AF190224 Jomantiene et al.
disease (MD) (1998)
VI-C Illinois elm yellows AF409069 Jacobs et al.
(EY-IL1) (2003)
VI-D Periwinkle little leaf AF228053 Siddique et al.
(PLL-Bd) (2001)
VI-E Centaurea solstitialis AY270156 Faggioli et al.
virescence (CSVI) (2004)
VI-F Catharanthus phyllody EF186819 Martini et al.
(CPS) (2007)
VI-H Portulaca little leaf EF651786 Samad et al.
phytoplasma (PLL-Ind) (2008)
VI-I Passionfruit (WB-Br4) ‘Ca. P. GU292081 Davis et al.
sudamericanum’ (2012)
16SrVII – ash yellows
VII-A Ash yellows (AshY) ‘Ca. P. fraxini’ AF092209 Griffiths et al.
(1999)
VII-B Erigeron witches’ broom AY034608 Barros et al.
(ErWB) (2002)
VII-C Argentinian alfalfa AY147038 Conci et al.
witches’ broom (2005)
(ArAWB)
VII-D Erigeron witches’ broom KJ831066 Flôres et al.
(EboWB-Br0) (2015)
16SrVIII – loofah witches’ broom
VIII-A Loofah witches’ broom ‘Ca. P. luffae’ AF086621 Davis et al.
(LufWB) (2017)
16SrIX – pigeon pea witches’ broom
IX-A Pigeon pea witches’ AF248957 Gundersen et al.
broom (PPWB) (1996)
IX-B Almond witches’ broom ‘Ca. P. AF515636 Verdin et al.
(AlWB) phoenicium’ (2003)
IX-C Naxos periwinkle HQ589191 Duduk et al.
virescence (NAXOS) (2008)
IX-D Almond witches’ broom AF515637 Verdin et al.
(AlWB) (2003)
IX-E Juniperus witches’ GQ925918 Davis et al.
broom (2010)
(continued)

GenBank
16Sr accession
IX-F Almond and stone fruit HQ407532 Molino Lova
witches’ broom (N27-2) et al. (2011)
IX-G Almond and stone fruit HQ407514 Molino Lova
witches’ broom (A1-1) et al. (2011)
IX-H Sarson phyllody KU892213 Ahmad et al.
(2017)
16SrX – apple proliferation
X-A Apple proliferation (AP) ‘Ca. P. mali’ AJ542541 Seemüller and
Schneider (2004)
X-B European stone fruit ‘Ca. P. prunorum’ AJ542544 Seemüller and
yellows (ESFY) Schneider (2004)
X-C Pear decline (PD) ‘Ca. P. pyri’ AJ542543 Seemüller and
Schneider (2004)
X-D Spartium witches’ ‘Ca. P. spartii’ X92869 Marcone et al.
broom (SpaWB) (2003a)
X-E Black alder witches’ X76431 Seemüller et al.
broom (BAWB(BWB) (1994)
16SrXI – rice yellow dwarf
XI-A Rice yellow dwarf ‘Ca. P. oryzae’ AB052873 Jung et al.
(RYD) (2003b)
XI-B Sugarcane white leaf X76432 Lee et al. (1998a,
(SCWL) b)
XI-C Leafhopper-borne X76429 Lee et al. (1998a,
(BVK) b)
XI-D Sugarcane white leaf KR020685 Zhang et al.
(SCWL) (2016)
XI-E Cirsium phytoplasma ‘Ca. P. cirsii’ KR869146 Šafárová et al.
(2016)
XI-F Sugarcane grassy shoot HF586636 Yadav et al.
(SCGS) (2017)
16SrXII – “stolbur”
XII-A “Stolbur” (STOL11) ‘Ca. P. solani’ AF248959 Quaglino et al.
(2013)
XII-B Australian grapevine ‘Ca. P. L76865 Davis et al.
yellows (AUSGY) australiense’ (1997)
XII-C Strawberry lethal AJ243045 Padovan et al.
yellows (StrawLY) (2000)
XII-D Japanese hydrangea ‘Ca. P. japonicum’ AB010425 Sawayanagi et al.
phyllody (1999)
XII-E Yellows diseased ‘Ca. P. fragariae’ DQ086423 Valiunas et al.
strawberry (StrawY) (2006)
XII-F “Bois noir” (BN-Op30) EU836630 Quaglino et al.
(2009)
(continued)

