New hosts of 16SrI phytoplasma group associated with edible Opuntia ficus-indica crop and its pests in Mexico

Abstract

In Mexico in the region of Nopaltepec in Mexico State, the edible Cactus crop, Opuntia ficus-indica is mainly cultivated for prickly pear fruit production. This crop has problems with common pests (insects, mollusks and weeds) which may serve as reservoirs and together with the named Cactus with phytoplasma-like symptoms which through the time inhibit the fruit production, and for this reason, the farmers called these Opuntia plants as Planta Macho (male plant). For molecular identification of the probably involved phytoplasma during 2005 and 2006, 38 samples of Opuntia plant tissues, fruit and some pests were collected for DNA extraction. By direct and nested PCR, 16S rRNA gene was amplified and sequenced. PCR products were analyzed by RFLP with restriction enzymes and in the sequences restriction sites were mapped. Phylogenetic analysis showed that the same phytoplasma was associated with Opuntia crop and its pests (the weeds Argemone mexicana, a grass and Lupinus sp.; a chinch bugs, Chelinidea sp. and the brown garden snail, Helix aspersa). Thus the edible Cactus crop and pests represented novel hosts of Cactus male plant Phytoplasma, and was classified as 16SrI Aster yellows group, of the species Candidatus phytoplasma asteris. This is the first report of this phytoplasma in Mexico and elsewhere.

Full text

African Journal of Microbiology Research Vol. 5(5) pp. 910-918, 18 April, 2011
Available online http://www.academicjournals.org/AJMR
ISSN 1996-0808 ©2011 Academic Journals
Full Length Research Paper
New hosts of 16SrI phytoplasma group associated with
edible Opuntia ficus-indica crop and its pests in Mexico
Leopold Fucikovsky Zak*, María de Jesús Yáñez-Morales*, Iobana Alanis-Martínez and
Enrique González-Pérez
Colegio de Postgraduados, Campus Montecillo, Fitosanidad, Mexico State, Mexico.
Accepted 1 March, 2011
In Mexico in the region of Nopaltepec in Mexico State, the edible Cactus crop, Opuntia ficus-indica is
mainly cultivated for prickly pear fruit production. This crop has problems with common pests (insects,
mollusks and weeds) which may serve as reservoirs and together with the named Cactus with
phytoplasma-like symptoms which through the time inhibit the fruit production, and for this reason, the
farmers called these Opuntia plants as Planta Macho (male plant). For molecular identification of the
probably involved phytoplasma during 2005 and 2006, 38 samples of Opuntia plant tissues, fruit and
some pests were collected for DNA extraction. By direct and nested PCR, 16S rRNA gene was amplified
and sequenced. PCR products were analyzed by RFLP with restriction enzymes and in the sequences
restriction sites were mapped. Phylogenetic analysis showed that the same phytoplasma was
associated with Opuntia crop and its pests (the weeds Argemone mexicana, a grass and Lupinus sp.; a
chinch bugs, Chelinidea sp. and the brown garden snail, Helix aspersa). Thus the edible Cactus crop
and pests represented novel hosts of Cactus male plant Phytoplasma, and was classified as 16SrI Aster
yellows group, of the species Candidatus phytoplasma asteris. This is the first report of this
phytoplasma in Mexico and elsewhere.
Key words: Argemone, brown garden snail, chinch bugs, edible Cactus, grasses, Lupinus, male plant
phytoplasma, Mexico.
INTRODUCTION
In the region of the great Mexican pyramids in the
Teotihuacan Valley, especially near the community of
Nopaltepec inside the municipality of Nopaltepec and
whose ubication is in the northeast region of Mexico
State (Lat. 19º28´20´´ to 19º52´24´´, Long. 98º38´20´´ to
98º46´45´´, at 2300 m asl and weather classified as Bs1k;
and which means a semiarid and temperate climate, with
rain season during the months of June until October)
(SEDUV, 2003), exist approximately 20,000 ha of
cultivated and edible Cactus, Opuntia ficus-indica (L.)
Mill. (Cactaceae family).
This crop is grown mostly for its sweet and refreshing
fruits called “tunas” (prickly pear) that are consumed
locally and a great quantity is exported to United States
of America during the months of August and September
*Corresponding authors. E-mail: fucikovs@colpos.mx,
yanezmj@colpos.mx. Tel: + 55-595-95-20200.
each year. The young cladodes free of spines are also
consumed locally in different, very tasty preparations.
