Limited morphological, physiological and genetic diversity of Phytophthora palmivora from cocoa in Papua New Guinea
Abstract
In Papua New Guinea (PNG) cocoa (Theobroma cacao) is one of the most important cash crops grown in the tropical lowland and island regions. As in most cocoa-growing areas, phytophthora black pod and canker cause significant yield losses. Cocoa breeding activities in PNG are focused in East New Britain province where disease control recommendations are also developed. This study tested the hypothesis that there was no diversity in the Phytophthora palmivora population causing black pod on cocoa by characterizing the variation in pathogen populations within and between the five major cocoa-growing areas. Diseased pods were sampled hierarchically from the five locations and additional isolates were collected from soil, stem and leaf lesions, or retrieved from culture collections. Morphological characters showed continuous variation within the range described for P. palmivora. Genetic analysis revealed that the isolates belonged to one dominant clonal lineage, with restricted distributions of several other subpopulations. Lowest diversities were found in the geographically isolated Karkar Island and East Sepik province. Soil isolates showed greater genetic diversity than isolates from cocoa lesions. Intra-farm variation was as much as inter-farm or inter-province variation. Both mating types were detected, although no strong evidence of sexual recombination was observed. The analysis revealed limited geographic, temporal or host specialization, suggesting continuous selection for pathogenicity from a genetic pool of P. palmivora. These findings have significant implications on the deployment of cocoa genotypes, enforcement of inter-province quarantine and sustainable disease management strategies.
Introduction
For any plant disease to be managed and controlled successfully the causal organism must be identified and its origin, diversity and potential for variation need to be understood. The negative economic and ecological impacts of plant disease are caused by populations of pathogens that evolve and constantly adapt to changes in their environment (McDonald & Linde, 2002; McDonald, 2004). In cocoa ecosystems in Papua New Guinea (PNG) these changes have included the introduction of new pathogen populations, exotic hosts and host genotypes, and agricultural practices such as intercrops and shade trees (Konam, 1999).
Phytophthora palmivora occurs as a pathogen in every cocoa-growing region of the globe (Guest, 2007). Its biogeographical origin remains unknown, but the significant variation found in southeast Asia is consistent with its evolution there (Mchau & Coffey, 1994). There have been many opportunities to introduce cocoa pathogens into PNG since European colonization in the late nineteenth century. German colonists introduced cocoa seeds to Madang province from Ceylon (now Sri Lanka) in 1889 and from Amboina or Makassar in 1896, from Singapore to Massava, East New Britain province (ENB) in 1897, and from Makassar and Batavia to Gunanur, ENB (Sack & Clark, 1979; Firth, 1982). Trinitario cocoa was introduced from Java, Indonesia in 1932; however, the original parent trees at the Rabaul Botanical Gardens and the plantings at Keravat were completely destroyed during the Second World War (Bridgland, 1960; Anonymous, 1961). After the war, the selection of elite Trinitario clones from surviving trees in ENB led to their widespread planting across the country (Anonymous, 1963). In the 1960s, Upper Amazonian cocoa was introduced from Trinidad and crossed with Trinitario clones to produce hybrid cocoa for distribution throughout the country (Tan, 1981; Tan & Tan, 1990). Colonists also introduced many other known hosts of P. palmivora, including coconut, durian, mango, breadfruit, pineapple, papaya, oil palm, vanilla and ornamental plants from Samoa, Australia, the Dutch East Indies, the Philippines and Africa (Parkinson, 1907; Sentinella, 1975; Robson, 1979; Sack & Clark, 1979; Firth, 1982).
