America is the center of origin of cacti plants being Mexico a rich region
in their biodiversity. In the last years stems and fruits are used as nutritional
source but its demand as ornamental plants has been grown substantially. Recently,
an increase in field diseases has been observed and a number of new varieties
with unusual anatomical characteristics are offered to cacti collectors in national
nurseries. These cacti plants show abnormal growth with excessive proliferation
of buds, yellow stems, yellow mosaics and purple colorations (Fig.
1). Some of these syndromes have been previously associated to phytoplasmas
by present group in scientific meetings (Leyva-López
et al., 1999).
Phytoplasmas are nonculturable degenerate gram-positive prokaryotes related
with mycoplasmas and spiroplasmas. Lee et al. (2000)
have classified these pathogens in 14 main groups according to restriction enzyme
patterns of their 16SrRNA genomic operons (16SrI-XIV). These pathogenic agents
are usually transmitted by insect vectors and are associated to more than 700
diseases worldwide (Weintraub and Beanland, 2006; Bertaccini,
Phytoplasmas are associated to a high number of plant diseases in Mexico, being
the so-called potato purple top the most notorious due to its financially impact
on the Mexican potato industry. Present studies led to conclude that two different
phytoplasmas were involved in the potato purple top syndrome (Leyva-López
et al., 2002). One of them was identified as potato hair sprouts
phytoplasma and is a member of the group 16SrII, being this parasite the first
one found in Mexico, since all the reported members have been described affecting
plants is Asia. Recently, Lee et al. (2006) reported
the presence of another member of this group that might be the same phytoplasma
reported in 2002.
The potato hair sprouts phytoplasma was found infecting cacti growing as weed
or barriers in the potato fields as well (Leyva-López
et al., 2002). Very few phytoplasmas associated to cacti species
have been described so it was of interest to investigate its presence in other
It is important to emphasize that these pathogens confer unusual characteristics
that make cacti plants attractive in commercial nurseries. Objectives
of this study included the detection and molecular analysis of any phytoplasma
related to the observed syndromes affecting ornamental cacti being commercialized
in Mexican nurseries.
||Cacti displaying phytoplasma-associated symptoms. Panel (A)
Opuntia sp. showing phyllody, (B) Opuntia sp. showing witches
broom, (C) Opuntia sp. showing cladode mosaics
MATERIALS AND METHODS
DNA isolation: Cacti samples of affected plants were collected from
plant nurseries located in the Mexican States of Guanajuato and Colima during
2006-2007. Fresh tissue in active growth was collected and used for DNA isolation
and processing using the method reported by Lopes and Larkins
PCR assays: Universal primer pairs were used for the amplification of
the 16SrRNA gene and the 16S/23S spacer region. The universal phytoplasma primers
P1 (5-aagagtttgatcctggctcaggatt-3) and tint (5-tcaggcgtgtgctctaaccagc-3)
were used to amplify the 16S rRNA and tRNA operon region in a first PCR (Smart
et al., 1996). PCR was performed in a 25 μL total volume of
reaction, containing 100 ng of total DNA, 10 pmol of each primer, 1x of buffer
solution for PCR, 2 mM MgCl2, 200 mm of dNTPs and 2.5 units of Taq
DNA polymerase (PROMEGA Corp. Madison WI). PCR rounds were 3 min to 90°C
followed by 30 cycles of three steps: 1 min to 90°C, 1 min at 55°C and
1 min at 37°C. Products obtained in the first amplification were used to
made 1:20 dilutions and 1 μL was taken from each dilution like DNA template
to make the second amplification for samples that could have low titer of phytoplasmas,
where the pair of internal initiators R16F2 (5-catgcaagtcgaacgga-3)/R16R2
(5-tgacgggcggtgtgtacaaaccccg-3) (Lee et al.,
1994) were used with the conditions of reaction mentioned previously. Products
were visualized in agarose 1% gels. PCR amplification was conducted in an automated
thermocycler (PT-100, MJ Research, San Francisco CA, USA).