Approximately 15,000 families depend on this plant in this
area. There are many larger or smaller areas in Mexico
where this Cactus is grown for the same purpose.
It has been estimated (Fucikovsky and Yáñez, 2006)
that about 60% of the plants of six years of age and on,
do not produce any fruits, or the fruits are very small
(Figure 1a) and without taste, born frequently on the flat
part of the cladode surface (Figure 1b) instead on the
crest. Many cladodes increase in thickness, compared to
normal ones (Figure 1c) on the same plant. Plants grow
slow and some may be very stunted (Figure 1d). Many
plants turn yellowish without production and occupy an
unproductive space (Figure 1e). Besides, the affected
plants have young or older cladodes in form of a heart
(Figure 1f) (Fucikovsky and Yáñez, 2006). The incubation
time of the disease and the appearance of the symptoms
is unknown, although it was observed in the field that
detached diseased cladodes with symptoms give rise to

Zak et al. 911
Figure 1. Associated Cactus male plant phytoplasma symptoms on edible O. ficus-indica crop. Panel shows: (a)
atrophied fruits (left), (b) fruits on the flat part of the cladodes surface, (c) cladodes increase in thickness (from center to
the left), (d) stunted plant, (e) plants turn yellowish and without fruits, (f) cladodes in form of a heart.
new ones with thick or a heart shape formation in few
months.
Besides the above problem, among the Opuntia crop
there are common pests (such as insects, molluscs and
weeds). Some of them are Argemone mexicana L.
(Papaveraceae), Ipomoea sp. (Convolvulaceae), a grass
(Gramineae) and Lupinus sp. (Leguminosae) (Sánchez-
Sánchez, 1980); plus chinch bugs, Chelinidea sp.
(Hemiptera: Coreidae) and the brown garden snail, Helix
aspersa Müller (Gastropoda) (Nobel, 2002; Hernández-
Gutiérrez, 1993; Pimienta, 1990) which cause problems.
Anteriorly worldwide, ornamental cacti were studied
with similar symptoms and phytoplasma was associated.
Some of these were ornamentals such as Opuntia

912 Afr. J. Microbiol. Res.
monacantha in Lebanon (Choueiri et al., 2005); and
Opuntia sp. and Zygocactus truncatus in China (Cai et
al., 2007, 2008), and on Cactus in USA (Hodgetts et al.,
2008). Also in Mexico phytoplasmas were associated
with ornamental cacti such as Echinopsis subdenudata
and Opuntia sp. (Leyva-Lopez et al., 1999; Avina-Padilla
et al., 2009). Furthermore on O. ficus-indica crop,
phytoplasma causing disease was reported in Italy
(Granata et al., 2006); and also in Argentina, Chile and
South Africa (Granata et al., 2006) and in USA
(Bertaccini et al., 2007).
In Mexico phytoplasma-like symptoms on Opuntia crop
has been described by several authors (Hernández-
Gutiérrez, 1993; Nobel, 2002; Pimienta, 1990).
This phytoplasma problem (Fucikovsky and Yáñez,
2006; Hernández-Pérez et al., 2009a, 2009b) has
preoccupied the farmers for many years and for this
reason, this work was done in order to determine the
presence of some microorganism, possibly phytoplasma,
in these sterile plants, where the farmers gave it the
name Planta Macho (male plant), which indicates that it
will not produce practically any fruit when the symptoms
are advanced. About the above pest, in addition weeds,
sucking insects and snails were also analyzed for
phytoplasma detection. We hypothesize that these
organism-pests are related to the male plant disease on
the edible Cactus crop.
MATERIALS AND METHODS
Collection of samples
In the Opuntia crop, during 2005 and 2006, 38 samples from
healthy and diseased looking plants plus some samples of common
pests were collected in the area of Nopaltepec as follows: 15
samples were of cladodes, spines, and pear fruits (seed with the
pulp, and peel); 18 samples of weeds (representing four genera),
plus three snails and two winged chinch bugs (green and brown).
All the samples were preserved at -85°C before laboratory
processing.
DNA extraction
From each sample, DNA was extracted from 0.3 g of previously
lyophilized tissues and the protocol of Ahrens and Seemüller (1992)
was followed. The DNA quality was verified by electrophoresis on a
1% agarose gel (Ultrapure, Gibco, USA) using TBE buffer, stained,
and visualized in a transilluminator (Gel Doc 2000, BIO RAD®,
USA). The concentration was quantified in a Perking-Elmer
spectrophotometer (Lambda BIO 10, USA).
