New hosts of “Candidatus Phytoplasma australiense” in New Zealand

Article (PDF Available)inAustralasian Plant Pathology 40(3):238-245 · May 2011with 150 Reads
DOI: 10.1007/s13313-011-0036-z
Abstract
“Candidatus Phytoplasma australiense” occurs in New Zealand and Australia where it is associated with plant diseases in native, weed and crop plants. In New Zealand, this phytoplasma is historically associated with the diseases, Phormium yellow leaf, Strawberry lethal yellows, Cordyline sudden decline and Coprosma lethal decline. Between January 2009 and July 2010, four new hosts of “Ca. P. australiense” have been identified in New Zealand: potato (Solanum tuberosum), Jerusalem cherry (Solanum pseudocapsicum), swan plant (Gomphocarpus fruticosa) and celery (Apium graveolens), as well as a new disease association in boysenberry (Rubus hybrid). A 1.2kb region of the 16S rRNA gene of the phytoplasma amplified from the new hosts was identical to each other. Partial tuf gene sequence analysis of 32 isolates from potato, Jerusalem cherry, swan plant, celery, boysenberry as well as from the Zeoliarus planthopper vector, revealed that they belonged to two separate subgroups, tuf variant VII and tuf variant IX. Two of the isolates, one from potato and the other from celery, contained a mixed infection of both phytoplasma subgroups. KeywordsMollicute–PCR–Detection–Epidemiology
New hosts of Candidatus Phytoplasma australiense
in New Zealand
Lia W. Liefting &Stella Veerakone &Gerard R. G. Clover
Received: 15 December 2010 / Accepted: 30 January 2011 / Published online: 18 March 2011
#Australasian Plant Pathology Society Inc. 2011
Abstract Candidatus Phytoplasma australienseoccurs in
New Zealand and Australia where it is associated with plant
diseases in native, weed and crop plants. In New Zealand,
this phytoplasma is historically associated with the dis-
eases, Phormium yellow leaf, Strawberry lethal yellows,
Cordyline sudden decline and Coprosma lethal decline.
Between January 2009 and July 2010, four new hosts of
Ca. P. australiensehave been identified in New Zealand:
potato (Solanum tuberosum), Jerusalem cherry (Solanum
pseudocapsicum), swan plant (Gomphocarpus fruticosa)
and celery (Apium graveolens), as well as a new disease
association in boysenberry (Rubus hybrid). A 1.2 kb region
of the 16S rRNA gene of the phytoplasma amplified from
the new hosts was identical to each other. Partial tuf gene
sequence analysis of 32 isolates from potato, Jerusalem
cherry, swan plant, celery, boysenberry as well as from the
Zeoliarus planthopper vector, revealed that they belonged
to two separate subgroups, tuf variant VII and tuf variant
IX. Two of the isolates, one from potato and the other from
celery, contained a mixed infection of both phytoplasma
subgroups.
Keywords Mollicute .PCR .Detection .Epidemiology
Introduction
Phytoplasmas are obligate, intracellular prokaryotes (class
Mollicutes) associated with diseases in hundreds of plant
species worldwide including economically important food,
fibre and forage crops (Lee et al. 2000). They are
characterised by their lack of a cell wall, a pleiomorphic
or filamentous shape, normally with a diameter less than
1μm, small genome size and a low guanine plus cytosine
content. Phytoplasmas are restricted to the phloem cells of
the host plant and are transmitted by phloem-feeding
insects, primarily leafhoppers, and less commonly by
planthoppers and psyllids. They replicate in both their
insect and plant hosts. Phytoplasmas have not been cultured
successfully in vitro and are classified using the Candida-
tusconcept where each of the major clades established by
16S rRNA sequence analysis represent a Candidatus
species of the Phytoplasma genus (The IRPCM Phyto-
plasma/Spiroplasma Working TeamPhytoplasma taxono-
my group 2004).
Candidatus Phytoplasma australiense(16SrXII-B) is
found in New Zealand and Australia where it is associated
with a range of host plants. In New Zealand, it is associated
with the diseases, Phormium yellow leaf (Liefting et al.
1998), Strawberry lethal yellows (Andersen et al. 1998b),
Cordyline sudden decline (Andersen et al. 2001)and
Coprosma lethal decline (Beever et al. 2004). During an
investigation into the cause of Boysenberry decline disease,
Ca. P. australiensewas also detected in boysenberry
(Wood et al. 1999). However phytoplasma detection was
not consistent with affected plants and boysenberry decline
was eventually determined to be caused by the fungus
Cercosporella rubi. Most recently, in January of 2009, Ca.
This paper is in memory of Dr Ross Beever (19462010), APPS
Fellow and authority on phytoplasmas in New Zealand.
