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Keywords:

  • basidiomycetes;
  • biological control;
  • cocoa;
  • endophytes;
  • rDNA phylogeny;
  • Theobroma cacao

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Endophytic fungi were isolated from healthy stems and pods of cacao (Theobroma cacao) trees in natural forest ecosystems and agroecosystems in Latin America and West Africa. These fungi were collected for screening as a potential source of biocontrol agents for the basidiomycetous pathogens of cacao in South and Central America, Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom). Many of these isolates were morphologically unidentifiable as they failed to form fruiting structures in culture, or only produced arthrosporic stages. Affinities with basidiomycetes were suspected for many of these based on colony morphology. Fifty-nine of these morphologically unidentifiable isolates were selected for molecular identification by DNA extraction and sequence analysis of nuclear ribosomal DNA (rDNA). The large subunit (LSU) was chosen for initial sequencing because this region has been used most often for molecular systematics of basidiomycete fungi, and comprehensive LSU datasets were already available for sequence analyses. Results confirmed that the majority of the isolates tested belonged to the Basidiomycota, particularly to corticoid and polyporoid taxa. With LSU data alone, identification of the isolates was resolved at varying taxonomic levels (all to order, most to family, and many to genus). Some of the isolates came from rarely isolated genera, such as Byssomerulius, whilst the most commonly isolated basidiomycetous endophyte was a member of the cosmopolitan genus Coprinellus (Agaricales). The role of these fungi within the host and their potential as biological control agents are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chocolate is produced from the fermented and dried beans of the ‘chocolate tree’ (Theobroma cacao, Malvaceae), which has its origins in the tropical rain forests of Amazonia (Motomayor et al., 2002; Bartley, 2005). Cacao (cocoa) is now cultivated in most tropical regions throughout the world and is an economically important crop for smallholder farmers (Holmes et al., 2004). The main biological constraint to cacao production worldwide is fungal disease (Gotsch, 1997; Bowers et al., 2001). The dominant pathogens of cacao in Latin America are Moniliophthora roreri, causal agent of frosty pod rot (Evans, 1981) and the closely related Moniliophthora perniciosa (= Crinipellis perniciosa; Aime & Phillips-Mora, 2005), the causal agent of witches’ broom disease (Pound, 1938). In Latin America both diseases are still in an invasive phase (Evans, 2002) and conventional control methods have failed to halt their progress (Evans, 1981; Rubini et al., 2005). An effective mechanism of control is urgently required. One alternative strategy being investigated is that of biological control (Holmes et al., 2004).

Endophytic fungi exist asymptomatically within host plant tissues for at least part of their life cycle (Wilson, 1995), occupying leaves, stems and branches (Arnold et al., 2003; Photita et al., 2004; Suryanarayan & Thennarasan, 2004). In woody perennials they are thought to protect the plants in which they live by one or more mechanisms (antibiosis, mycoparasitism, induced resistance and/or competitive exclusion), and are thought to develop from environmental or background inoculum and are not transferred from generation to generation (Johnson & Whitney, 1992). Therefore, plants that have been removed from their natural environment and cultivated are thought to become depleted in their specific or coevolved endophytes (Taylor et al., 1999) and, as a result, may become more susceptible to pests and diseases.

In this study, fungal endophytes of T. cacao were isolated and identified as a potential source of novel biocontrol agents for M. roreri and M. perniciosa. In parallel, studies are being carried out to survey and assess the endophytes within another South American Theobroma species, T. gileri, the purported original forest host of the frosty pod rot pathogen (Evans et al., 2003b). A diverse range of endophytes, primarily anamorphic Hypocreales (Ascomycota) and basidiomycetes, were isolated from the woody stems and fruits of T. gileri (Evans et al., 2003b). These taxonomic assemblages differ from previous studies that found ascomycetes belonging to the Diaporthales, Dothideales, Pezizales and Xylariales to be the predominant endophytic groups (Carroll, 1988; Bills, 1996; Taylor et al., 1999; Arnold et al., 2001; Vujanovic & Brisson, 2002; Schulz & Boyle, 2005). However, the same profile of fungal endophytes is now being encountered in present studies of cacao as within T. gileri, i.e. predominantly anamorphic Hypocreales and basidiomycetes, with the greatest proportion of unidentified morphospecies belonging to the Basidiomycota.

