Genetic variation, morphology and
pathogenicity of Ceratocystis fimbriata on
Hevea brasiliensis in Brazil
Denise C. O. F. Valdetaro, Leonardo
S. S. Oliveira, Lúcio M. S. Guimarães,
Thomas C. Harrington, Maria
A. Ferreira, et al.
Tropical Plant Pathology
e-ISSN 1983-2052
Trop. plant pathol.
DOI 10.1007/s40858-015-0036-6
1 23
Your article is protected by copyright and
all rights are held exclusively by Sociedade
Brasileira de Fitopatologia. This e-offprint is
for personal use only and shall not be selfarchived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.
1 23
Author's personal copy
Trop. plant pathol.
DOI 10.1007/s40858-015-0036-6
Genetic variation, morphology and pathogenicity of Ceratocystis
fimbriata on Hevea brasiliensis in Brazil
Denise C. O. F. Valdetaro 1 & Leonardo S. S. Oliveira 1 & Lúcio M. S. Guimarães 1 &
Thomas C. Harrington 2 & Maria A. Ferreira 3 & Rodrigo G. Freitas 1 & Acelino C. Alfenas 1
Received: 16 October 2014 / Accepted: 10 February 2015
# Sociedade Brasileira de Fitopatologia 2015
Abstract Ceratocystis fimbriata causes diseases on a wide
variety of plants in Brazil, including rubber tree (Hevea
brasiliensis), on which it causes gray mold or moldy rot on
tapping panels affecting latex yield. However, C. fimbriata
isolated from rubber tree have not been critically studied. Phylogenetic analyses of sequences of ITS rDNA and a mating
type gene placed rubber tree isolates from Acre and Bahia
among Brazilian isolates of C. fimbriata from other hosts. In
the analyses of 14 microsatellite loci, the rubber tree isolates
from Bahia were identical to each other and had alleles similar
to those of Brazilian isolates from mango and eucalyptus. The
microsatellite alleles of the Acre rubber tree isolates were
identical to each other but distinct from other Brazilian isolates. The rubber tree isolates were morphologically indistinguishable from each other and very similar to the isolates of
C. fimbriata on Ipomoea batatas, on which the species was
originally described. Based on inoculation experiments results, the Bahia and Acre rubber tree isolates do not appear
to be host specialized, which is typical for Brazilian isolates of
C. fimbriata sensu stricto.
Keywords Ceratocystis wilt . Gray mold . Microsatellite .
Moldy rot . Pathogen variation . Rubber tree
Section Editor: Silvaldo Silveira
* Acelino C. Alfenas
aalfenas@ufv.br
1
Dep. de Fitopatologia, Universidade Federal de Viçosa,
Viçosa, MG 36570-900, Brazil
2
Department of Plant Pathology and Microbiology, Iowa State
University, Ames 50011, IA, USA
3
Dep. de Fitopatologia, Universidade Federal de Lavras,
Lavras, MG 37200-000, Brazil
Introduction
Among the many hosts of Ceratocystis fimbriata Ell. & Halst.
in Brazil, one of the least studied is rubber tree, Hevea
brasiliensis, on which the fungus causes a gray mold or moldy
rot on the tapping panel (Albuquerque et al. 1972). In addition
to rubber tree, C. fimbriata has been found infecting numerous
crops, including Gmelina arborea (Muchovej et al. 1978;
Ribeiro 1982), Mangifera indica (Carvalho 1938; Pyenson
1938), Eucalyptus spp. (Ferreira et al. 2006; Alfenas and Mafia 2007), Ficus carica (Figueiredo and Pinheiro 1969), and
Cassia fistula (Ribeiro et al. 1984, 1987). Rubber tree is one of
the few hosts species of C. fimbriata complex which is native
to Brazil and of significant economic importance. In Brazil,
C. fimbriata was first reported on rubber tree in the Amazon
region (Albuquerque et al. 1972) and subsequently in Bahia
(Pereira and Santos 1985) and São Paulo (Silveira et al.
1985). The symptoms usually start with small cankers on
the tapping panel, uneven renewal of the outer bark, and
the formation of pustules with exudation of latex above the
wounded region. A grayish white layer of fungal growth and
sporulation may be seen on the affected areas of the tapping
panel. The infection can progress internally and cause death
of the tree (Albuquerque et al. 1972). If fungicides are not
applied on the tapping panel, the fungus can cause necrosis
of large portions of the inner bark and xylem, and the panel
may be covered with callus growth that limits latex extraction (Furtado 2007).
Ceratocystis fimbriata was originally described as the
causal agent of black rot of sweet potato (Ipomoea batatas)
in New Jersey, USA (Halsted 1890; Halsted and Fairchild
1891). C. fimbriata is a complex of many species, but the
sweet potato pathogen and South American strains of
C. fimbriata, including those studied in Brazil, are very closely related and have been placed in the Latin American Clade
Author's personal copy
Trop. plant pathol.