Successful breeding and deployment of host plant resistance depends on a thorough understanding of variation in the pathogen population (Peever et al., 2000). Laboratory strains or isolates used in routine screens may not be representative of individuals present in field populations. Currently, all cocoa planting material for PNG is bred at the Cocoa Coconut Institute (CCI), Tavilo in ENB and distributed throughout the country. Phytophthora isolates from cocoa are traditionally identified on the basis of colony morphology, temperature tolerance and growth rate on agar, growth rate on cocoa pods, mating type, antheridial type, sporangia shape and size, and pedicel length (Brasier & Griffin, 1979; Erwin & Ribeiro, 1996). It was long assumed that the cocoa pathogen was P. palmivora (Waterhouse, 1963); however, Turner (1960) separated West African cocoa isolates into ‘G’ (Ghanaian) and ‘N’ (Nigerian) based on the appearance of lesions on cocoa pods, and Sansome et al. (1975) reported further physiological, pathogenic and chromosome differences between these groups. Brasier & Griffin (1979) later distinguished four morphotypes in a global survey of Phytophthora from cocoa: MF1 and MF2 were confirmed as P. palmivora, MF3 (Turner's ‘N’ isolates) as the new species P. megakarya, and MF4 as being related to P. capsici. Phytophthora palmivora remains the most common species isolated from lesions on cocoa, with localized populations of P. citrophthora and P. capsici in the Americas and P. megakarya in West Africa (Guest, 2007).
In PNG variations in disease symptoms on cocoa pods and in sporangial shape and size led to speculation that a significant amount of pathogen diversity was present in the P. palmivora population from cocoa (Saul, 1993). The current study was carried out to support the cocoa-breeding programme by testing the hypothesis that there is no diversity in P. palmivora causing black pod of cocoa in the main cocoa-growing areas. Hierarchical sampling was conducted to collect isolates from the five main cocoa-growing regions of PNG. This population of P. palmivora was analysed using morphological and physiological characters, together with random amplified microsatellites (RAMS). RAMS, which are useful genetic markers that are highly polymorphic, locus-specific and co-dominant (Hantula et al., 1996), were used to give an indication of the level of clonality in the pathogen population.
Materials and methods
Sampling
Diseased cocoa pods were sampled hierarchically from five locations: Bougainville province, ENB, East Sepik province (ESP), Karkar Island and Madang province (Fig. 1), including eight farms chosen arbitrarily at each location and eight diseased pods randomly sampled at each farm. Two hundred and sixty-six isolates were collected and analysed (Table S1). Of these isolates, 53 were from Bougainville, 60 from ENB, 48 from ESP, 41 from Karkar and 64 from Madang. Additionally, eight isolates from pod tissue, 28 from leaf, 11 from stem and two soil isolates from one cocoa plot at CCI were compared in order to obtain an indication of the level of diversity of isolates from different cocoa tissues (Table S2). These isolates were all obtained from diseased tissue, whereas the soil isolates were baited using cocoa pods. Temporal changes in the pathogen population were also estimated by comparing 66 isolates collected between 1988 and 2005 (Tables S1 & S3) from ENB, Karkar and Madang.
Morphological studies
Carrot agar (CA) plates were centrally inoculated with a 7 mm diameter agar plug taken from the leading edge of actively growing 4-day-old cultures of the respective isolates. The plates were sealed with Parafilm and incubated in the dark at 25 °C for 10–14 days. After 4 days, the cultures were examined for colony morphology and colony diameter was measured; the presence of chlamydospores was noted after 14 days. Five blocks of agar from each isolate (2.5 × 1 cm) were cut and mounted on five microscope slides. Images of 10 sporangia per slide were taken under the light microscope and measurements of sporangial length and breadth, and pedicel length were recorded. Using these measurements the sporangial length to breadth (L:B) ratio was calculated. Sporangial shape and sporangiophore branching were also recorded. Measurements were recorded for three replicate plates for each isolate, giving a total of 150 sporangia per isolate.
Mating type determination
Each isolate was paired on V8 juice/red kidney bean extract agar (Duncan, 1988) with tester strains P. palmivora type cultures A1 (UQ3694) and A2 (UQ3738) and P. cinnamomi A1 (UQ4856) and A2 (UQ4827) obtained from Professor André Drenth, University of Queensland, Australia. Isolates Mag17 (A1) and Mag28 (A2) were used for further repeat pairings to confirm results. The paired cultures were incubated as described above for the morphological studies. Determination of mating type, A1 or A2, was made by observing the presence of oospores at the interface of each pair of cultures under the light microscope. An isolate was designated A1 if oospores were present when paired with a known A2 tester and vice versa.