L. W. Liefting (*):S. Veerakone :G. R. G. Clover
Plant Health and Environment Laboratory,
MAF Biosecurity New Zealand,
P.O. Box 2095, Auckland 1140, New Zealand
e-mail: lia.liefting@maf.govt.nz
Australasian Plant Pathol. (2011) 40:238245
DOI 10.1007/s13313-011-0036-z
P. australiensewas detected in potato in New Zealand
(Liefting et al. 2009). In Australia, this phytoplasma species
has been associated with Australian grapevine yellows,
Papaya dieback (Liefting et al. 1998), as well as diseases in
a range of other hosts including strawberry, pumpkin and
bean (Streten and Gibb 2006).
Sequence analysis of the tuf gene of different isolates of
Ca. P. australiensein New Zealand determined that there
are nine different tuf variant groups (I-IX) that cluster into
two distinct clades (Andersen et al. 2006). Out of 36
isolates there was no obvious correlation between the tuf
group, host or geographic location. Andersen et al. (2006)
also analysed the available tuf gene sequences from
Australian isolates and determined that some isolates
formed a third distinct tuf gene clade while other isolates
clustered into one of the clades formed by the New Zealand
isolates.
The insect vectors of Ca. P. australiensein New
Zealand are Zeoliarus (Oliarus)atkinsoni (Cumber 1953;
Liefting et al. 1997) and Zeoliarus oppositus (Beever et al.
2008). Both of these insect species are planthoppers in the
family Cixiidae and are endemic to New Zealand where
they are widespread. Z. atkinsoni is restricted to Phormium
and therefore has only been demonstrated to transmit the
phytoplasma from Phormium to Phormium. In contrast, Z.
oppositus is polyphagus and has been reported from many
different plants. To date, the insect vector responsible for
the spread of Ca. P. australiensein Australia is unknown.
This manuscript describes three new diverse hosts of
Ca. P. australiensein New Zealand: Jerusalem cherry,
swan plant and celery. A new association of Ca.P.
australiensein boysenberry that differs from Boysenberry
decline disease is also described. The tuf gene sequences of
Ca. P. australiensefrom these new hosts as well as from
different potato and boysenberry isolates were analysed
extending the work of Andersen et al. (2006) to gain
insights into the epidemiology of this phytoplasma.
Materials and methods
Plant and insect material
Potato (Solanum tuberosum) samples were received from
commercial potato growers between January 2009 and
January 2010. A single symptomatic Jerusalem cherry
(Solanum pseudocapsicum) sample was collected from the
edge of a native bush remnant in February 2009. This bush
remnant was adjacent to a potato paddock with plants that
had tested positive for phytoplasma. Symptomatic swan
plant (Gomphocarpus fruticosa) and celery (Apium grave-
olens) were received from the same home garden in May
2009 and March 2010, respectively. Symptomatic boysen-
berry (Rubus hybrid) samples were received from a
commercial grower in July and August 2010. The geo-
graphic origins of each of the samples are shown in Table 1.
Adults of Zeoliarus spp. were collected from within and
around potato paddocks during January 2010.
DNA isolation
Total plant DNA was extracted from leaf midribs and
petioles, stems or tubers using an InviMag Plant DNA Mini
Kit (Invitek, Berlin, Germany) and a KingFisher mL
workstation (Thermo Scientific, Waltham, MA, USA)
according to the manufacturers instructions.
DNA was extracted from Zeoliarus planthoppers by the
method of Goodwin et al. (1994). Individual insects were
macerated in 150 μL extraction buffer (2% CTAB, 1.4 M
NaCl, 1% PVP-40, 0.02 M EDTA and 0.1 M TrisHCl, pH
8.0) in a microfuge tube using a microtube pestle. The tube
was briefly vortexed and then incubated at 65°C for 5 min.
The suspension was extracted once with an equal volume of
chloroform:isoamyl alcohol (24:1 v/v), the nucleic acids
were ethanol precipitated and finally resuspended in 30 μL
of sterile distilled water.
PCR amplification, cloning and sequencing
Initial screening of samples for phytoplasma infection was
performed using the TaqMan real-time PCR assay of
Christensen et al. (2004). PCR reactions were set up in a
volume of 20 μL containing a final concentration of 1 ×
LightCycler 480 Probes Master (Roche Diagnostics, Man-
nheim, Germany), 300 nM of each primer, 120 nM of the
TaqMan probe, 0.4 mg/mL bovine serum albumin Fraction
V (BSA) and 2 μL of DNA template. Real-time PCR
cycling was performed on a Rotorgene 3000 real-time
rotary analyser (Qiagen, Valencia, CA, USA) or a CFX96
real-time PCR detection system (Bio-Rad, Hercules, CA,
USA). Cycling conditions were: an initial cycle at 95°C for
5 min, then 40 or 50 cycles of 95°C for 10 s and 60°C for
45 s. Real-time PCR results were analysed with the
manufacturers software.