(LAC) of the complex (Harrington et al. 2011). Although
there is limited variation in phenotype, numerous species have
been described recently in the LAC (Engelbrecht and Harrington 2005; van Wyk et al. 2007, 2009, 2010, 2011a, b, 2012),
but Harrington et al. (2014) only recognize the host specialized species C. platani (cause of canker stain of plane tree),
C. cacaofunesta (Ceratocystis wilt of cacao) and
C. colombiana (Ceratocystis wilt of coffee and citrus). With
the exception of C. cacaofunesta, all strains in Brazil are considered to be a single species (C. fimbriata), which has a very
broad host range and includes isolates that vary greatly in
aggressiveness to many cultivated hosts.
Strains of C. fimbriata from rubber tree have not been
studied for genetic variation or host range, so the aim of this
study was to elucidate whether isolates from this host were
host specialized and belong to a cryptic species within the
C. fimbriata complex. We collected isolates from tapping
panels in Acre and Bahia, where the disease is currently
found, and determined their morphological characteristics, genetic relationships, and pathogenicity to rubber tree and other
hosts.
Materials and methods
DNA extraction
The fungus was grown on MYEA and incubated at 28 °C for
about 15 days before extracting DNA using the Wizard Genomic DNA Purification kit (Promega) with the following
modifications: initially the mycelium was transferred to
2 mL tube containing 200 μL of nuclei lysis solution, the
mycelium was homogenized in an extractor (Tissuelyser III,
Qiagen) at 30Hz for 2 min, incubated at −20 °C for 5 min,
400 μL of nuclei lysis solution was added, again macerated,
and incubated 65 °C for 15 min (tubes inverted every 5 min).
After centrifugation for 5 min at 13,000 g, the supernatant was
incubated for 5 min at room temperature, 300 μL of protein
precipitation solution was added and centrifuged at 13,000 g
for 10 min. A mixture of 500 μL of the supernatant and
500 μL of phenol:chloroform:isoamyl alcohol (25:24:1) was
stirred and centrifuged at 13,000 g for 5 min, and the aqueous
phase was added to 600 μL of cold isopropanol. After 12 h at
−20 °C and centrifugation (7 min at 13,000 g), the supernatant
was discarded and the pellet washed three times with 600 μL
of cold ethanol (70 %). The resulting pellet was dried at room
temperature, resuspended in 50 μL of DNA rehydration solution plus l μL of RNase solution, and incubated at 37 °C
overnight, followed by 65 °C for 10 min. The concentration
of purified DNA was quantified with a Nanodrop 2000c
(Thermo) and adjusted to 50 ng/μL.
Collection of isolates
ITS and mating type gene sequences
The characterization of disease symptoms was based on observations of rubber tree tapping panels at two plantations near
two different cities in Acre and at five plantations near Ituberá,
Bahia (Table 1). Samples of infected wood below whitishgray mycelia (with no signs of perithecia) on the surface of
tapping panels were collected in February 2011, during the
raining season, and stored in paper bags. Soil samples were
also collected from near the base of trees showing typical
symptoms of the disease. The samples were processed at the
Laboratory of Forest Pathology at the Federal University of
Viçosa (UFV). The fungus was baited from diseased wood
tissue by placing the pieces of discolored tissue between two
discs of fresh carrot root and incubated at 25 °C (Laia et al.
2000). Five to 10 days later, ascospore masses from perithecia
that formed on the carrot discs were transferred to MYEA
media (2 % malt extract, 0.2 % yeast extract, and 2 % agar)
for purification. The isolation of the fungus from soil samples
was attempted by placing 16 pieces of carrot (2 cm × 1 cm) into
gerboxes (11 × 11 × 3.5 cm) with approximately 200 g of soil.
Single ascospore strains were derived from the original
field isolates by dispersing an ascospore mass in a light oil
and spreading the spore suspension over the plate with MYEA
media; individual germlings were subcultured to fresh plates
(Harrington and McNew 1997). Only one isolate per tree was
stored in 15 % glycerol at −80 °C.
For amplification and sequencing of the ITS rDNA region the
primers ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA
A-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)
were used (Harrington et al. 2011). For the MAT-2 gene region, primers X9978a (5′-GCT AAC CTT CAC GCC AAT
TTT GCC-3′) and CFM2-1 F (5′-AGT TAC AAG TGT TCC
CAA AAG- 3′) were used to amplify and sequence a product
of about 1150 bp (Harrington et al. 2014). The sequencing
was performed at the Iowa University DNA Facility using
the sequencer AB3730 (Applied Biosystems).
Phylogenetic analyses
Sequences were analyzed and edited using Sequence Navigator (Applied Biosystems) software and subsequently aligned
using Clustal W v.1.5 (Thompson et al. 1992), followed by
manual adjustments in MEGA v5.0 (Tamura et al. 2011). Sequences of isolates obtained in this study were compared to
those of 30 isolates of the C. fimbriata complex from the Latin
American clade. Ceratocystis variospora was used as the
outgroup taxon (Harrington et al. 2011, 2014).