Morphological data were analysed using one-way anova in GenStat v. 8.1 and confirmed using Bonferroni's t-test (P < 0.05) where significant differences were indicated between the groups (Ashcroft & Pereira, 2003). Nonparametric data were analysed using the Kruskal–Wallis test (Dytham, 1999; Ashcroft & Pereira, 2003). Where there were differences, pairwise comparisons between isolate subpopulations were made using Kruskal–Wallis anova.
Genetic analysis
Isolates were grown at 25 °C in 20% V8 broth amended with calcium carbonate, either in 250 mL flasks on an orbital shaker (100 rpm) for 14 days, or in still plastic Petri dishes for 10 days (Erwin & Ribeiro, 1996). Mycelium was harvested by filtration through sterile gauze and stored at −20 °C. For each culture, 1 g of frozen mycelium was transferred to a sterile microtube with sand and a sterile ceramic bead. One millilitre of extraction buffer was added and the mycelium was homogenized for 30 s, centrifuged for 5 min at 8000 g and the supernatant was transferred to a clean sterile 1.5 mL tube. Subsequently, 125 μL of protein precipitate solution (PPS) was added and the tubes were inverted several times, centrifuged for 5 min at 11 000 g, and the supernatant was carefully transferred into clean sterile tubes. Then, 700 μL of binding matrix was added to each tube, mixed by inverting the tubes for 3–5 min and centrifuged for 1 min at 11 000 g. The pellet was dried with lint-free tissue (Kimwipes), resuspended in 800 μL salt/ethanol wash solution (SEWS), centrifuged for 1 min at 11 000 g, and washed again in SEWS. The pellet was air dried for 15–30 min, then resuspended in 120 μL Tris EDTA (TE) + RNase A (10 μg mL−1). After 5–10 min with occasional swirling, the tubes were centrifuged for 2 min at 11 000 g and the supernatant (DNA) transferred into fresh sterile tubes. The supernatant in the tubes was centrifuged a second time then transferred into another set of fresh sterile tubes. After this the tubes were incubated at 37 °C for 1 h to digest residual RNA. The tubes of DNA were stored at −20 °C for subsequent analysis.
PCR amplification was carried out in a 20 μL reaction volume containing PCR buffer (Promega), 25 mm MgCl2 (Sigma), 1 U Taq DNA polymerase (Bioline), 25 mm dNTPs (Astral), 50 μm primer (Sigma) and c. 20 ng DNA extract. Thermocycling reactions were performed in a Corbett DNA thermocycler according to the following temperature profile: an initial denaturation of 10 min at 95 °C; then 35 (primers ACA, CGA, CA) or 37 (primer CCA) cycles of denaturation for 30 s at 95 °C, annealing for 45 s at 51 °C (ACA), 62 °C (CGA) 43 °C (CA) or 64 °C (CCA) and extension for 2 min at 72 °C; and a final extension for 7 min at 72 °C (Hantula et al., 1996, 1997). The primer sequences were ACA: BDB (ACA)5; CGA: DHB (CGA)5; CA: DBDA (CA)7; and CCA: DDB (CCA)5, where B = G, T or C; D = G, A or T; H = A, T or C.
Amplification products were separated by electrophoresis in 2% agarose gels run in TBE buffer at room temperature at 100 V for 1 h. After staining in TBE buffer containing ethidium bromide (10 mg L−1) for 30–45 min, the gels were visualized under a UV transilluminator (TFX-20M; EEC) and photographed using a digital camera (C-370 Zoom; Olympus). The amplification product size was estimated by comparison with a 100 bp ladder (Promega). Clear bands were scored as either present (1) or absent (0). The number and percentage of polymorphic loci, Shannon's diversity index (H) and Shannon's equitability (EH) were calculated (Beals et al., 2000).
A binary matrix from all four primers was constructed and a cluster analysis was performed using DICE similarity coefficient and unweighted pair group method using arithmetic averages (UPGMA) agglomeration in the software NTSYSpc v. 2.1 (Rohlf, 2000).
