It is widely accepted that cacao tree (Theobroma cacao L.) is native to tropical forests of northern South America and was later introduced by man in Central America(Motamayor et al., 2002). Most cultivated varieties are generally susceptible to pests and diseases that cause large losses and limit cacao sustainability (Acebo et al., 2012; Gutiérrez et al., 2016; Marelli et al., 2019). Cacao black pod rot disease (BPR), caused by various species ofPhytophthora, occurs in most cacao growing areas around the world (Ploetz, 2016). It is estimated that about 30% of cacao production is lost by BPR disease (Drenth & Guest, 2004; Ndoumbe et al., 2004), corresponding to US$ 1.6 billion in 2017 (Bymolt et al., 2018).

Of the various species of the microbial oomycete Phytophthora that cause BPR, only P. palmivora Butler, shows a global distribution. P. megakarya Brasier and Griffin, which is confined to the African continent, is considered the most destructive pathogen of BPR. P. capsici Leonian and P. citrophthora Leonian are present in Central and South America, and P. capsici has been identified also in Camerún, Africa (Zentmyer et al., 1981; Kellam & Zentmyer, 1986); P. hevea Thompson has been reported in Malaysia (Turner, 1961) and México (Lozano & Romero, 1974); P. megasperma Drechsler in Venezuela and Cuba (Reyes & Capriles, 2000); and P. arecae Coleman, in India and Sri Lanka (Stamps et al., 1990).

BPR may cause 100% of crop losses in the tropics during environmental conditions favourable to disease development or poor management practices (Guest, 2007). Cacao is susceptible to BPR during all stages of plant development. Main symptoms include necrotic spots of grizzly color in seedlings, stem canker and foliar spots in mature trees, brown necrotic spots in the pod Surface and rot of beans reducing production and affecting cacao quality (Guest, 2007).

Phytophthora identification at the species level is usually made by morphological characteristics and the length to width ration of sporangia, pedicel length and other structures of the microorganism and corresponding colonies (Wilson, 1914; Al-Hedaithy & Tsao, 1979; Brasier & Griffin, 1979; Appiah et al., 2003; Erwin & Ribeiro, 2005). In cacao, colony pattern and growth rate have been used as an approach for identification of Phytophthora species causing BPR (Appiah, 2001; Appiah et al., 2003). However, accurate identification based only in morphology is sometimes difficult because of the large variability observed in the populations of different species associated to cacao trees (Brasier et al., 1981; Erwin & Ribeiro, 1996).

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 this important genus that affects not only crops but plants in natural ecosystems and natural evolution of new species (Ersek & Ribeiro, 2010). A number of biochemical and molecular techniques have been used as support for morphological identification of Phytophthora species such as electrophoretic patterns of proteins (Bielenin et al., 1988), isozyme analysis (Oudemans & Coffey, 1991), restriction fragment length polymorphisms (RFLP) and other molecular approaches (Förster et al., 1990). Recently, sequence analysis of mitochondrial and genomic genes and regions or whole genome has become widely used, mainly due to the availability of high throughput techniques andlower costs. The internal transcribed spacer (ITS) region of rRNA, the elongation factor (EF1a), β-tubulin, CoxI, Cox II and NADH subunit I genes and regions have shown to be useful for identification of different species of Phytophthora (Coulibaly et al., 2018; Maizatul-Suriza et al., 2019).

Genetic resistance is the most cost-effective method for disease control; therefore, reliable quantification of BPR progress rate is very important for cacao breeding. BPR development may be measured using inoculation of pods in the laboratory or under field conditions. Since a positive correlation has been observed when comparing results between both methods, it is more appropriate to perform evaluations in the laboratory to avoid disease dissemination and contamination of other trees in cacao orchards (Nyassé, 1997; Pokoua et al., 2008).

Recently, P. megakarya has dispersed and displaced P. palmivora in Africa, where 55% of cacao is produced in the world, posing a threat to the industry not only in Africa but in other places of cacao production such as Asia and America (Drenth & Sendal, 2004). For this reason, prompt and accurate disease diagnosis and management is key for cacao crop sustainability. In addition, more research on basic biology of causal agents addressing their origin, diversity, biological fitness, epidemiology, ecological relationships and adaptive abilities, is needed to prevent and manage BPR disease appropriately.

In Colombia, Phytophthora spp. associated to BPR have not yet been accurately identified and virulence and diversity of pathogen populations is unknown. This knowledge is crucial for cacao breeding for resistance to BPR and disease management. In the present research, morphological characteristics together with molecular sequencing of the ITS-rRNA and Cox DNA regions of Colombian isolates were used to identify the species of Phytophthora causing BPR disease in cacao and to determine phenotypic and genotypic diversity. In addition, virulence of selected isolates of Phytophthora sp. on different genotypes of cacao, was measured.