Maximum parsimony (MP) and Bayesian inference (BI)
were used to construct phylogenetic trees. The MP analysis
was performed with PAUP * 4.0b10 (Swofford 2003) using
Author's personal copy
Trop. plant pathol.
Table 1
Collection sites of 20 isolates of Ceratocystis fimbriata from rubber tree (Hevea brasiliensis) tapping panels
State
City
Farm
Isolate codes
Geographic coordinates
Bahia
Ituberá
Acre
Capixaba
Sen. Guiomar
Parcela 112
PMB
Maçaranduba
Mudururu
Jatobá
Gema
Bonal
A01, A05
A16, A21
A35, A40
A50, A58
A64, A74
RB02, RB05, RB08, RB09, RB11, RB13, RB16
RB17, RB19, RB21
S48°31′09″ W84°77′29.0″
S48°13′09″ W84°71′07.8″
S47°96′80″ W84°73′78.7″
S47°82′62″ W84°64′18.3″
S48°05′84″ W84°68′93.7″
S64°29′12″ W88°43′51.4″
S69°98′43″ W88°96′53.4″
heuristic searches with the Tree Bisection and Reconnection
algorithm and stepwise addition with 1000 random repetitions. The stability of branches was checked with
bootstrapping using 1000 replications. Bayesian inference
was performed using MrBayes 3.1.2 (Ronquist and
Huelsenbeck 2003). The substitution model was chosen based
on the Akaike Information Criterion of MrModelTest 3.2
(Nylander 2004). A posterior probability distribution of trees
was calculated using Metropolis-coupled Markov chain Monte Carlo (MCMC) and two chains initiated from a random
tree, with 25 million generations and discarding the first
25 % of the trees. The convergence of the MCMC and the
effective sample size were checked using Tracer 1.4 (Rambaut
and Drummond 2007). Phylogenetic trees were viewed and
edited in Figtree 1.3.1 (http://tree.bio.ed.ac.uk/software).
Morphological characterization
Microsatellite markers
Pathogenicity test on different hosts
Fourteen microsatellite loci developed from an isolate of
C. cacaofunesta (Steimel et al. 2004) were analyzed, as used
previously in studies of C. cacaofunesta (Engelbrecht et al.
2007), C. platani (Engelbrecht et al. 2004; Ocasio-Morales
et al. 2007), and C. fimbriata (Ferreira et al. 2010, 2011; Harrington et al. 2015). For each primer pair specific to the
flanking regions of 14 simple sequence repeat regions, one
of the primers was fluorescently labeled. PCR amplifications
of all microsatellite loci were performed using a PTC-100 96well thermal cycler (MJ Research) following the earlier described conditions (Ferreira et al. 2010). Band sizes of the
product were determined using a four-capillary ABI Prism
3100-Avant Genetic Analyzer (Applied Biosystems) and
ABI Peak Scanner v1.0 Analysis Software (Life Technologies). Each product length (within 1 bp) was considered to
be a different allele. Most of the microsatellite loci contained
trinucleotide repeats, and most alleles of a given locus differed
by increments of 3 bp. Relationships among the Hevea genotypes of C. fimbriata and representative genotypes from other
hosts of the LAC were examined in PAUP* (Swofford 2003)
using genetic distance (Nei’s) matrices and UPGMA trees,
with 1000 bootstrap replications.
Representative isolates from Acre (RB17) and Bahia (A50)
were selected for the first inoculation study (1 March 2012) on
clones of rubber tree (8-months old clones of grafted FX3864),
mango tree (Mangifera indica, cultivar Espada, 11-month old),
acacia (Acacia mearnsii seedlings, 14-months old), crotalaria
(Crotalaria juncea, 45-days old seedlings), eucalyptus
(3-months old hybrid clone of E. urophylla x E. grandis), kiwifruit (Actinidia deliciosa, cultivar Monty, 12-months old), edible
fig (Ficus carica, 8-months old), teak (Tectona grandis, 12months old), and andiroba (Carapa guianensis, 6-months old
seedlings). The plants were transplanted into 2 L pots containing
MecPlant substrate supplemented with 6 kg/m3 of superphosphate and 1.5 kg/m3 of slow release fertilizer (Osmocote 19-612). The plants were wounded (3-mm deep) with a downwardslanting cut from the outer bark into the wood with a sterile
scalpel at 3 cm above groundline. A volume of 500 μL of the
inoculum (2.5×106 spores/mL) was applied into the wound and
the inoculation site wrapped with parafilm. The control plants
were wounded and treated with the same volume of sterile distilled water. The plants were incubated in a greenhouse for
60 days, when the stem of each plant was vertically split and
the length of xylem discoloration above and below
Two isolates from Bahia state (A05 and A50) and two from
Senador Guiomar and Capixaba, Acre state (RB08 and
RB17, respectively) were used for morphological studies.