Results
Morphological analysis
Sporangiophores of all isolates developed simple sympodial branching (Fig. 2) and shed their sporangia at maturity. The caducous, papillate sporangia measured 24.93–77.60 μm long (Fig. S1) and 16.48–37.04 μm wide (Fig. S2), with mean dimensions of 50.10 × 27.23 μm. Pedicels were occluded and ranged from 0.99 to 7.37 μm, with an average length of 4.66 μm (Table 1; Figs 3, S1, S2 & S3). Although mean pedicel lengths from the five locations were significantly different, variation was continuous and there was no significant correlation between location and pedicel length (P = 0.1326, r = 0.0982). The sporangial L:B ratio ranged from 1.29 to 2.39 (Table 1; Fig. S4). There were significant correlations between geographic region and sporangial length (P = 0.0001, r = 0.3724), sporangia breadth (P = 0.001, r = 0.2939) and L:B ratio (P = 0.001, r = 0.2896). Both terminal and intercalary chlamydospores were observed (Table 2).
| Location | Chla | n b | Ic | Sporangia length (μm) | Sporangia breadth (μm) | Pedicel length (μm) | L:B |
|---|---|---|---|---|---|---|---|
| Bougainville | 51/52 | 2550 | 17 | 43.16 ± 0.18 a | 25.78 ± 0.10 a | 4.75 ± 0.05 b | 1.69 ± 0.005 c |
| ENB | 48/60 | 2350 | 16 | 57.46 ± 0.27 d | 30.48 ± 0.10 d | 4.51 ± 0.04 e | 1.90 ± 0.008 d |
| ESP | 46/49 | 2250 | 15 | 44.94 ± 0.18 c | 26.28 ± 0.10 c | 4.11 ± 0.04 c | 1.73 ± 0.005 e |
| Karkar | 35/41 | 1700 | 12 | 52.25 ± 0.28 e | 29.28 ± 0.13 b | 4.74 ± 0.05 b | 1.79 ± 0.008 b |
| Madang | 61/64 | 2950 | 20 | 52.95 ± 0.17 b | 28.18 ± 0.09 e | 5.09 ± 0.03 d | 1.90 ± 0.005 d |
| Mean | 50.10 | 27.23 | 4.66 | 1.81 | |||
| LSD (α = 0.05) | 0.43 | 0.23 | 0.10 | 0.01 |
- ENB, East New Britain; ESP, East Sepik.
- Values within columns with same letters are not significantly different at P < 0.05.
- a Proportion of isolates with chlamydospores present/total number of isolates examined.
- b Total number of sporangia examined.
- c Total number of isolates examined for sporangial morphology per location.
| Province | Clonal group | ||||||
|---|---|---|---|---|---|---|---|
| P1 | P2 | P3 | P4 | P5 | P6 | P7 | |
| Bougainville | 0 | 6 | 6 | 8 | 0 | 0 | 1 |
| ENB | 2 | 8 | 5 | 8 | 1 | 0 | 0 |
| ESP | 0 | 4 | 2 | 8 | 0 | 0 | 0 |
| Karkar | 0 | 2 | 2 | 8 | 2 | 0 | 0 |
| Madang | 0 | 6 | 6 | 8 | 0 | 2 | 0 |
- ENB, East New Britain; ESP, East Sepik.
The majority (98.5%) of isolates developed stellate/striate colonies on CA, with a low percentage from ENB and Madang forming stoloniferous colonies. Growth rates ranged from 5.87 to 20.43 mm per day (Fig. S5), with significant differences between them (P < 0.05), but no significant correlation with location.
Mating type
Isolates from Bougainville, ENB, ESP and Karkar and were all of the A2 mating type. Isolates of the A1 mating type were found in very low proportion, i.e. only six out of the total population analysed in the study were A1 isolates. These A1 isolates (Mag 17, Mag 18, Mag 19, Mag 20, Mag 21 and Mag 24) were found on only one farm at Gonoa (Madang) where seven isolates were obtained (86% A1). All the other sites/farms in Madang had only isolates of mating type A2.
