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Characterization of Thielaviopsis paradoxa Isolates from Oil Palms in

Colombia, Ecuador and Brazil

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DOI: 10.1111/jph.12012

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J Phytopathol doi: 10.1111/jph.12012
© 2012 Blackwell Verlag GmbH

Plant Pathology Program, Centro Internacional de Agricultura Tropical, Cali, Colombia

Characterization of Thielaviopsis paradoxa Isolates from Oil Palms in Colombia,

Ecuador and Brazil
Elizabeth Álvarez1, Germán A. Llano1, John B. Loke1 and Maria I. Chacon2
Authors’ addresses: 1Plant Pathology Program, Centro Internacional de Agricultura Tropical, AA 6713, Cali, Colombia;
2
Universidad Nacional, Bogotá, Colombia (correspondence to E. Álvarez. E-mail: e.alvarez@cgiar.org)
Received March 7, 2012; accepted August 11, 2012

Keywords: Elaeis guineensis, Ceratocystis spp., molecular markers, genetic diversity, Colombia, Ecuador, Brazil

Abstract biggest producer, with 50% of the world production.

Ceratocystis paradoxa (Anamorph: Thielaviopsis parad-
oxa) is parasitic on a range of economic and food
crops and is the cause of dry basal rot, a limiting dis-
ease in oil palm. The objective of this study was to
determinate the pathogenic and genetic diversity of
Thielaviopsis isolates from oil palms in Colombia,
Ecuador and Brazil. A total of 164 strains of Thielavi-
opsis paradoxa were characterized using pathogenicity
tests, random amplified polymorphic DNA (RAPD)
markers and PCR sequencing of the internal tran-
scribed spacer (ITS) region of 5.8 S ribosomal DNA.
Oil palm seedlings were inoculated by injecting the
base of stems in the seedling stage with a fungal sus-
pension and severity scores of disease reactions were
evaluated. PCR amplification of the ITS region
resulted in a 590 base pair (bp) product. Digestion of
the PCR product with two restriction enzymes pro-
duced three restriction patterns, which according to
ITS sequences could be classified as T. paradoxa. Six
RAPD primers gave polymorphic bands in T. paradox-
a. Population structure analyses of the RAPD data
suggested that most of the isolates obtained in this
study belonged to a single population. The genetic
diversity of the isolates from South America was inter-
mediate, and therefore, T. paradoxa is likely to be pre-
dominantly clonal compared with Ceratocystis species.
Sporadic sexual reproduction may occur for T. parad-
oxa but is secondary to clonal reproduction. Data on
pathogen diversity will provide information on breed-
ing strategies and population structures.
Introduction
Oil palm [Elaeis guineensis Jacq. and, to a lesser
extent, E. oleifera (H.B.K)] are the world′s most valu-
able oil crop. High yields have led to a rapidly expand-
ing worldwide industry, currently in many tropical
areas of Asia, Africa and the Americas (Corley and
Tinker 2003). To date, the tree crop covers 14.9 mil-
lion ha worldwide (FAO 2009), and Malaysia is the
Colombia is the fourth producer worldwide and the
first in the Americas (165 000 ha in production), fol-
lowed by Ecuador (135 000 ha) and Brazil (91 000 ha)
(Corley and Tinker 2003).
Thielaviopsis diseases of palm have been reported
worldwide, just approximately everywhere palms can
be grown (Garofalo and McNillan 2004). Dry basal
rot, caused by the vascular pathogen Thielaviopsis
paradoxa (Teleomorph: Ceratocystis paradoxa), can
attack the stem, leaves and fruits, causing premature
fruit drop (Kile 1993) and has the potential to devas-
tate global palm resources (Garofalo et al. 2004).
Thielaviopsis paradoxa is a soil-borne fungus widely
distributed worldwide (Al-Onazi et al. 2011) that can
be efficiently spread by nature and by man in soil and
plants. The disease is characterized by dry rot of the
bud tissue with necrotic lesions of different colours
(Buitrago and Nieto 1995). It can be economically sig-
nificant, for example, in northern Colombia where
70% of plantations are affected (Tovar and Nieto
1998). It is also economically important in the south-
ern region of Bahia state, Brazil. It affects palms of
any age and is prevalent in lowland areas where the
average annual rainfall is 2500 mm/year or above.
The genus Thielaviopsis, now synonymous with the
genus Chalara, is largely composed of asexual states of
Ceratocystis species. The sexual state of Thielaviopsis
paradoxa (de Seynes) Höhnel, namely Ceratocystis
paradoxa (Dade) Moreau (Chalara paradoxa (de Sey-
nes) Sacc.), is a weak pathogen that is restricted to
monocotyledonous plants that are stressed and plant
organs that are senescing. The sexual stage of the fun-
gus usually only invades the parenchymous tissue
rather than more densely packed cells of meristems or
bundle sheaths (Streets 1933; Kile 1993).
Isolates of T. paradoxa are difficult to differentiate
with other Thielaviopsis spp. because of similarity in
morphological characteristics. Knowledge of genetic
diversity and population structure of plant pathogens
2 Álvarez et al.
may contribute to the design of effective control mea-
sures. Molecular tools provide data sets that are inde-
pendent of morphological features that can be used to
objectively evaluate the presence of genetically diver-
gent groups of isolates within fungal populations. In
addition, molecular fingerprints can validate the signif-
icance of morphological characteristics in distinguish-
ing fungal taxa (Hausner et al. 1993). The efficiency of
management strategies for dry basal rot disease in oil
palm may be improved by the narrow diversity, both
pathogenic and genetic, found among T. paradoxa
populations in the regions.
Ribosomal genes (rDNA) consist of a mixture of
highly conserved sequences that offer a means of
assessing affinities above the species level (Kurtzman
1985) and also less conserved internal transcribed
spacer (ITS) sequences which are highly variable even
among closely related taxa (Dvorak and Appels 1982;
Hausner et al. 1993; Hirata and Takamatsu 1996). For
example, Parsimony analyses of ITS region and partial
large subunit (LSU) ribosomal sequences placed four
anamorphic Thielaviopsis species as a monophyletic
group within the teleomorph genus Ceratocystis.
Three of these species (T. ovoidea, T. thielavioides
and T. populi) were morphologically similar to each
other but distinct by rDNA sequences, and one (T. ele-
gans) was more distant (Paulin-Mahady et al. 2002).
Therefore, the rDNA repeat unit offers an ideal system
for studying relatedness at various taxonomic levels in
fungal species (Blackwell et al.1993; Geldenhuis et al.
2006).
Amplification of anonymous fragments of DNA,
such as those found by using the RAPD (Random
Amplified Polymorphic DNA) system, has several
advantages to fungal strain classification. First, RAPD
markers do not require previous knowledge of any
specific sequence and secondly the system is easy to
implement for the assessment of genetic diversity and
genetic structure in various fungal, plant or animal
populations that do not have a wide array of genetic
tools developed for them. For example, various decam-
er primers can be used for amplification of different
sets of RAPD bands which provide complementary evi-
dence for population structure. The RAPD technique,
therefore, can be used to generate a well-supported
phylogenetic tree, especially at an intraspecific level,
and the large number of bands obtained make it a
useful technique in genetic variability studies (Welsh
et al. 1991).
Our objective was to contribute to the understand-
ing of the pathogenic and genetic diversity of 164
