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Morphological, Molecular and Pathogenic Characterization of Phytophthora Palmivora Isolates Causing Black Pod Rot of Cacao in Colombia
Morphological, Molecular and Pathogenic Characterization of Phytophthora Palmivora Isolates Causing Black Pod Rot of Cacao in Colombia
Abstract
Aim of study: To characterize isolates of Phytophthora sp. causing black pod rot (BPR) of cacao (Theobroma cacao L.).
Area of study: Eight cocoa-growing regions in Colombia.
Material and methods: Sixty isolates of Phytophthora sp. were obtained from tissues of cacao pods showing symptoms of BPR. Isolates
were characterized using the morphology of sporangia and chlamydospores, molecular sequencing of regions of nuclear DNA (rDNA-ITS)
and mitochondrial (COX) and virulence in different genotypes of cocoa pods.
Main results: A high phenotypic variability between the isolates was determined, being the pedicel length and the length/width ratio
(L/W) the most stable characters for species identification. Short pedicels with an average of 3.13 μm ± 0.28 and a length/width ratio of
sporangia (L/W) with an average of 1.55 μm ± 0.11 were established as the most consistent morphological characteristics within palmivora
species.
Research highlights: Phytophthora pamivora was the only species associated to BPR, identified using morphology together with
sequence analyses.
Additional keywords: Theobroma cacao L.; Phytophthora sp.; sporangia; pedicel; chlamydospores.
Abbreviations used: ADL (average diameter of the lesion); AV8 (agar-V8 juice); BPR (cacao black pod rot disease); ITS (internal
transcribed spacer); L/W (length to width ratio)
Authors’ contributions: ERP: conceived the research project, conceived and designed the experiments, wrote the article. JGM:
performed genetic and statistical analyses, critical revision of the manuscript for important intellectual content. MMA: molecular sequence
analysis of ITS and Cox regions. MLCG: morphology of the colonies and biometric characteristics of sporangia and chlamydospores
and BPR severity of selected Phytophthora isolates. JDSA: morphology of the colonies and biometric characteristics of sporangia and
chlamydospores BPR severity of selected Phytophthora isolates. All authors read and approved the final manuscript.
Citation: Rodriguez-Polanco, E; Morales, JG; Muñoz-Agudelo, M; Segura, JD; Carrero, ML (2020). Morphological, molecular and
pathogenic characterization of Phytophthora palmivora isolates causing black pod rot of cacao in Colombia. Spanish Journal of Agricultural
Research, Volume 18, Issue 2, e1003. https://doi.org/10.5424/sjar/2020182-15147
Supplementary material (Table S1) accompanies the paper on SJAR’s website
Received: 10 May 2019. Accepted: 03 Jun 2020
Copyright © 2020 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
(CC-by 4.0) License.
Ministerio de Agricultura y Desarrollo Rural de Colombia # 404: Estudio de la diversidad genética, virulencia y mecanismos
de defensa en el patosistema Phytophthora sp. - Cacao
Competing interests: The authors have declared that no competing interests exist.
Correspondence should be addressed to Eleonora Rodríguez: lrodriguezp@agrosavia.co
Phytophthora, occurs in most cacao growing areas around lower costs. The internal transcribed spacer (ITS) region
the world (Ploetz, 2016). It is estimated that about 30% of of rRNA, the elongation factor (EF1a), β-tubulin, CoxI,
cacao production is lost by BPR disease (Drenth & Guest, Cox II and NADH subunit I genes and regions have
2004; Ndoumbe et al., 2004), corresponding to US$ 1.6 shown to be useful for identification of different species
billion in 2017 (Bymolt et al., 2018). of Phytophthora (Coulibaly et al., 2018; Maizatul-Suriza
Of the various species of the microbial oomycete et al., 2019).