Sixty isolates of Phytophthora spp. were collected from eight departments of Colombia where cacao is grown (Table 1 and Table S1 [suppl.]). Tissues of cacao pods showing symptoms of BPR, were incubated in P5ARPH (cornmeal agar amended with 10 mg/L pimaricin, 250 mg/L, ampicillin, 5 mg/L rifampicin, 100 mg/L PCNB and 50mg/L hymexazol) culture media for microorganism isolation and purification following procedures described in Jeffers & Martin (1986).

Fifty isolates were grown in agar-V8 juice (AV8) in petri dishes (20% v/v V8 vegetable juice, Campbell; 0.3% w/v CaCO3; 1.8 w/v agar, pH adjusted to 6.0 with NaOH 10% w/v). Morphology of the colonies was determined according to the growth pattern and the format of the edge of each colony in AV8, after 4 days of incubation in darkness at 28 ± 2 °C (Brasier & Griffin, 1979; Erwin & Ribeiro, 1996). In addition, other variables of each colony were described such as texture, aerial development of mycelia and color. Evaluation for morphology determination was performed four times for each colony.

For microorganism morphometrics, microorganisms were grown placing a disc of fresh AV8 media in the center of a sterile microscope slide (~ 13 mm), that is placed on a bended glass road inside a Petri dish with a humid f ilter paper. Growing mycelia were collected with an inoculation loop and were spread around the disc of AV8 media in the microscope slide. Another sterile microscope slide was put on top of the disc of media AV8 and the Petri dish was covered with the top and was sealed with parafilm and incubated at 25 °C under continuous white 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 covered with a glass cover slip. Sporangia obtained by this method in 4 or 5 days were clean and mature enough for further use in morphometric measurements (Appiah et al., 2003). Thirty measurements of each structure were performed per isolate. Length, width and length to width ratio (L/W) of sporangia were measured, diameter of chlamydospores, length of the pedicel and presence or absence of papilla and chlamydospores were determined. Sporangia dehiscence was determined following the test reported by Cerqueira et al. (1999). All measurements were performed using a light microscope with an objective of 40X (Carl Zeiss Primo star, coupled with camera Axio CamERc5s Zeiss and software Zem 2011).

DNA sequence analysis of the genomic ITS and the mitochondrial Cox regions was used as support for identification of isolates at the species level (Martin & Tooley, 2003; Kroon et al., 2004) in 60 isolates. DNA was purif ied using the Plant/Fungi DNA isolation kit following the manufacturer instructions (Norgen Biotek, Corp.). Quality and quantity were determined by spectrophotometry in a Nanodrop equipment (NanoDrop Thermo Scientific) and agarose (0.8% w/v) gel electrophoresis (5 v/cm) in TBE buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.3)stained with EZVision (Amresco) following the manufacturer guidelines and visualized under UV in a transilluminator (BioRad). Purified DNA was stored at -20 °C for further use. Best amplification conditions for PCR reaction were determined for each pair of primers of a combination of six primers for ITS and three primers for the Cox regions (Table 2). Once the best primer pair/PCR condition was determined, it was used for amplification of ITS and Cox regions of each isolate evaluated.

A reaction mix was prepared containing the following components at final concentrations: buffer 1X (Thermo Scientific), 1.5 mM MgCl2, 200 μM dNTPs, 0.4 μM of each forward and reverse primers, 1.5 U Taq Polymerase enzyme (Fermentas), 2 ng/µL of DNA template, and molecular biology grade water to a final volume of 25 μL. Amplification was carried out in a thermalcycler (LabNet) with the following program: initial denaturation 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C for 1 min, extension at 72°C for 1 min; after the 40 cycles finished, a final extension at 72°C for 10 min was performed.

A reaction mix was prepared containing the following components at final concentrations: buffer 1X (Thermo Scientific), 3 mM MgCl2, 100 μM dNTPs, 1 μM of each forward and reverse primers, 2 U Taq Polymerase enzyme (Fermentas), 2 ng/µL of DNA template, and molecular biology grade water to a final volume of 25 μL. Amplif ication was carried out in a thermocycler (LabNet) with the following program: initial denaturation 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, primer annealing at 60 °C for 1 min, extension at 72°C for 1 min;after the 40 cycles finished a final extension at 72°C for 10 min was performed.

Amplified fragments of ITS and Cox regions were analyzed in agarose (2% w/v) gel electrophoresis as described before. PCR products from 3-4 reactions showing a single and clear band were purified using the GeneJET PCR purification kit (Thermo Scientific) following the manufacturer instructions. Purified products were quantified by spectrophotometry using a Nanodrop equipment and verified by agarose gel electrophoresis (2% w/v) as described. Purified fragments were sent for sequencing in both forward and reverse sense following the company guidelines (Macrogen, Republic of Korea). Obtained sequences were manually assembled, cleaned and edited using BioEdit software. Sequences were aligned using the Clustal W algorithm implemented in BioEdit software (Larkin et al., 2007). Homologies were identified by comparison with databases using the algorithm BLASTn (http://blast.ncbi.nlm.nih.gov/blast.cgi).