Thielaviopsis isolates from infected tissue of oil palms
and surrounding soil samples from Colombia, Ecuador
and Brazil by means of pathogenicity tests, RAPD
marker fingerprinting and ITS molecular analyses.
Materials and Methods
Collection and isolation of fungal strains
Samples were collected in 1996 and 1997 from roots,
stems, meristem, bud tissue (under the meristem),
spear leaves, leaflets from old leaves, petiole bases of
young leaves, inflorescences, fruit peduncles and soils
near oil palms (E. guineensis) affected by dry rot dis-
ease in Colombia, Ecuador and Brazil.
Different culture media were used for isolations such
as potato dextrose agar (39 g PDA, Difco, Detroit,
MI per litre), carrot agar (200 g cooked and blended
carrot, and 20 g agar, Difco per litre) and V-8 juice
agar (200 ml V8 juice, 3 g calcium carbonate, 20 g
Difco agar per litre). The carrot agar was modified
with thiamine hydrochloride at a concentration of 300
parts per million (ppm) (Sigma, St. Louis, MO, USA).
After tissue collection, surface sterilization was per-
formed by the immersion of the previously washed
plant tissue in hypochlorite (1%) for 1 min, then in
ethanol (70%) for 1 min which was then rinsed with
water. Tissue sterilization was followed by incubation
in small polyethylene bags for 5 days. Infected tissue
fragments were then transferred to carrot agar for fun-
gal cultivation.
Isolates from soil samples were obtained by serial
dilution (10 1, 10 2 and 10 3) with sterilized distilled
water; 0.1 ml of each dilution was plated on carrot
agar and incubated at 25 °C. Where fungal growth
was detected, subsequent 0.1-ml spore dilutions were
transferred into water agar (20 g Difco agar, 1 l dis-
tilled water) plates and incubated at 25°C until isola-
tions of single germinated endoconidia were obtained
using microscopy (9100 magnification). Monoconidial
isolates were transferred to modified carrot agar med-
ium and incubated at 25°C without light. After identi-
fication, monosporic cultures were grown on 1-cm2
sterilized filter paper squares placed on the carrot agar;
filter papers of each isolate were dried at 28°C for
7 days and stored in envelopes at 4°C.
Pathogenicity testing of fungal isolates
Isolates were inoculated onto approximately 4-month-
old oil palm [E. guineensis (highly susceptible species)]
seedlings, which were at the 2–4 true-leaf stage, by
injecting the base of the seedling stem with 1 ml of a
conidial suspension (c. 1 9 106 conidia/ml) using a
sterile syringe (needle gauge 21 G 9 38 mm or
0.80 mm 9 1½″). The control plants were similarly
injected with sterile distilled water.
This injection method for testing pathogenicity cor-
responded with fungal wound infections that occur
naturally in the field. The inoculated and control
plants were incubated at 27–31°C, 80–100% relative
humidity with 12 h light per day (2200 lux measured
with a LI-1000, Li-Cor Inc., Lincoln, Nebraska, USA)
provided by six lamps (400 W, General Electrics,
Castle Hayne, NC, USA) in a growth chamber (Perci-
val Mfg.Co., Boone, IA 50036, USA) in the grounds
of the International Center for Tropical Agriculture.
For the evaluation of symptoms, the length of stem
necrosis in cm was recorded 1 month after inoculation.
Isolates (111) were arranged in a complete random
block design with three repetitions, with five inoculated
seedlings per isolate per replicate. All treatments were
Characterizing Thielaviopsis paradoxa in Oil Palms
evaluated simultaneously in two growth rooms kept
under the same conditions. Infection data were analy-
sed using SAS software, isolates were grouped accord-
ing to their pathogenicity using analysis of variance
with Duncan’s multiple range test of mean separation,
a = 5%.
Measurements of genetic diversity and population structure
Isolates were grown in a V-8 liquid medium on a
rotary shaker at room temperature in the dark for
5 days. Mycelium was harvested by filtration and
ground with liquid nitrogen. The mycelium was used
for DNA extraction following the Dellaporta method
(Dellaporta et al. 1983), modified with two additional
chloroform–isoamyl alcohol washes to remove pheno-
lic compounds and to improve DNA quality.
Random amplified polymorphic DNA reactions
used 20 ng of DNA and a set of 20 oligonucleotide
(decamer or 10-nucleotide) primers obtained from
Operon Technologies Inc., Alameda, CA, USA. The
thermal cycler was programmed for initial denatur-
ation at 94°C for 4 min, followed by 40 cycles of 45 s
at 94°C, 1 min at 36°C (annealing) and 1 min at 72°C
(extension), with a final extension of 5 min at 72°C.
PCR reactions were performed in a final total volume
of 25 ll containing 1.5 units of Taq polymerase (Pro-
mega, Madison, WI, USA), the 1X buffer supplied
with the enzyme, 2.5 mM MgCl2, 0.2 mM dNTPs and
0.5 lM primer. PCR products and amplified DNA
band were separated electrophoretically in 1.4% aga-
rose gels and detected by staining with ethidium bro-
mide. Gels were photographed after electrophoresis
using Stratagene Eagle Eye® II video system (La Jolla,
CA, USA). To study the genetic structure of the Thiel-
aviopsis isolates, RAPD polymorphisms were analysed
by means of two approaches as described below.
In the first approach to diversity analyses, no a priori
populations were assumed, and genetic relationships
among all isolates were studied by means of a hierar-
chical cluster analysis and a correspondence analysis
(CA) as implemented in the software MVSP v. 3.13
(Kovach Computing Services) and the software NTSys
v. 1.70 (Rohlf 1992). For the cluster analysis, a pairwise
genetic distance matrix based on Nei and Li’s coeffi-
cient was used, and the UPGMA algorithm was
applied. The statistical support of UPGMA clusters
was assessed by means of bootstrap analyses (1000
replicates) carried out by the Winboot (Yap and
Nelson 1996) and PAUP (Phylogenetic Analysis Using
Parsimony) (Swofford 1998).
In the second approach, populations of isolates were
defined a priori according to their geographic source.
Total and pairwise population differentiation indexes
(u, which is analogous to Sewall Wright’s fixation
indexes FST (Wright 1951) and applied to dominant
markers) were calculated via AMOVA as the proportion
of the molecular variance among populations relative
to the total molecular variance (within and between
populations) using the population genetic program
GENALEX v. 6.41 (Peakall and Smouse 2006). Statisti-
3
cal significance was tested by means of 10 000 random
permutations. Genetic diversity within populations was
calculated by Nei’s gene diversity indexes (Nei 1987)
again using GENALEX v. 6.41 (Peakall and Smouse
2006).
Molecular evaluation of the fungal isolates by sequencing and
restriction analyses
To establish the taxonomic status of the isolates
obtained from E. guineensis in this study, PCR amplifi-
cation of internal transcribed spacers of the rDNA
genes (ITS1 and ITS2) was carried out for all isolates
followed by restriction digestion. For this purpose, iso-
lates were grown for 15 days at room temperature in
25 ml of liquid culture media with 2% malt and 1%
yeast extracts. The mycelium was used for DNA
extraction following the Dellaporta method (Dellapor-
ta et al. 1983) to obtain a pure DNA for both restric-
tion analysis and sequencing.
Amplification of the ITS regions and the 5.8 S ribo-
somal gene was obtained using the ITS 4 and ITS 5
universal primers (White et al. 1990). Amplifications
were performed in a thermal cycler (PTC-100, MJ
Research Inc., Watertown, MA, USA) at an initial
denaturation of 95°C for 3 min and programmed for