Phytophthora that cause BPR, only P. palmivora Butler, Genetic resistance is the most cost-effective method
shows a global distribution. P. megakarya Brasier and for disease control; therefore, reliable quantification of
Griffin, which is confined to the African continent, is con- BPR progress rate is very important for cacao breeding.
sidered the most destructive pathogen of BPR. P. capsici BPR development may be measured using inoculation of
Leonian and P. citrophthora Leonian are present in Cen- pods in the laboratory or under field conditions. Since a
tral and South America, and P. capsici has been identified positive correlation has been observed when comparing
also in Camerún, Africa (Zentmyer et al., 1981; Kellam & results between both methods, it is more appropriate to
Zentmyer, 1986); P. hevea Thompson has been reported perform evaluations in the laboratory to avoid disease
in Malaysia (Turner, 1961) and México (Lozano & Ro- dissemination and contamination of other trees in cacao
mero, 1974); P. megasperma Drechsler in Venezuela and orchards (Nyassé, 1997; Pokoua et al., 2008).
Cuba (Reyes & Capriles, 2000); and P. arecae Coleman, Recently, P. megakarya has dispersed and displaced
in India and Sri Lanka (Stamps et al., 1990). P. palmivora in Africa, where 55% of cacao is produ-
BPR may cause 100% of crop losses in the tropics du- ced in the world, posing a threat to the industry not
ring environmental conditions favourable to disease de- only in Africa but in other places of cacao production
velopment or poor management practices (Guest, 2007). such as Asia and America (Drenth & Sendal, 2004). For
Cacao is susceptible to BPR during all stages of plant this reason, prompt and accurate disease diagnosis and
development. Main symptoms include necrotic spots of management is key for cacao crop sustainability. In ad-
grizzly color in seedlings, stem canker and foliar spots dition, more research on basic biology of causal agents
in mature trees, brown necrotic spots in the pod Surface addressing their origin, diversity, biological fitness,
and rot of beans reducing production and affecting cacao epidemiology, ecological relationships and adaptive
quality (Guest, 2007). abilities, is needed to prevent and manage BPR disease
Phytophthora identification at the species level is usua- appropriately.
lly made by morphological characteristics and the length to In Colombia, Phytophthora spp. associated to BPR
width ration of sporangia, pedicel length and other structures have not yet been accurately identified and virulence
of the microorganism and corresponding colonies (Wilson, and diversity of pathogen populations is unknown. This
1914; Al-Hedaithy & Tsao, 1979; Brasier & Griffin, 1979; knowledge is crucial for cacao breeding for resistance to
Appiah et al., 2003; Erwin & Ribeiro, 2005). In cacao, co- BPR and disease management. In the present research,
lony pattern and growth rate have been used as an approach morphological characteristics together with molecular
for identification of Phytophthora species causing BPR (Ap- sequencing of the ITS-rRNA and Cox DNA regions of
piah, 2001; Appiah et al., 2003). However, accurate iden- Colombian isolates were used to identify the species of
tification based only in morphology is sometimes difficult Phytophthora causing BPR disease in cacao and to de-
because of the large variability observed in the populations termine phenotypic and genotypic diversity. In addition,
of different species associated to cacao trees (Brasier et al., virulence of selected isolates of Phytophthora sp. on
1981; Erwin & Ribeiro, 1996). different genotypes of cacao, was measured.
In recent decades the number of species classified in
the genus Phytophthora have increased at an accelerated
rate, mainly because more researchers are interested in Material and methods
this important genus that affects not only crops but plants
in natural ecosystems and natural evolution of new species Isolates
(Ersek & Ribeiro, 2010). A number of biochemical and
molecular techniques have been used as support for mor- Sixty isolates of Phytophthora spp. were collected
phological identification of Phytophthora species such as from eight departments of Colombia where cacao is grown
electrophoretic patterns of proteins (Bielenin et al., 1988), (Table 1 and Table S1 [suppl.]). Tissues of cacao pods
isozyme analysis (Oudemans & Coffey, 1991), restriction showing symptoms of BPR, were incubated in P5ARPH
fragment length polymorphisms (RFLP) and other mole- (cornmeal agar amended with 10 mg/L pimaricin, 250
cular approaches (Förster et al., 1990). Recently, sequen- mg/L, ampicillin, 5 mg/L rifampicin, 100 mg/L PCNB
ce analysis of mitochondrial and genomic genes and re- and 50mg/L hymexazol) culture media for microorganism
gions or whole genome has become widely used, mainly isolation and purification following procedures described
due to the availability of high throughput techniques and in Jeffers & Martin (1986).