Alignment was used to identify the best substitution model and to perform the dendrogram. The model showing the lowest value of Bayesian information criteria (BIC) was selected as the best substitution pattern of each gene. Phylogenetic reconstructions were performed with the Maximum likelihood method with a Bootstrap of 1000 iterations. All calculations were performed with the computational package MEGA X (Kumar et al., 2018).

Incidence and severity of BPR caused by isolates ANYA 228, SARIO 189, ARAR 153, TOVR 01 and HURV 19 (Table 1), on cacao clones CCN51, ICS 95, EET 8, TSH 565, IMC 67 and PA 46, were measured. Isolates were grown in AV8 media incubated at 28 ± 2 °C for 10 days, 4 days in darkness and 6 days under a photoperiod of 12 h white light and 12 h in darkness. For zoospore release, sterile distilled water at 10 °C was added to Petri dishes and were incubated for 25 min at 5 °C and then incubated for 30 min at 25°C. A disc of filter paper of 0.5 cm of diameter impregnated with a suspension of zoospores at a concentration of 1,5 x 105 zoospores/mL, was placed in the equatorial zone of each of 10 cacao pods of 4.5 months old per each clone tested. As control, discs impregnated with sterile distilled water were placed in pods of each clone evaluated (Rodríguez & Vera, 2015). Pods were incubated in a humid chamber and incidence and the average diameter of the lesion (ADL) were measured at 6 and 10 days after inoculation. The diameter of the lesion was measured in two perpendicular directions and with values obtained, the average was calculated and used for rating the disease development according to the scale proposed by Phillips-Mora & Galindo (1989) (Table 3). A completely randomized design with ten replicates per treatment was used and experiments were performed twice through time.

Isolates were grouped by homogeneous characteristics by multivariate analysis of conglomerates using the squared Euclidian distance and the minimum variance grouping method of Ward. Comparison of groups with quantitative variables (length, width, sporangia length to width ratio) was performed by analysis of variance (ANOVA) (p ≤0.05) followed by the multiple mean comparison test of Tukey (p ≤0.05). Contingency tables and Chi-squared test were applied for analysis of qualitative variables and the generated groups. ADL data were analyzed by ANOVA followed by the Tukey test (p ≤ 0.05) for identification of differences between the mean of treatments. ADL data from the two experiments performed through time were combined after variance homogeneity was verified by the Cochran test (Gomez & Gomez, 1983). All statistical analyses were performed using the software SAS 9.3.

Three groups of isolates were formed; 14% of isolates in the first group (I), 50% in the second (II) and 36% in thethird (III) (Fig. 1). In group I, seven isolates from Arauca (ARTA 161), four from Huila (HUAL07, HUGAR60, HUGAR70, HURV19) and two from Tolima (TORB104, TOCHA78) grouped together. Less diversity was found in I than in II and III, and similarly, I showed the lowest values of length and width of sporangia and chlamydospore diameter (p ≤ 0.05) (Fig. 2). No significant differences were found between groups for L/W ratio of sporangia and pedicel 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 intermediate 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).

Significant differences (p ≤ 0.001) were observed between groups for edge and growth pattern, texture (p ≤ 0.0243) and aerial development of mycelia (p ≤ 0.0033)(Table 4). In cluster I, 47.62% of isolates showed regular edge and 42.86% wavy edge; 52.38% exhibited starry growth pattern, 71.43% plush texture, 76.19% scarce aerial development of mycelia and 100% of isolates showed two concentric rings. In cluster II 98.67% of isolates showed regular edge; 37.33% exhibited starry growth pattern, 53.33% plush texture, 76.19% scarce aerial development of mycelia and 100% of isolatesshowed two concentric rings. In cluster III, 81.48% of isolates showed regular edge; 53.70% exhibited starry growth pattern, 62.96% plush texture, 70.37% scarce aerial development of mycelia and 69.23% of isolates showed two concentric rings (Table 4). Only the aerial development of mycelia was present in the three clustersidentified. Growth pattern and number of concentric rings showed a higher variation between groups.

All sequences analyzed showed high homology with sequences from P. palmivora available in GenBank, using the Blast algorithm (Table 1). For ITS, the best substitution model was Hasegawa-Kishino-Yano + Gamma distribu-tion with five categories and for Cox sequences best model was Tamura 3-parameter + Gamma distribution with five categories. Non-uniformity of evolutionary rates among sites were modeled by using a discrete Gamma distribution (+G) with five rate categories (Nei & Kumar, 2000; Kumar et al., 2018). In Fig. 4 (ITS sequences) and Fig. 5 (Cox sequences), phylogenetic relationships between Phytoph-thora sp. isolates may be observed. Phylogenetic analyses for both Cox and ITS regions showed that sequences of P. palmivora grouped together confirming the identification of all isolates. Sequences of P. arecae from the palm trees Areca catechu and Cocos nucifera were found in the same group with P. palmivora for both Cox and ITS regions. Inaddition, sequences from other Phytophthora-related spe-cies such as P. niederhauserii, P. novaeguinee, P. megakarya, P. quercetorum and P. alticola, formed independent groups for both Cox and ITS regions.