Genetic analysis
There were clear polymorphisms with primers CA and CGA, but not with primers ACA and CCA. The genetic analysis incorporated 12 loci from all four primers of which six (50%) were polymorphic (non-clonal). All seven RAMS fingerprinting phenotypes identified were clonal and UPGMA cluster analysis showed that the isolates clustered into seven clonal groups (P1 to P7) independent of location, and distinct from the out-group species P. capsici (Fig. 4).
Clonal group 1 (P1) was present in low frequencies on two farms in ENB (Table 2). The second clonal group (P2) was present in all eight farms in ENB, four farms in ESP, six in Madang, two on Karkar and six on Bougainville. Clonal group 3 (P3) was present in six farms in Bougainville, five in ENB, six in Madang, two in Karkar and four in ESP, while clonal group 4 (P4) was present in all farms in all provinces. The fifth clonal group (P5) was found in very low frequencies only in ENB and Karkar, while the sixth group (P6) was present only in Madang. The seventh clonal group (P7) was present only in one farm in Bougainville. Mating type A1 was found in clonal groups 3 (Mag 21 and Mag 23), 4 (Mag 17, Mag 19 and Mag 20) and 6 (Mag 18).
Shannon's index of diversity (H) for P. palmivora phenotypes on cocoa in PNG was calculated as 1.10 and the evenness (E) was 0.57, confirming that the pathogen population is highly clonal but unevenly distributed. Sixty-two percent of the individuals belonged to P3 and 38% were distributed between the other six clonal groups. ENB had the highest number of different clonal groups (five), followed by Madang (four) and Bougainville (four). Samples from ESP (three) and Karkar (four) were the least diverse.
Isolates from the same cocoa tree were mostly clonal; however, samples from some individual trees had multiple fingerprinting phenotypes, suggesting a certain level of diversity within trees. Within-tree genetic diversity was greatest for isolates obtained from the high yielding, but highly susceptible, cocoa clone 73-14/1.
Soil isolates were more diverse than those from other substrates (H = 1.39) and the different fingerprinting phenotypes more evenly distributed. Leaf isolates were the least diverse (H = 0.34) and not equally distributed between the fingerprinting phenotypes present (E = 0.49) with 89% of the individuals belonging to one phenotype. However, because of low numbers of isolates from some tissue substrates these results should be regarded with caution.
Fifteen RAMS fingerprinting phenotypes, nine clonal and six unique, were identified in the isolates collected between 1988 and 2005, revealing a level of temporal diversity (H = 2.13). These fingerprinting phenotypes were not evenly distributed (E = 0.78), with 30% of the individuals belonging to one group. The 1997 (H = 1.39, E = 1.00) and 2004 (H = 1.37, E = 0.76) isolates were more diverse; however, 2004 isolates were unevenly distributed. The 1994 isolates from Karkar were the least diverse, but fingerprinting phenotypes were equally distributed (H = 0.64, E = 0.92) (data not shown).
Discussion
All 263 of the Phytophthora isolates analysed in the current study belong to a continuously variable population of P. palmivora, as shown by their colony morphology, amphigynous antheridia, sympodial sporangiophore morphology, sporangial caducity and dimensions, and chlamydospore production (Waterhouse et al., 1983). The mean pedicel length, sporangial lengths and breadths, and L:B ratio of isolates in this study fell within the range for P. palmivora (Turner, 1961; Griffin, 1977; Zentmyer et al., 1977; Brasier & Griffin, 1979). Variation in sporangial dimensions was very high within farms, between farms within locations and between locations, suggesting that there is one continuous population affecting cocoa in PNG. The range of growth rates of isolates in this study was higher than that reported for a smaller sample of isolates of P. palmivora from cocoa (Brasier & Griffin, 1979).