Morphology of the colonies and biometric Table 2. Primers used for sequencing the ITS and Cox regions
characteristics of sporangia and chlamydospores (based on Martin & Tooley, 2003; Kroon et al., 2004)
Primer Sequence 5’→3’
Fifty isolates were grown in agar-V8 juice (AV8) in
ITS1 TCC GTA GGT GAA CCT GCG G
petri dishes (20% v/v V8 vegetable juice, Campbell; 0.3%
ITS1F CTT GGT CAT TTA GAG GAA GTA A
w/v CaCO3; 1.8 w/v agar, pH adjusted to 6.0 with NaOH
ITS4 TCC TCC GCT TAT TGA TAT GC
10% w/v). Morphology of the colonies was determined
according to the growth pattern and the format of the edge ITS4B CAG GAG ACT TGT ACA CGG TCC AG
of each colony in AV8, after 4 days of incubation in dark- ITS5 GGA AGT AAA AGT CGT AAC AAG G
ness at 28 ± 2 °C (Brasier & Griffin, 1979; Erwin & Ribei- ITS6 GAA GGT GAA GTC GTA ACA AGG
ro, 1996). In addition, other variables of each colony were FMPh-8 dAAG GTG TTT TTT ATG GAC AAT GTA
described such as texture, aerial development of mycelia FMPhy-8b AAA AGA GAA GGT GTT TTT TAT GGA
and color. Evaluation for morphology determination was FMPhy-10b GC AAA AGC ACT AAA AAT TAA ATA TAA
performed four times for each colony.
For microorganism morphometrics, microorganisms
were grown placing a disc of fresh AV8 media in the cen- stained with EZVision (Amresco) following the manufac-
ter of a sterile microscope slide (~ 13 mm), that is placed turer guidelines and visualized under UV in a transillu-
on a bended glass road inside a Petri dish with a humid minator (BioRad). Purified DNA was stored at -20 °C
filter paper. Growing mycelia were collected with an for further use. Best amplification conditions for PCR
inoculation loop and were spread around the disc of AV8 reaction were determined for each pair of primers of a
media in the microscope slide. Another sterile microscope combination of six primers for ITS and three primers for
slide was put on top of the disc of media AV8 and the the Cox regions (Table 2). Once the best primer pair/PCR
Petri dish was covered with the top and was sealed with condition was determined, it was used for amplification of
parafilm and incubated at 25 °C under continuous white ITS and Cox regions of each isolate evaluated.
light. After 5 days of incubation, the microscope slide was
withdrawn and a drop of stain (lactophenol / cotton blue
/ lactofucsin) was placed on growing mycelia and was Internal Transcribed Spacer (ITS)
covered with a glass cover slip. Sporangia obtained by genomic region
this method in 4 or 5 days were clean and mature enough
for further use in morphometric measurements (Appiah A reaction mix was prepared containing the following
et al., 2003). Thirty measurements of each structure were components at final concentrations: buffer 1X (Thermo
performed per isolate. Length, width and length to width Scientific), 1.5 mM MgCl2, 200 μM dNTPs, 0.4 μM of
ratio (L/W) of sporangia were measured, diameter of each forward and reverse primers, 1.5 U Taq Polymerase
chlamydospores, length of the pedicel and presence or enzyme (Fermentas), 2 ng/µL of DNA template, and mo-
absence of papilla and chlamydospores were determined. lecular biology grade water to a final volume of 25 μL.