GenBank
16Sr accession
XII-G “Bois noir” (BN-Fc3) 47EU8366 Quaglino et al.
(2009)
XII-H Bindweed yellows ‘Ca. P. convolvuli’ JN833705 Martini et al.
(BY-S57/11) (2012)
XII-I Potato strain from China EU338445 Cheng et al.
(169/Hezuo 88) (2015)
16SrXIII – Mexican periwinkle virescence
XIII-A Mexican periwinkle ‘Ca. P. AF248960 Davis et al.
virescence (MPV) hispanicum’ (2016)
XIII-B Strawberry green petal U96616 Jomantiene et al.
(STRAWB2) (1998)
XIII-C Chinaberry yellows AF495882 Harrison et al.
(CBY1) (2002)
XIII-D Mexican potato purple FJ914647 Santos-Cervantes
top (SINPV) et al. (2010)
XIII-E Papaya apical curl EU719111 Melo et al.
necrosis (PACN) (2013)
XIII-F Strawberry red leaf KJ921641 Fernández et al.
2015
XIII-G Chinaberry yellowing ‘Ca. P. meliae’ KU850940 Fernández et al.
(ChTY) (2016)
16SrXIV – Bermuda grass white leaf
XIV-A Bermuda grass white ‘Ca. P. AJ550984 Marcone et al.
leaf (BGWL) cynodontis’ (2003b)
XIV-B Bermuda grass white EF444485 Salehi et al.
leaf Iran strain (2009)
XIV-C Bermuda grass white KP019339 Mitrovic et al.
leaf (RS304/13) (2015)
16SrXV – hibiscus witches’ broom
XV-A Hibiscus witches’ broom ‘Ca. P. brasiliense’ AF147708 Montano et al.
(HibWB) (2001)
XV-B Guazuma witches’ HQ258882 Villalobos et al.
broom (GWB) (2011)
16SrXVI – sugarcane yellow leaf
XVI-A Sugarcane yellow leaf ‘Ca. P. graminis’ AY725228 Arocha et al.
(2005)
16SrXVII – papaya bunchy top
XVII-A Papaya bunchy top ‘Ca. P. caricae’ AY725234 Arocha et al.
(2005)
16SrXVIII – American potato purple top wilt
XVIII-A American potato purple ‘Ca. P. DQ174122 Lee et al. (2006a,
top wilt americanum’ b)
(continued)

GenBank
16Sr accession
16SrXIX – chestnut witches’ broom
XIX-A Chestnut witches’ broom ‘Ca. P. castaneae’ AB054986 Jung et al. (2002)
16SrXX – Rhamnus witches’ broom
XX-A Rhamnus witches’ ‘Ca. P. rhamni’ AJ583009 Marcone et al.
broom (2003a)
16SrXXI – Pinus phytoplasma
XXI-A Pinus phytoplasma ‘Ca. P. pini’ AJ310849 Schneider et al.
(PinP) (2005)
16SrXXII – lethal yellowing-type
XXII-A Lethal yellowing ‘Ca. P. palmicola’ KF751387 Harrison et al.
Mozambique (LYDM (2014)
178)
XXII-B Cape Saint Paul Wilt Y13912 Tymon et al.
disease Ghana (LDG) (1998)
16SrXXIIIa
XXIII-A Buckland valley AY083605 Wei et al. (2007)
grapevine yellows
16SrXXIVa
XXIV-A Sorghum bunchy shoot AF509322 Wei et al. (2007)
16SrXXVa
XXV-A Weeping tea witches’ AF521672 Wei et al. (2007)
broom
16SrXXVIa
XXVI-A Sugarcane phytoplasma AJ539179 Wei et al. (2007)
D3T1
16SrXXVIIa
XXVII-A Sugarcane phytoplasma AY539180 Wei et al. (2007)
D3T2
16SrXXVIIIa
XXVIII-A Derbid phytoplasma AY744945 Wei et al. (2007)
16SrXXIX – cassia witches’ broom
XXIX-A Cassia witches’ broom ‘Ca. P. omanense’ EF666051 Al-Saady et al.
(CaWB) (2008)
XXIX-B Bindweed witches’ KY047493 Esmailzadeh
broom (RBiWB) Hosseini et al.
(2016)
16SrXXX – salt cedar witches’ broom
XXX-A Salt cedar witches’ ‘Ca. P. tamaricis’ FJ432664 Zhao et al.
broom (2009a)
16SrXXXI – soybean stunt
XXXI-A Soybean stunt ‘Ca. P. HQ225630 Lee et al. (2011)
(SoyST1c1) costaricanum’
(continued)