PCR amplification
With DNA of each sample, direct PCR followed by nested PCR
were conducted. At first PCR universal primers were used, P1 (5`-
AAG AGT TTG ATC CTG GCT CAG GAT T-3`) and Tint (5`-TCA
GGC GTG TGC TCT AAC CAG C-3`) for amplification of the 16S
rRNA gene and the 16S-23S spacer region (Smart et al., 1996).
The reaction mixture was 80 ng of DNA, 20 pmols of each primer
and a PCR bead (PuRe Taq Ready To-Go, PCR Beads; Amersham
Biosciences) in 17 µL of ultrapure sterile water. For nested PCR,
the primers were R16F2n (5`-GAA ACG ACT GCT AAG ACT GG-
3`) and R16R2 (5`-TGA CGG GCG GTG TGT ACA AAC CCC G-3`)
(Lee et al., 1993). The reaction mixture was 1 µL from the dilution
1:30 (1 µL of the direct PCR product in 30 µL of ultrapure sterile
water), 20 pmols of each primer and a PCR bead in 20 µL of
ultrapure sterile water. The positive control was DNA from
Catharanthus roseus with Aster yellows phytoplasma and the
negative control was ultrapure sterile water.
The PCR amplification was done using a programmable
thermocycler (Gene Amp. PCR System mod. 2400, Perkin-Elmer®,
USA) with a cycle of initial denaturalization at 94°C for 1 min; 35
cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2
min and an extension at 72°C for 3 min; and a final extension cycle
at 72°C for 10 min (White et al., 1990). The DNA quality was
verified by electrophoresis on a 1% agarose gel (Ultrapure, Gibco,
USA) using TBE buffer, stained, and visualized in a transilluminator
(Gel Doc 2000, BIO RAD®, USA). The concentration was quantified
in a Perking-Elmer spectrophotometer (Lambda BIO 10, USA). The
PCR fragments were visualized as mentioned above. The marker 1
Kb, DNA ladder was used.
RFLP analyses
Twelve of the nested PCR products with the primers
R16F2n/R16R2 were digested with the following restriction
enzymes: AluI, HhaI, HpaII, KpnI and MseI. The restriction products
were separated in 8% acrylamid gels and stained with etidium
bromide. The DNA patterns of RFLP obtained were compared with
those already reported (Lee et al., 1993, 1998). The same above
DNA positive control and øX174 RFI DNA HaeIII digest marker
were used.
Restriction sites map
With four of the restriction endonucleases (AluI, HhaI, KpnI and
MseI) and five representative sequences, two from this study and
tree of the 16SrI phytoplasma group aligned, restriction sites were
determined by MapDraw of the DNASTAR program (DNASTAR,
Inc.).
DNA sequencing
All the PCR products were cleaned with PCR purification QIAquick
kit (Qiagen, USA) before sequencing. The sequence was in two
directions with primers R16F2 and R16R2 in a sequenciator ABI
PRISM 3700 (Applied Biosystems, USA). Later the sequences were
deposited in the NCBI GenBank.
Sequence similarity
The sequences were analysed by the Lasergene 2001, V. 5
Software (DNASTAR Inc., USA) with the profile mode of one pair by
Martinez-NW and multiple aligment by Clustal W Methods. All the
sequences were aligned and also the most related sequences were
obtained by Blasting in the GenBank data base.
