J Gen Plant Pathol (2015) 81:166–168
DOI 10.1007/s10327-014-0571-x
DISEASE NOTE
Rickettsia-related bacteria associated with papaya plants showing
bunchy top disease in Cuba
M. Luis-Pantoja • P. L. Ramos-González •
M. Naranjo • L. Hernández-Rodrı́guez •
J. Rodrı́guez • E. Pérez-López
Received: 7 April 2014 / Accepted: 3 September 2014 / Published online: 14 December 2014
Ó The Phytopathological Society of Japan and Springer Japan 2014
Abstract Papaya bunchy top (PBT) disease has been
associated with Rickettsia-related proteobacterium. To
study the occurrence of this proteobacterium in Cuba,
during 2012, we collected 264 samples from asymptomatic
and symptomatic papaya trees in three regions of Cuba.
Symptomatic tissue evaluated by transmission electron
microscopy revealed Rickettsia-like morphology. PCR
using rickettsial sdhA-specific primers amplified a single
band with expected size from 95 % of symptomatic plants.
Phylogeny confirmed the presence of Rickettsia-related
proteobacterium in plant showing PBT disease. This work
confirms Rickettsia-related proteobacterium is a general-
ized pathogen in papaya plant affected by bunchy top
disease in Cuba.
Keywords Bacteria
PBT
Phytopathology

Non-cultivable
Papaya (Carica papaya L.) is extensively cultivated in
Cuba to consume as fresh or processed fruits (Alonso et al.
2009). Systemic diseases caused by viruses, bacteria and
phytoplasmas are the main constraint on papaya yields in
the American continent (Teixeira et al. 2007). In Cuba,
papaya is mainly affected by a nonviral disease termed
M. Luis-Pantoja
P. L. Ramos-González
M. Naranjo

L. Hernández-Rodrı́guez
J. Rodrı́guez
E. Pérez-López (&)
Research Institute on Tropical Fruit Crops,
7th Ave., # 3005, Playa, P.O. Box 11 300, Havana, Cuba
e-mail: edellopez1987@gmail.com
E. Pérez-López
Instituto de Biotecnologı́a y Ecologı́a Aplicada (INBIOTECA),
Universidad Veracruzana, Avenida de las Culturas Veracruzanas
No 101, Colonia Emiliano Zapata, CP 91090 Xalapa, Veracruz,
Mexico
123
bunchy top symptom (BTS), with symptoms similar to
those of Papaya bunchy top (PBT) disease (Arocha et al.
2005; Davis et al. 1996). Two phytoplasmas, Candidatus
Phytoplasma aurantifolia group16SrII, and Candidatus
Phytoplasma caricae group 16SrXVII have been identified
in BTS-showing plants and in its most likely vector, Em-
poasca spp. (Arocha et al. 2005, 2006, 2009).
The causal agent of PBT-affected plants in Florida has
been correlated with the invariable presence of a fastidious,
laticifereous-inhabiting, rod-shaped, small Gram-negative
bacterium member of the a-subdivision of the Proteobac-
teria, genus Rickettsia (Davis 1998). Specific PBT symp-
toms include diffuse chlorosis; rigid, downward cupping of
leaves; necrosis; reduced expansion of leaf blades and
elongation of internodes; hard petioles; discrete, water-
soaked spots; and defoliation have been reported for PBT
disease. Also, flowering and fruit set are rare in plants with
PBT, but when fruits are produced, they are small and
deformed (Davis et al. 1996).
Based on PCR evidence, Rickettsia-related bacteria was
detected in some PBT symptomatic plants from western
Cuba during 2003 (Arocha et al. 2003). However, Rick-
ettsia was not detected in extensive surveys beyond the
same region from November 2005 to June 2006 (Arocha
et al. 2007).
During 2012, we collected 13 samples from asymp-
tomatic and 251 samples from PBT-diseased papaya trees
in commercial areas in the western, central and eastern
regions of the country to confirm the association of Rick-
ettsia-related proteobacterium with bunchy top diseased
plants. PBT symptoms included the entire range of typical
symptoms. Also we took into account the ‘‘frog skin’’ in
the base of the petioles and floral set abortion (Fig. 1a–d).