For sequence analysis of the 16S rRNA gene,
conventional PCR was performed with the primer pairs,
P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995),
R16F2/R16R2 (Lee et al. 1995) and NGF/NGR (Andersen
et al. 1998a) in both direct and nested-PCR. The tuf gene
was amplified using the fTufAY/rTufAY primer pair
(Schneider et al. 1997). All conventional PCR reactions
were performed in a 20 μL volume containing a final
concentration of 1×GoTaq Green Master Mix (Promega,
Madison, WI, USA), 250 nM of each primer, 0.5 mg/mL
BSA and 2 μL of DNA template. Cycling conditions were:
an initial cycle at 94°C for 5 min, then 40 cycles of 94°C
New hosts of Candidatus Phytoplasma australiense239
for 30 s, 53°C (P1/P7), 50°C (R16F2/R16R2), 54°C (NGF/
NGR) or 52°C (fTufAY/rTufAY) for 30 s, and 72°C for
1 min, with a final extension at 72°C for 10 min.
Amplifications were performed in a GeneAmp PCR System
9700 (Applied Biosystems, Foster City, CA, USA). The
amplified DNA was analysed by agarose gel electrophoresis
and bands were visualised with SYBR® Safe (Invitrogen,
Carlsbad, CA, USA).
Conventional PCR products were either gel purified
using the Quantum Prep Freeze N Squeeze DNA Gel
Extraction Spin Columns (Bio-Rad) or using the QIAquick
PCR Purification Kit (Qiagen). The purified products were
either sequenced directly or cloned into the pCR 4-TOPO
vector (Invitrogen) followed by transformation into One
Shot TOP10 chemically competent Escherichia coli accord-
ing to the manufacturers instructions. Plasmid DNA was
purified using the QIAprep Spin Miniprep Kit (Qiagen).
The DNA fragments were sequenced in both directions
using the M13 forward and reverse primers for cloned
inserts or the relevant PCR primer for direct sequencing of
PCR products. DNA was sequenced by EcoGene (Auck-
land, New Zealand) on an ABI Avant 3100 Genetic
analyser using BigDye 3.2 chemistry.
Sequence analysis
The sequences were assembled and edited using Geneious
Pro (Drummond et al. 2010). Searches of the GenBank
database for homologous sequences were performed using
the BLASTn network service available at the National
Centre for Biotechnology Information (Bethesda, MD,
USA).
Host Geographical origin Site Sample no. Tuf variant group
Potato Te Kauwhata, Waikato A 1 IX
Potato Te Kauwhata, Waikato A 2 VII
Jerusalem cherry Te Kauwhata, Waikato A 3 IX
Zeoliarus Te Kauwhata, Waikato A 4 VII
Zeoliarus Te Kauwhata, Waikato A 5 IX
Zeoliarus Te Kauwhata, Waikato A 6 IX
Potato Te Kauwhata, Waikato B 1 VII
Potato Te Kauwhata, Waikato B 2 VII
Potato Te Kauwhata, Waikato B 3 VII+IX
Potato Te Kauwhata, Waikato B 4 VII
Potato Te Kauwhata, Waikato B 5 IX
Potato Te Kauwhata, Waikato B 6 VII
Potato Te Kauwhata, Waikato B 7 IX
Potato Te Kauwhata, Waikato B 8 VII
Potato Te Kauwhata, Waikato B 9 IX
Potato Matamata, Waikato A 1 VII
Potato Matamata, Waikato B 1 VII
Potato Matamata, Waikato C 1 VII
Potato Manawatu, Wanganui A 1 VII
Potato Manawatu, Wanganui A 2 VII
Potato Waituna, Wanganui A 1 VII
Potato Kiwitea, Wanganui A 1 VII
Potato Dannyvirke, Hawkes Bay A 1 VII
Potato Norsewood, Hawkes Bay A 1 VII
Potato Norsewood, Hawkes Bay A 2 VII
Potato Ashley Clinton, Hawkes Bay A 1 VII
Swan plant Clevedon, Auckland A 1 VII
Celery Clevedon, Auckland A 1 VII+ IX
Boysenberry Whakatane, Bay of Plenty A 1 VII
Boysenberry Whakatane, Bay of Plenty A 2 VII
Boysenberry Whakatane, Bay of Plenty A 3 VII
Boysenberry Whakatane, Bay of Plenty A 4 VII
Table 1 New hosts and isolates
of Candidatus Phytoplasma
australienseshowing their geo-
graphical origin and tuf variant
group
240 L.W. Liefting et al.
Results and discussion
Description of symptomatic plants
Jerusalem cherry (Christmas/Winter cherry) belongs to the
family Solanaceae and is a common weed in New Zealand
and Australia. In other countries, cultivated dwarf varieties
of Jerusalem cherry are decoratively grown as house or
garden plants. During a site visit to a phytoplasma-infected
potato field in the Waikato region, a symptomatic Jerusalem
cherry plant was observed on the edge of a native bush
remnant. Symptoms included witchesbroom, foliar yel-
lowing and reduced leaf size (Fig. 1a). Numerous Jerusalem
cherry plants were present in the bush remnant, however
only one was symptomatic. The only known host of Ca.P.
australiensein the bush remnant was cabbage tree (Cordy-
line australis) which appeared asymptomatic.