Phylogenetic analyses
Two evolutionary trees were constructed with 43 sequences (35
download from the NCBI GenBank plus 10 sequences from this
study). In one tree (Figure 2A) were included our 10 selected
sequences (2 from the crop and 8 from its pests) and which were

Zak et al. 913
16SrI. Periwinkle virescence P. EF050085
Cactus witches-broom P. EF190970
Chinch bug-Cactus male plant phytoplasma. HM579897
Grass-Cactus male plant phytoplasma. HM579902
Argemone-Cactus male plant phytoplasma. HM579899
Snail-Cactus male plant phytoplasma. HM579895
Chinch bug-Cactus male plant phytoplasma. HM579896
Argemone-Cactus male plant phytoplasma. HM579898
Lupinus-Cactus male plant phytoplasma. HM579901
Cactus male plant phytoplasma. HM579894
16SrI. Periwinkle little leaf P. EU375834
16SrI. Aster yellows P. AY549311
Argemone-Cactus male plant phytoplasma. HM 579900
Cactus male plant phytoplasma. HM579893
Cactus P. M.S. 2001. AF356846
16SrI. Ca. P. japonicum. AB010425
16SrII. Ca. P. aurantifolia. U15442
Echinopsis witches-broom P. EU183345
Cactus P. M.-S. 1999. AF200718
Opuntia sp. mosaic-inducing P. DQ535899
Opuntia sp. P. AY995133
P. sp. pv. mosaic-inducing. AF320575
Cactus witches-broom P. EU099573
Lebanese cactus P. AY939815
Christmas cactus witches-broom P. AY647459
Echinopsis sp. yellow patch P. DQ535900
A. abactoclasticum. NR 029167
20
17
17
29
19
45
59
85
100
98
100
60
0.02
Figure 2a. Phylogenetic trees that show the relationships among representative 16S rRNA phytoplasma strains gene sequences and
constructed by the neighbour-joining method, Anaeroplasma (A.) abactoclasticum as outgroup and numbers on the branches are
bootstraps confidence values. It shows the relationships among 10 strains from this study (all in boldface) of Cactus male plant
phytoplasma detected on edible Cactus crop (HM579893, HM579894), and its pests; the weeds Argemone mexicana (HM579898,
HM579899, HM579900), a grass (HM579902) and Lupinus sp. (HM579901); the Hemiptera-Coreidae insect, Chelinidae sp.
(HM579896, HM579897) and the molluscs, Helix aspersa (HM579895); and the reference strains of 16SrI phytoplasma group from
GenBank; which included Cactus witches-broom (EF190970) and Cactus phytoplasma (P.) Martínez (M.) Soriano (S.) 2001
(AF356846), plus the species Candidatus (Ca.) Phytoplasma japonicum (AB010425) all of them of the 16SrI group. The reference
strains of 16SrII phytoplasma group also from GenBank clustered in a different branch.
deposited in the NCBI GenBank, plus 16 sequences retrieved (9
from cacti and Opuntia, three aligned and representing one species
of the 16SrI group and one more of a second species of this same
group; and one sequence of the 16SrII phytoplasma group and two
more from Echinopsis, an ornamental Cactaceae, of this same last
group). In a second tree (Figure 2B) were included only two of our
sequences with high nucleotide fragment length, the ones above
mentioned, and 16 additional sequences from each one of 15
representative 16SrI-XV groups (IRPCM, 2004). The trees were
constructed with the neighbor-joining algorithm and the confidence
was assessed by bootstrap analysis based on 5000 strap
replications using MEGA 4.1 software (Kumar et al., 2004).
Anaeroplasma abactoclasticum (an obligately anaerobic Mollicutes)
was the out-group to root each one of the trees (GenBank

914 Afr. J. Microbiol. Res.
Christmas cactus witches-broom P. AY647459
Echinopsis sp. yellow patch P. DQ535900
Lebanese cactus P. AY939815
P. sp. pv. mosaic-inducing. AF320575
Cactus witches-broom P. EU099573
Echinopsis witches-broom P. EU183345
Opuntia sp. mosaic-inducing P. DQ535899
Opuntia sp. P. AY995133
Cactus P. M.-S. 1999. AF200718
16SrII. Ca. P. aurantifolia. U15442
16SrXV. Ca. P. brasiliense. AF147708
16SrIII. Soybean veinal necrosis P. AF177383
16SrIX. Ca. P. phoenicium. AF515637
16SrIV. Yucatan coconut l. d. P. U18753
16SrXI. Ca. P. oryzae. D12581
16SrXIV. Ca. P. cynodontis. AF248961
16SrVIII. Loofah witches-broom P. AF248956
16SrV. Ca. P. ziziphi. AF305240
16SrVI. Ca. P. trifolii. AY390261
16SrVII. Ca. P. fraxini. AF189215
16SrX. Ca. P. mali. AF248958
16SrXII. Ca. P. australiense. L76865
16SrXIII. Mexican periwinkle v. P. AF248960
16SrI. Ca. P. japonicum. AB010425
16SrI. Aster yellows P. B. AF503568
16SrI. Aster yellows P. A. AF268403
Cactus P. M.S. 2001. AF356846
16SrI. Aster yellows P. AY549311
16SrI. Periwinkle virescence P. EF050085
Argemone-Cactus male plant phytoplasma. HM579899
Cactus male plant phytoplasma. HM579893
Cactus witches-broom P. EF190970
16SrI. Aster yellows P. O. AF268405
16SrI. Periwinkle little leaf P. EU375834
A. abactoclasticum. NR 029167
54
100
89
93
52
66
69
24
20
18
29
14
15
65
93
100
100
98
99
61
61
44
100
100
41
47
0.01
Figure 2b. Phylogenetic tree that shows the relationships among representative 16S rRNA phytoplasma
strains gene sequences and constructed by the neighbour-joining method, Anaeroplasma (A.)