This paper reports on the molecular identification of morphologically unidentifiable endophytic isolates from stems and pods of T. cacao in both forest and agro-ecosystems. Greater emphasis has been placed, at this time, on the classification of the basidiomycetous endophytes, as they occupy a similar ecological niche to that of the basidiomycetous pathogens of cacao (M. roreri and M. perniciosa) and therefore may be useful as a potential source of biocontrol agents.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Collection and isolation of endophytic fungi

Surveys for endophytes were carried out between 1999 and 2003, during which time samples were taken from cacao trees (T. cacao) in Latin America (Mexico, Costa Rica, Brazil and Ecuador) and West Africa (Cameroon), from both natural forest (Latin America) and cacao farms/germplasm collections (Latin America and West Africa). The method used for sampling the cacao trees and subsequent isolation of endophytes was that described previously by Evans et al. (2003a). In brief, a section of living tissue from the inner bark (approx. 8 × 6 cm) was removed from each tree at around chest height (∼1·5 m) using a machete and surface-sterilized by flaming with 90% alcohol. The cut surface was then pared further to clean it before 10 triangular slivers were excised from the inner bark using a scalpel blade (Swann-Morton blade no. 11). Each individual sliver was then transferred onto a plate of selective medium: five onto one-fifth strength potato dextrose agar (20% PDA) supplemented with 10 ml L−1 penicillin-streptomycin solution (Sigma P0781); and five onto malt extract agar (MEA) supplemented with 0·05 g L−1 chloramphenicol. Samples were also taken from cacao pods in the field by the method described above after sterilizing the pod surface by flaming with 90% ethanol. On return to the UK, the samples were incubated at 25°C and monitored over an 8-week period. As the fungal isolates began to emerge, hyphal tips or spores were transferred onto 20% PDA or potato carrot agar (PCA) and incubated at 25°C with a near-UV cycle to promote sporulation. Those isolates that failed to form fruiting structures in culture, or only produced arthrosporic stages, were chosen for molecular characterization. All isolates were stored as DIS codes at the CABI UK Centre (Tables 1 and 2). Isolates submitted to GenBank were also deposited in the CABI Genetic Resource Collection (IMI numbers).

Table 1.  Identification of Basidiomycota isolates based on large subunit (LSU) rDNA sequence data with geographic location, ecosystem and cacao tissue type from which they were collected, and GenBank accession number
Tentative ID based on phylogenetic analysis of LSU sequenceIsolate codeIMI numberGenBank numberGeographic locationEcosystemdTissue
  • a

    Centro Agronómico Tropical de Investigación y Enseñanza.

  • b

    Commissão Executiva do Plano da Lavoura Cacaueira.

  • c

    Empresa Brazileira de Pesquisa Agropecuária.

  • d

    Exotic, cultivated cacao (farm, germplasm collection) outside the centre of origin; exotic/forest, naturalized cacao in a forest habitat outside the centre of origin, e.g. Mayan cacao (brought from the Amazon, centuries, if not millennia ago) is now feral in Mexico and, seemingly, part of the indigenous forest ecosystem (Bartley, 2005); forest, wild cacao within the centre of origin, growing as an understorey tree; forest/exotic, cultivated cacao within the centre of origin, but outside the forest ecosystem.

