SUSTAINABLE COCOA
THE FUNGAL COMMUNITY COMPONENT

A contributed integrated pest management paper

Overview

Sustainable cocoa cultivation in the Neotropics is significantly challenged not only by the limits to production imposed by detrimental fungi, but by the costly and variably effective means currently employed to control fungal pathogens. Progress is being made in overcoming these challenges through a diversity of projects currently supported by the American Cocoa Research Institute, including a new approach to sustainable cocoa now underway at the Smithsonian Tropical Research Institute. Based in Panamá, our team of forest ecologists and evolutionary biologists currently is examining the myriad mutualistic and symbiotic fungi that occur naturally with rainforest trees. In isolating, recording, and cataloguing previously unknown fungal symbionts of tropical species, we have begun to understand the intricate ways in which naturally occurring, beneficial fungal symbionts may help keep plant diseases in check. Applying the principles and approaches we first designed in lowland forests to the chocolate tree, we are now focusing not only on the fungal pathogens that affect cocoa productivity, but on the ecological importance and diversity of mycorrhizal and endophytic fungal symbionts in natural forest, agroforest, and monoculture settings. In so doing, we hope to understand the interplay between beneficial and pathogenic fungi in and on cocoa trees, and to understand how the former might help keep cocoa diseases such as Moniliophthora (monilia), Phytophthora (black pod), and Crinipellis perniciosa (witches’ broom) in check. Here, I present an overview of our rationale and work to date.

Witches broom is new to Panama. Click on the thumbnail to see the full size picture.

The Research Context: The Fungal Community

Tropical forest trees interact with a remarkable diversity of fungal species. The exposed surfaces and interiors of living or dead roots, stems, leaves, flowers and fruits provide rich substrates for a variety of beneficial and antagonistic fungi, which interact directly and indirectly in their microhabitat on or within the host. Although little is known about the true scale of fungal diversity with which any tropical tree species has contact, a focused approach to the genetic and species diversity of tropical fungi is now underway at the Smithsonian Tropical Research Institute (Panamá). Researchers there are collecting, quantifying and manipulating the fungal symbionts, such as root inhabiting mycorrhizae, or leaf inhabiting endophytes, that interact with tree species in the lowland tropical forest understory and canopy. Understanding how pathogens or beneficial fungi might be harbored by conspecific, congeneric, or even unrelated trees, how fungal pathogens and mutualists might be specialized on the levels of locality, species, or genotype, and how fungal community dynamics might be influenced by temporal or spatial changes in forest structure, drive aspects of the larger question: what is the nature, and the ecological importance, of the fungal community in a tropical forest?

Our international and interdisciplinary collaborators have begun to apply these questions to the fungi that inhabit and interact with Theobroma cacao across the suite of forest and agricultural settings. Concentrating on mycorrhizae, fungal endophytes, and Oomycete pathogens (see below), we are seeking to assess, and then to comprehend, the patterns and processes that underlie the fungal communities we observe, as well as the ecological importance of these fungal groups, which to date have been little studied both in tropical forests in general, and especially in cacao.

Mycorrhizal Fungi

Arbuscular mycorrhizal associations (AM) between specialized Zygomycete fungi and host plants have been documented in the majority of terrestrial plant families. AM fungi grow around and within root tissues, may influence nutrient uptake, water flow, and other critical parameters of plant health, and may compete directly with invasive pathogens for root colonization space. Although the importance of mycorrhizae for plant growth and survival is well understood in some temperate zone plants, their roles are little known in tropical forests. For example, how many fungal species form mycorrhizal associations with a single host species, or a single individual host? How are mycorrhizal populations structured in space and time? How might a single mycorrhizal fungus species interact with different host species? What determines the composition of the mycorrhizal community of a forest tree like cacao? And how might the mycorrhizal community be impacted by nonsustainable agricultural practices, and how might this, in turn, influence cocoa productivity? Within the context of sustainable cocoa, could forest mycorrhizae be manipulated or enhanced to improve the productivity of forest grown cocoa?