Mean general distance observed for ITS sequences was of 0.011 (SE = 0.001) and for Cox sequences was of 0.011 (SE = 0.002) (Fig. 6). As expected, low genetic variability was observed between isolates because sequences used were conserved and confirmed taxonomy of isolates.

Incidence was 100% for all genotypes inoculated. ANOVA results indicated significant differences in lesion development in cacao pods between genotypes tested at 6 days after inoculation (p ≤ 0.05) (Table 5). Genotype CCN51 showed the highest value for lesion size with46.08% and 46.39% higher compared to genotypes IMC 67 and PA 46, respectively. Genotype CCN51 was classi-fied as susceptible together with ICS 95, EET8 and TSH 565; and PA 46 and IMC 67 were classified as moderately susceptible (Table 5). Ten days after inoculation, geno-types CCN51, ICS 95 and EET8 exhibited the highest values of lesion size in the same group of significance (21.86, 19.29 and 19.25 cm, respectively) (p ≤ 0.05) (Table 5). Lowest scores of diseases were registered for genotypes TSH 565, IMC 67 and PA 46 (15.30, 13.95 and 13.25 cm, respectively).

Significant differences (p ≤ 0.05) were identified be-tween five isolates of P. palmivora tested for virulence on pods of cacao genotypes (Table 6). More virulent isolate was HURV19 (in Cluster I) with a lesion value of 13.79 cm and 45.98% higher than value found for ANYA 228 (in Cluster II), which exhibited the lowest virulence of all isolates tested. Isolates SARIO 189 (Cluster II) and ARAR 153 (Cluster II) showed intermediate values of le-sion size with 10.84 and 12.11 cm, respectively (Table 6).

In the present work, morphological and DNA variabili-ty and virulence of P. palmivora isolates collected from different cacao growing regions in Colombia, were studied. Morphology of P. palmivora has been extensively studied since was identified as a plant pathogen. As early as in 1924, Gadd described that isolates obtained from rubber, orchids and bread fruit, exhibited smaller sporangia and chlamydospores than those isolated from cacao plants. Morphometric analyses were also used for identification of P. palmivora subgroups according to geographical region of origin (Chee, 1971; Mchau & Coffey, 1994) and host (Brasier & Griffin, 1979; Chee, 1969). Our results indicate that morphology of various microbial structures was significantly variable between isolates in agreement of what has been reported before (Erwin & Ribeiro, 1996; Kroon et al., 2012; Martin et al., 2012). Morphometry may be influenced not only by natural variation but by environmental conditions as well, thereupon species identif ication based solely in morphological characters had caused confusion when characterizing within and between Phytophthora sp. populations, generating the need to find stable characters (Erwin & Ribeiro, 1996). Morphological analyses of sporangia, pedicel and chlamydospores grouped isolates in three main clusters. In these three clusters, only the pedicel length and length to width ratio characters showed stability, pointing to pedicel length as a consistent characteristic within this species. Similar results were reported by Al-Hedaithy & Tsao (1979), who identified pedicel length as both intra and interspecific stable diagnostic character in the genus Phytophthora. This character has been useful for initial identification of two Phytophthora sp. pathogenic on cacao (P. palmivora and P. capsici) (Griffin, 1977; Cerqueira et al., 1999). In P. palmivora a short pedicel is usually observed (2.0–4.0 µm), in clear contrast to P. capsici where a deciduous long pedicel is registered (18.3–40.4 µm) (Cerqueira et al., 1999). Other studies of P. palmivora on cacao populations have shown similar findings with a continuous range of variation of morphological characteristics (Torres, 2016; Maora et al., 2017).

Length to width ratio did not show significant differences between groups suggesting that, despite the large variation observed in sporangia measurements, the proportion is consistent in P. palmivora. Erwin & Ribeiro (1996), pointed that the L/W ratio may be useful as a species characteristic to circumvent difficulties associated to subjective descriptions such as ovoid, obvoid and others. A high variability was found for colony characteristics and shape of sporangia. Similar findings have been reported widely, so many authors do not recommendusing these characteristics for species identification using it as supplementary information only (Erwin & Ribeiro, 1996). Identification of isolates as P. palmivora sensu Buttler, using morphology was confirmed by molecular sequencing of nuclear rDNA-ITS and mitochondrial Cox regions (Griffin, 1977; Brasier & Griffin, 1979). Sequence results have been used to define clear limits between species of Phytophthora (Robideau et al., 2011). Rahman et al. (2014) found that a phylogenetic tree obtained using the Cox region only resulted in a similar tree constructed with combined sequences from five genes (ITS, LSU, COX I, β-tubulin and EF1-a), confirming its use for species identification in oomycetes.