Swan plant (milkweed) is commonly grown in New
Zealand home gardens to attract the monarch butterfly
because it is the preferred food of the larva. All six swan
plants growing in a home garden in Auckland exhibited
symptoms of abnormal foliar yellowing and slight upward
rolling of the leaves (Fig. 1b) that resulted eventually in
plant death.
Fig. 1 Symptoms associated
with infection by Candidatus
Phytoplasma australiense.(a)
Witchesbroom, foliar yellow-
ing and reduced leaf size in a
Jerusalem cherry shoot on the
right compared to a healthy
shoot on the left. (b) Leaf
yellowing in swan plant. (c)
Pink discolouration of celery
foliage. (d) Yellowing of celery
foliage. (e) Leaf distortion as
well as pink and yellow disco-
louration of the leaf margins in
celery. (f) Overall chlorosis and
reduced leaf size in boysenberry
with foliage from neighbouring
healthy plants on either side. (g)
Bronzing of older boysenberry
leaves
New hosts of Candidatus Phytoplasma australiense241
Celery plants growing in the same home garden in
Auckland that had symptomatic swan plants were observed
to be showing unusual symptoms of pink and yellow
foliage and leaf deformation (Fig. 1c, d and e). As far as we
are aware, these symptoms have not yet been observed by
commercial celery growers in New Zealand.
In the early 1980s a new disease of boysenberry called
Boysenberry declineseriously affected boysenberry pro-
duction in New Zealand (Wood et al. 1999). Symptoms of
Boysenberry decline include witchesbroom and deformed
flowers which fail to develop fruit. With the identification of
the fungus Cercosporella rubi as the causal agent of
Boysenberry decline, this disease is now successfully
controlled by fungicide treatment (Langford et al. 2003).
Boysenberry plants exhibiting symptoms different from
those of Boysenberry decline were observed by a commer-
cial grower in the Bay of Plenty region in October 2009. The
symptoms become obvious close to flowering when the
lateral branches become stunted. Young leaves are smaller
than normal and chlorotic (Fig. 1fg). As the disease
progresses, the older leaves become purple-bronze in colour,
particularly towards the margin (Fig. 1g). The fruit set as
usual but remain small. New canes fail to grow and the plant
is dead by the following winter. Approximately 1% of the
plants in the orchard were showing these symptoms.
Phytoplasma detection and identification
The symptomatic plants, Jerusalem cherry, swan plant,
celery and boysenberry, produced positive real-time PCR
results (data not shown). The primers and probe used in the
real-time PCR assay of Christensen et al. (2004) detect all
phytoplasma species and isolates. In order to identify the
phytoplasma present in these samples, a 1.2 kb region of
the 16S rRNA gene was sequenced directly. This 1.2 kb
fragment was amplified by conventional nested-PCR using
the P1/P7 primers as the first stage PCR followed by the
primers R16F2/R16R2 as the second stage PCR. In order to
achieve sequence coverage in both directions, the amplicon
produced by the NGF/NGR primer pair with either P1/P7
or R16F2/R16R2 as the first stage PCR primers was also
sequenced. BLAST analysis of the 1.2 kb region of the 16S
rRNA gene amplified from symptomatic Jerusalem cherry,
swan plant, celery and boysenberry showed 100% identity
to Ca. P. australiense16S rRNA gene sequences in the
GenBank database.
As far as we are aware this is the first report of a
phytoplasma infecting Jerusalem cherry. Although Jerusa-
lem cherry is classified as a weed in New Zealand, its status
as a host of Ca. P. australienseis an important discovery
in order to understand the epidemiology of this phyto-
plasma and to help devise management strategies in
commercial crops.
Several different phytoplasmas have been detected in
swan plant and its close relative, the balloon cottonbush
(Gomphocarpus physocarpus). In Australia, Ca.P.austral-
iense(16SrXII-B), Tomato big bud (16SrII-E) and Sweet
potato little leaf strain V4 (16SrII-D) were all detected in
balloon cottonbush (Streten et al. 2005). Phytoplasmas
belonging to subgroups 16SrI-B (Aster yellows), 16SrIII-B
(Clover yellow edge) and 16SrXII-A (Stolbur) were detected
in swan plants in Italy (Bertaccini et al. 2006;dAquilio et al.