Coprinellus sp. 1Dis 129aIMI 393905DQ327642Cabiria, CATIEa, Turrialba, Costa RicaExoticStem
Coprinellus sp. 2Dis 238aIMI 393906DQ327649Garzacocha, Rio Napo, Orellana Province, east EcuadorForest/exoticStem
Coprinellus sp. 2Dis 112i  Rio Añangu Orellana Province, east EcuadorForestStem
Coprinellus sp. 2Dis 222a  Rio Caoni, Puerto Quito, Pichincha Province, west EcuadorExoticStem
Coprinellus sp. 2Dis 233d  Rio Añangu, Orellana Province, east EcuadorForestStem
Coprinellus sp. 2Dis 251i  Mocache Road, Los Rios Province, west EcuadorExoticStem
Gloeosterioid sp.Dis 181cIMI 393907DQ327647CEPLACb, Medici Landia, Pará State, BrazilForest/exoticStem
Corticioid sp. 1Dis 296aIMI 393908DQ327656Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 1Dis 296c  Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 1Dis 296h  Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 1Dis 298c  Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 2Dis 168c  Almirante Cacau, Itabuna, Bahia State, BrazilExoticStem
Corticioid sp. 2Dis 168d  Almirante Cacau, Itabuna, Bahia State, BrazilExoticStem
Corticioid sp. 2Dis 168jIMI 393909DQ327645Almirante Cacau, Itabuna, Bahia State, BrazilExoticStem
Corticioid sp. 3Dis 296eIMI 393910DQ327657Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Phlebioid sp.Dis 178aIMI 393911DQ327646EMBRAPAc, Belém, Pará State, BrazilForest/exoticPod
Podoscypha sp.Dis 296fIMI 393912DQ327658Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 4Dis 125bIMI 393913DQ327639Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Corticioid sp. 5Dis 298eIMI 393914DQ327659Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Corticioid sp. 6Dis 245eIMI393915DQ327650Achidona, Napo Province, EcuadorForest/exoticStem
Corticioid sp. 7Dis 292 gIMI 393916DQ327655Mbalmayo, nr. Yaounde, Centre Province, CameroonExoticStem
Phanerochaete sp.Dis 267cIMI 393917DQ327652Rio Caoni, Puerto Quito, Pichincha Province, west EcuadorExoticStem
Corticioid sp. 8Dis 267bIMI 393918DQ327651Rio Caoni, Puerto Quito, Pichincha Province, west EcuadorExoticStem
Oxyporus sp.Dis 099cIMI 393919DQ327635Chajul, Rio Lacantun, Chiapas, MexicoExotic/forestStem
Corticioid sp. 9Dis 267eIMI 393920DQ327653Rio Caoni, Puerto Quito, Pichincha Province, west EcuadorExoticStem
Byssomerulius sp.Dis 233hIMI 393921DQ327648Rio Añangu, Orellana Province, east EcuadorForestStem
Inonotus sp.Dis 126eIMI 393922DQ327641Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Hymenochaetoid sp. 1Dis 140hIMI 393923DQ327643Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Hymenochaetoid sp. 1Dis 129b  Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Hymenochaetoid sp. 1Dis 131a  Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Hymenochaetoid sp. 1Dis 140b  Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Hymenochaetoid sp. 2Dis 109dIMI 393924DQ327636El Descanso, Rio Quincha – Rio Napo confluence, Orellana Province, east EcuadorForestStem
Lentinus sp.Dis 113eIMI 393925DQ327637Pañacocha – Panayacu Forest, Rio Napo, Orellana Province, east EcuadorForestStem
Polyporaceae sp. 1Dis 141dIMI 393926DQ327644Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Pycnoporus sp. 1Dis 343dIMI 393927DQ327660Maldonado, Pichincha Province, west EcuadorExoticPod
Pycnoporus sp. 2Dis 343fIMI 393928DQ327661Maldonado, Pichincha Province, west EcuadorExoticPod
Pycnoporus sp. 2Dis 343c  Maldonado, Pichincha Province, west EcuadorExoticPod
Polyporaceae sp. 2 (phylotype 1)Dis 126aIMI 393929DQ327640Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Polyporaceae sp. 2 (phylotype 1)Dis 260f  Caluma-Guaranda Road, Bolivar Province, west EcuadorExotic/forestStem
Polyporaceae sp. 2 (phylotype 2)Dis 124aIMI 393930DQ327638Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Polyporaceae sp. 2 (phylotype 2)Dis 124d  Cabiria, CATIE, Turrialba, Costa RicaExoticStem
Auriculariales sp.Dis 290eIMI 393931DQ327654Mbalmayo, nr. Yaounde, Centre Province, CameroonExoticStem
Table 2.  Identification of Ascomycota isolates based on large subunit (LSU) rDNA sequence data with geographic location, ecosystem and cacao tissue type from which they were collected, and GenBank accession number
Tentative ID based on blast analysis of LSU sequenceIsolate codeIMI numberGenBank numberGeographic locationEcosystemaTissue
  • a

    Exotic, cultivated cacao (farm, germplasm collection) outside the centre of origin; exotic/forest, naturalized cacao in a forest habitat outside the centre of origin, e.g. Mayan cacao (brought from the Amazon, centuries, if not millennia, ago) is now feral in Mexico and, seemingly, part of the indigenous forest ecosystem (Bartley, 2005); forest, wild cacao within the centre of origin, growing as an understorey tree; forest/exotic, cultivated cacao within the centre of origin, but outside the forest ecosystem.

