Tropical Plant Pathology, vol. 36, 2, 110-115 (2011)
Copyright by the Brazilian Phytopathological Society. Printed in Brazil
www.sbfito.com.br 
SHORT COMMUNICATION / COMUNICAÇÃO
Guazuma ulmifolia (Sterculiaceae), a new natural host of 
16SrXV phytoplasma in Costa Rica
William Villalobos1, Marta Martini2, Laura Garita1, Melania Muñoz1, Ruggero Osler2 & Lisela Moreira1,3 
1Centro de Investigación en Biología Celular y Molecular, Universidad de Costa Rica, 11501-2060, San José, Costa Rica; 
2Dipartimento di Scienze Agrarie e Ambientali, Università di Udine, 33100, Udine, Italy; 3Escuela de Agronomía, Universidad 
de Costa Rica, 11501-2060, San José, Costa Rica
Author for correspondence: Lisela Moreira, e-mail: lisela.moreira@ucr.ac.cr; Marta Martini, e-mail: marta.martini@uniud.it
ABSTRACT
Guacimo trees (Guazuma ulmifolia, Sterculiaceae) showing witches’ broom symptoms (GWB), small leaves, short internodes, 
stunting and no flower and fruit production were observed on side roads and fences in different areas of Costa Rica. The occurrence of 
phytoplasma infection in GWB trees was evaluated by transmission electron microscopy (TEM), and by molecular analyses based on 16S 
rDNA: nested-PCR/RFLP, sequencing and phylogenetics. Phytoplasmas were observed only in the sieve cells of symptomatic trees by 
TEM. The infection was confirmed by nested-PCR; amplicons of about 1.2 kb were obtained from all DNA samples from symptomatic 
trees. The RFLP analysis generated patterns identical among GWB samples and showed a relationship of this phytoplasma to hibiscus 
witches’ broom group (16SrXV). The 16S rDNA sequence (1460 nt), obtained from the P1A/16S-SR semi-nested-PCR products of two 
phytoplasma strains, shared 98.8% similarity with ‘Candidatus Phytoplasma brasiliense’ (GenBank accession: AF147708). The virtual 
RFLP pattern indicated a similarity coefficient of 0.95 with 16Sr group XV-A (AF147708), suggesting that the GWB phytoplasma may 
represent a new subgroup within this group. This is the first report of a phytoplasma infecting the neotropical tree species G. ulmifolia and 
the natural occurrence of a phytoplasma strain closely related to ‘Ca. Phytoplasma brasiliense’ in Costa Rica.
Key words: ‘Ca. Phytoplasma brasiliense’, 16S rRNA, guacimo witches’-broom, nested-PCR, RFLP.
RESUMEN
Guazuma ulmifolia (Sterculiaceae), un nuevo hospedero natural de fitoplasmas del grupo 16SrXV en Costa Rica
En varias zonas de Costa Rica se observaron árboles de guácimo (Guazuma ulmifolia, Sterculiaceae) con síntomas de escoba de 
bruja (GWB), hoja pequeña, acortamiento de entrenudos, dando al árbol un aspecto general de enanismo. La infección por fitoplasmas en 
los árboles de guácimo se evaluó mediante microscopia electrónica de transmisión (TEM), análisis moleculares del 16S rDNA mediante 
PCR anidado, RFLP´s, secuenciación y filogenia. En la TEM, los fitoplasmas se observaron sólo en las células del floema de los árboles 
sintomáticos. La infección se confirmó por PCR anidada, los productos amplificados de aproximadamente 1,2 kb se obtuvieron para todas 
las muestras sintomáticas evaluadas. El análisis de RFLP generó patrones idénticos entre las muestras con GWB y mostró relación de 
este fitoplasma con el de la “escoba de bruja del hibisco” (16SrXV). La secuencia de ADNr 16S (1460 nt) de los productos obtenidos por 
PCR semi-anidado (P1A/16S-SR) de dos muestras de GWB mostraron 98,8% de similitud con “Candidatus Phytoplasma brasiliense” 
(GenBank, registro AF147708). El patrón RFLP virtual reveló 95% de similitud con el grupo 16Sr XV-A (AF147708), lo que sugiere que 
el fitoplasma GWB puede representar un nuevo subgrupo dentro del 16Sr XV. Este es el primer informe de un fitoplasma infectando a la 
especie neotropical G. ulmifolia y de la ocurrencia natural de un fitoplasma estrechamente relacionado con “Ca. Phytoplasma brasiliense” 
en Costa Rica.