2002). A subgroup 16SrI-B phytoplasma was also found
infecting swan plant in Iran (Babaie et al. 2007)andGriffiths
et al. (1994) characterised a subgroup 16SrIII-B phytoplasma
of this host plant in the United States.
This paper reports for the first time celery as a host of
Ca. P. australiense, although other phytoplasmas affect
this crop worldwide, including the closely related phyto-
plasma, Stolbur (16SrXII-A), that causes a severe epidemic
of celery in Italy (Carraro et al. 2008). Aster yellows
phytoplasma (16SrI-B) affects celery crops commonly in
North America and Europe (Lee et al. 2000). Geographi-
cally closer, Tran-Nguyen et al. (2003) detected Tomato big
bud phytoplasma (16SrII-E) in celery in Australia.
Although boysenberry is strictly not a new host of Ca.P.
australiense, here we report a new phytoplasma association
in plants exhibiting symptoms that differ from Boysenberry
decline. During a study to determine the cause of Boysen-
berry decline in the late 1990s, 4 out of 20 symptomatic
boysenberry plants tested positive for Ca. P. australiense
(Wood et al. 1999). It was concluded that the fungus C. rubi
causes Boysenberry decline, however the possibility that Ca.
P. australiensemay be involved in the disease was not totally
ruled out. These recent results suggest that Ca. P. austral-
ienseis associated with a disease different than Boysenberry
decline. Perhaps the symptoms associated with the phyto-
plasma were previously being masked by the more dominant
Boysenberry decline symptoms (M. Andersen, personal
communication). Further work is required to demonstrate
association between the new symptoms and the phytoplasma.
Other phytoplasmas that have been recorded in Rubus spp.
include Rubus stunt (16SrV) in the UK, North America and
Europe (Lee et al. 2000), Aster yellows (16SrI) in the UK
(Reeder et al. 2010) and Pakistan (Fahmeed et al. 2009), a
group 16SrIII phytoplasma in the UK (Davies 2000), and
Black raspberry witches-broom phytoplasma (16SrIII-Q) in
North America (Davis et al. 2001). The relationship between
the group 16SrIII phytoplasmas detected in Rubus spp. in the
UK and North America is unknown as the 16S rRNA gene
sequence is available for only one of them.
Tuf gene sequence analysis
Partial sequence (~800840 bp) of the tuf gene of Ca.P.
australienseamplified from the single symptomatic sam-
242 L.W. Liefting et al.
ples of Jerusalem cherry, swan plant and celery and from 22
potato and 4 boysenberry isolates were determined (Table 1).
These samples were determined previously to be phyto-
plasma positive by PCR amplification of the 16S rRNA
gene. Twenty-two of these sequences were identical to tuf
variant VII and five sequences were identical to tuf variant
IX of Andersen et al. (2006). Interestingly, these two tuf
variants belong to distinct subgroups: tuf clade 1 (includes
variants VIII and IX) isolates originate from both New
Zealand and Australia whereas tuf clade 2 (includes variants
I to VII) consists exclusively of isolates from New Zealand
(Andersen et al. 2006). None of the isolates in this study
belonged to tuf variants I to VI and VIII and no new variants
were discovered.
The sequences of the PCR products from celery and
from one of the potato isolates indicated that these hosts
contained a mixed phytoplasma population due to the
presence of ambiguous bases. The tuf gene PCR amplicon
from these two samples were cloned and the sequence was
determined from 10 resulting clones. Sequence analysis of
the clones from celery revealed that 7 were identical to
variant VII and 3 were identical to variant IX, and for
potato, 2 clones were of variant VII and 8 were of variant
IX, thereby confirming the presence of two tuf gene types
in these samples (Table 1). Andersen et al. (2006) did not
encounter mixed infections of tuf clade 1 and tuf clade 2
subgroups of Ca. P. australiensealthough this phenom-
enon has been reported for other phytoplasma species. For
example, Seemüller et al. (2010) determined that a single
apple tree could be infected with one, two or three distinct
strains of Ca. P. mali(Apple proliferation phytoplasma).
Furthermore, these researchers suggest that multiple infec-
tions may be of pathological significance due to the finding
that the severity of the symptoms induced by Apple
proliferation phytoplasma varies depending on the number
and types of strains present. To date, we have not observed
any differences in symptoms between different tuf variants
and single or double infections.