Cultures were grown on MYEA and incubated at 28 °C with
a photoperiod of 12 h. Measurements were made after
21 days. Fungal structures were mounted into lactic acid
and observed with a compound microscope (Olympus
BX53, Olympus), and the images were captured with Olympus Q-Color 5 digital camera and Image-Pro Plus version
7.0 (Media Cybernectics, Inc). Ascospores, endoconidia,
aleuroconidia, and endoconidiophores were measured at
400× magnification and perithecia with 100× magnification.
At least 20 measurements were made of each structure for
each isolate.
Author's personal copy
Trop. plant pathol.
the point of inoculation was measured. Plants that died before
60 days were similarly evaluated at the time of death.
The experiment was performed in a completely randomized design in a factorial arrangement consisting of two factors
(host and isolate) with seven replicates (plants) for each isolate. Data were submitted to analysis of variance (ANOVA)
and the means of each treatment were compared by the Fisher’s test (p < 0.01) using SAS statistical software (SAS
Institute).
In a second experiment (12 February 2014), three isolates
from Acre state (RB09, RB17, and RB19) and three from
Bahia state (A05, A50, and A66) were tested for aggressiveness on rubber tree (grafted clone FX3864, 8-months old).
Inoculations were conducted and evaluations were made as
described for first inoculation experiment. The experiment
was in a completely randomized design, with seven replicates
per isolate. Data were submitted to analysis of variance
(ANOVA) and the means of each treatment were compared
by the Fisher’s test (p<0.01) using SAS statistical software.
Results
A total of 101 wood samples (75 from Bahia and 26 from
Acre) from tapping panels of trees showing gray mold
Fig. 1 Symptoms of gray mold
on rubber tree (Hevea
brasiliensis) caused by
Ceratocystis fimbriata. a, Rubber
tree plantation in Bahia state; b,
Gray mycelial growth on a
tapping panel; c, Detail of
superficial gray mycelial growth
on the panel
symptoms (Fig. 1) were collected. Fifty-one samples from
Bahia and 18 samples from Acre yielded the fungus. The
pathogen was not recovered from the 21 soil samples.
Phylogenetic analyses
Ten sampled rubber tree isolates from Bahia had the same ITS
sequence, while 10 sampled isolates from Acre had a different
sequence. These two sequences were compared with a dataset
with 40 ITS sequences (Harrington et al. 2011) in an alignment of 689 bp. The number of variable characters was 171,
and 135 of those characters were parsimony informative.
Maximum parsimony analysis found 100 trees of 733 steps,
with the rubber tree isolates grouping with other Brazilian
isolates from the Latin American clade (trees not shown).
The branch of the ITS sequence of the Acre isolates was
moderately-supported (72 % bootstrap and posterior probability value=0.98). The branch of the ITS sequence of the Bahia
isolates had no bootstrap support and was separated by a low
posterior probability value (0.72). The branches of the rubber
tree genotypes were separated from each other, with the Bahia
genotype most similar to the isolates from Eucalyptus in Bahia, while the Acre genotype was more similar to the isolates
from mango in São Paulo.
Author's personal copy
Trop. plant pathol.
For MAT-2 gene sequences, an alignment of 1139 bp
showed limited variation among the 40 isolates, with 276
variable characters and 56 parsimony informative characters.
The single most parsimonious tree (Fig. 2) had very similar
topology to that found by Bayesian inference. Brazilian isolates of the LAC were separated by a moderately-supported
(77 % bootstrap) branch and a high posterior probability value
(=0.98). Two rubber tree MAT-2 genotypes (Bahia vs. Acre
isolates, respectively) differed at only one base position, and
these two sequences were closely related found close related
to each other and placed along with the other Brazilian isolates
from the LAC.
Microsatellite diversity
Only two microsatellite genotypes were identified among the
20 sampled rubber tree isolates, with uniformity among isolates from each state. Of the 14 microsatellite loci, six loci
were monomorphic (estimated allele size in bp: AAG9=397,
CAA15 = 321, CAT1 = 258, CAT12 = 374, CAG900 = 194,
GACA60=187). The eight polymorphic microsatellite loci
had the following respective allele sizes (Acre, Bahia):
AAG8 = 180, 174; CAA9 = 220, 194; CAA10 = 131, 125;
Fig. 2 Single most parsimonious
tree of 298 steps using 1139
characters from a portion of the
MAT1-2 gene (MAT-2 mating
type gene) of Ceratocystis
fimbriata and other members of
the Latin American Clade
(C. cacaofunesta, C. colombiana,
and C. platani) The tree was
rooted to C. variospora, a
member of the North American
Clade of the C. fimbriata
complex. The host genus and
state of origin (AC, Acre; BA,
Bahia; DF, Distrito Federal; PA,
Pará; PE, Pernambuco; PR,
Paraná; RJ, Rio de Janeiro; and
SP, São Paulo) or country of
origin are given for each isolate.