abactoclasticum as outgroup and numbers on the branches are bootstraps confidence values. The 34
sequences reconfirmed the phylogenetic relationship in 16SrI Aster yellows phytoplasma group of two
selected strains of this study (HM579893, HM579899) (in boldface) which were grouped in a last branch,
while the reference strains of 16SrII group were in the first branch, and the other representative reference
strains of 16SrIII-XV phytoplasma groups were also in different branches. GenBank accession numbers are
indicated. Bars, 0.01 (B) and 0.02 (A) are substitutions per nucleotide position.
Accesssion NR 029167).
RESULTS
DNA amplification
From the 38 samples analyzed, 24 of them were
phytoplasma positive. The nested PCR amplification
generated DNA fragments of 1.2 kb which were observed
by electrophoresis and corresponding to the amplification
of the 16S rRNA gene according with the DNA positive
control. No band was observed in the negative control.
The DNA of these samples were deposited in the Colegio
de Postgraduados, Fitosanidad, bacteriology laboratory.
On O. ficus-indica crop, the phytoplasma was detected

on six symptomatic samples (on a young cladode center
with cuticle and a margin with a deformed zone, transition
zone between old and young cladode, a cladode, peel
and seed with the pulp of an atrophied fruit), plus two
samples from apparently healthy plants (apex of a young
cladode with true leaves and spines, and base of a
cladode joined with an old cladode).
Related to the pests, 16 samples were phytoplasma
positive: five of A. mexicana (on small apical leaves,
young leaves, deformed green-thick petals, and young
elongated capsule with seeds), plus three samples of
apparently healthy plants (young leaves and young
capsule), on Lupinus sp. two samples (leaves of chlorotic
and stunted plant, and from apparently healthy plant), on
grass one positive sample from healthy appearing leaves
with apical part of the stem; and on the chinch bugs the
three samples were positive and also the two samples of
the brown garden snail. No phytoplasma was detected on
Convolvulaceae samples.
PCR amplification and sequence similarity
The nested PCR products amplified the 16S ribosomal
phytoplasma gene of 22 DNA samples. Twenty of them
were of 914 to 1233 bp and the other two of 441 bp
(nested PCR product from transition zone between old
and young cladode) and 454 bp (product from seed with
the pulp). The sequences from Opuntia and pests shared
99.9 and 100% of similarity index among them. Ten of
the high length nucleotide sequences (2 from the crop
and 8 from its pests) were deposited in the NCBI
GenBank (Accessions numbers HM579893, HM579894,
HM579895, HM579896, HM579897, HM579898,
HM579899, HM579900, HM579901, HM579902) and
used for the construction of trees (Figures 2A and B).
Phylogenetic analyses
A selected largest nucleotide sequence of 1233 bp
fragment length (Accsession # HM579899) from this
study blasted in the GenBank only with 100
phytoplasmas sequences belonging to 16SrI group with
99 and 100% homology. Four aligned representative
sequences of this 16SrI group were:
Aster yellows phytoplasma (AY549311), Cactus witches'-
broom phytoplasma (EF190970) (of our host family);
Periwinkle little leaf phytoplasma (EU375834) and
Periwinkle virescence phytoplasma (EF050085) (both of
two last sequences related to our DNA positive control).
All of them showed 99.6 to 100% homology with our
sequence. The phylogenetic tree (864 nucleotide portion
of fragment length) (Figure 2A) had two clusters. Cluster
one had two groups and the first group had two
subgroups.
Zak et al. 915
In one subgroup (Cluster 1), all the phytoplasma
sequences from this study (in boldface) were grouped
together; plus the four sequences from the 16SrI
phytoplasma group (AY549311, EF050085, EF190970,
EU375834) (including the sequence from the ornamental
Cactus). In the other subgroup was the sequence of an
ornamental Cactus (AF356846) with 98.2% of similarity.