Different tuf variants were present in plants growing in
the same potato paddock as indicated at sites A and B from
Te Kauwhata in the Waikato region (Table 1). At site A, the
phytoplasma isolate in one potato plant was variant VII and
the other plant was variant IX. The tuf gene was also
sequenced from the phytoplasma amplified from three
individual Zeoliarus planthoppers collected from site A
(Table 1) and were of either variant VII or IX. These
planthoppers were collected from a small strip of vegetation
between the potato plots that consisted of cabbage trees,
native flax and weeds (mainly bindweed). The cabbage
trees and flax were not showing obvious phytoplasma
disease symptoms but it is possible that the weeds could
harbour the phytoplasma which is then spread into the
potato crop by the Zeoliarus planthopper. In this paper we
have shown that the Jerusalem cherry weed is a host of the
phytoplasma and is present in native bush remnants, a
common feature next to potato paddocks in New Zealand.
Transmission of the phytoplasma into potato by Zeoliarus
oppositus has not been demonstrated experimentally but
these planthoppers were observed on the potato foliage and
in one instance exhibited mating behaviour.
In this study, the most abundant subgroup of Ca.P.
australiensewas tuf variant VII with 23 out of 30 isolates
being of this type (77%). The remaining seven isolates were
of tuf variant IX (23%), with the final two isolates being
mixed infections of these two types. As with the work by
Andersen et al. (2006) there was no obvious correlation
between variants and host or geographic location. The
original work by Andersen et al. (2006)ontuf subgroups
also recorded variants VII (46%) and IX (22%) as the most
common subgroups occurring in all four plant hosts known
at that time with variants I to VI being reported only from
naturally growing stands of Phormium spp.
Conclusion
Between January 2009 and July 2010, four new hosts (potato,
Jerusalem cherry, swan plant and celery) of Ca.P.
australiensehave been identified in New Zealand, as well
as a new disease association in boysenberry. These are the
first new hosts of Ca.P.australienseto be recognised in
New Zealand since Coprosma in 1998 (Beever et al. 2004),
although they may have gone unnoticed for several years.
Potato was the first of this new series of phytoplasma hosts
to be recognised, not long after the detection of Candidatus
Liberibacter solanacearumin this crop (Liefting et al.
2008). The outbreak of the new liberibacter disease may
have resulted in closer vigilance of the potato crops leading
to the detection of the phytoplasma. The second host,
Jerusalem cherry, was noticed by chance during an inspec-
tion of a phytoplasma-infected potato paddock. The other
two new hosts, swan plant and celery, were observed by a
scientist involved in liberibacter research.
The economic impact that Ca. P. australiensewill have
on these newly identified hosts is potentially significant.
Already, phytoplasma-infected potato tubers have failed
quality control checks at the processing factory. In
boysenberry, fruit from infected plants are unmarketable
and the plants die within a year from when the symptoms
are first noticeable. Although there have been no reports of
the phytoplasma in commercially grown celery, the dis-
colouration of the foliage that the phytoplasma induces in
this crop would also render it unmarketable.
The polyphagous feeding behaviour of Zeolarius oppo-
situs, the insect responsible for transmitting the phyto-
plasma into Cordyline and Coprosma, suggests that it may
New hosts of Candidatus Phytoplasma australiense243
also be moving the phytoplasma into the new hosts
described here. Z. oppositus is especially abundant in
grasses and sedges that commonly grow around crops in
New Zealand. These hosts may act as symptomless
reservoirs of the phytoplasma and along with weed hosts
such as Jerusalem cherry, play an important role in the
spread of the phytoplasma. The diversity of the new hosts
described in this paper emphasises the potential that Ca.P.
australiensehas to spread into additional native plants and
horticultural crops.
Acknowledgements We thank Dr Ross Beever and Mr Mark
Andersen for valuable discussions and Mr Craig Julian for the
photographs and descriptions of symptomatic boysenberry.
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  • Article
    Full-text available
    Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.
  • Chapter
    Grapevine yellows diseases occur in most viticultural regions worldwide and they are associated with at least 24 different phytoplasmas. Their epidemiology is often different, and it can be strongly linked to the environment, particularly to factors such as the presence of alternative plant hosts and the biology of the insect vector (s). Sometimes the epidemiology of grapevine yellows diseases associated with the phytoplasmas also differs at the regional level. Therefore, it is important to understand every aspect of the disease biology and epidemiology, so that specific management practices can be designed to reduce the risk of the spread of grapevine phytoplasmas and the associated disease. In this chapter, an overview of the biology and epidemiology of the different grapevine yellows diseases is presented.