GenBank
16Sr accession
16SrXXXII – Malaysian periwinkle virescence and phyllody
XXXII-A Malaysian periwinkle ‘Ca. P. EU371934 Nejat et al.
virescence (MaPV) malaysianum’ (2013)
XXXII-B Malayan yellow dwarf EU498727 Nejat et al.
(MYD) (2013)
XXXII-C Malayan oil palm EU498728 Nejat et al.
(MOP) (2013)
16SrXXXIII – Allocasuarina muelleriana phytoplasma
XXXIII-A Allocasuarina ‘Ca. P. AY135523 Marcone et al.
phytoplasma allocasuarinae’ (2003a)
a
Groups designed only on the basis of GenBank deposited sequences
Hodgetts et al. 2008; Mitrović et al. 2011, 2015) and are useful for epidemiological
studies. However using only genetic differentiation to characterize phytoplasmas
could end in producing just lists of genotypes if the knowledge of phytoplasma biol-
ogy is not accompanying the appropriate taxonomy. Biological characteristics of
phytoplasmas are needed in particular to help finding management solutions to
reduce phytoplasma disease impact on worldwide agriculture.
1.4 Transmission and Epidemiology
In nature phytoplasmas infect numerous plant species and as equally numerous

insects that serve as their vectors in a successful three-way interaction.
Phytoplasmas are mainly spread between plants by insects in the families
Cicadellidae (leafhoppers) and Psyllidae (psyllids) and superfamily Fulgoroidea
(plant hoppers), which feed on the phloem sap of infected plants; therefore their
host range is dependent upon feeding habits of their insect vectors (Bertaccini
2007). Phytoplasmas overwinter in insect vectors or in perennial plant hosts and
interact with insect hosts also reducing or enhancing their fitness (Sugio et al.
2011a). Transovarial transmission (Alma et al. 1997; Kawakita et al. 2000;
Hanboonsong et al. 2002; Tedeschi et al. 2006) and seed transmission (Khan et al.
2002; Botti and Bertaccini 2006; Calari et al. 2011; Chung and Jeong 2014; Satta
et al. 2016) were also demonstrated in some plant species/insect-phytoplasma
combinations. Phytoplasmas are also efficiently spread via vegetative propagation
such as cuttings, grafting, and micropropagation practices (Bertaccini et al. 1992;
Jarausch et al. 1996; Bertaccini 2007).
1.5 Genomic Sequencing and Metabolic Features
A lot of information has been achieved by full genome sequencing especially

related to putative biochemical pathways showing that phytoplasmas are very spe-
cial microorganisms because they lack a lot of relevant features such as cell wall,
mobility, key enzymes, and pathways. Phytoplasmas possess the smallest genome
among bacteria; however, gene duplication and redundancy are well represented
(Oshima et al. 2004). Moreover, extrachromosomal DNA or plasmids of various
sizes have also been found in several phytoplasma groups together with sequences
of variable mosaic (SVM) termed potential mobile units or PMUs (Schneider et al.
1992; Kuboyama et al. 1998; Rekab et al. 1999; Oshima et al. 2001; Jomantiene and
Davis 2006; Jomantiene et al. 2007; Wei et al. 2008; Ishii et al. 2009; Toruno et al.
2010). Microarray analysis of ‘Ca. P. asteris’ strain OY-M revealed that expression
of approximately 33% of the genes change during host switching between plant and
insect (Oshima et al. 2011) and the phytoplasma may use transporters, secreted
proteins, and metabolic enzymes in a host-specific manner. Several factors, namely,
tengu, SAP11, SAP54, and P38, that could modulate possible phytoplasma patho-
genicity were reported to induce symptoms similar to those observed in phytoplasma-
infected plants when inserted into transgenic plants (Hoshi et al. 2009; Sugio et al.
2011a, b; MacLean et al. 2011; Neriya et al. 2014). Phytoplasmas possess two
secretion systems, YidC and Sec, the latter seems to be common to most or all phy-
toplasmas (Kakizawa et al. 2004, 2009). It was also demonstrated that phytoplas-
mas lack ATP synthase genes suggesting a metabolism strongly dependent on
glycolysis. While two sets of glycolytic enzymes were encoded in a duplicated
genomic region of a strongly pathogenic strain of ‘Ca. P. asteris’, mild strains do
not possess this duplication, suggesting this as a possible pathogenicity mechanism
(Oshima et al. 2007). The glycolysis genes are completely absent in the full genome
of ‘Ca. P. mali’ in which a gene-encoding 2-dehydro-3-deoxy-phosphogluconate
aldolase was retrieved leading to the hypothesis that in this phytoplasma pyruvate is
formed independently from glycolysis (Kube et al. 2012). It is possible that patho-
genic mechanisms may differ according to the strain genomic content and/or the
diverse environmental conditions such as different host species. The occurrence of
major surface epitopes that are unique to each phytoplasma group or ‘Candidatus
species’, suggests their role in specific interactions with host/insect cells. The Amp
protein of the OY phytoplasma forms a complex with insect microfilaments corre-
lated with their phytoplasma-transmitting capacity (Suzuki et al. 2006; Galetto
et al. 2011).
1.6 Cultivation in Artificial Media
After preliminary evidence that phytoplasmas can be grown in cell-free media