24 cycles of 30 s at 95°C (denaturation), 30 s at 57°C
(annealing) and 2 min at 72°C (extension), with a final
extension of 10 min at 72°C. PCR reactions were per-
formed in a 25 ll final volume containing 0.625 units
of Taq polymerase (Promega), 1X buffer supplied with
the enzyme, 3 mM MgCl2, 0.2 mM dNTPs, 0.5 lM of
each primer (ITS 4 and ITS 5) and 5 ng of template
DNA.
Restriction profiles of the amplified DNA were gen-
erated using the endonucleases AluI and MspI (Pro-
mega). For sequencing, the non-digested PCR product
was cleaned using Quiagen’s Clean DNA Kit and sent
for sequencing at the Iowa State University’s DNA
Sequencing and Synthesis Facility. Sequences from
both DNA strands were obtained directly from PCR
products using the automated dideoxy sequencing
method (Sanger and Coulson 1975) carried out on an
Applied Biosystems Prism 377 DNA sequencer.
Sequencing reactions involved the Applied Biosystems
(Foster City, CA, USA) Prism Big Dye terminator-
cycle-sequencing kit with AmpliTaq DNA polymerase.
For sequence analysis, each individual sequence was
aligned against a Thielaviopsis spp. database managed
by the National Center for Biotechnology information
(NCBI http://www.ncbi.nlm.nih.gov) using the
SEQUENCE NAVIGATOR Program (v. 1.0.1 Perkin-Elmer
Applied Biosystems, Inc.). Phylogenetic analysis was
performed under the parsimony criterion using PAUP
(Phylogenetic Analysis Using Parsimony) (Swofford
1998).
Results
Collection of the fungal isolates
We isolated 164 strains of Thielaviopsis paradoxa from
oil palms affected by dry basal rot disease and sur-
4 Álvarez et al.
rounding soil. Isolates were collected at four different
sites in Colombia: nine isolates from Barrancabermeja
(Department of Santander); 58 isolates from the Lla-
nos region (Manavire, Manuelita, Bucarelia, Casanare,
Guaicaramo, Unipalma, Meta); 58 isolates from
Tumaco (Nariño) and one isolate from Fundación
(Magdalena) (Table 1). Twenty-eight isolates were also
obtained from one site in Ecuador (Lago Agrio,
Sushifindi) and ten isolates from one site in Brazil
(Denpasa).
The number of successful isolations varied between
sites and between plant organs especially. For exam-
ple, 35.4% of the isolates were obtained from stem tis-
sues (stipe, bud and meristem), 49.4% from leaf and
reproductive tissues (petiole base, rachis, leaflet, spear
leaf, inflorescence and peduncle), 3.0% from root and
2.4% from soil samples. The rest of the samples came
from unidentified tissues or from soils within the same
plantations as the oil palms.
Pathogenicity levels defined
Pathogenicity data were obtained from a total of 111
fungal isolates collected over the 2-year period. During
the pathogenicity testing, symptoms of the disease
were evident within a week after inoculation. All iso-
lates caused brown necrotic lesions, and in some cases,
leaf chlorosis was also observed extending along leaves
of the plant. The symptoms caused by individual iso-
lates of T. paradoxa from the different sites and coun-
tries were similar, although symptom severity differed
between isolates. Table 1 summarizes pathogenic char-
acterization evaluated across three separate experi-
ments (the highest values of each isolate from the
different experiments were used for the analysis of
pathogenicity).
The isolates were classified into four groups or levels
according to their pathogenicity on the E. guineensis,
oil palm seedlings using Duncan’s multiple range test
(a = 5%). The controls remained healthy during the
evaluation period while the T. paradoxa strains showed
high pathogenic variation between isolates. Pathoge-
nicity level - group 1 consisted of 13 highly pathogenic
isolates, which caused stem lesions that were between
50 and 73 mm in length, all of which had typical
necrotic symptoms. The second level - group consisted
of 20 intermediate pathogenicity isolates, causing stem
lesions of 27–49 mm in length. The third pathogenicity
level - group included 68 poor disease causing isolates,
which showed 3–26 mm length stem lesions; while, the
fourth pathogenicity level – group included 10 non-
pathogenic isolates which showed lesions between 0
and 2.8 mm in length.
Genetic diversity and population structure
Among the 20 RAPD primers screened, only six of
them (OPAO2, OPAO3, OPAO4, OPCO1, OPHO5
and OPN10) amplified consistent PCR products and
were reproducible across 98 of the 111 isolates. These
primers produced a total of 66 scorable bands. The
phenogram obtained by the UPGMA method and
the results of the correspondence analysis identified 5
outlier isolates: CECITH 4, CECITH 36, CECITH 37,
CECITH 40 and CECITH 95 (Figs 1 and 2a).
Apart from these outliers, isolates were very similar
genetically, and although they seemed to group into
several clusters, bootstrap analyses only supported the
presence of a single genetic cluster (P value of 87%).
After removing outliers, correspondence analyses were
performed again and the results are shown in Fig. 2b.
Variation explained by the first three axes were 22.8%,
13.6% and 9.2%, respectively.
The correspondence analyses results did not show
the presence of well-differentiated groups based on
their geographic distribution, a result that was consis-
tent with the cluster analysis where isolates from dif-
ferent geographic regions were intermingled.
Population structure was further studied, therefore,
through an AMOVA analyses (data not shown) and by
pairwise comparisons as shown in Table 2.
In the AMOVA analysis, isolates were assigned a priori
to five different populations based on geographic ori-
gin: three populations in Colombia (from the depart-
ments of Meta, Nariño and Santander), one in
Ecuador and one in Brazil. According to these analy-
ses, molecular variance of isolates showed that genetic
diversity was distributed mainly within populations
(90%) rather than between populations (10%). The
statistical significance of population differentiation
(average u value was calculated as 0.096) was tested
by 10 000 random permutations. The results suggested
that population differentiation was low but statistically
significant (P = 0.010). Pairwise u values (see Table 2)
show that populations located in different countries
were slightly more divergent genetically (u values rang-
ing from 0.071 to 0.364) than populations located
within the same country.
Within Colombia, only populations from Meta and
Santander were significantly different (u = 0.136,
P = 0.020). Gene diversity indexes (He) were calculated
only for populations from Meta and Nariño, which
contained higher and similar number of isolates
(n = 43 and 37, respectively). The values for these two
sites were very similar (He = 0.228 and 0.211, respec-
tively) which suggested that relatively high levels of
diversity are maintained in both regions. These diver-
sity values were approximately 1.5–3 times higher than
in the other sites (Santander, Ecuador and Brazil) but
this may have been an effect of the smaller number of
samples analysed from these latter regions.
Molecular taxonomy
Amplification products of rDNA sequences from the
ITS region were obtained for 121 isolates. The PCR
product size for all Thielaviopsis spp. isolates was
approximately 590 bp in length. Restriction digestions
with AluI and MspI revealed three different restriction
patterns (I, II and III). Pattern I was predominant and
was present in 116 isolates, which also showed a high
genetic similarity based on RAPD data. Pattern II was
present only in two isolates (CECITH 35 and
Characterizing Thielaviopsis paradoxa in Oil Palms 5