Bootstrap values greater than
50 % are indicated on appropriate
branches and posterior probability
values greater than 0.8 are in
parenthesis. Scale bar indicates
base pair differences
CAA38=305, 208; CAA80=314, 311; CAG5=320, 317;
CAG15=286, 283; and GACA6k=221, 215.
A UPGMA dendrogram based on the alleles of 14 microsatellite loci showed that the Bahia rubber tree genotype
grouped with Brazilian isolates from Mangifera and
Eucalyptus (Fig. 3). The Acre rubber tree genotype was
unique but somewhat similar to the genotype from Ipomoea
found worldwide.
Morphological characterization
The rubber tree isolates were morphologically indistinguishable from each other and similar to isolates of C. fimbriata
from Ipomoea (Engelbrecht and Harrington 2005). The rubber
tree isolates and Ipomoea isolates do not produce wide-mouth
endoconidiophores with doliiform endoconidia. Perithecia of
rubber tree isolates superficial or embedded in the substrate,
dark brown to black, globose, 120–250 μm tall and 115–
240 μm wide. Necks with the same coloration, 170–670 μm
long, 23–60 μm wide at base, 15–25 μm wide at apex.
Ostiolar hyphae divergent, hyaline, with no septum, 55–
100 μm long. Ascospores hyaline, hat shaped, 5–7 × 3–
4 μm, accumulating in a cream-colored ascospore mass at
Author's personal copy
Trop. plant pathol.
Fig. 3 UPGMA dendrogram of
genotypes of Ceratocystis
fimbriata, C. cacaofunesta and
C. platani based on alleles of 14
microsatellite loci. The first three
letters indicate the host genus
(Col, Colocasia; Euc, Eucalyptus;
Fic, Ficus; Gme, Gmelina; Hev,
Hevea; Ipo, Ipomoea; Man,
Mangifera; Pla, Platanus; The,
Theobroma) and next two letters
indicate the Brazilian state (AC,
Acre; BA, Bahia; MG, Minas
Gerais; PA, Pará; RJ, Rio de
Janeiro; and SP, São Paulo) or
country (US, United States; EC,
Ecuador; PG, Papua New Guinea;
WW, world wide) of origin.
Bootstrap values are shown
alongside the branches. Scale bar
indicates genetic distance
the apex of perithecia. Flask-shaped endoconidiphores with
1–11 septa, 50–240 μm long and 2–5 μm wide at base.
Phialides pale brown to hyaline, 35–70 μm long, 3–8 μm
wide in the middle, 2–5 μm wide at apex. Endoconidia unicellular, hyaline, smooth, cylindrical, 10–22×3–5 μm, in
chains. Aleurioconidiophores with 0–6 septa, 7–90 μm long,
2–5 μm wide at base. Aleurioconidia brown, globose to ovoid,
13–18×8–14 μm, singly or in short chains (Fig. 4).
Pathogenicity
Isolates A50 and RB17 were pathogenic on rubber tree and on
most of the other tested hosts, with the exception of
C. guianenesis and T. grandis (Table 2). The ANOVA showed
significant variation in the length of xylem discoloration between
the two isolates and among the nine inoculated host species, and
there was significant isolate ×host interaction (F =13.57,
p<0.001). Hevea, Crotalaria, Acacia, and Ficus were the most
susceptible hosts for both isolates. None of the plants were killed
at the end of the experiment, however, wilting symptoms were
observed on some inoculated plants. The controls remained
asymptomatic and had only a trace of xylem discoloration at
the inoculation point. At the end of the experiment, each isolate
was re-isolated from infected tissue of each host.
The six isolates inoculated on rubber tree plants differed
significantly in aggressiveness (F=4.62, p<0.001). The three
isolates from Acre caused greater xylem discoloration compared to the three isolates from Bahia (Table 3). Isolate RB09
was the most aggressive, followed by RB19, but the extent of
xylem discoloration of the other tested isolates did not differ
from that of the controls.
Discussion
Phylogenetic analyses using sequences of ITS rDNA and a
portion of the MAT-2 gene placed the isolates from rubber tree
among other Brazilian isolates of C. fimbriata from Gmelina,
Mangifera, Eucalyptus, and Ficus, as well as the Ipomoea
strain of C. fimbriata. Thus, isolates from rubber tree are
Author's personal copy
Trop. plant pathol.