In our work, P. arecae grouped together with P. palmivora for both regions sequenced (i.e., ITS and Cox). However, P. arecae is similar to P. palmivora in morphology, isozyme analysis and DNA markers (SSCP), and now is considered a synonymous species of P. palmivora (Oudemans & Coffey, 1991; Mitchell & Kannwischer-Mitchell, 1992; Cooke et al., 2000). Therefore, it can be concluded that all sequences obtained in the present research from Cox and ITS regions corresponded to P. palmivora (Gallegly & Hong, 2008). No clear grouping was identif ied within P. palmivora sequences according to country of origin or host. P. palmivora is a redoubtable, ubiquitous and pantropical plant pathogen with over a thousand host species reported and thrives in a range of different environmental conditions causing severe diseases not only on cacao but on oil palm, mango, black pepper, Hevea sp., ornamentals, pineapple, papaya, citrus, coconut and many others (Widmer, 2014). Consequently, it is understandable that P. palmivora sequences of isolates from diverse countries and host plants grouped together using conserved molecular markers that has proven useful for taxonomical identification at the species level (Cooke et al., 2000; Robideau et al., 2011). Further analysis about molecular identification of pathogenicity factors such as effector proteins or host preferences is crucial for a better understanding of P. palmivora populations.

Genetic analyses in Papua New Guinea showed that P. palmivora strains isolated from cacao lesions belonged to one clonal lineage with limited variability; however, isolates from soil in the same regions showed higher genetic diversity, suggesting continuous selection for pathogenicity from a genetic pool of P. palmivora (Maora et al., 2017). It is not known whether or not there are in Colombia genetic differences between plant or soil populations of P. palmivora. Therefore, further research is required to answer this question. In a similar finding, P. palmivora populations obtained from oil palm crops in Colombia and Malaysia were not separated by phylogenetic analysis, supporting previous results and highlighting the importance to continue research expanding host species, number of isolates from different geographical regions and environmental conditions, looking for a better understanding of P.palmivora populations as a key tool for disease management programs (Maizatul-Suriza et al., 2019).

Virulence varied between five isolates tested in cacao genotypes with 45.98% of difference between the most (HURV 19, cluster I) and the less aggressive (ANYA 228, cluster III) isolate in the mean diameter of the lesion. It is considered that 90% of known cacao genotypes are susceptible to BPR disease (Iwaro et al., 1997). In our research, cacao genotype PA 46 showed the smallest disease score. High levels of resistance to cacao BPR have been usually associated to the named ˈForasteroˈ of Amazonian cacao genotypes such as SCA 6, PA 150, P7 and P 46 (Tahi et al., 1999; Bartley, 2005). ˈTrinitarioˈ genotypes such as CCN 51, ICS 95, EET 8 and TSH 565, have been reported more frequently as susceptible (Paulin et al., 2008). No clear relationship was established between morphological, virulence and genetic groups, identified in the present research. Contrasting results have been reported around the world on aggressiveness of P. palmivora isolates from different hosts and countries (Surujdeo-Maharaj et al., 2001; Thevenin et al., 2012; Torres, 2016; Fuzitani et al., 2018). Usually, plant breeding programs use a narrow sample of the genetic variability of pathogens for disease resistance selection. Pathogen populations exhibit complex dynamics in constant evolution, new genotypes are constantly emerging posing a great challenge for sustainable agriculture, as evidenced for a morphotype identification in Central America (Johnson et al., 2007; Cooke et al., 2012). In the present research, high variation was identified, therefore indicating that accurate and prompt identification of Phytophthora sp. attacking cacao is of crucial importance in an effective and efficient integrated crop management program. As most pathogen populations may be complex, wide studies and higher representation of pathogen diversity should be used in breeding programs to increase the possibility of selection of cacao genotypes, which may adapt to a wider range of pathogen variability in different environments.

Our knowledge about the origin and evolution of P. palmivora populations in Colombia with the whole host range is poor, more basic research is needed to understand the complex ecological relationships, sources of variation, mating type, gene flow, and many other aspects of pathogen biology to design better or new management tools for this important microorganism.

Authors wish to express sincere thanks to Anyela Gicela Vera for technical support at laboratory procedures.