Table 1
Thielaviopsis paradoxa isolates from Oil Palm (Elaeis guineensis) plantations in Colombia, Ecuador and Brazil collected in 1996 and 1997

Isolate – straina Collection siteb Tissuec Pathogenicity level – groupsc,d

CECITH 1 Nariño, COL Bud 3

CECITH 2 Nariño, COL Bud 3
CECITH 3 Llanos, COL NI 3
CECITH 4 Llanos, COL NI 3
CECITH 5 Llanos, COL NI 3
CECITH 6 Llanos, COL NI 3
CECITH 7 Llanos, COL NI 1
CECITH 8 Llanos, COL NI 1
CECITH 9 Llanos, COL Stipe 2
CECITH 10 Llanos, COL Stipe 2
CECITH 11 Llanos, COL Spear Leaf 3
CECITH 12 Llanos, COL Spear Leaf 3
CECITH 13 Santander, COL Spear Leaf 3
CECITH 14 Santander, COL Spear Leaf 3
CECITH 15 Santander, COL Spear Leaf 3
CECITH 16 Santander, COL Spear Leaf 3
CECITH 17 Santander, COL Leaflet 2
CECITH 18 Santander, COL Leaflet 1
CECITH 19 Nariño, COL Bud NT
CECITH 20 Llanos, COL Bud 3
CECITH 21 Llanos, COL Bud 3
CECITH 22 Llanos, COL Leaflet 2
CECITH 23 Llanos, COL Leaflet 2
CECITH 24 Llanos, COL Leaflet 1
CECITH 25 Llanos, COL Bud 1
CECITH 26 Llanos, COL Bud 3
CECITH 27 Llanos, COL Stipe 1
CECITH 28 Llanos, COL Stipe 3
CECITH 29 Llanos, COL Stipe 1
CECITH 30 Llanos, COL Stipe 3
CECITH 31 Llanos, COL Stipe 2
CECITH 32 Llanos, COL Bud 1
CECITH 33 Llanos, COL Bud 3
CECITH 34 Sushifindi, ECU NI 3
CECITH 35 Sushifindi, ECU NI 3
CECITH 36 Sushifindi, ECU Bud 1
CECITH 37 Sushifindi, ECU Inflorescence 3
CECITH 38 Nariño, COL Petiole base 3
CECITH 39 Nariño, COL Petiole base 2
CECITH 40 Nariño, COL Bud 2
CECITH 41 Nariño, COL Bud 2
CECITH 42 Nariño, COL Bud 3
CECITH 44 Nariño, COL Petiole base 2
CECITH 45 Nariño, COL Meristem 1
CECITH 46 Nariño, COL Soil 3
CECITH 47 Nariño, COL Leaflet NT
CECITH 48 Nariño, COL NI 3
CECITH 49 Nariño, COL Bud 2
CECITH 50 Nariño, COL Rachis 3
CECITH 51 Nariño, COL Leaflet 3
CECITH 52 Nariño, COL Leaflet 3
CECITH 53 Nariño, COL Soil 2
CECITH 54 Nariño, COL Petiole base 1
CECITH 55 Nariño, COL Spear leaf 2
CECITH 56 Nariño, COL NI 3
CECITH 57 Llanos, COL Bud NT
CECITH 58 Llanos, COL Bud 2
CECITH 59 Llanos, COL Soil 3
CECITH 60 Llanos, COL Bud 3
CECITH 61 Llanos, COL Bud 3
CECITH 62 Llanos, COL Stipe 3
CECITH 63 Llanos, COL Bud 2
CECITH 64 Llanos, COL Bud 3
CECITH 65 Nariño, COL Spear leaf 1
CECITH 66 Nariño, COL Leaflet 1
CECITH 67 Nariño, COL Bud 3
CECITH 68 Nariño, COL NI 2
CECITH 69 Nariño, COL Leaflet 3
CECITH 70 Nariño, COL NI 3
CECITH 71 Nariño, COL Leaflet 3
6 Álvarez et al.