Fig. 4 Morphological
characteristics of an isolate of
Ceratocystis fimbriata (isolate
RB08) from rubber tree (Hevea
brasiliensis). a, Perithecia; b,
Ostiolar hyphae and ascospores;
c, Flask-shaped
endoconidiophore producing
cylindrical endoconidia; d, Hatshaped ascospores; e,
Conidiophore producing an
aleurioconidium. f, Cylindrical
endoconidia
C. fimbriata sensu stricto (Harrington et al. 2011). Though the
rubber tree isolates were from two different regions of Brazil
Table 2 Xylem discoloration (cm) caused by two isolates of
Ceratocystis fimbriata from rubber tree (Hevea brasiliensis) inoculated
into wounds of nine plant species known to be hosts of C. fimbriata in
Brazil
Host
Isolates
A50
RB17
Control
Crotalaria juncea
Ficus carica
Hevea brasiliensis
Acacia mearnsii
Actinidia deliciosa
17.25
5.35
6.35
6.00
2.15
Aaab
BCb
Ba
Ba
Dab
6.15
10.20
5.15
4.95
6.15
Bb
Aa
BCa
BCa
Ba
1.05
1.00
1.00
1.00
1.00
Ac
Ac
Ab
Ab
Ab
Mangifera indica
Eucalyptus sp.
Tectona grandis
Carapa guianensis
2.55
1.95
1.35
1.25
CDa
Da
Da
Da
2.90
1.75
1.35
0.95
CDa
Dab
Da
Da
1.00
1.00
0.95
0.95
Ab
Ab
Aa
Aa
a
Means within a column followed by the same upper case letter are not
significantly different from each other (p< 0.01) based on Fisher’s
protected least significant difference
b
Means within a row followed by the same lower case letter are not
significantly different from each other (p< 0.01) based on Fisher’s
protected least significant difference
and differed in microsatellite markers and DNA sequences, no
morphological difference was found among the rubber tree
isolates or from the isolate from Ipomoea. Isolates from rubber
tree do not form wide-mouthed endoconidiophores with
doliform endoconidia, nor does the Ipomoea strain of
C. fimbriata (Engelbrecht and Harrington 2005).
Fourteen new species of C. fimbriata complex have been
described in the LAC in recent years (Engelbrecht and Harrington 2005; van Wyk et al. 2007, 2009, 2010, 2011a, b, 2012).
However, only three species (C. platani, C. cacaofunesta and
C. colombiana) have distinguishing phenotype, and the other
Table 3 Aggressiveness
(extent of xylem
discoloration) of six
isolates of Ceratocystis
fimbriata woundinoculated into a
susceptible clone
(FX3864) of rubber tree
(Hevea brasiliensis)
Isolates
Xylem descoloration (cm)
RB09
RB19
RB17
A50
A66
A05
Control
12.36
6.50
5.71
5.14
3.57
2.43
1.00
a
Aa
B
BC
BC
BC
BC
C
Means within a column followed by the
same letter are not significantly different
from each other (p<0.01) based on Fisher’s protected least significant difference
Author's personal copy
Trop. plant pathol.
new species are based solely on ITS rDNA sequence variation,
which is too variable to delineate lineages or species (Harrington et al. 2014). Intersterility tests to delimit biological species
and phylogenetic analyses of gene coding sequences group all
Brazilian isolates except those from Theobroma cacao with the
Ipomoea pathogen (Ferreira et al. 2010; Harrington et al. 2014).
Microsatellite markers developed for C. fimbriata (Steimel
et al. 2004) have been used for population studies on
C. cacaofunesta (Engelbrecht et al. 2007), C. platani
(Engelbrecht et al. 2004; Ocasio-Morales et al. 2007),
C. pirilliforms (Nkuekam et al. 2009), and C. fimbriata (van
Wyk et al. 2006; Ferreira et al. 2010, 2011; Harrington et al.
2015). In the present work, two genotypes were found according the region of origin, Acre or Bahia states, among the
Brazilian rubber tree isolates studied. Uniformity of markers
in each location suggests that the strains may have been
moved from site-to-site by humans and the tapping panels
may have been infected via contaminated tools rather than
infection from natural soilborne inoculum. The latter is typical
for C. fimbriata in Brazil (Ferreira et al. 2010, 2011, 2013;
Rossetto and Ribeiro 1990). Further, the fungus was not recovered from soil samples below symptomatic rubber trees.
The Bahia genotype was similar to other genotypes of
C. fimbriata found on Mangifera and Eucalyptus in Bahia,
Minas Gerais, Rio de Janeiro, and São Paulo (Ferreira et al.
2010). The Acre genotype differed from the Bahia genotype,
and according to the microsatellites markers the Acre genotype was closely related to the genotypes of isolates obtained
from sweet potato, on which the species was originally
described.
Although rubber tree is native to Brazil and has been reported to be a host of C. fimbriata (Albuquerque et al. 1972;
Pereira and Santos 1985; Silveira et al. 1985), pathogenicity
of isolates to rubber tree following Koch’s postulates has now
been demonstrated for the first time. The tested isolates varied
substantially in aggressiveness to rubber tree, and two isolates
from Acre were particularly aggressive, but strong evidence
of specialization to the host was not observed. The tested
rubber tree isolates differed in their aggressiveness to common hosts of C. fimbriata in Brazil, as typical for
other Brazilian isolates of C. fimbriata (Baker et al. 2003;
Harrington et al. 2011). For successful breeding programs
focusing on disease resistance, it is essential to understand
the genetic variability in the virulence and aggressiveness of
the pathogen (McDonald and Linde 2002). If resistance to
gray mold is thought to be important in rubber crop, it is
highly recommended that the most aggressive isolates be
used in artificial inoculations for resistance screening of genotypes. Additionally, the gray mold on rubber tree is the
only disease caused by C. fimbriata that can be partially
controlled by the use of fungicides (Furtado 2007), and currently represents the only available method to control the
disease on rubber tree.