Acebo-Guerrero Y, Hernández-Rodríguez A, Heydrich-Pérez M, El Jaziri M, Hernández-Lauzardo A, 2012. Management of black pod rot in cacao (Theobroma cacao L.): A review. Fruits 67 (1): 41-48. https://doi.org/10.1051/fruits/2011065Al-Hedaithy SSA, Tsao PH, 1979. Sporangium pedicel length in Phytophthora species and the consideration of its uniformity in determining sporangium caducity. Trans Br Mycol Soc 72: 1-13. https://doi.org/10.1016/S0007-1536(79)80001-7Appiah AA, 2001. Variability of Phytophthora species causing black pod disease of cocoa (Theobroma cacao, L.) and implications for assessment of host resistance. PhD Thesis. University of London, UK.Appiah A, Flood J, Bridge PD, Archer SA, 2003. Inter and intra specific morphometric variation and characterization of Phytophthora isolates from cocoa. Plant Pathol 52: 168-180. https://doi.org/10.1046/j.1365-3059.2003.00820.xBartley BGD, 2005. The genetic diversity of cacao and its utilization. CABI Publ, Wallingford. https://doi.org/10.1079/9780851996196.0000Bielenin A, Jeffreys SN, Wilcox WF, Jones AL, 1988. Separation by protein electrophoresis of six species of Phytophthora associated with deciduous fruit crops. Phytopathology 78: 1402-1408. https://doi.org/10.1094/Phyto-78-1402Brasier CM, Griffin MJ, 1979. Taxonomy of Phytophthora palmivora on cocoa. Trans Br Mycol Soc 72: 111-143. https://doi.org/10.1016/S0007-1536(79)80015-7Brasier CM, Griffin MJ, Maddison AC, 1981. Cocoa black pod Phytophthoras. In: Epidemiology of Phytophthora on cocoa in Nigeria; Gregory PH & Maddison AC, eds. Phytopathological Paper 25: 18-30. Commonwealth Mycological Institute. Kew, UK.Bymolt R, Laven A, Tyszler M, 2018. Demystifying the cocoa sector in Ghana and Côte d'Ivoire. Chapter 11, Cocoa marketing and prices. The Royal Tropical Institute (KIT). https://www.kit.nl/wp-content/uploads/2018/12/Demystifying-cocoa-sector-chapter11-cocoa-marketing-and-prices.pdf Cerqueira AO, Luz EDMN, Rocha CSS, 1999. Caracterização morfológica e biométrica de alguns isolados de Phytophthora spp. da micoteca do Centro de Pesquisas do Cacau. Fitopatologia Brasileira 24: 114-119.Chee KH, 1969. Variability of Phytophthora species from Hevea brasiliensis. Trans Br Mycol Soc 52: 425-436. https://doi.org/10.1016/S0007-1536(69)80126-9Chee KH, 1971. Host adaptability to strains of Phytophthora palmivora. Trans Br Mycol Soc 57: 175-178. https://doi.org/10.1016/S0007-1536(71)80098-0Cooke D, Drenth A, Duncan J, Wagels G, Brasier C, 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fung Genet Biol 30: 17-32. https://doi.org/10.1006/fgbi.2000.1202Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, 2012. Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen. PLOS Pathog 8 (10): 14. https://doi.org/10.1371/journal.ppat.1002940Coulibaly K, Aka AR, Camara B, Kassin KE, Kouakou K, Kebe BI, Koffi KN, Tahi GM, Walet NP, Guiraud SB, et al., 2018. Molecular identification of Phytophthora palmivora in the cocoa tree orchard of Côte d'Ivoire and assessment of the quantitative component of pathogenicity. Int J Sci 7 (8): 7-15. https://doi.org/10.18483/ijSci.1707Drenth A, Guest DI, 2004. Phytophthora in the tropics. In: Diversity and management of Phytophthora in Southeast Asia; Drenth A & Guest DI, eds. ACIAR Monograph No. 114, 238 pp. ACIAR, Canberra, Australia.Drenth A, Sendall B, 2004. Isolation of Phytophthora from infected plant tissue and soil, and principles of species identification. In: Diversity and management of Phytophthora in Southeast Asia; Drenth A & Guest DI, eds, pp: 94-102. Aust Centr Int Agr Res, Canberra, Australia).Ersek T, Ribeiro OK, 2010. Mini review: An annotated list of new Phytophthora species described post 1996. Acta Phytopathologica et Entomologica Hungarica 45: 251-266. https://doi.org/10.1556/APhyt.45.2010.2.2Erwin DC, Ribeiro OK, 1996. Phytophthora diseases worldwide. Am Phytopathol Soc Press, St Paul, MN, USA.Erwin DC, Ribeiro OK, 2005. Phytophthora diseases worldwide, 562 pp. APS Press, St. Paul, MN, USA.Förster H, Oudemans P, Coffey MD, 1990. Mitochondrial and nuclear DNA diversity within six species of Phytophthora. Exp Mycol 14: 18-31. https://doi.org/10.1016/0147-5975(90)90083-6Fuzitani EJ, Santos AF, Damatto R, Nomura ES, Kalil AN, 