We used molecular and morphology evidence to identify
the pathogen.
J Gen Plant Pathol (2015) 81:166–168
Fig. 1 Symptoms caused by Rickettsia-related bacteria in PBT
diseased papaya and pathogen morphology. a–d Natural symptoms.
a Reduction of internode length. b Absence of latex efflux. c Frog
skin at base of petioles. d Chlorotic leaves with interveinal mosaic.
e, f TEM of morphology of pathogen in laticifer cells. e Collapsed
To examine plant tissues for Rickettsia-related bacte-
ria, tissues were fixed in 4 % (v/v) glutaraldehyde in
0.1 M sodium phosphate buffer pH 7.2, washed with
buffer, dehydrated in a graded ethanol series, and
embedded in Spurr’s resin (Spurr 1969). Thin sections
(50 nm) were cut using an Ultratome 2128 microtome
(LKB). Tissue sections were contrast-stained as described
by Reynolds (1963) and examined in a JEOL 2000 EX
transmission electron microscope at 25 kV.
In the four samples from symptomatic plants examined
by transmission electron microscopy (TEM), the laticifer
cells were collapsed, and rod-shaped bacteria were pres-
ent in the cytoplasm of laticifer cells (Fig. 1e). The bac-
teria measured *0.3–0.5 lm long and 0.09–0.2 lm wide
and contained electron-lucent inclusion bodies. These
bacterial cells were delimited by a double membrane with
a periplasmic space between the cell wall and the cyto-
plasm (Fig. 1f).
For the molecular identification, DNA was extracted
from 0.5 g of leaf midribs of papaya plants collected in
every region of the country (Murray and Thompson 1980)
and used as a template for the PCR assay. Specific primers
that target rickettsial sdhA PBTF1/PBTR1 (Davis 1998)
were used. PCR products were separated using 1 % (w/v)
agarose gel electrophoresis, and a band of the expected size
(*750 bp), obtained from 239 of the symptomatic plants
167
laticifer cells with rod-shaped bacteria (bar 500 nm). f Bacteria
delimited by a double membrane with a periplasmic space (Ps)
between cell wall and cytoplasm, and electron-lucent inclusion
bodies (Ib) (bar 200 nm)
(95 %), was then purified (Wizard SV Gel and PCR Clean-
Up System, Promega, Madison, USA) and cloned (pGEMT-
Easy Vector, Promega). Two individual clones per region of
the country (six total samples) were sequenced by Macro-
gene, South Korea. The PBTF1/PBTR1 sequences were
trimmed, assembled into a consensus using the Staden
Package (Bonfield and Whitwham 2010) and compared
with reference sequences from GenBank using the BLAST
program (http://www.ncbi.nlm.nih.gov).
The PBTF1/PBTR1 sequences of the rickettsial sdhA
detected in the symptom-bearing papaya plants were
100 % identical. The consensus sequence (705 nt) of the
rickettsial sdhA (GenBank accession FN825675) showed
the highest sequence identity (100 %) with the rickettsial
sdhA (AF018287) and rickettsial sdhA (U76909) reported
by Davis (1998) in Puerto Rico.
PBT disease etiology has been under study for some
time, with virus and phytoplasma among the candidates.
Attempts to isolate this bacterium in axenic culture have
been unsuccessful, impeding verification of its pathoge-
nicity (Davis 1991; Davis et al. 1996).
The use of electron microscopy, PCR with specific
primers, cloning, sequencing and alignment with the
reference rickettsial sdhA sequence permitted us to
demonstrate that Rickettsia-relative bacteria are strongly
correlated with typical PBT disease in Cuba.
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Acknowledgments This research was supported by project 0578
financed by the Cuban Fruit Group (Grupo Empresarial Frutı́cola,
GEF). E. Pérez-López thanks CONACYT for a scholarship (CVU:
517835).
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