Acknowledgments This work was supported by CNPq, FAPEMIG
and CAPES. We would like to thank Dr. Rivadalve Goncalves (Embrapa
Acre) and Dr. Carlos Mattos (Michelin) for technical assistance during the
collection of isolates. Also, we are thankful to Dr. Dalmo L. Siqueira and
the companies Michelin and Tecnoplanta for providing plant material for
the inoculations and Clonar Resistência a Doenças Florestais for the plant
growth and inoculation facilities.
References
Albuquerque FC, Duarte MLR, Silva HM (1972) Ocorrência do mofo
cinzento (Ceratocystis fimbriata) da seringueira. In: Seminário
Nacional da Seringueira. Cuiabá, MS. p. 125–128
Alfenas AC, Ferreira FA, Mafia RG, Gonçalves RC (2007) Isolamento de
fungos fitopatogênicos. In: Alfenas AC, Mafia RG. Métodos em
Fitopatologia. Editora UFV, Viçosa
Baker CJ, Harrington TC, Krauss U, Alfenas AC (2003) Genetic variability and host specialization in the Latin American clade of
Ceratocystis fimbriata. Phytopathology 93:1274–1284
Carvalho MB (1938) Sobre dois insetos nocivos à mangueira. Bol Secr
Agric Ind Comércio Estado Pernamb 3:130–132
Engelbrecht CJ, Harrington TC (2005) Intersterility, morphology and
taxonomy of Ceratocystis fimbriata on sweet potato, cacao and sycamore. Mycologia 97:57–69
Engelbrecht CJ, Harrington TC, Steimel J, Capretti P (2004) Genetic variation in eastern North American and putatively introduced populations
of Ceratocytsis fimbriata f. platani. Mol Ecol 13:2995–3005
Engelbrecht CJ, Harrington TC, Alfenas AC, Suarez C (2007) Genetic
variation in populations of the cacao wilt pathogen, Ceratocystis
cacaofunesta. Plant Pathol 56:923–933
Ferreira FA, Maffia LA, Barreto RW, Demuner NL, Pigatto S (2006)
Sintomatologia da murcha de Ceratocystis fimbriata em eucalipto.
Rev Árvore 2:155–162
Ferreira EM, Harrington TC, Thorpe DJ, Alfenas AC (2010) Genetic
diversity and interfertility among highly differentiated populations
of Ceratocystis fimbriata in Brazil. Plant Pathol 59:721–735
Ferreira MA, Harrington TC, Alfenas AC, Mizubuti ESG (2011)
Movement of genotypes of Ceratocystis fimbriata within and
among Eucalyptus plantations in Brazil. Phytopathology 101:
1005–1012
Ferreira MA, Harrington TC, Gongora-Canul CC, Mafia RG, Zauza EAV,
Alfenas AC (2013) Spatial-temporal patterns of Ceratocystis wilt in
Eucalyptus plantations in Brazil. For Pathol 43:153–164
Figueiredo P, Pinheiro ED (1969) Uma nova doença da figueira (Ficus
carica L.) na região de Valinhos, SP. Biológico 35:227–203
Furtado EL (2007) Doenças da seringueira e seu manejo no Brasil.
Informe Agrop 237:78–87
Halsted BD (1890) Some fungus diseases of the sweet potato. The black
rot. N J Agric Exp Station Bull 76:7–14
Halsted BD, Fairchild DG (1891) Sweet-potato black rot. J Mycol 7:1–11
Harrington TC, McNew DL (1997) Self-fertility and unidirectional mating type switching in Ceratocystis coerulescens, a filamentous ascomycete. Curr Genet 32:52–59
Harrington TC, Thorpe DJ, Alfenas AC (2011) Genetic variation and
variation in aggressiveness to native and exotic hosts among
Brazilian populations of Ceratocystis fimbriata. Phytopathology
101:555–566
Harrington TC, Kazmi MR, Al-Sadi AM, Ismail SI (2014) Intraspecific
and intragenomic variability of ITS rDNA sequences reveals taxonomic problems in Ceratocytsis fimbriata sensu stricto. Mycologia
106:224–242
Harrington TC, Huang Q, Ferreira MA, Alfenas AC (2015) Genetic analyses trace the Yunnan, China population of Ceratocystis fimbriata
Author's personal copy
Trop. plant pathol.
on pomegranate and taro to populations on Eucalyptus in Brazil.