Table 1
Continued

Isolate – straina Collection siteb Tissuec Pathogenicity level – groupsc,d

CECITH 72 Santander, COL Bud NT

CECITH 72-B Santander, COL Bud 4
CECITH 72-D Santander, COL Bud 4
CECITH 73 Nariño, COL Bud 3
CECITH 73-B Nariño, COL Bud NT
CECITH 73-D Nariño, COL Bud NT
CECITH 74 Nariño, COL Rachis 3
CECITH 74-A Nariño, COL Rachis NT
CECITH 74-E Nariño, COL Rachis NT
CECITH 75 Nariño, COL Leaflet 3
CECITH 75-A Nariño, COL Leaflet NT
CECITH 75-E Nariño, COL Leaflet NT
CECITH 76 Nariño, COL Bud 3
CECITH 76-B Nariño, COL Bud NT
CECITH 76-D Nariño, COL Bud NT
CECITH 77 Nariño, COL Rachis 3
CECITH 77-A Nariño, COL Rachis NT
CECITH 78 Nariño, COL Rachis 3
CECITH 78-A Nariño, COL Rachis NT
CECITH 79 Nariño, COL Rachis NT
CECITH 79-B Nariño, COL Rachis 4
CECITH 79-D Nariño, COL Rachis 4
CECITH 80 Nariño, COL Bud 3
CECITH 80-B Nariño, COL Bud NT
CECITH 80-D Nariño, COL Bud NT
CECITH 81 Nariño, COL Bud 3
CECITH 81-A Nariño, COL Bud NT
CECITH 81-E Nariño, COL Bud NT
CECITH 82 Nariño, COL Peduncle 3
CECITH 82-A Nariño, COL Peduncle NT
CECITH 83 Sushifindi, ECU Meristem 3
CECITH 84 Sushifindi, ECU Meristem 3
CECITH 84-E Sushifindi, ECU Meristem NT
CECITH 85-B BRA Root NT
CECITH 86 Llanos,COL Leaflet 2
CECITH 86-A Llanos, COL Leaflet NT
CECITH 86-E Llanos, COL Leaflet NT
CECITH 87-A Sushifindi, ECU Meristem NT
CECITH 87-B Sushifindi, ECU Meristem NT
CECITH 87-D Sushifindi, ECU Meristem NT
CECITH 88 Sushifindi, ECU Leaflet 3
CECITH 88-B Sushifindi, ECU Leaflet NT
CECITH 88-D Sushifindi, ECU Leaflet 4
CECITH 89 Sushifindi, ECU Spear Leaf NT
CECITH 90 Llanos, COL Spear Leaf 2
CECITH 90-B Llanos, COL Spear Leaf NT
CECITH 90-D Llanos, COL Spear Leaf NT
CECITH 91 Llanos, COL Spear Leaf 3
CECITH 91-B Llanos, COL Spear Leaf NT
CECITH 91-D Llanos, COL Spear Leaf NT
CECITH 92 Sushifindi, ECU Spear Leaf 2
CECITH 93 Sushifindi, ECU Rachis 3
CECITH 94 Sushifindi, ECU Spear Leaf NT
CECITH 95 Llanos, COL Leaflet 4
CECITH 96 Sushifindi, ECU Stipe 3
CECITH 97 Sushifindi, ECU Spear Leaf 3
CECITH 97-A Sushifindi, ECU Spear Leaf NT
CECITH 97-E Sushifindi, ECU Spear Leaf NT
CECITH 98-B Nariño, COL Leaflet NT
CECITH 98-E Nariño, COL Leaflet NT
CECITH 99 Sushifindi, ECU Spear Leaf NT
CECITH 99-A Sushifindi, ECU Leaflet 4
CECITH 100 Llanos, COL Leaflet 4
CECITH 100-A Llanos, COL Leaflet 3
CECITH 100-E Llanos, COL Leaflet 3
CECITH 101 Sushifindi, ECU Spear Leaf 3
CECITH 101-A Sushifindi, ECU Spear Leaf NT
CECITH 101-E Sushifindi, ECU Spear Leaf NT
CECITH 102 Llanos, COL Leaflet 3
CECITH 102-A Llanos, COL Leaflet 3
Characterizing Thielaviopsis paradoxa in Oil Palms 7

Table 1
Continued

Isolate – straina Collection siteb Tissuec Pathogenicity level – groupsc,d

CECITH 102-E Llanos, COL Leaflet 4

CECITH 103-B Llanos, COL Leaflet NT
CECITH 103-D Llanos, COL Leaflet NT
CECITH 106 Sushifindi, ECU Meristem 3
CECITH 106-D Sushifindi, ECU Meristem NT
CECITH 107 Llanos, COL Rachis 3
CECITH 107-B Llanos, COL Rachis NT
CECITH 107-D Llanos, COL Rachis NT
CECITH 108 Nariño, COL Meristem NT
CECITH 109 Llanos, COL Leaflet NT
CECITH 110 Llanos, COL Leaflet 4
CECITH 111 BRA Root NT
CECITH 112 BRA Spear Leaf 3
CECITH 113 BRA Root 3
CECITH 114 BRA Root NT
CECITH 115 BRA Soil 3
CECITH 116 BRA Stipe 3
CECITH 117 BRA Meristem 3
CECITH 118 BRA Bud 3
CECITH 119 BRA Root 3
CECITH 120 Llanos, COL NI NT
CECITH 121 Llanos, COL NI NT
CECITH 122 Llanos, COL NI NT
CECITH 123 Magdalena, COL NI 2
a
All isolates were monosporic. Isolates were designated with a letter indicating a secondary monosporic of the similarly-named isolate.
b
Country abbreviations: BRA, Brazil; COL, Colombia; and ECU, Ecuador.
c
Other abbreviations: NI, not identifided; NT, not-tested.
d
Analysis of variance and separation of means with Duncan’s multiple range test (5%). Grouping: 1: 50–73 mm; 2: 27–49 mm; 3: 3–26 mm;
4: 0–2.8 mm; NT, not tested.