Sporangia dehiscence was determined following the test Amplification was carried out in a thermalcycler (LabNet)
reported by Cerqueira et al. (1999). All measurements with the following program: initial denaturation 95°C for
were performed using a light microscope with an objec- 3 min; 40 cycles of denaturation at 95°C for 30 s, primer
tive of 40X (Carl Zeiss Primo star, coupled with camera annealing at 55°C for 1 min, extension at 72°C for 1 min;
Axio CamERc5s Zeiss and software Zem 2011). after the 40 cycles finished, a final extension at 72°C for
10 min was performed.
DNA sequence analysis of the genomic ITS and the A reaction mix was prepared containing the following
mitochondrial Cox regions was used as support for iden- components at final concentrations: buffer 1X (Thermo
tification of isolates at the species level (Martin & Tooley, Scientific), 3 mM MgCl2, 100 μM dNTPs, 1 μM of each
2003; Kroon et al., 2004) in 60 isolates. DNA was puri- forward and reverse primers, 2 U Taq Polymerase enzyme
fied using the Plant/Fungi DNA isolation kit following the (Fermentas), 2 ng/µL of DNA template, and molecular
manufacturer instructions (Norgen Biotek, Corp.). Quality biology grade water to a final volume of 25 μL. Ampli-
and quantity were determined by spectrophotometry in a fication was carried out in a thermocycler (LabNet) with
Nanodrop equipment (NanoDrop Thermo Scientific) and the following program: initial denaturation 95°C for 3
agarose (0.8% w/v) gel electrophoresis (5 v/cm) in TBE min; 40 cycles of denaturation at 95°C for 30 s, primer
buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.3) annealing at 60 °C for 1 min, extension at 72°C for 1 min;
Table 3. Scale of resistance / susceptibility to BPR disease based on mean diameter of lesion (cm)
caused by Phytophthora sp. isolates in cacao pods (Phillips-Mora & Galindo, 1989).
Level of resistance/susceptibility Mean diameter of the lesion (cm)[a]
of cacao genotypes 6 days 10 days
Resistant (R) 0-2 0-3
Moderately resistant (MR) 2.1 - 4 3.1 - 6
Moderately susceptible (MS) 4.1 - 6 6.1 - 9
Susceptible (S) >6 >12
[a]
Mean diameter of cacao pod lesion calculated from length and width measurements.
after the 40 cycles finished a final extension at 72°C for °C. A disc of filter paper of 0.5 cm of diameter impregnated
10 min was performed. with a suspension of zoospores at a concentration of 1,5 x 105
Amplified fragments of ITS and Cox regions were zoospores/mL, was placed in the equatorial zone of each of 10
analyzed in agarose (2% w/v) gel electrophoresis as cacao pods of 4.5 months old per each clone tested. As con-
described before. PCR products from 3-4 reactions trol, discs impregnated with sterile distilled water were placed
showing a single and clear band were purified using in pods of each clone evaluated (Rodríguez & Vera, 2015).
the GeneJET PCR purification kit (Thermo Scientific) Pods were incubated in a humid chamber and incidence and
following the manufacturer instructions. Purified products the average diameter of the lesion (ADL) were measured at
were quantified by spectrophotometry using a Nanodrop 6 and 10 days after inoculation. The diameter of the lesion
equipment and verified by agarose gel electrophoresis was measured in two perpendicular directions and with values
(2% w/v) as described. Purified fragments were sent for obtained, the average was calculated and used for rating the
sequencing in both forward and reverse sense following disease development according to the scale proposed by
the company guidelines (Macrogen, Republic of Korea). Phillips-Mora & Galindo (1989) (Table 3). A completely ran-
Obtained sequences were manually assembled, cleaned domized design with ten replicates per treatment was used and
and edited using BioEdit software. Sequences were experiments were performed twice through time.
aligned using the Clustal W algorithm implemented in
BioEdit software (Larkin et al., 2007). Homologies were
identified by comparison with databases using the algori- Statistical analyses
thm BLASTn (http://blast.ncbi.nlm.nih.gov/blast.cgi).