In group two was the sequence of a different species
(Accession # AB010425) of this same 16SrI phytoplasma
group. In Cluster 2, all the sequences of the 16SrII
phytoplasma group were grouped (U15442). Here
sequences of ornamental cacti and Opuntia, plus the only
one of O. ficus-indica crop in Italy (AY995133) were
included.
The other phylogenetic tree constructed with 1072
nucleotide portion of fragment length (Figure 2B)
reconfirmed the finding in Figure 2A. One time more the
16SrI phytoplasma group included our sequences
(HM579893, HM579899) (boldface) and the ones from
ornamental Cactus (AF356846, EF190970); and showed
that all of them belong to the same species namely, Aster
yellows phytoplasma.
RFLP analyses
The restriction patterns with the enzyme KpnI of our
Opuntia and pests samples and the positive control of
16SrI phytoplasma group were close to the ones
previously described (Lee et al., 1993).
Restriction sites map
The map (Figure 3) showed equal restriction sites with
the three sequences of the 16SrI phytoplasma group
(Gundersen et al., 1996) and our sequences (HM579893,
HM579899). They had the same restriction site patterns
(eight with MseI, four with AluI, and two with HhaI and
KpnI).
DISCUSSION
In Mexico, by direct sequence analysis we identified on
O. ficus-indica crop and its pests the 16SrI phytoplasma
group. Worldwide there are other reports of this
phytoplasma group mainly on ornamental Cactaceae in
countries such as: China (Accession # EF190970) (Wei
et al., 2007; Cai et al., 2008), even in Mexico (Acc. #
AF356846) (IRPCM, 2004; Leyva-López et al., 1999); UK
(Aster yellows Cactus) (Lee et al., 1998) and in USA
(Cactus aster yellows) (Hodgetts et al., 2008). On
Opuntia crop, in USA a 16SrI-B phytoplasma subgroup
was identified on a fruit of O. ficus-indica (Bertaccini et
al., 2007) and this 16SrI phytoplasma group agrees with
our results.
In relation to the 16SrII phytoplasma group, in Italy,

916 Afr. J. Microbiol. Res.
Figure 3. Map of the restriction sites in 16SrI phytoplasma group of five representative sequences. HM579893 strain from
O. ficus-indica crop and HM579899 from the pest-weed Argemone mexicana; and the aligned ones (NCBI GenBank) of
the 16SrI group, EU375834 (Periwinkle little leaf phytoplasma), EF050085 (Periwinkle virescence phytoplasma) and
AY549311 (Aster yellows phytoplasma). Recognition sites were with the endonuclases AluI, HhaI, KpnI and MseI. Map
was generated by DNASTAR program and MapDraw option.
16SrII-C phytoplasma subgroup was identified on O.
ficus-indica crop (AY995133) (Granata et al., 2006) which
differs from our findings in Mexico. However, in this same
Opuntia crop and a close area to the region of our study,
a 16SrII phytoplasma group was recently reported
(Hernández-Pérez et al., 2009a, b), although no
sequence was available in the NCBI GenBank or indirect
proofs such as RFLP were showed. The authors
mentioned some Accessions numbers which belong to
other authors.
Again this 16SrII group was reported mainly on
ornamental cacti in several countries. These are China
(Acc. # AY647459; 16SrII-C, Acc. # EU099573) (Cai et
al., 2008), Lebanon (Acc. # AY939815) (Choueiri et al.,
2005), and in Mexico (Acc. # AF200718, AF320575)
(Granata et al., 2006; IRPCM, 2004), (Acc. # DQ535899)
(Avina-Padilla et al., 2009), plus one unclassified strain
(EU183345) also in Mexico. On the Opuntia crop, this
phytoplasma group appears to affect the crop fertility
gradually, through time (Fucikovsky and Yáñez, 2006),
however apparently it is unknown if the fertility on
ornamental Cactaceae is also affected.
All these mentioned sequences of both 16SrI and
16SrII groups were in agreement with our phylogenetic
100 200 300 400 500 600 700 800 900 1000 1100 1200
bp

analyses (Figures 2A and B). The fact that in the same
host crop we report 16SrI group in Mexico and 16SrII
group in Italy should be due to the different geographic
regions as it was thought before, that each group has
specific geoubication area (Lee et al., 1998). In the cases
of two 16Sr phytoplasma groups in a same area (one
group in the crop and other in an ornamental Cactaceae),
perhaps it means that different groups are adapting in a
same area (Wei et al., 2007) or possibly because of the
phytoplasma genetic diversity (Cai et al., 2008). In fact,
two phytoplasma groups can have the same symptoms,
as reported in USA on Cactus pear (Bertaccini et al.,
2007) and in China on ornamental cacti (Cai et al., 2008).