Cloning and nucleotide sequencing: RFLP-PCR and DNA sequences
analysis were used for molecular characterization. PCR products were digested
with Kpn I, Hinf I and Alu I (Promega Corporation,
Madison, WI, USA) and visualized in agarose 1.5% gels for enzymatic restriction
analysis. Intact amplified products were purified with Wizard PCR preps
Kit and inserted into pGEM-T vector Easy Vector System II (Promega, Madison,
WI) later used to transform Escherichia coli DH5alfa cells. Plasmids
containing the expected sizes were chosen for automatic sequencing using
an ABI PRISM 377 PERKIN-ELMER DNA sequencer.
Sequence alignment and phylogenetic analysis: Restriction site maps
of the 16S rRNA gene of the phytoplasmas found here were generated using the
CLC Free Workbench 4.5.1 program for the Macintosh operating system (Cambridge,
MA, USA). DNA sequences were compared to each other and with the existing sequences
at the GenBank database using the same software and the NCBI Blast program (Zhang
et al., 2000).
RESULTS AND DISCUSSION
PCR assays using the P1/Tint pair allowed the amplification of a 1650
bp fragment in a first reaction, followed by the R16F2/R16R2 pair that
yielded the a 1250 bp amplified fragment. PCR analysis demonstrated that
cacti plants are important phytoplasma hosts because they maintain a high
titre apparently (data not shown).
||Dendrogram based on sequence analysis showing the cacti phytoplasma
sequences presented in this report
These plants may be important natural hosts for the phytoplasmas to be dispersed
by insects to other plants of agronomic importance (Weintraub
and Beanland, 2006).
RFLP analysis of the amplified fragments revealed the presence of a single
phytoplasma group. Present results showed the presence of single phytoplasma
DNA in each screened plant and no mixed RFLPs patterns were found in any case.
Different titres of phytoplasmas were observed as suggested by Firrao
et al. (2007) and Marzachi and Bosco (2005).
Restriction analysis of amplified fragments showed a clear difference between
two clones. DNA sequencing of the R16F2/R16R2 amplified region yielded two different
sequences belonging to the 16SrII group according to their RFLP patterns (Wei
et al., 2007). 16S RNA RFLP comparison between Opuntia sp.
Mosaic-inducing phytoplasma and Christmas Cactus Witches broom phytoplasma
shows that both maps are identical, but the comparison of nucleotide sequences
indicated that they are different phytoplasmas (Fig. 2).
DNA sequence analysis indicated that two of the phytoplasmas described
here have not been reported previously at the GenBank database. As result
of this work the 16S rDNA sequences of the phytoplasmas associated with
cacti were registered as Opuntia sp. Mosaic-inducing phytoplasma
(accession No. DQ535899) and Echinopsis sp. yellow patch phytoplasma
(accession No. DQ535900).
An important profit in this study is the identification of phytoplasmas
associated to two types of syndromes of cacti plants becoming in the first
report of phytoplasmas of the 16SrII group present in the Americas. Their
presence in Mexico is difficult to explain but it could be speculated
that these phytoplasmas arrived to our country via., ornamental plants
imported from Asia, where this group (16SrII) has been repeatedly reported.
Thus, the phytoplasmas named here as Opuntia sp. mosaic-inducing
phytoplasma (DQ535899) and Echinopsis sp. yellow patch phytoplasma
(DQ535900) belong to the 16SrII group. It is remarkable to emphasize that
cacti species that are alternating hosts for phytoplasma are common in
commercial nurseries and they have a great demand because of the unusual
characteristics that make them more attractive may involve involuntary
risk of dispersion of diseases to crops of agronomic importance.
KAP and FPC have Ph. D scholarships from Consejo Nacional de Ciencia
y Tecnología (CONACYT), Mexico. We also wish to thank to Consejo
de Ciencia y Tecnología del Estado de Guanajuato (CONCYTEG) for
providing the grant Fitoplasmas de importancia agrícola presentes
en viveros del Estado de Guanajuato code 08-03-k662-076. We are grateful
with Verónica Sánchez-Briseño, Maribel Moreno-Valenzuela
and Claudia Castro-Transviña for their technical support.