  • Article
    Medicinal and aromatic plants include a broad array of wild and cultivated plants which contain many biologically- active compounds, known as phytochemicals, that are of great interest for their ability to promote human and animal health. The present review provides a literature overview of phytoplasma diseases affecting medicinal and aromatic plants, with an emphasis on phytoplasma taxa associated. An overview of studies that examined the effect of phytoplasma infections on phytochemical content and other secondary metabolites of affected plants is also included. Phytoplasma diseases of medicinal and aromatic plants occur worldwide; however, the majority of reports are from Europe and southeastern Asian countries. These diseases affect plant species belonging to over 70 families, mostly to Apiaceae and Asteraceae. They differ considerably in geographic distribution and size of the various taxonomic groups and subgroups of the associated phytoplasmas. Subgroup 16SrI-B phytoplasmas are the prevalent agents occurring mainly in Europe, North America and Asia. Phytoplasma presence induces changes in the amount and composition of secondary metabolites in diseased plants in which, however, the concentrations of valuable phytochemicals are greatly affected. An exception is represented by phytoplasma diseases of periwinkle in which an accumulation of pharmaceutically important compounds occurs upon phytoplasma infections. Prospects for future research are presented and critically discussed.
  • Article
    Full-text available
    A complete review of the records of plant virus, viroid, liberibacter and phytoplasma in New Zealand has found evidence for 220 viruses, seven viroids, two liberibacters and two phytoplasmas. Of these, 80 viruses, one viroid and two species of liberibacter have been reported as new to New Zealand since the last review in 2006. Ten viruses and two viroids, which were previously placed in the unconfirmed category, have now been confirmed. Based on insufficient evidence, 25 virus, three viroid, three mollicute and 36 disease records are considered unconfirmed.
  • Article
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  • Article
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  • Article
    Sunshine trees (Senna surattensis) exhibiting unusual stem fasciation symptoms were observed in Yunnan, China. Morphological abnormalities of the affected plants included enlargement and flattening of stems and excessive proliferation of shoots. An electron microscopic investigation revealed presence of single membrane bound mycoplasma-like bodies in sieve elements of symptomatic plants. With DNA templates extracted from diseased plants and phytoplasma universal primers P1/P7 and P1A/R16S-SR, nested polymerase chain reactions produced amplicons of 1.5 kb. Subsequent restriction fragment polymorphism and nucleotide sequence analyses of the amplicons indicated that the diseased plants were infected by distinct phytoplasmas affiliated with two phylogenetically distant taxa classified in two 16Sr groups (16SrXII and 16SrV). This is the first report that sunshine tree is a natural host of two evolutionarily divergent phytoplasmas and the first report that a ‘Candidatus Phytoplasma australiense’-related strain is present in China. The findings signal a significant expansion of both geographical distribution and host range of 16SrXII and 16SrV phytoplasmas.
  • Article
    Asclepias physocarpa (sin. Gomphocarpus physocarpus; Asclepiadaceae) is a perennial ornamental plant, mainly distributed in tropical and subtropical areas and it is reported to be infected by a few viruses, such as tobacco streak and tomato spotted wilt viruses. In August 2002, plants one and two years old showing severe stunting, associated with rosette-like symptoms were observed; in other plants symptoms of yellows and vein yellowing were also present. High percentages (up to 30-40%) of diseased plants were present in many fields in Imperia area (Liguria, North-Western Italy). Mechanical inoculations on herbaceous plants, 'leaf-dip' preparations for electron microscopy, and DAS-ELISA tests gave negative results for virus presence. Molecular analyses (PCR/RFLP) were performed on nucleic acid extracted from phloem tissue collected from symptomatic and asymptomatic plants in November, and provided evidence of phytoplasma presence in the majority of samples examined. RFLP on 16S ribosomal gene indicated that 16SrI-B (aster yellows), sometimes in mixed infection with 16SrXII-A (stolbur) phytoplasmas, and 16SrIII-B (clover yellow edge) phytoplasmas were present in symptomatic material; in asymptomatic plants stolbur phytoplasmas were identified. This disease is seriously affecting A. physocarpa cultivations and it is likely that the plants become infected during cultivation cycles since leafhopper presence was quite spread in all the fields examined. It was not possible to attribute a certain phytoplasma or mixture of phytoplasmas to the different symptoms observed, except for stolbur phytoplasmas detected in single infection only in asymptomatic plants. Probably, the type of symptoms could be influenced by the plant stage at the moment of infection: i.e., infected young plants show stunting and resetting, while those infected at older stages (2 years) only react with yellows to the presence of the same pathogen/s.