CECITH95*
CECITH004*

8 6 CECITH037*
CECITH036*
CECITH040*
CECITH002
5 CECITH018
CECITH001
CECITH017
CECITH022
CECITH015
CECITH070
CECITH023
CECITH071
CECITH013
CECITH009
CECITH014
CECITH024
CECITH069
CECITH053
CECITH051
CECITH067
CECITH052
CECITH020
CECITH003
CECITH007
CECITH055
CECITH006
CECITH008
CECITH044
CECITH72
CECITH106
CECITH042
CECITH041
CECITH039
CECITH035
CECITH034
CECITH038
CECITH005
CECITH011
CECITH063
CECITH060
CECITH010
CECITH030
CECITH031
CECITH027
CECITH033
CECITH054
CECITH064
CECITH049
CECITH014
CECITH062
CECITH061
CECITH068
CECITH058
CECITH059
CECITH92
CECITH93
CECITH94
CECITH101
CECITH102
CECITH100
CECITH108
CECITH107
CECITH99
CECITH103
CECITH109
CECITH91
CECITH97
CECITH96
CECITH80
CECITH83
CECITH82
CECITH81
CECITH87
CECITH85
CECITH84
CECITH117
CECITH119
CECITH120
CECITH86
CECITH75
CECITH74
CECITH112
CECITH76
CECITH115
CECITH118
CECITH88
CECITH89
CECITH90
CECITH79
CECITH77
CECITH78
CECITH73
CECITH113
CECITH122
CECITH116
CECITH121

0,4 0,5 0,6 0,7 0,8 0,9 1

Nei & Li’s Coefficint

Fig. 1 Phenogram from hierarchical cluster analysis of data from 98 Thielaviopsis strains. Clusters were fused using the UPGMA method
and Nei and Li’s similarity coefficient. The scale shown corresponds to the average similarity at which clusters fuse. Numbers indicate boot-
strap support values and asterisks indicate isolates identified as outliers
8 Álvarez et al.

1.5
(a)
1.0

0.5

Axis 2 –2.4 –1.9 –1.5 –1.0 –0.5 0.5 1.0 1.5

–0.5
CECITH004
–1.0

–1.5
CECITH36
CECITH40
CECITH37 –1.9
CECITH95

–2.9
Axis 1

(b) 3.01

2.41

1.80
Axis 2

1.20

0.60

–1.80 –1.20 –0.60 0.60 1.20 1.80 2.41 3.01

–0.60

–1.20

–1.80
Brazil Ecuador Llanos Santander Nariño Axis 1

Fig. 2 Correspondence Analysis of genetic similarity based on RAPD data among 98 isolates of T. paradoxa. Geographic origin of isolates is
shown. The per cent of variation explained by the first three axes is axis 1 (22.82%), axis 2 (13.55%) and axes 3 (9.15%). (a) Scatter plot with
outliers. (b) Scatter plot without outliers

Table 2 C. paradoxa from different plant species were used (see

Pairwise population differentiation indexes (upt). Significant proba- Table 3) to perform a phylogenetic analysis of rDNA
bilities are shown in bold.
sequence data. The reference isolates were obtained
Population 1a Population 2a upt Prob from Dr. Thomas Harrington, Pathology Department,
Iowa State University, USA.
Llanos, COL Nariño, COL 0.018 0.085
Santander, COL Nariño, COL 0.068 0.104
Sequencing of the ITS region was successful for 9 of
Suhifindi, ECU Nariño, COL 0.071 0.005 10 randomly selected isolates that showed restriction
Llanos, COL Santander, COL 0.136 0.020 pattern I (Table 3) and for the isolate CECITH 35,
Sushifindi, ECU Llanos, COL 0.108 0.000 which showed restriction pattern II. All of these desig-
BRA Nariño, COL 0.145 0.002
BRA Llanos, COL 0.174 0.001 nated references isolates had highly similar ITS
BRA Sushifindi, ECU 0.216 0.000 sequences (data not shown) which in turn were highly
Sushifindi, ECU Santander, COL 0.232 0.002 similar to the reference sequence for C. paradoxa iso-
BRA Santander, COL 0.364 0.002 lates from date palm and pineapple. These 10 isolates
a
Country abbreviations: BRA, Brazil; COL, Colombia; and ECU, were then classified as T. paradoxa, whose sexual stage
Ecuador. was identified as Ceratocystis paradoxa sensu stricto
based on the groupings of the dendrogram shown in
Fig. 3. The source of the restriction digest differences
was evident in the sequences of the reference strains.
CECITH 59). Pattern III was observed in the single
isolate CECITH 95, which was identified as an outlier Discussion
based on RAPD data (Figs 1 and 2a). The isolation of T. paradoxa from many different
Based on the digestion results and the RAPD data, organs of oil palm trees and from different agro-
11 isolates were selected, and 10 reference isolates of ecological zones in different countries can be considered
Characterizing Thielaviopsis paradoxa in Oil Palms 9

Table 3
Reference species used in this study and reference strains of Ceratocystis paradoxa sensu stricto as defined by the phylogenetic analysis shown
in Fig. 3

Reference isolatesa Host Location Species

C869 Phoenix dactylifera California, USA Ceratocystis radicicola

C870 Phoenix dactylifera California, USA Ceratocystis radicicola
C907 Musa sp. Unknown Ceratocystis paradoxa (?)
C914 Cocos nucifera Maranhao, BRA Ceratocystis sp.
C915 Musa AAA Belem, BRA Ceratocystis paradoxa (?)
C926 Ipomoea batatas New Jersey, USA Ceratocystis fimbriata
C1001 Ananas comosus BRA Ceratocystis paradoxa sensu stricto
C1002 Air BRA Ceratocystis paradoxa (?)
C1003 Phoenix carariensis Spain Ceratocystis paradoxa sensu stricto
C1107 Elaeis guineensis Nariño, COL Ceratocystis paradoxa sensu stricto
C953 (CECITH 61) Elaeis guineensis Llanos, COL Ceratocystis paradoxa sensu stricto
C954 (CECITH 62) Elaeis guineensis Llanos, COL Ceratocystis paradoxa sensu stricto
C956 (CECITH 54) Elaeis guineensis Nariño, COL Ceratocystis paradoxa sensu stricto
C957 (CECITH 63) Elaeis guineensis Llanos, COL Ceratocystis paradoxa sensu stricto
C959 (CECITH 56) Elaeis guineensis Nariño, COL Ceratocystis paradoxa sensu stricto
C1021 (CECITH 43) Elaeis guineensis Magdalena, COL Ceratocystis sp.(?)
C1090 (CECITH 37) Elaeis guineensis Sushifindi, ECU Ceratocystis paradoxa sensu stricto
C1091 (CECITH 35) Elaeis guineensis Sushifindi, ECU Ceratocystis paradoxa sensu stricto
C1092 (CECITH 68) Elaeis guineensis Nariño, COL Ceratocystis paradoxa sensu stricto
C1093 (CECITH 44) Elaeis guineensis Nariño, COL Ceratocystis paradoxa sensu stricto
C1094 (CECITH 72) Elaeis guineensis Santander, COL Ceratocystis paradoxa sensu strict
a
Reference isolates obtained from different plant species provided by Thomas Harrington, Pathology Department, Iowa State University,
USA.