2018. Inoculation methods, aggressiveness of isolates and resistance of peach palm progenies to Phytophthora palmivora. Summa Phytopathologica 44 (3): 213-217. https://doi.org/10.1590/0100-5405/178476Gadd CH, 1924. The swarming of zoospores of Phytophthora faberi. Ann Bot 38: 394-397. https://doi.org/10.1093/oxfordjournals.aob.a089904Gallegly MC, Hong C, 2008. Phytophthora: Identifying species by morphology and DNA fingerprints. APS Press, St. Paul, MN, USA, 158 pp.Gomez KA, Gomez AA, 1983. Statistical procedures for agricultural research, 2nd Ed, Wiley, NY.Griffin MJ, 1977. Cocoa Phytophthora Workshop, Rothamsted Experimental Station, England, 24-26 May, 1976. Pest News Summaries Cent. Overseas Pest Res 23: 107-110. https://doi.org/10.1080/09670877709412411Guest D, 2007. Black pod: diverse pathogens with a global impact on cocoa yield. Phytopathology 97: 1650-1653. https://doi.org/10.1094/PHYTO-97-12-1650Gutiérrez OA, Campbell AS, Phillips-Mora W, 2016. Breeding for disease resistance in cacao. In: Cacao diseases; Bailey B & Meinhardt L (eds). Springer, Cham. https://doi.org/10.1007/978-3-319-24789-2_18Hasegawa M, Kishino H, Yano T, 1985. Dating the human-ape split by a molecular clock of mitochondrial DNA. J Mol Evol 22: 160-174. https://doi.org/10.1007/BF02101694Jeffers SN, Martin SB, 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Dis 70: 1038-1043. https://doi.org/10.1094/PD-70-1038Johnson ES, Aime MC, Crozier J, Flood J, Iwaro DA, Schnell RJ. 2007. A new morpho-type of Phytophthora palmivora on cacao in Central America, under IPM-Advances in conventional methods. Proc 5th INCOPED Int Sem on Cocoa Pest and Diseases, Akrofi A & Baah F (Eds). INCOPED Secretariat Cocoa Res Inst of Ghana.Kellam MK, Zentmyer GA, 1986. Morphological, physiological, ecological and pathological comparisons of Phytophthora species isolated from Theobroma cacao. Phytopathology 76: 159-164. https://doi.org/10.1094/Phyto-76-159Kroon LPNM, Bakker FT, Van den Bosch GBM, Bonants PJM, Flier WG, 2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences. Fungal Genet Biol 41: 766-782. https://doi.org/10.1016/j.fgb.2004.03.007Kumar S, Stecher G, Li M, Knyaz C, Tamura K, 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. https://doi.org/10.1093/molbev/msy096Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al., 2007. ClustalW and ClustalX version 2.0. Bioinformatics 23 (21): 2947-2948. https://doi.org/10.1093/bioinformatics/btm404Lozano TZE, Romero CS, 1974. Estudio taxonómico de aislamientos de Phytophthora patogenos de cacao. Agrociencia 56: 176-182.Maizatul-Suriza M, Dickinson M, Idris, AS, 2019. Molecular characterization of Phytophthora palmivora responsible for bud rot disease of oil palm in Colombia. World J Microbiol Biotechnol 35: 44. https://doi.org/10.1007/s11274-019-2618-9Maora S, Liew J, Guest D, 2017. Limited morphological, physiological and genetic diversity of Phytophthora palmivora from cocoa in Papua New Guinea. Plant Pathol 66: 124-130. https://doi.org/10.1111/ppa.12557Marelli J, Guest D, Bailey B, Evans H, Brown J, Junaid M, Barreto R, Lisboa D, Puig A, 2019. Chocolate under threat from old and new cacao diseases. Phytopathology 109 (8): 1331-1343. https://doi.org/10.1094/PHYTO-12-18-0477-RVWMartin FN, Tooley, PW, 2003. Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II Genes. Mycologia 95 (2): 269-284. https://doi.org/10.1080/15572536.2004.11833112Martin FN, Abad ZG, Balci Y, Ivors K, 2012. Identification and detection of Phytophthora: Reviewing our progress, identifying our needs. Plant Dis 96: 1080-1103. https://doi.org/10.1094/PDIS-12-11-1036-FEMchau GRA, Coffey MD, 1994. Isozyme diversity in Phytophthora palmivora: evidence for a Southeast Asian centre of origin. Mycol Res 98: 1035-1043. https://doi.org/10.1016/S0953-7562(09)80430-9Mitchell D, Kannwischer-Mitchell M, 1992. Phytophthora. In: Methods for research on soilborne phytopathogenic fungi; Singleton L, Mihail J, Rush C (eds). APS, St. Paul, MN, USA, pp: 31-38.Motamayor JC, Risterucci AM, López PA, Ortiz CF, Moreno A, Lanaud C, 2002. Cacao domestication I: the origin of the cacao cultivated by the Mayas. Heredity 89: 380-386. https://doi.org/10.1038/sj.hdy.6800156Ndoumbe-Nkeng M, Cilas C, Nyemb E, Nyasse