Palabras-clave: ‘Ca. Phytoplasma brasiliense’, 16S rRNA, escoba de bruja del guacimo, PCR anidada, RFLP.
Guazuma ulmifolia Lam. (Sterculiaceae) is a diseases, including gastrointestinal disorders and stomach 
middle-sized tree, which can reach 20 m in height. It is aches; diabetes; malaria and syphilis (Magos et al., 2008). 
pantropical, semideciduous, heliophytic, a characteristic The phytochemical studies carried out with G. ulmifolia 
pioneer of second-growth broad-leaf forests. This species have led to the isolation of different substances, some of 
occurs naturally throughout Latin American, being found them related to antihypertensive activities (Hoer et al., 
from Mexico to the Northern Region of Argentina (Francis, 1996; Magos et al., 2008), others to in vitro antimicrobial 
1991; Tapia-Pastrana, 2007). It is commonly known as (Camporonese et al., 2003) or antiviral properties (Felipe 
“guacimo”, “guacima”, “mutamba” and other names. The et al., 2006).
fruits and foliage are eaten by domestic animals and wildlife, Recently, several trees of G. ulmifolia exhibited 
and timber is an important source of firewood in rural symptoms of witches’-broom (WB): reduced leaf size, 
areas. Additionally, this tree is widely used in neotropical short internodes and proliferation of axillary shoots (Figure 
American folk-medicine for the treatment of a variety of 1-A), as well as reduced overall size (Figure 1-B), and no 
110 Tropical Plant Pathology 36 (2) March - April 2011
Guazuma ulmifolia (Sterculiaceae), a new natural host of 16SrXV phytoplasma in Costa Rica
production of flowers and fruit. Symptomatic guacimo trees 9,9910/ -84,4160; 10,5648/ -85,1038; 10,6497 /-85,0895; 
were found beside fences and on side roads in Costa Rica 8,9507/ -83,3603). Petioles and leaf midribs were used for 
along the Pacific coast from the border with Nicaragua to analysis in the present study. DNA extracted from Sechium 
nearly the border with Panama [provinces of Guanacaste edule infected with aster yellows phytoplasma (16SrI-B) 
(La Cruz, Liberia, Cañas) and Puntarenas (Jaco, Caldera, (Villalobos et al., 2002) was used as a positive control.
Parrita, Palmar Sur)], in the Northern and South-Western Pieces of midrib and petiole (1-2 mm long) from 
areas of Alajuela Province (Upala, Atenas, Tacares) and the symptomatic and healthy guacimo leaves were fixed 
Western area of San Jose Province (Santa Ana) (Figure 1- with Karnovsky solution (Karnovsky, 1965) in 0.05 M 
C). The symptoms were reminiscent of diseases caused by cacodylate buffer and post-fixed with osmium tetraoxide 
phytoplasmas (Bertaccini, 2007); therefore, to determine the (1%) for transmission electron microscopy (TEM). The 
association of phytoplasmas with guacimo witches’-broom samples were dehydrated using an ethanol/propilen oxide 
(GWB) disease, analyses of samples based on transmission series and finally embedded and polymerized using epoxy 
electron microscopy, nested-PCR, RFLP, sequencing and resin (Spurr´s medium). A double-staining of sections was 
phylogenetics were carried out. performed with uranyl acetate and lead citrate to be observed 
Samples were collected from symptomatic guacimo with a Hitachi H-7100 electron microscope (Tokyo, Japan) 
trees in October 2009 in Costa Rica at different points at 100kV.
below 1000 masl (Latitude/ Longitude data of some DNA was extracted from leaf midribs, following in 
sampling points: 10,9720/ -85,6210; 10,0810/ -84,7840; the initial steps a protocol modified from Lee et al. (1993). 
10,0180/ -84,6000; 10,9080/ -85,0220; 9,9070/ -84,5110; Approximately 1 g of leaf midribs, frozen in liquid nitrogen, 
FIGURE 1 - A-B Witches’ 
broom symptoms observed in 
guacimo trees (Guazuma ulmifolia 
Sterculiaceae). A. proliferation of 
axillary shoots; B. short internodes 
and small leaves; C. Distribution 
map of the disease (white dots) in 
Costa Rica; D. Phloem elements 
(*) of a symptomatic guacimo tree 
showing pleomorphic phytoplasma 
bodies inside.