The genetic structure of the P. palmivora population on cocoa in PNG was generally clonal, forming seven clonal groups, between the five locations sampled, between farms within the five locations, between trees within farms and within individual trees. However, there were cases where the isolates within trees were genetically different. Phytophthora palmivora isolates from the different host tissues were also clonal, but the soil isolates were unique. An apparent change in temporal structure was observed as the 2005 isolates were genetically different from the isolates collected previously. Given the presence of diverse subpopulations in cocoa-growing areas, the high level of variation in the DNA fingerprinting phenotypes observed within some individual trees most probably reflects the multiple methods of inoculum dispersal by rain-splash, ants and flying beetles (McGregor & Moxon, 1985; Prior, 1986; Konam & Guest, 2004). The only previous study on the genetic diversity of P. palmivora on cocoa in PNG, comparing 31 PNG isolates with those from other parts of southeast Asia, also showed very limited diversity and suggested a predominance of asexual reproduction (C. Blomley, University of Queensland, Australia, personal communication).
Most P. palmivora isolates from cocoa in this study were found to be of mating type A2, with a very small proportion of A1 isolates also found in Gonoa in Madang. This is the first report of both mating types associated with cocoa in PNG, although both mating types have been reported in forest soils (Arentz, 1986). While the greatest diversity of fingerprinting phenotypes were found in Madang, the continuous morphological variation and clonal population structures provide no strong evidence for sexual recombination. To obtain a clearer understanding of the level of recombination within the pathogen population, further testing using a more sophisticated genetic marker system would be warranted in future research.
No patterns of geographic specialization were observed. The most common clone included 164 isolates from all locations except Karkar Island. A further two fingerprinting phenotypes, with 43 isolates each, were found in all five locations surveyed. A fourth clonal group was only present in ENB and Madang, a fifth in ENB and Karkar, and a sixth in ESP and Bougainville. Three isolates from Madang, including one A1, one A2 and one isolate of unidentified mating type, were grouped into a distinct clonal group found in that region only. A more probable explanation for the diversity in Madang is that the long history of cocoa cultivation and multiple introductions of planting material, as well as its proximity to subpopulations in forest soils in the Highlands, have provided more opportunities for diversity compared to the island locations of Karkar, ENB, Bougainville and the isolated ESP.
These results indicate the presence of a large clonal subpopulation together with a series of small isolated genetic groups of P. palmivora causing disease on cocoa in PNG. This suggests that most of the isolates associated with cocoa spread with the host, resulting in less diversity than would be expected if indigenous populations adapted to new cocoa plantings. Pearson's correlation test discriminated between two subpopulations based on sporangial length and L:B ratio, with Madang and ENB forming one dominant subpopulation and Bougainville isolates forming the other. Karkar and ESP isolates were mixed.
From these results it seems as though P. palmivora was introduced separately into Bougainville and the rest of PNG, with subsequent intermixing of isolates. Prior to 1886, Bougainville was part of the British-administrated Solomon Islands, and a number of plantations on the island were established using cocoa from there. Between 1906 and 1914, a boom in copra prices encouraged the expansion of coconut and cocoa plantations into northern Bougainville from Madang, Gazelle Peninsula and Northern New Ireland (Firth, 1982). Future analysis of the phylogenetic relationships of local and global strains of P. palmivora is required to confirm whether the pathogen was indeed introduced into PNG together with the host material or whether indigenous strains adapted to new cocoa plantings.
The data presented in this study show that P. palmivora from cocoa in PNG forms a single, continuous largely asexual population. Although some diversity exists in the pathogen population, its extent was observed to be limited. This justifies the current programme of cocoa breeding for resistance or tolerance to P. palmivora in one location for distribution throughout the country. However, strict internal quarantine measures should be placed on movements of soil and plants, especially from Madang province where both mating types are present and diversity is relatively higher. A more comprehensive screening of P. palmivora isolates from ENB, including both mating types and representative isolates from other locations, is necessary to eliminate the risk of spreading new isolates to other regions along with planting material.
Acknowledgements
The scholarship provided by AusAid to undertake this study is highly appreciated. The authors acknowledge the assistance of CCIPNG staff of the Industrial Services Division with collecting isolates outside ENB, the Agronomy and Pathology staff for collecting isolates within ENB and the assistance provided by Dr Rose Daniel at the University of Sydney. Mating type reference isolates were generously provided by Professor André Drenth, University of Queensland, Australia.