Isolates were grouped by homogeneous characteristics
by multivariate analysis of conglomerates using the squared
Genetic analyses Euclidian distance and the minimum variance grouping
method of Ward. Comparison of groups with quantitative
Alignment was used to identify the best substitu- variables (length, width, sporangia length to width ra-
tion model and to perform the dendrogram. The model tio) was performed by analysis of variance (ANOVA) (p
showing the lowest value of Bayesian information crite- ≤0.05) followed by the multiple mean comparison test of
ria (BIC) was selected as the best substitution pattern of Tukey (p ≤0.05). Contingency tables and Chi-squared test
each gene. Phylogenetic reconstructions were performed were applied for analysis of qualitative variables and the
with the Maximum likelihood method with a Bootstrap of generated groups. ADL data were analyzed by ANOVA
1000 iterations. All calculations were performed with the followed by the Tukey test (p ≤ 0.05) for identification
computational package MEGA X (Kumar et al., 2018). of differences between the mean of treatments. ADL data
from the two experiments performed through time were
combined after variance homogeneity was verified by
BPR severity of selected Phytophthora isolates the Cochran test (Gomez & Gomez, 1983). All statistical
analyses were performed using the software SAS 9.3.
Incidence and severity of BPR caused by isolates ANYA
228, SARIO 189, ARAR 153, TOVR 01 and HURV 19 (Ta-
ble 1), on cacao clones CCN51, ICS 95, EET 8, TSH 565, Results
IMC 67 and PA 46, were measured. Isolates were grown in
AV8 media incubated at 28 ± 2 °C for 10 days, 4 days in Biometric characteristics of sporangia
darkness and 6 days under a photoperiod of 12 h white light and chlamydospores
and 12 h in darkness. For zoospore release, sterile distilled
water at 10 °C was added to Petri dishes and were incuba- Three groups of isolates were formed; 14% of isolates
ted for 25 min at 5 °C and then incubated for 30 min at 25 in the first group (I), 50% in the second (II) and 36% in the
Figure 1. Groups of isolates obtained by multivariate analysis of conglomerates using the squared Euclidian
distance and the minimum variance grouping method of Ward. Isolates characteristics used in the analysis:
length, width and L/W ratio of sporangia, and pedicel length. Fifty isolates were used for the analysis. Yellow,
cluster I; black, cluster II; and red, cluster III.
third (III) (Fig. 1). In group I, seven isolates from Arau- (Table 4). In cluster I, 47.62% of isolates showed re-
ca (ARTA 161), four from Huila (HUAL07, HUGAR60, gular edge and 42.86% wavy edge; 52.38% exhibited
HUGAR70, HURV19) and two from Tolima (TORB104, starry growth pattern, 71.43% plush texture, 76.19%
TOCHA78) grouped together. Less diversity was found scarce aerial development of mycelia and 100% of iso-
in I than in II and III, and similarly, I showed the lowest lates showed two concentric rings. In cluster II 98.67%
values of length and width of sporangia and chlamydospore of isolates showed regular edge; 37.33% exhibited sta-
diameter (p ≤ 0.05) (Fig. 2). No significant differences were rry growth pattern, 53.33% plush texture, 76.19% scar-
found between groups for L/W ratio of sporangia and pedi- ce aerial development of mycelia and 100% of isolates
cel length (Fig. 3). Cluster II grouped 25 isolates and was
the most diverse. This cluster had isolates from all regions
studied with a higher percentage from Caldas and Bolivar.