Tropical Plant Pathology 36 (2) March - April 2011 111
W. Villalobos et al.
was pulverized in a pre-chilled mortar. Then 7 mL of grinding were performed using PAUP version 4.0 (Swofford, 
buffer and 6 mL of ethylene glycol monomethyl ether were 2002). A. palmae was used as the outgroup. Bootstrap 
added. The samples were clarified by centrifugation using analyses (1000 replicates) were performed to estimate the 
a Sorvall rotor SS-34 at 2500 g for 5 minutes (min). The stability and support for the inferred clades. Virtual RFLP 
supernatant was collected and centrifuged at high speed analysis of 16S rDNA F2nR2 fragments was conducted 
24000 g for 30 min. The pellet was re-suspended in 2 using the iPhyclassifier program (Zhao et al., 2009) 
mL of CTAB 2% and two aliquots (about 600 µL) were available on the web site of USDA in Beltsville, MD, USA 
collected and incubated at 60ºC for 30 min, with sporadic (http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/
agitation. The next steps of DNA extraction were carried iphyclassifier.cgi).
out according to Martini et al. (2009). The extracted DNA Numerous wall-less structures, round to pleomorphic 
was quantified by Nanodrop ND1000 spectrophotometer with diameter between 200 and 500 nm, resembling 
(Thermo Scientific, Wilmington, DE, USA) and diluted in phytoplasma (Bertaccini, 2007) were only observed in the 
sterile water to obtain a concentration of about 20 ng/μL. phloem tissue of leaf midrib and petioles from symptomatic 
A nested PCR amplification was carried out to guacimo trees (Figure 1-D). The phytoplasma infection was 
confirm the presence of phytoplasmas, with universal confirmed by nested-PCR with primer pairs P1/16S-SR 
primer pairs, P1/16S-SR (Lee et al., 2004), followed followed by R16F2n/R2 showing positive results with the 
by R16F2n/R2 (Gundersen & Lee, 1996) after a 1:30 amplification of 1.2 kb DNA fragments from all the tested 
dilution of the direct PCR products. Amplifications were symptomatic samples. No amplification was obtained from 
performed with an automated thermal cycler PTC-100 asymptomatic samples tested. 
(MJ Research, Cambridge, MA, USA) in 25 µL reactions The DNA extraction protocol using ethylene glycol 
containing 200 µM of each of the four dNTPs, 0.4 µM of monomethyl ether in the initial steps permitted to obtain 
each primer, 1.5 mM MgCl2, 0.625 units of GoTaq Flexi good quality DNAs for successful PCR amplifications, 
DNA Polymerase (Promega, Madison, WI, USA) and 1µL which were not achieved using the DNeasy Plant Mini Kit 
of diluted DNA. The PCR program consisted of 38 cycles: (Qiagen, Valencia, CA, USA) and Martini et al. (2009) 
denaturation at 94°C for 1 min (2 min for the first cycle), protocol without modified initial steps. This plant species 
annealing at 55°C for 1 min, and extension at 72°C for 2 produces a gummy product during the DNA extraction 
min (10 min for the last cycle). Five µL of the amplified process and that may be the cause of unsuccessful 
products were electrophoresed through a 1% agarose amplification.
gel, stained in ethidium bromide, and visualized on a UV The actual RFLP analysis with the endonucleases 
transilluminator. AluI, HaeIII, HpaII, Tru1I, TaqI (Figure 2-A), HinfI and 
Restriction fragment length polymorphism (RFLPs) Tsp509I (data not shown) of R16F2n/R16R2 PCR products 
of nested PCR products obtained from three GWB generated patterns identical among the phytoplasmas under 
phytoplasma infected samples were analysed by single study but different from the RFLP pattern of phytoplasma 
restriction endonuclease digestion with AluI, HaeIII, HinfI infecting Sechium edule (subgroup 16SrI-B, Villalobos et 
HpaII, Tru1I, TaqI, Tsp509I (Fermentas, St. Leon-Rot, al., 2002). The RFLP results indicated that the phytoplasma 
Germany) according to the manufacturer’s instructions. The associated with GWB belonged to hibiscus witches’ broom 
restriction products were then separated by electrophoresis group (16SrXV), represented by ‘Candidatus Phytoplasma 
through 5% polyacrylamide gel stained and visualized as (Ca. P.) brasiliense’ (Montano et al., 2001b; Silva et al., 
described above. Two semi-nested PCR products obtained 2009). The 16S rDNA sequence (about 1460 nt), obtained 
with P1A/16S-SR primer pair (Lee et al., 2004) were purified from the P1A/16S-SR semi-nested-PCR products of 
using a Wizard® SV Gel and the PCR Clean-Up System Kit two phytoplasma strains (GenBank accession numbers: 
(Promega, Madison, WI, USA). Sequencing was performed HQ258882 and HQ258883), shared 98.8% similarity with 
with an automated DNA sequencer (ABI Prism Model that of the ‘Ca. P. brasiliense’ reference strain (GenBank 
3730) at the Genelab (ENEA Casaccia, Rome, Italy) using accession: AF147708). 