  • Article
    Full-text available
    The trivial name 'phytoplasma' has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus 'Candidatus (Ca.) Phytoplasma'. Given the diversity within 'Ca. Phytoplasma', several subtaxa are needed to accommodate organisms that share < 97-5% similarity among their 16S rRNA gene sequences. This report describes the properties of 'Ca. Phytoplasma', a taxon that includes the species 'Ca. Phytoplasma aurantifolia' (the prokaryote associated with witches'-broom disease of small-fruited acid lime), 'Ca. Phytoplasma australiense' (associated with Australian grapevine yellows), 'Ca. Phytoplasma fraxini' (associated with ash yellows), 'Ca. Phytoplasma japonicum' (associated with Japanese hydrangea phyllody), 'Ca. Phytoplasma brasiliense' (associated with hibiscus witches'-broom in Brazil), 'Ca. Phytoplasma castaneae' (associated with chestnut witches'-broom in Korea), 'Ca. Phytoplasma asteris' (associated with aster yellows), 'Ca. Phytoplasma mali' (associated with apple proliferation), 'Ca. Phytoplasma phoenicium' (associated with almond lethal disease), 'Ca. Phytoplasma trifolii' (associated with clover proliferation), 'Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), 'Ca. Phytoplasma ziziphi' (associated with jujube witches'-broom), 'Ca. Phytoplasma oryzae' (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence doree, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97.5% similar to those of other 'Ca. Phytoplasma' species, are characterized by distinctive biological, phytopathological and genetic properties. These include 'Ca. Phytoplasma pyri' (associated with pear decline), 'Ca. Phytoplasma prunorum' (associated with European stone fruit yellows), 'Ca. Phytoplasma spartii' (associated with spartium witches'-broom), 'Ca. Phytoplasma rhamni' (associated with buckthorn witches'-broom), 'Ca. Phytoplasma allocasuarinae' (associated with allocasuarina yellows), 'Ca. Phytoplasma ulmi' (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being < 97-5% similar to that of any other 'Ca. Phytoplasma' species, are not presently described as Candidatus species, due to their poor overall characterization.
  • Chapter
    This chapter presents a phytoplasma taxonomic scheme that is based on the analysis of two evolutionary markers: the 16S ribosomal ribonucleic acid (rRNA) gene and the spacer region that separates the 16S from the 23S rRNA genes. This chapter provides an outline of the procedures that are available for phylogenetically classifying an unknown phytoplasma strain. Information presented in this chapter includes phytoplasma phylogenetic relationships based on restriction fragment length polymorphisms (RFLP) analysis and sequence analysis of the 16/23S spacer regions. Phylogenetic analyses have provided a coherent framework for the classification of diverse taxa, including the Mollicutes. Another very attractive aspect of this type of analysis is the ability to analyze these phylogenetic markers from non-culturable prokaryotes, such as plant pathogenic mycoplasma-like organisms (MLOs). There are advantages and disadvantages associated with both RFLP and sequence analysis of rDNA for classifying phytoplasmas. RFLP analysis of the polymerase chain reaction (PCR)-amplified 16S rRNA gene is a rapid method to assess the potential affinity of an unknown phytoplasma.
  • Article
    Full-text available
    Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as `Candidatus Phytoplasma australiense'.
  • Article
    Plants of Rubus occidentalis (black raspberry) 'Munger' exhibiting symptoms of black raspberry witches'-broom (BRWB) disease were observed in commercial fields in Oregon (1). Symptoms were often severe, leading to death of infected plants, and a phytoplasma (mycoplasmalike bodies) was observed in ultrathin sections of diseased plants (1). In the current work, the association of phytoplasma with BRWB was assessed using the polymerase chain reaction (PCR) for specific amplification of phytoplasmal rDNA. DNA template for use in the PCR was extracted from plants as described elsewhere (2). Phytoplasmal 16S rDNA was amplified from diseased black raspberry plants in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) by a method described previously (2). These results indicated the presence of a phytoplasma, designated BRWB phytoplasma, in the diseased plants. Identification of BRWB phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2. Phytoplasma classification was done according to the system of Lee et al. (3). On the basis of collective RFLP patterns of the amplified 16S rDNA, the BRWB phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of BRWB 16S rDNA differed from that of its close relative, clover yellow edge (CYE) phytoplasma. The RsaI RFLP pattern of BRWB rDNA differed from that of rDNA from all phytoplasmas previously described in group III. Based on these results, BRWB phytoplasma was classified in a new subgroup, designated subgroup Q (III-Q) within group III. The 1.8 kbp DNA product of PCR primed by primer pair P1/P7 was cloned and its nucleotide sequence determined. The sequence was deposited in GenBank under Accession no. AF302841. Results from putative restriction site analysis of the cloned and sequenced rDNA were in excellent agreement with the results from enzymatic RFLP analysis of uncloned rDNA amplified from BRWB diseased black raspberry. Sequence similarity between the 1.8 kbp rDNA of BRWB phytoplasma and that of CYE phytoplasma was 99.4%. The nucleotide sequence data support the conclusion that the BRWB phytoplasma is related to, but is distinct from, other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and provide information to aid the diagnosis of BRWB disease. References: (1) R. H. Converse et al. Plant Dis. 66:949, 1982. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.