Pleosporales sp.Dis 343gIMI 393932DQ327633Maldonado, Pichincha Province, west EcuadorExoticPod
Pleosporaceae sp.Dis 298dIMI 393933DQ327632Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem
Hypocreales sp. 1Dis 256bIMI 393934DQ327628Rio Vinces, Mocache-Vinces Road, Los Rios Province, west EcuadorExoticStem
Hypocreales sp. 2 (cf. Leucosphaerina sp.)Dis 267aIMI 393935DQ327630Rio Caoni, Puerto Quito, Pichincha Province, west EcuadorExoticStem
Hypocreaceae sp.Dis 110gIMI 393936DQ327622Rio Añangu, Orellana Province, east EcuadorForestStem
Clavicipitaceae sp.Dis 108hIMI 393937DQ327621El Descanso, Rio Quincha-Rio Napo, Orellana Province, east EcuadorForestStem
Bionectria sp.Dis 114eIMI 393938DQ327624Pañacocha – Panayacu Forest, Rio Napo, Orellana Province, east EcuadorForestStem
Nectriaceae sp. (cf. Stephanonectria sp.)Dis 098aIMI 393939DQ327619Chajul, Rio Lacantun, Chiapas, MexicoExotic/forestStem
Xylariaceae sp. 1Dis 190aIMI 393940DQ327625CEPLAC, Medici Landia, Pará State, BrazilExoticPod
Xylariaceae sp. 2Dis 343jIMI 393941DQ327634Maldonado, Pichincha Province, west EcuadorExoticPod
Xylaria sp. 1Dis 099aIMI 393942DQ327620Chajul, Rio Lacantun, Chiapas, MexicoExotic/forestStem
Xylaria sp. 2Dis 112oIMI 393943DQ327623Rio Añangu, Orellana Province, east EcuadorForestStem
Xylaria sp. 2Dis 255i  Rio Vinces, Mocache-Vinces Road, Los Rios Province, west EcuadorExoticStem
Xylaria sp. 3Dis 233aIMI 393944DQ327626Rio Añangu, Orellana Province, east EcuadorForestStem
Xylaria sp. 4Dis 255jIMI 393945DQ327627Rio Vinces, Mocache-Vinces Road, Los Rios Province, west EcuadorExoticStem
Xylaria sp. 5Dis 258gIMI 393946DQ327629Rio Vinces, Mocache-Vinces Road, Los Rios Province, west EcuadorExoticStem
Xylaria sp. 6Dis 298bIMI 393947DQ327631Mwellye, Idenao to Mbenge Road, Western Province, CameroonExoticStem

DNA extraction, PCR amplification and rDNA sequencing of endophytic morphospecies

Provenances of cultures are detailed in Tables 1 and 2. Hyphal tips from 59 isolates, each representing individual morphospecies groupings based on colony characteristics, were used to inoculate conical flasks containing 60 mL liquid glucose yeast medium (GYM; Mugnai et al., 1989), which were incubated at 25°C in an orbital shaker at 100 rpm for 5 days. The resulting mycelium was vacuum-filtered on filter paper (Whatman no. 3) with three washes in sterile distilled water. The mycelium was then freeze-dried before being ground in a pestle and mortar containing liquid nitrogen. The powdered mycelium was stored at −20°C until required. DNA was extracted from the mycelium of the individual isolates and resuspended in 50 µL−1 of rehydration solution (1% TE buffer) using a Promega Wizard Genomic DNA Purification Kit.

The first 1 kb of the 28S nuclear ribosomal large subunit (LSU) DNA was chosen for initial amplification and sequencing because this region has been used most frequently in basidiomycete systematics and comprehensively sampled LSU datasets are available for phylogenetic reconstruction and analyses (e.g. Moncalvo et al., 2002). Methods for PCR amplification and sequencing followed Aime & Phillips-Mora (2005). Sequences were deposited in GenBank (Tables 1 and 2).

Sequences obtained were initially blasted in GenBank (http://www.ncbi.nlm.nih.gov) to predict the family and/or order for each isolate. For closer phylogenetic placement, a data matrix of LSU sequences was then constructed in the following manner: (i) a skeletal LSU dataset was constructed by pruning that of Moncalvo et al. (2002) to exclude redundant taxa from lineages not related to any of the fungal endophytes as indicated by blast analyses; (ii) additional LSU sequences were then added to this dataset by including all close (> 97% similarity) blast results for the isolates; (iii) additional exemplar sequences were included from families and orders of Hymenomycetes to which blast analyses indicated the majority of endophytes had taxonomic affinities; and (iv) several heterobasidiomycete sequences were included as outgroups. GenBank accession numbers for additional sequences used in these analyses are shown on Fig. 1. Sequences were manually aligned in Se-Al: sequence alignment editor (Rambaut, 1996). The assembled dataset contained 192 taxa aligned across 928 bp; a total of 118 bp were considered too ambiguous to align confidently and were excluded from analyses. Maximum parsimony analyses were conducted in paup* 4·0b10 (Swofford, 2002) as heuristic searches with 100 random addition replicates and tree bisection-reconnection (TBR) branch swapping; gaps were coded as missing data. Names for the resulting fungal clades followed Moncalvo et al. (2002) and Lutzoni et al. (2004).