From this study, the same 16SrI phytoplasma group
was associated with the host crop and its pests. Then
there are concerns because, even the tissues of crop and
weed plant with healthy appearance gave positive
results. This indicates that the phytoplasma is present in
both healthy appearing and disease plants. In Opuntia
crop this finding is important, because the farmers in this
region replace diseased or old plants, or plant new crop,
by using cladodes from other healthy-looking Opuntia
plants although it may harbour phytoplasma and then
dispersing the microorganism. This possibility was also
discussed in Italy on Opuntia crop management (Granata
et al., 2006). The phytoplasma on weeds also indicates
that many plants may act as a reservoir for the
phytoplasma that affects Cactus.
Besides, a surprise was encountered when the snails
and the chinch bugs resulted also positive for the same
phytoplasma. Snails have rasping tongues, feed at night
and together with the chinch bugs that are sucking
insects (Nobel, 2002), both may well transmit the
phytoplasma to the Cactus. In the case of the insect
chinch bugs, Chelinidea sp. of the Hemiptera order was
mentioned that this Hemiptera order is the most
successful of insect phytoplasma vectors (Weintraub and
Beanland, 2005). Furthermore these chinch bugs were
also found frequently on the grass which may also serve
as a reservoir of the phytoplasma.
Because of these worldwide cacti phytoplasma
diseases among ornamental and crop plantations, the
Cactaceae species biodiversity are probably at great risk.
We suggest that quarantine officials should fix rules to
avoid dispersing this disease and have in mind that some
potential vectors mentioned, may also be a problem
inside and also among countries because of the
movement of the plants.
Conclusion
In Mexico, Cactus male plant phytoplasma is associated
with the edible Cactus crop, O. ficus-indica and its pests,
and all of these represent a novel phytoplasma hosts.
This Cactus male plant phytoplasma was classified as
Aster yellows phytoplasma 16SrI group, of the species
Candidatus phytoplasma asteris. On bases of our
Zak et al. 917
knowledge, this is the first report in Mexico and
elsewhere, relating this microorganism with edible Cactus
and pests, and is an approach to understand this disease
and its possible future control.
ACKNOWLEDGMENTS
Sincere thanks are due to Group Produce, State of
Mexico, Project no. 7230, who financed the work.
REFERENCES
Ahrens U, Seemuller E (1992). Detection of plant pathogenic
mycoplasma like organisms by polimerasa chain reaction that
amplifies a sequence of the 16S rRNA gene. Phytopathol., 82: 828-
832.
Avina-Padilla K, Parra-Cota F, Ochoa-Sanchez JC, Perales-Segovia C,
Martinez-Soriano JP (2009). Phytoplasmas associated to diseases of
ornamental cacti in Mexico. J. Biol. Sci., 9: 268-271.
Bertaccini A, Calari A, Felker P (2007). Developing a method for
phytoplasma identification in Cactus pear samples from California. B.
Insectol., 60(2): 257-258.
Cai H, Chen HR, Kong BH, Yang GH, Liu T (2007). First report of a
phytoplasma associated with Christmas Cactus witches’ broom. Plant
Pathol., 56(2): 346.
Cai H, Wei W, Davis RE, Chen H, Zhao Y (2008). Genetic diversity
among phytoplasmas infecting Opuntia species: virtual RFLP
analysis identifies new subgroups in the peanut witches'-broom
phytoplasma group. Int. J. Syst. Evol. Microbiol., 58: 1448-1457.
Choueiri E, Massad R, Jreijiri F, Danet JL, Salar P, Bové JM, Foissac X
(2005). First report of a 16SrII group phytoplasma associated with
shoot proliferation of a Cactus (Opuntia monacantha) in Lebanon.
Plant Dis., 89(10): 1129-1129.
Fucikovsky ZL, Yáñez MMJ (2006). Presence of phytoplasma in edible
Cactus, insects, snails, and weeds. Proceedings of the 11th
International Conference on Plant Pathogenic Bacteria. Edinburgh,
Scotland. United Kingdom. Abstract A- p. 29.
Granata G, Paltrinieri S, Botti S, Bertaccini A (2006). Aetiology of
Opuntia ficus-indica malformations and stunting disease. Ann.
Applied. Biol., 149(3): 317-325.