In cluster II, the highest values for all groups (p ≤ 0.05) of
length (53.069 ± 0.650 µm) and width of sporangia (35.211
± 0.493 µm) and diameter of chlamydospore (36.859 ±
0.611 µm), were observed (Fig. 2). In cluster III, 18 isolates
were grouped with seven isolates from Antioquia, five from
Santander and four from Arauca. From Tolima and Nariño
only one isolate from each department were found in this
cluster (Fig. 1). Isolates in this group showed significant in-
termediate values (p ≤ 0.05) for length of sporangia (42.766
± 0.765 µm), width of sporangia (26.808 ± 0.581 µm) and
diameter of chlamydospore (31.564 ± 0.720 µm) (Fig. 2).
Morphology of the colony Figure 2. Mean biometric characteristics: length and width
of sporangia and diameter of chlamydospores, in µm. Mean
of each characteristic was calculated from 30 measurements.
Significant differences (p ≤ 0.001) were observed be- Different letters represent significant differences between each
tween groups for edge and growth pattern, texture (p ≤ biometric characterization of different cluster identified by
0.0243) and aerial development of mycelia (p ≤ 0.0033) Tukey test (p ≤ 0.05)
Figure 4. Molecular phylogenetic analysis of rADN-ITS sequences from P. palmivora (Pp) isolates. The evolutionary history was
inferred by using the Maximum Likelihood method and Hasegawa-Kishino-Yano model (Hasegawa et al., 1985). The tree with the
highest log likelihood (-2998.64) is shown. The percentage of trees in which the associated taxa clustered together is shown next to
the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a
matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology
with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5
categories (+G, parameter = 0.7314)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per
site. This analysis involved 145 nucleotide sequences. There were a total of 941 positions in the final dataset. Evolutionary analyses
were conducted in MEGA X (Kumar et al., 2018).
Figure 5. Molecular phylogenetic analysis of COX sequences from isolates of P. palmivora (Pp). The evolutionary history was
inferred by using the Maximum Likelihood method and Tamura 3-parameter model (Tamura, 1992). The tree with the highest log
likelihood (-12449.39) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the bran-
ches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix
of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with
superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 cate-
gories (+G, parameter = 0.2104)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
This analysis involved 66 nucleotide sequences. There were a total of 473 positions in the final dataset. Evolutionary analyses were
conducted in MEGA X (Kumar et al., 2018).
0.014
46.08% and 46.39% higher compared to genotypes IMC
0.012 67 and PA 46, respectively. Genotype CCN51 was classi-
fied as susceptible together with ICS 95, EET8 and TSH
0.01
Mean of base substitutions
Table 5. Level of resistance / susceptibility of cacao pods of different genotypes to P. palmivora (isolates ANYA 228, SARIO 189,
ARAR 153, TOVRO1, HURV19).
6 days after inoculation 10 days after inoculation
Table 6. Virulence of selected isolates of P. palmivora on five using these characteristics for species identification using
genotypes of cacao pods (CCN51, ICS95, EET8, TSH 565, it as supplementary information only (Erwin & Ribeiro,
IMC 67, PA 46). 1996). Identification of isolates as P. palmivora sensu
Buttler, using morphology was confirmed by molecular
Department Mean diameter
Code of isolate
of collection of lesion (cm) [a]
sequencing of nuclear rDNA-ITS and mitochondrial Cox
regions (Griffin, 1977; Brasier & Griffin, 1979). Sequence
ANYA 228 Antioquia 7.45 A results have been used to define clear limits between spe-
SARIO 189 Santander 10.84 B cies of Phytophthora (Robideau et al., 2011). Rahman et
ARAR 153 Arauca 12.11 B al. (2014) found that a phylogenetic tree obtained using
TOVRO1 Tolima 12.6 C the Cox region only resulted in a similar tree constructed
HURV19 Huila 13.79 C with combined sequences from five genes (ITS, LSU,
[a]
Different letters in the same column represent significant COX I, β-tubulin and EF1-a), confirming its use for spe-
differences identified by the Tukey test (p ≤ 0.05). cies identification in oomycetes.