C1090 - Sushlfindl, ECU

C1091 - Sushlfindl, ECU

C1094 - Santander, COL

C1093 - Nariño, Col

C1092 - Nariño, COL

C1003 - Spain
ITS Sequences C1001 - BRA
Ceratocystis
1 paradoxa
C954 - Llanos, COL sensu
C953 - Llanos, COL stricto
C959 - Nariño, COL

C956 - Nariño, COL

7 C957 - Llanos, COL
100
C1107 - Nariño, COL

C907 - Unknown
3 4 Ceratocystis
95 1 C915 - Belem, BRA paradoxa ??
79
2 C1002 - BRA

6 C1021 - Nariño, COL Ceratocystis

100 C914 - Maranhao, BRA sp.
54 4 C869 - California, USA Ceratocystis
100 C870 - California, USA radicicola
C926 - New Jersey, USA
Ceratocystis
fimbriata

Fig. 3 One of the four most parsimonious trees of 82 steps based on 537 aligned bases from the ITS-1, ITS-2 and 5.8 rDNA sequences of
Ceratocystis paradoxa and relatives. The tree is rooted to C. fimbriata. Numbers above branches indicate number of base substitutions, and
numbers below branches are bootstrap values. CI = 0.939, HI = 0.061, RI = 0.909.