Plant Dis 99:106–111
Laia ML, Alfenas AC, Harrington TC (2000) Isolation, detection in soil,
and inoculation of Ceratocystis fimbriata, causal agent of wilting,
die-back and canker in Eucalyptus. Fitopatol Bras 25:384
McDonald BA, Linde C (2002) Pathogen population genetics, evolutionary potential and durable resistance. Annu Rev Phytopathol 40:349–
379
Muchovej JJ, Albuquerque FC, Ribeiro GT (1978) Gmelina arborea – a
new host of Ceratocystis fimbriata. Plant Dis Report 62:717–719
Nkuekam GK, Barnes I, Wingfield MJ, Roux J (2009) Distribution and
population diversity of Ceratocystis pirilliformis in South Africa.
Mycologia 101:17–25
Nylander JAA (2004) MrModeltest Version 2. Program distributed by the
author. Evolutionary Biology Centre, Uppsala University, Uppsala
Ocasio-Morales RG, Tsopelas P, Harrington TC (2007) Origin of
Ceratocystis platani on native Platanus orientalis in Greece and
its impact on natural forests. Plant Dis 91:901–904
Pereira JCR, Santos AF (1985) Controle químico do mofo cinzento no
painel de sangria da seringueira. Comunicado técnico. n.46.
Manuas, AM. Embrapa.
Pyenson L (1938) Problems of applied entomology in Pernambuco. II A
survey of some of the pests of the crops of Pernambuco. Rev
Entomol 9:16–31
Rambaut A, Drummond AJ (2007) Tracer v1.4.. Available at: http://beast.
bio.ed.ac.uk/Tracer. Accessed date: 15 October, 2014.
Ribeiro GT (1982) Avaliação preliminar da resistência de árvores de
Gmelina arborea Lineaus, mediante inoculações do fungo
Ceratocystis fimbriata El. & Halst., causador do cancro em gmelina.
Fitopatol Bras 7:517
Ribeiro IJA, Ito MF, Rossetto CJ (1984) Cassia renigera Wall., um novo
hospedeiro de Ceratocystis fimbriata Ell. & Halst. Fitopatol Bras 9:
322
Ribeiro IJA, Ito MF, Rossetto CJ (1987) Cassia renigera Wall.: novo
hospedeiro de Ceratocystis fimbriata Ell. & Halst. Bragantia 46:
417–423
Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574
Rossetto CJ, Ribeiro IJA (1990) Mango wilt. XII. Recommendations for
control. Rev Agric 65:173–180
Silveira AP, Cardoso RMG, Neto FB, Oliveira DA (1985) Ocorrência e
controle químico do mofo cinzento (Ceratocystis fimbriata Ell. &
Hast.) da seringueira. Fitopatol Bras 10:281
Steimel J, Engelbrecht CJB, Harrington TC (2004) Development and
characterization of microsatellite markers for fungus Ceratocystis
fimbriata. Mol Ecol Notes 4:215–218
Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony
(*and Other Methods). Version 4. Sinauer Associates, Sunderland
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011)
MEGA5: molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony
methods. Mol Biol Evol 28:2731–2739
Thompson JD, Higgins D, Gibson TJ (1992) CLUSTAL versionW: a
novel multiple sequence alignment program. Nucleic Acids Res
22:4673–4680
van Wyk M, van der Merve NA, Roux I, Wingfield BD, Kamgan GN,
Wingfield MJ (2006) Population genetic anlyses suggest that the
Eucalyptus fungal pathogen Ceratocystis fimbriata has been introduced into South Africa. S Afr J Sci 102:259–263
van Wyk M, Al Adawi AO, Khan IA, Deadman ML, Al Jahwari AA,
Wingfield BD, Ploetz R, Wingfield MJ (2007) Ceratocystis
manginecans sp. nov., causal agent of a destructive mango wilt
disease in Oman and Pakistan. Fungal Divers 27:213–230
van Wyk M, Wingfield BD, Mohali S, Wingfield MJ (2009) Ceratocystis
fimbriatomima, a new species in the C. fimbriata sensu lato complex
isolated from Eucalyptus trees in Venezuela. Fungal Divers 34:175–
185
van Wyk M, Wingfield BD, Marin M, Wingfield MJ (2010) New
Ceratocystis species infecting coffee, cacao, citrus and native trees
in Colombia. Fungal Divers 40:103–117
van Wyk M, Wingfield BD, Al-Adawi AO, Rossetto CJ, Ito MF,
Wingfield MJ (2011a) Two new Ceratocystis species associated
with mango disease in Brazil. Mycotaxon 117:381–404
van Wyk M, Wingfield BD, Wingfield MJ (2011b) Four new
Ceratocystis spp. associated with wounds on Eucalyptus,
Schizolobium and Terminalia trees in Ecuador. Fungal Divers 46:
111–163
van Wyk M, Roux J, Nkuekam GK, Wingfield BD, Wingfield MJ (2012)
Ceratocystis eucalypticola sp. nov. from Eucalyptus in South Africa
and comparison to global isolates from this tree. IMA Fungus 3:45–
58