the primers P1A, 16S-SR and the internal primer 16S(RT) The virtual RFLP pattern derived from the query 
F1 (Martini et al., unpublished). The obtained nucleotide 16S rDNA F2nR2 fragment (data not shown) demonstrated 
sequences (about 1460 nt) were compared with those that the most similar is the reference pattern of the 16SrXV 
present in GenBank using the BLAST program (http://blast. group, subgroup A (AF147708), with a similarity coefficient 
ncbi.nlm.nih.gov/Blast.cgi). The nucleotide sequences were of 0.95, suggesting that this strain may represent a new 
deposited in GenBank. subgroup within the 16SrXV group, 16SrXV-B. The enzymes 
Both 16S rRNA gene sequences of GWB HaeIII and HpaII distinguished GWB phytoplasma from the 
phytoplasma strains along with 26 previously described and closely related ‘Ca. P. brasiliense’ (Figure 2-B). The actual 
seven incidentally cited ‘Candidatus Phytoplasma’ species, laboratory restriction digestion with the key enzyme HpaII 
and Acholeplasma palmae (ATCC 49389T) were aligned confirmed the new subgroup pattern (Figure 2-A). The 
using CLUSTAL V (Higgins & Sharp, 1989) from the phytoplasma nature of the GWB disease-associated agent 
Lasergene software MegAlign program. Cladistic analyses was further confirmed by a phylogenetic analysis of its 16S 
112 Tropical Plant Pathology 36 (2) March - April 2011
Guazuma ulmifolia (Sterculiaceae), a new natural host of 16SrXV phytoplasma in Costa Rica
FIGURE 2 - RFLP and phylogenetic analyses based on 16S rRNA gene of GWB phytoplasma strains, 16SrXV-B. A. Actual restriction 
patterns derived from digestions using enzymes: AluI, HaeIII, HpaII, TruI and TaqI. Lanes GWB #4, #7, #10: three different guazuma 
witches’ broom (GWB) phytoplasma strains; 16SrI-B: phytoplasma infecting Sechium edule; MW: Ф174 HaeIII digested; B. Virtual 
RFLP patterns derived from in silico digestions using HaeIII and HpaII restriction enzymes of GWB #4 phytoplasma strain (16SrXV-B) 
and ‘Ca. P. brasiliense’ (AF147708, 16SrXV-A) sequences. MW: Ф174 HaeIII digested; C. Phylogenetic tree constructed by parsimony 
analysis of nearly full-length 16S rRNA gene sequences from GWB phytoplasma strains and previously described and incidentally cited 
‘Candidatus Phytoplasma (Ca. P)’ species. In bold GWB phytoplasma 16S rDNA sequences obtained in this work; other sequences used 
in this study were retrieved from GenBank. 
Tropical Plant Pathology 36 (2) March - April 2011 113
W. Villalobos et al.
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(Figure 2C) clearly demonstrated that the GWB disease- Ethnopharmacology 87:103-107.
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This finding contributes knowledge about the phytoplasmas by nested-PCR assay using two universal primer 
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Previous reports of phytoplasmas in a Sterculiaceae weed of high osmolality for use in electro microscopy. Journal Cell 
species, Waltheria indica, were done in Brazil (Kitajima & Biology 27:137-138.
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different geographical origins in Costa Rica on the basis of Leaf Disease in Costa Rica. Plant Disease 83:77D.
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as ribosomal protein and secY genes. Additionally, studies associados a diversas moléstias de plantas, do grupo amarelo, no 
will be carried out to investigate epidemiological aspects estado de São Paulo. Ciência e Cultura (São Paulo) 23:285-291.
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information that is currently available does not allow us to (2011) ‘Candidatus Phytoplasma costaricanum’ a new phytoplasma 
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TPP 214 - Received 12 December 2010 - Accepted 29 April 2011
Section Editor: F. Murilo Zerbini
Tropical Plant Pathology 36 (2) March - April 2011 115