S, Bieysse D, Flori A, Sache I, 2004. Impact of removing diseased pods on cocoa black pod caused by Phytophthora megakarya and on cocoa production in Cameroon. Crop Prot 23: 415-424. https://doi.org/10.1016/j.cropro.2003.09.010Nei M, Kumar S, 2000. Molecular evolution and phylogenetics. Oxford Univ Press, Oxford, England.Nyassé S, 1997. Étude de la diversité de Phytophthora megakarya et caractérisation de la résistance du cacaoyer (Theobroma cacao L.) à cet pathogène. Thèse, Inst. Nat. Polytech. de Toulouse, 145 pp.Oudemans P, Coffey MD, 1991. A revised systematics of twelve papillate Phytophthora species based on isozyme analysis. Mycol Res 95: 1025-1046. https://doi.org/10.1016/S0953-7562(09)80543-1Paulin D, Ducamp M, Lachenaud PH, 2008. New sources of resistance to Phytophthora megakarya identified in wild cocoa tree populations of French Guiana. Crop Prot 27: 1143-1147. https://doi.org/10.1016/j.cropro.2008.01.004Phillips-Mora W, Galindo JJ, 1989. Métodos de inoculación y evaluación de la resistencia a Phytophthora palmivora en frutos de cacao (Theobroma cacao). Turrialba 39 (4): 488-496.Ploetz R, 2016. The impact of diseases on cacao production: a global overview. In: Cacao diseases: A history of old enemies and new encounters; Bailey PA & L. W. Meinhardt LW (eds). Springer Int Publ, NY. pp: 33-59. https://doi.org/10.1007/978-3-319-24789-2_2Pokoua ND, N'Gorana JAK, Kebea I, Eskesb A, Tahia M, Sangarea A, 2008. Levels of resistance to Phytophthora pod rot in cocoa accessions selected on-farm in Cote d'Ivoire. Crop Prot 27: 302-309. https://doi.org/10.1016/j.cropro.2007.07.012Rahman MZ, Uematsu S, Coffey MD, Uzuhashi S, Suga H, Kageyama K, 2014. Re-evaluation of Japanese Phytophthora isolates based on molecular phylogenetic analyses. Mycoscience 55: 314-327. https://doi.org/10.1016/j.myc.2013.11.005Reyes H, Capriles L, 2000. El cacao en Venezuela. Moderna tecnología para su cultivo. El Rey C.A., Caracas.Robideau GP, de Cock AWAM, Coffey MD, Voglmayr H, Brouwer H, Bala K, Chitty DW, Désaulniers N, Eggertson QA, Gachon CMM, et al., 2011. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer. Mol Ecol Resour 11: 1002-1011. https://doi.org/10.1111/j.1755-0998.2011.03041.xRodríguez Polanco E, Vera Rodríguez AG, 2015. Identificación y manejo de la pudrición parda de la mazorca (Phytophthora sp) en cacao. Corporación Colombiana de Investigación Agropecuaria. http://hdl.handle.net/20.500.12324/13138. https://doi.org/10.21930/978-958-740-197-4Stamps DJ, Waterhouse GM, Newhook FJ, Hall GS, 1990. Revised tabular key to the species of Phytophthora. Mycological paper 162. Commonwealth Mycological Institute, Kew, Surrey, UK.Surujdeo-Maharaj S, Umaharan P, Iwaro A, 2001. A study of genotype-isolate interaction in cacao (Theobroma cacao L.): resistance of cacao genotypes to isolates of Phytophthora palmivora. Euphytica 118 (3): 295-303. https://doi.org/10.1023/A:1017516217662Tahi M, Kebe I, Eskes A, Ouattara S, Sangare A, Mondeil F, 1999. Rapid screening of cacao genotypes for field resistance to Phytophthora palmivora using leaves, twigs and roots. Eur J Plant Pathol 106: 87-94. https://doi.org/10.1023/A:1008747800191Tamura K, 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Mol Biol Evol 9: 678-687.Thevenin JM, Rossi V, Ducamp M, Doare F, Condina V, et al., 2012. Numerous clones resistant to Phytophthora palmivora in the ''Guiana'' genetic group of Theobroma cacao L. PLoS ONE 7 (7): e40915. https://doi.org/10.1371/journal.pone.0040915Torres G, 2016. Morphological characterization, virulence, and fungicide sensitivity evaluation of Phytophthora palmivora. PhD Thesis-dissertation, Michigan State Univ, USA.Turner PD, 1961. Complementary isolates of Phytophthora palmivora from cocoa and rubber, and their taxonomy. Phytopathology 51: 161-164.Widmer T, 2014. Phytophthora palmivora. Forest Phytophthoras 4 (1). https://doi.org/10.5399/osu/fp.4.1.3557Wilson GW, 1914. Studies of North American Peronosporales. V. A review of the genus Phytophthora. Mycologia 6: 54-83. https://doi.org/10.1080/00275514.1914.12020951Zentmyer GA, Kaosiri T, Idosu GO, Kellman MK, 1981. Morphological forms of Phytophthora palmivora. Proc 7th Int Cocoa Res Conf, 4-12 Novr 1979, Douala, Cameroun, pp: 291-296.