imageimage

Figure 1. Parsimony analysis of large subunit (LSU) rDNA sequences showing phylogenetic positions of endophytic basidiomycetes within the major lineages of homobasidiomycetes (tree length = 4177, CI = 0·184, RI = 0·603). Clade names for lineages are from Moncalvo et al. (2002) and Lutzoni et al. (2004). Bold type indicates fungal isolates cultured from inner bark of Theobroma cacao.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sequence data for the first 1 kb of the 5′ end of the 28S nuclear LSU gene for 59 fungal endophyte morphospecies were generated. blast analysis revealed that 42 of the isolates were Basidiomycota (Table 1), representing 27 different taxa; the remaining 17 isolates belonged to the Ascomycota and were not analysed further (Table 2).

Basidiomycota were identified by additional parsimony analyses of the basidiomycete sequence data. Of 810 included characters, 103 were variable but parsimony-uninformative and 337 were parsimony-informative. Analyses yielded a single most parsimonious tree [length = 4177, consistency index (CI) = 0·184, retention index (RI) = 0·603] in which all major clades of Basidiomycota were resolved (Fig. 1). Although the topology presented in Fig. 1 was supported by low bootstrapping values, all recovered clades were fully supported in the multigene analyses of Binder & Hibbett (2002) and Lutzoni et al. (2004). A single endophyte, DIS 181c, belonged to a clade containing two other taxa currently classified in the Meruliaceae, but which in these analyses appeared associated with the euagarics with weak support. All the endophytic basidiomycetes belonged to the Hymenomycetes; one, DIS 290e, with heterobasidiomycetous affinities, the remainder belonging to various lineages of homobasidiomycetes. Six of these isolates represented two different taxa of euagarics, both belonging to the genus Coprinellus, one of which was the most commonly isolated basidiomycetous endophyte in this study; the remainder of the homobasidiomycete isolates belonged to the polyporoid, hymenochaetoid and corticioid lineages. Interestingly, no endophytes were recovered from predominantly ectomycorrhizal russuloid, thelephoroid and boletoid lineages.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Of the 854 individual endophyte isolates from the woody tissue of stems or fruits of cacao, 556 isolates (65%) could not be identified on the basis of traditional taxonomic techniques and were grouped into 59 morphospecies. Morphospecies are artificial groupings that are thought not normally to reflect taxonomic relationships (Guo et al., 2003). However, Lacap et al. (2003) verified, on the basis of ribosomal DNA sequence analysis, the validity of morphospecies as taxonomic groups. Preliminary identification of the morphospecies in the present study showed that they comprised both Ascomycota and Basidiomycota. The Basidiomycota were the largest group, with 42 of the isolates (representing individual morphospecies), corresponding to 27 different taxa. The basidiomycetes were further characterized as a potential source of biocontrol agents that may occupy similar ecological niches as the basidiomycetous pathogens of cacao M. roreri and M. perniciosa. The 17 ascomycete morphospecies were not characterized further in this study. Within the Basidiomycota, all but one of the isolates belonged to the homobasidiomycetes, with Coprinellus sp. being the most commonly isolated endophyte.

There are limitations to the identification of the basidiomycetes and sterile mycelia using DNA (Guo et al., 2003; Promputtha et al., 2005; Wang et al., 2005). Even if similarities in sequences are high between an isolate and a reference sequence, sufficient data for full resolution are often unavailable. Many of the isolates presented here may be new species or even genera. However, in the absence of fructification it is nearly impossible to confirm the systematic placement of any homobasidiomycete. Additionally, even for a comparatively well-sampled group of fungi such as the homobasidiomycetes, LSU sequence data exist for perhaps less than 10% of known species. Thus, until more reference sequences are available, a confident generic determination for many of these isolates cannot be made. Sequencing of additional genes is under way to aid further identification of the basidiomycete isolates. Identification to genus will be important for determining which isolates may be potential biocontrol agents.