of important for the understanding of stem rot disease
in this crop species. The fact that T. paradoxa was iso-
lated from both new and old oil palm plantation show-
ing symptoms of stem rot disease in three countries and
in very distant regions within Colombia demonstrates
how widespread the fungus is.
The reason for this wide distribution is unknown
because oil palm seeds are mostly free from T. parad-
oxa, so transmission through seedlings is low and
some other mechanism of disease inoculum dispersal
must be postulated. In this sense, the relatively fre-
quent isolation of this pathogen from different plant
tissues of oil palm trees indicated to us that dispersal
might be by airborne spores or by infected harvesting
and pruning equipment. Because old leaves of oil
palms are carefully pruned during their growth cycle,
10
this second dispersal mechanism may be common, as it
is in other crops according to Kile (1993).
Although T. paradoxa is an opportunistic pathogen
that affects susceptible host genotypes under stress,
epidemics of the disease were not observed in the culti-
vation zones of the three countries studied. The proba-
ble reason is that the sexual state is not readily found
under field conditions, even though it can be obtained
under in vitro conditions.
In terms of the diversity in pathogenicity levels
caused by the isolates, there was no correlation with
isolate origin (r = 0.08; P > 0.05), nor with tissue ori-
gin (r = 0.07). Consequently, we observed isolates of
different levels of pathogenicity, regardless of site or
tissue source. The failure of T. paradoxa to cause stem
rot in unwounded seedlings (unpublished data) sug-
gests the need for the presence of wounds for the suc-
cessful entry of the fungus.
Reports indicated that T. basicola isolated from Jap-
anese holly (Ilex crenata) shows pathogenicity on vari-
ous palm species (Wills and Lambe 1978) in addition
to T. paradoxa. Considering that T. paradoxa is widely
distributed and is found in soil samples as well as in
infected trees and seedlings, it is surprising that dry
basal rot of oil palm is not a more common disease
throughout the production zone. Therefore, it may be
likely that this fungus interacts with the palm as a sap-
rophyte and as an opportunistic pathogen (Abbas and
Abdulla 2003) when plants are damaged.
The hierarchical and multivariate analyses of RAPD
data suggested to us that, in general, T. paradoxa iso-
lates are genetically very similar across the geographic
regions studied, and all isolates may be considered to
belong to a single population. Genetic diversity could
be assessed for only the geographic regions in Colom-
bia where sample size was greater than 30, but ranged
from 0.228 to 0.211 (average = 0.22). Although these
diversity values in absolute terms may be considered
moderate, they are relatively higher than other Thiel-
aviopsis species such as T. basicola in South Africa
which is almost exclusively clonal in propagation
(He = 0.077) (Geldenhuis et al. 2006). Meanwhile, the
levels of genetic diversity in both Thielaviopsis species
are relatively lower than in the corresponding sexual
stage Ceratocystis species, such as C. fimbriata from
Colombia (He = 0.506) (Marin et al. 2003).
Because of the limited pathogenic and genetic diver-
sity existing among T. paradoxa populations in the
regions, results obtained from isolates collected during
1996 to 1997 are relevant to the current situation.
They facilitate and validate similar management strate-
gies for dry basal rot disease in oil palm in Colombia,
Brazil and Ecuador.
Five outliers (CECITH 4, CECITH 36, CECITH
37, CECITH 40 and CECITH 95) were identified on
the basis of RAPD data, which indicated that their
banding patterns differed from those of the main
group of isolates. The outliers come from different
geographic regions (Llanos and Nariño of Colombia
and Ecuador) and include different pathogenicity
Álvarez et al.
groups (groups 2, 3, 4 and 5). From the data so far,
interpreting the significance or relevance of these out-
liers in terms of disease incidence in oil palm is diffi-
cult. For example, outlier isolate CECITH 36 belongs
to the second most pathogenic group (pathogenicity
group 2), whereas the outlier isolate CECITH 95
belongs to the least pathogenic group (pathogenicity
group 5). Other outliers belong to pathogenicity
groups 3 and 4.
Although no appropriate marker is available to
study the reproductive mode in Thielaviopsis species, it
can be assumed that T. paradoxa may be predomi-
nantly clonally propagated with sporadic presence of
sexual reproduction. This hypothesis is further sup-
ported by the fact that most isolates clustered into a
single genetic group that there was a low level of dif-
ferentiation among isolates across geographic regions
in the correspondence and AMOVA analyses (Average
upt = 0.065), and that only moderate levels of genetic
diversity were found within Colombia. The lack of
geographic population structuring among isolates from
different countries may be a combined result of the
mode of reproduction in this species and cultural prac-
tices which may favour the rapid spread of the patho-
gen not only among regions within Colombia but also
among regions in different countries.
After digestion of the ITS region with the two
restriction enzymes, three restriction patterns were
observed. As stated by Hausner et al. (1993), restric-
tion analysis of rDNA is a convenient method for rap-
idly screening and grouping large numbers of isolates.
The species primarily isolated from soil and diseased
palm tissue on oil palm was T. paradoxa, with its sex-
ual stage species being C. paradoxa sensu stricto. This
species forms thick-walled, pigmented conidia (chlamy-
dospores or aleuriospores) in chains on the top of con-
idiophores, and the spore walls are smooth. Further
morphological analyses are needed to determine the
biological status of the isolate CECITH 95, which was
identified as an outlier by the cluster and correspon-
dence analyses and showed a distinct restriction pat-
tern for the ITS region. An investigation of the
association of T. paradoxa with different palm geno-
types needs to be carried out in order to evaluate resis-
tance sources and strain x genotype interactions.
Knowledge of the genetic and pathogenic diversity,
and population structure, of C. paradoxa may help
when designing effective control measures such as
breeding for resistance or biological and chemical con-
trol. From the results, recommendations can be made
for collecting and analysing contemporary isolates of
the pathogen from plants that differ for genotype,
stage of development, location and conditions of crop
management and weather.
Acknowledgements
We are grateful to COLCIENCIAS for the financial support of one
of the authors (M. I. Chacón S.) to carry out research activities at
CIAT as a Visiting Scientist under the program CIAT- COLCIEN-
CIAS Junior Researcher.
 Characterizing Thielaviopsis paradoxa in Oil Palms
References
Abbas EH, Abdulla AS. (2003) First report of neck bending disease
on date palm in Qatar. Plant Pathol 52:790.
Al-Onazi M, Al-Dahain S, El-Ansary A, Marraiki N. (2011) Isola-
tion and Characterization of Thielaviopsis paradoxa L-alanine
Dehydrogenase. Asian J Appl Sci 4:702–711.
Blackwell M, Spatafora JW, Malloch D, Taylor JW. (1993) Consid-
eration of higher taxonomic relationships involving Pyxidiophora.
In: Wingfield MJ, Seifert KA, Webber JF. (eds) Ceratocystis and
Ophiostoma Taxonomy, Ecology, and Pathogenicity. The American
Phytopathological Society, St. Paul, Minnesota, APS Press, pp 105
–108.
Buitrago VM, Nieto LE. (1995) Hongos asociados con pudriciones
de flecha y cogollo en palma de aceite (Elaeis guineensis Jacq.) en
los Llanos Orientales. Revista Palmas (Colombia) 16:9–17.
Corley RHV, Tinker PBH. (2003) The Oil Palm. 4th edn. Blackwell
Publishing Company. 562 pp.
Dellaporta SL, Wood J, Hicks JR. (1983) A plant DNA miniprepa-
ration: version II. Plant Mol Biol Rep 1:19.
Dvorak J, Appels R. (1982) Chromosome and nucleotide sequence
differentiation in genomes of polyploid Triticum species. Theor
Appl Genet 63:349–360.
FAO. (2009) FAOSTAT. Internet Resource: http://faostat.fao.org/
DesktopDefault. aspx?PageID=567&lang=en (verified Aug 30,
2010).
Garofalo JF, McNillan RT. (2004) Thielaviopsis diseases of palms.
Proc Fla State Hort Soc 117:324–325.
Geldenhuis MM, Roux J, Cilliers AJ, Wingfield BD. (2006) Clonali-
ty in South African isolates and evidence for a European origin of
the root pathogen Thielaviopsis basicola. Mycol Res 110:306–311.
Hausner G, Reid J, Klassen GR. (1993) Grouping of isolates and
species of Ceratocystis sensu lato on the basis of molecular and
morphological characters. In: Wingfield MJ, Seifert KA, Webber
JF. (eds) Ceratocystis and Ophiostoma Taxonomy, Ecology, and
Pathogenicity. The American Phytopathological Society, St. Paul,
Minnesota, APS Press, pp 93–104.
Hirata T, Takamatsu S. (1996) Nucleotide sequence diversity of
rDNA internal transcribed spacers extracted from conidia and
cleistothecia of several powdery mildew fungi. Micoscience 37:
283–288.
Kile GA. (1993) Plant diseases caused by species of Ceratocystis
sensu stricto and Chalara. In: Wingfield MJ, Seifert KA, Webber
JF. (eds) Ceratocystis and Ophiostoma Taxonomy, Ecology, and
Pathogenicity. The American Phytopathological Society, St. Paul,
Minnesota, APS Press, pp 173–183.
View publication stats
11
Kurtzman CP. (1985) Gene Manipulations in Fungi/edited by J.W.
Bennett, Linda L. Lasure. Orlando, FL, Academic Press, pp 35–
63.
Marin M, Castro B, Gaitan A, Preisig O, Wingfield BD, Wingfield
MJ. (2003) Relationships of Ceratocystis fimbriata isolates from
Colombian coffee-growing regions based on molecular data and
pathogenicity. J Phytopathol 151:395–405.
Nei M. (1987) Molecular Evolutionary Genetics. Columbia University
Press, New York, NY, 512 pp.
Paulin-Mahady AE, Harrington TC, McNew D. (2002) Phylogenetic
and taxonomic evaluation of Chalara, Chalaropsis, and Thielaviop-
sis anamorphs associated with Ceratocystis. Mycologia 94:62–72.
Peakall R, Smouse PE. (2006) GENALEX 6.41: genetic analysis in
Excel. Population genetic software for teaching and research. Mol
Ecol Notes 6:288–295.
Rohlf FJ. (1992) NYSYS-pc Numerial Taxonomy and Multivariate
Analysis Systems. Version 1.70. Exerter Publications, Setauket,
NY, 250 pp.
Sanger F, Coulson AR. (1975) A rapid method for determining
sequences in DNA by primed synthesis with DNA polymerase.
J Mol Biol 94:441.
Streets RB. (1933) Heart rot of the date palm Caused by Thielaviop-
sis paradoxa (DeSeynes) von Höhn. Ariz Agric Exp Stn Tech Bull
48:162–169.
Swofford DL. (1998) PAUP*: Phylogenetic Analysis Using Parsi-
mony (and Other Methods), 4.0 ed. Sunderland, MA, Sinauer
Associates.
Tovar JP, Nieto LE. (1998) Pudrición de estipe seca Ceratocystis
paradoxa. Ceniavances 50:1–3.
Welsh L, Honeycutt RJ, McCelland M, Sobral BWS. (1991) Parent-
age determination in maize hybrids using the arbitrary primed
polymerase chain reaction. Theor Appl Genet 82:473–476.
White TJ, Bruns T, Lee S, Taylor W. (1990) Amplifications and
direct sequencing of fungal ribosomal RNA genes for phylogenet-
ics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. (eds) PCR
Protocols: A Guide to Methods and Applications. New York,
Academic Press, Inc., pp 315–322.
Wills WH, Lambe RC. (1978) Pathogenicity of Thielaviopsis basicola
from Japanese holly (Ilex crenata) to some other host plants. Plant
Dis Rep 62:1102–1106.
Wright S. (1951) The genetical structure of populations. Annals of
Eugenics 15:323–354.
Yap I, Nelson R. (1996) A program for performing bootstrap analy-
sis of binary data to determine the confidence limits of UPGMA-
based dendrograms. International Rice research Institute, 22 pp.