Gundersen DE, Lee IM, Schaff DA, Harrison NA, Chang CJ, Davis RE,
Kingsbury DT (1996). Genomic diversity and differentiation among
phytoplasma strains in 16S rRNA groups I (Aster yellows and related
phytoplasmas) and III (X-disease and related phytoplasmas). Int. J.
Syst. Bacteriol., 46: 64-75.
Hernández-Gutiérrez L (1993). Plagas y Enfermedades del Nopal en
México. Reporte de investigación No. 11. CIESTAAM, Universidad
Autonoma Chapingo, Chapingo, Edo. de México. México, p. 52.
Hernández-Pérez R, Noa-Carrazana JC, Gaspar R, Mata P, Flores-
Estévez N (2009a). Detection of phytoplasma on indian Fig (Opuntia
ficus-indica Mill) in Mexico central region. J. Biol. Sci., 9(3): 62-66.
Hernández-Pérez R, Noa-Carrazana JC, Gaspar R, Mata P, Flores-
Estévez N (2009b). First report of symptoms associated to a
phytoplasma affecting nopal (Opuntia ficusindica Mill) in the
“Pyramids” Indian, State of Mexico. J. Gen. Mol. Virol., 1(4): 046-047.
Hodgetts J, Boonham N, Mumford R, Harrison N, Dickinson M (2008).
Phytoplasma phylogenetics based on analysis of secA and 23S rRNA
gene sequences for improved resolution of candidate species of
‘Candidatus Phytoplasma’. Int. J. Syst. Evol. Microbiol., 58: 1826-
1837.
IRPCM (2004). Phytoplasma/spiroplasma working team–phytoplasma
taxonomy group. ‘Candidatus Phytoplasma’, a taxon f or the wall-less,
non-helical prokaryotes that colonize plant phloem and insects
‘Candidatus Phytoplasma’, a taxon f or the wall -less, non-helical
prokaryotes that colonize plant phloem and insects. Int. J. Syst. Evol.
Microbiol., 54: 1243-1255.
Kumar S, Tamura K, Nei M (2004). Mega 3: Integrated software for

918 Afr. J. Microbiol. Res.
molecular evolutionary genetics analysis and sequence alignment.
Brief. Bioinform., 5: 150-163.
Lee IM, Gundersen-Rindal DE, Davis RE, Bartoszyk IM (1998). Revised
classification scheme of phytoplasma based on RFLP analyses of
16S rDNA and ribosomal protein gene sequences. Int. J. Syst.
Bacteriol., 48: 1153-1169.
Lee IM, Hammond RW, Davis RE, Gundersen DE (1993). Universal
amplification and analysis of pathogen 16S rDNA for classification
and identification of mycoplasma like organisms. Phytopathol., 83:
834-842.
Leyva-Lopez NE, Aguilar-Rojas OI, Leal-Klevezas DS, Martinez-
Soriano JP (1999). Presence of phytoplasmas in Mexican cacti.
Phytopathology, 89(6): S45.
Nobel PS (2002). Cacti: Biology and Uses. University of California
Press. California, USA, p. 280.
Pimienta-Barrios E (1990). El Nopal Tunero. Universidad de
Guadalajara. Guadalajara, Jalisco. Mexico, p. 246.
Sánchez-Sánchez O (1980). La Flora del Valle de México. Sexta
Edición. Editorial Herrero, SA. Mexico, DF. Mexico, p. 519.
SEDUV (2003). Secretaría de Desarrollo Urbano y Vivienda. Plan
municipal de desarrollo urbano de Nopaltepec, Estado de México.
México. In:
http://seduv.edomexico.gob.mx/planes_municipales/nopaltepec. Jun 30,
2010.
Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens
U, Lorenz KH, Seemuller E, Kirkpatrick BC (1996). Phytoplasma-
specific PCR primers based on sequence of the 16-23S rRNA spacer
region. Appl. Env. Microbiol., 62: 2988-2993.
Wei W, Cai H, Chen H, Davis RE, Zhao Y (2007). First report of a
natural infection of Opuntia sp. by a 'Candidatus Phytoplasma
asteris'-related phytoplasma in China. Plant Dis., 91(4): 461.
Weintraub PG, Beanland LA (2005). Insect vectors of phytoplasmas.
Annu. Rev. Entomol., 51: 91-111.
White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: A
guide to Methods and Applications. Academic Press, San Diego, CA.
USA, pp. 315-322.