Other endophyte assemblage studies carried out have revealed unidentifiable fungi, as a result of lack of sporulation on artificial culture media (Promputtha et al., 2005; Wang et al., 2005). Many authors have disregarded these isolates and referred to them simply as ‘sterile mycelia’ or ‘unidentified’. Others have grouped such isolates into morphospecies (Fröhlich et al., 2000; Guo et al., 2003; Lacap et al., 2003), as was initially done in this study. Grouping in this way is a useful but limited tool, as comparisons cannot be made with other studies. Very few studies have used molecular techniques to identify these morphospecies further.

Basidiomycetes have only been identified in limited numbers in many of the endophyte studies undertaken, with the majority of endophytes being identified as ascomycetes or their anamorphs (Carroll, 1988; Sridhar & Raviraja, 1995; Bills, 1996; Wang et al., 2005). This could be because they have been overlooked, as most studies have focused on sporulating fungi only, or because of the type of plant tissues sampled. Few previous studies have targeted mature stems as a source of endophytes; other studies have isolated endophytes from leaves (Promputtha et al., 2005) and branches (Chapela & Boddy, 1988; Wang et al., 2005). More specifically, the sampling technique used may also influence the endophytes isolated. It is therefore difficult to estimate whether the number of basidiomycetes isolated from cacao in this study was greater than for other tropical hosts. Although most of the basidiomycetes belong to phylogenetic lineages comprised mainly of wood-rotting fungi, e.g. polyporoid and corticioid, it is thought that these fungi may have adopted an endophytic and asymptomatic phase in their life cycle, in which they exist as ‘active’ or quiescent colonizers before switching to a saprotrophic phase when the host tissues senesce. This would give them a clear temporal and spatial advantage over less ‘sophisticated’ wood-degraders.

The rationale for this study, of which this paper forms a part, was to compare and contrast endophytes from within and outside the native range of T. cacao in order to determine if there are any unique, potentially coevolved species that could be exploited as classical biocontrol agents for the control of cacao diseases (Evans et al., 2003b). In the case of ascomycetes, promising new species of Trichoderma have recently been described from wild cacao (Holmes et al., 2004; Samuels et al., 2006). However, from the data presented here, there appear to be no such clear and potentially exploitable differences in the basidiomycete assemblages between native and non-native cacao. Only Byssomerulius sp., Lentinus sp. and the gloeosterioid sp. warrant further investigation. Western and eastern (Amazonian) Ecuador are separated by the Andean Cordillera and it is not surprising therefore that there are marked differences between the basidiomycete colonizers. However, Coprinellus sp. 2, which was recorded from several locations in Amazonia, was also present in two distinct localities in western Ecuador (Table 1).

What is evident is that wherever cacao has been introduced, indigenous basidiomycetes have cryptically colonized the stems. In particular, from Costa Rica, where 15 cacao trees were sampled in a germplasm collection, data (unpublished) showed that the majority (65%) of endophytes isolated proved to be basidiomycetes representing a range of clades.

It is not surprising that nine of the isolates were identified as xylariaceous, as these are the most commonly isolated endophytes in tropical regions (Rodrigues & Petrini, 1997). In a similar molecular study by Guo et al. (2003), 13 of the 17 morphotypes sequenced were confirmed as members of the Xylariaceae with no basidiomycetes recorded.

Endophytic basidiomycetes could potentially be useful biocontrol agents of the main fungal diseases of cacao in Latin America, as the target organisms themselves are basidiomycetes with a distinct endophytic phase. One key mechanism by which these asymptomatic endophytic basidiomycetes could interfere with the activities of the pathogens is by competing for the same ecological niche. Such an interaction has been observed between Phlebiopsis gigantea and Heterobasidion annosum (Asiegbu et al., 2005). In this way, resident endophytes could prevent initial colonization or displace the invading pathogen. Basidiomycetes are also known to be prolific producers of bioactive metabolites that can be antagonistic towards fungi and other pathogens or pests (Ershova et al., 2001; Rosa et al., 2003; Zjawiony, 2004). It is possible that by utilizing these mechanisms the basidiomycetes in this study may be active as biocontrol agents. Further studies are required to determine their potential as biocontrol agents of the increasingly invasive and important basidiomycetous pathogens of cacao, M. roreri and M. perniciosa, in Latin America.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to the USDA-ARS for funding this study as part of the Alternative Crops Program, and in particular to Eric Rosenquist for his continued support. We also wish to thank Cindy Park, Malcolm DeCruise and Allison Kennedy for expert technical and laboratory assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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