(Bertaccini et al. 2010; Contaldo et al. 2012, 2013), a recent description of a suitable
and flexible medium was published (Contaldo et al. 2016). This is important infor-
mation to enable the study of their biology: substantially pure cultures can be
Fig. 1.2 Left. Ultrathin section of phytoplasma-like cells in agar medium embedded in Spurr resin
and observed under transmission electron microscope at 5,000 X magnification (A. Bertaccini and
A. Calzolari, 1984, unpublished). Right. Phytoplasma colonies growing in CB medium photo-
graphed under bifocal microscope 40 X (courtesy of N. Contaldo)
obtained to verify predicted metabolisms and biological properties of phytoplasmas

outside their hosts. Colonies with identical morphology and positive for phyto-
plasma DNA presence (Fig. 1.2) confirm that despite the reduced genome size, phy-
toplasmas retain an independent metabolism that allows them to survive as parasites
in environments as diverse as plant phloem and insect hemolymph and also in cell-
free media.
Although further research is needed in order to optimize the culture system, the
prospect of phytoplasma cultivation is now a real option. It will facilitate genome
sequencing of further phytoplasma species and strains to allow comparative genom-
ics which has been hampered by their intimate association with plant and insect
hosts. Moreover, along with genomics, biochemical and physiological studies, phy-
toplasma cultivation will define their taxonomy. Selection and screening of plants
resistant to phytoplasma infection as well as the study of the modes of colonization
by phytoplasmas of plant and insect vectors will also be possible. As a consequence,
strategies to manage and/or prevent phytoplasma-associated plant diseases more
efficiently will be prepared and employed.
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Chapter 1

Assunta Bertaccini and Ing-Ming Lee

Abstract A summary of the research carried out on phytoplasma-associated dis-

Keywords Taxonomy · Biology · Insect vector · Plant disease · Epidemiology

Many yellows-type diseases, including aster yellows and paulownia witches’

© Springer Nature Singapore Pte Ltd. 2018 1

1.2 Biological and Molecular Basis for Classification

1.3 16S Ribosomal DNA-Based Classification

The development of a robust and quite exhaustive classification system based on

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

Table 1.1 (continued)

1.4 Transmission and Epidemiology

In nature phytoplasmas infect numerous plant species and as equally numerous

1.5 Genomic Sequencing and Metabolic Features

A lot of information has been achieved by full genome sequencing especially

1.6 Cultivation in Artificial Media

After preliminary evidence that phytoplasmas can be grown in cell-free media

obtained to verify predicted metabolisms and biological properties of phytoplasmas

Acosta-Pérez KI, Piñol-Pérez BE, Zamora-Gutierrez L, Quiñones-Pantoja ML, Miranda-Cabrera

Kawakita H, Saiki T, Wei W, Mitsuhashi W, Watanabe K, Sato M (2000) Identification of mulberry

Phytoplasma rubi’. International Journal of Systematic and Evolutionary Microbiology 61,

Molino Lova M, Quaglino F, Abou-Jawdah Y, Choueiri E, Sobh H, Casati P, Tedeschi R, Alma

Pelletier C, Salar P, Gillet J, Cloquemin G, Very P, Foissac X, Malembic-Maher S (2009) Triplex

Schneider B, Ahrens U, Kirkpatrick BC, Seemüller E (1993) Classification of plant-pathogenic

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1.2 Biological and Molecular Basis for Classification

1.3 16S Ribosomal DNA-Based Classification

1.4 Transmission and Epidemiology

1.5 Genomic Sequencing and Metabolic Features

1.6 Cultivation in Artificial Media