In our work, P. arecae grouped together with P. palmi-
vora for both regions sequenced (i.e., ITS and Cox). Howe-
gion of origin (Chee, 1971; Mchau & Coffey, 1994) and ver, P. arecae is similar to P. palmivora in morphology,
host (Brasier & Griffin, 1979; Chee, 1969). Our results isozyme analysis and DNA markers (SSCP), and now is
indicate that morphology of various microbial structures considered a synonymous species of P. palmivora (Oude-
was significantly variable between isolates in agreement mans & Coffey, 1991; Mitchell & Kannwischer-Mitchell,
of what has been reported before (Erwin & Ribeiro, 1996; 1992; Cooke et al., 2000). Therefore, it can be conclu-
Kroon et al., 2012; Martin et al., 2012). Morphometry ded that all sequences obtained in the present research
may be influenced not only by natural variation but by en- from Cox and ITS regions corresponded to P. palmivora
vironmental conditions as well, thereupon species identi- (Gallegly & Hong, 2008). No clear grouping was identi-
fication based solely in morphological characters had cau- fied within P. palmivora sequences according to country
sed confusion when characterizing within and between of origin or host. P. palmivora is a redoubtable, ubiquitous
Phytophthora sp. populations, generating the need to find and pantropical plant pathogen with over a thousand host
stable characters (Erwin & Ribeiro, 1996). Morphologi- species reported and thrives in a range of different en-
cal analyses of sporangia, pedicel and chlamydospores vironmental conditions causing severe diseases not only
grouped isolates in three main clusters. In these three on cacao but on oil palm, mango, black pepper, Hevea
clusters, only the pedicel length and length to width ratio sp., ornamentals, pineapple, papaya, citrus, coconut and
characters showed stability, pointing to pedicel length as many others (Widmer, 2014). Consequently, it is un-
a consistent characteristic within this species. Similar re- derstandable that P. palmivora sequences of isolates from
sults were reported by Al-Hedaithy & Tsao (1979), who diverse countries and host plants grouped together using
identified pedicel length as both intra and interspecific conserved molecular markers that has proven useful for
stable diagnostic character in the genus Phytophthora. taxonomical identification at the species level (Cooke et
This character has been useful for initial identification of al., 2000; Robideau et al., 2011). Further analysis about
two Phytophthora sp. pathogenic on cacao (P. palmivora molecular identification of pathogenicity factors such as
and P. capsici) (Griffin, 1977; Cerqueira et al., 1999). In effector proteins or host preferences is crucial for a better
P. palmivora a short pedicel is usually observed (2.0–4.0 understanding of P. palmivora populations.
µm), in clear contrast to P. capsici where a deciduous long Genetic analyses in Papua New Guinea showed that P.
pedicel is registered (18.3–40.4 µm) (Cerqueira et al., palmivora strains isolated from cacao lesions belonged to
1999). Other studies of P. palmivora on cacao populations one clonal lineage with limited variability; however, iso-
have shown similar findings with a continuous range of lates from soil in the same regions showed higher genetic
variation of morphological characteristics (Torres, 2016; diversity, suggesting continuous selection for pathogenicity
Maora et al., 2017). from a genetic pool of P. palmivora (Maora et al., 2017).
Length to width ratio did not show significant It is not known whether or not there are in Colombia gene-
differences between groups suggesting that, despite the tic differences between plant or soil populations of P. pal-
large variation observed in sporangia measurements, the mivora. Therefore, further research is required to answer
proportion is consistent in P. palmivora. Erwin & Ribei- this question. In a similar finding, P. palmivora populations
ro (1996), pointed that the L/W ratio may be useful as a obtained from oil palm crops in Colombia and Malaysia
species characteristic to circumvent difficulties associa- were not separated by phylogenetic analysis, supporting
ted to subjective descriptions such as ovoid, obvoid and previous results and highlighting the importance to con-
others. A high variability was found for colony charac- tinue research expanding host species, number of isolates
teristics and shape of sporangia. Similar findings have from different geographical regions and environmen-
been reported widely, so many authors do not recommend tal conditions, looking for a better understanding of P.
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