We are accumulating evidence that AM fungal communities in tropical forests are composed of complex mixes of species, and, importantly, that individual fungal species can have a spectrum of effects on different hosts. We have found evidence for differences in mycorrhizal affinities for certain hosts, suggesting that host seedlings are differentially colonized by mycorrhizal fungi from different sources, and that there are differential effects on host growth depending on the source of the fungal inoculum. These effects appear to depend upon the AM species genotypes and community structure, their compatibility with host species, and on the environmental conditions in which the plant is growing, each of which has important implications for understanding how mycorrhizae might be important for sustainable cocoa cultivation. We are now in the preliminary stages of assessing each of these factors for cocoa mycorrhizae, with plans to expand our work in this area dramatically over the coming year.

Endophytic Fungi

Ascomycete fungi growing asymptomatically within aerial plant tissues such as stems and leaves have been found in every species of plant examined to date, including marine macroalgae, mosses, ferns, "gymnosperms", and herbaceous and woody angiosperms. Despite their ubiquity, however, their ecological importance is generally unknown. Endophytes, by definition, do not induce disease in their hosts, yet they do share space with, and may compete for resources with, pathogens that attack aerial tissues. How does a plant maintain a suite of fungal inhabitants without too dire a consequence for itself? Might endophytes be important in the susceptibility or resistance of hosts to pathogen infection?

In grasses, which have been relatively well studied, endophytic fungi of the Ascomycete family Clavicipitaceae act as mutualists with their host plants, deterring herbivores, enhancing host physiology, and in some cases, increasing resistance to fungal pathogens. These endophytes are closely related to pathogenic species, grow systemically, are maternally transmitted via seeds (or clonal reproduction of the host), and usually exist singly within the host tissues of a given species of grass. Relatively few workers have looked beyond the grasses in attempting to understand endophyte host symbioses, but recent examination of woody and herbaceous dicots has shown that these patterns are not consistent: endophytes of trees appear to be highly tissue specific within hosts, horizontally transmitted, and highly diverse within and among hosts, with many endophytic species coexisting in leaves and stems. We have found that endophytes are especially diverse in tropical trees, with individual leaves often containing more than a dozen endophytic morphospecies. Clearly, the interactions between fungal endophytes and their hosts will be far different in tropical trees than in grasses.

Cocoa leaves with endophytes.

Endophytes of tropical trees may act as parasites, as neutral inhabitants, or as mutualists, but such designations have been difficult in the past, as the costs and benefits of their presence in tree leaves are almost entirely unknown. In addition to collecting, counting, classifying, and vouchering the previously unexplored endophytes of cocoa in Panamá, we have begun to examine endophytic fungi for evidence of antipathogen activity. Corroborative evidence from leaves themselves, where endophyte and pathogen infection rates appear to be inversely related, suggests that endophytes may prove to be useful biocontrol agents for cocoa diseases in the field. Could naturally occurring fungal endophytes in agroforest and forest systems be manipulated to enhance host resistance? In the coming year, we hope to begin investigating this question directly.

Oomycete Pathogens

Much of the forest research carried out to date on Barro Colorado Island (BCI), Panamá, focuses on Oomycete caused diseases in lowland trees. Oomycetes, the fungus like group of pathogens including Phytophthora, the causal agent of black pod, occur frequently in the forests of Panamá. BCI, where access to well studied tropical forest and in situ microbiology labs are both available, presents an excellent arena for examining the spread, effects, and most importantly, the host specificity of such pathogens. Our work in the field, and lab, and through molecular analysis, has shown that infection of host plants by Oomycetes may be genotype specific, such that certain strains of pathogenic agents may be more virulent than others, and that given strains may coexist, and possibly compete, in natural systems. We hope to understand the specificity of Oomycetes both in terms of space and host species, and to examine, for example, the potential for shade tree species to harbor, or help defend cocoa from, these pathogenic organisms.

Research Methods

Study methods for mycorrhizae, endophytes, and Oomycete pathogens differ due to fundamental biological differences among them. AM mycorrhizae cannot be grown in culture, and thus are studied either directly, via counts and identifications of spores in soil, or by inference, using roots that are infected by given strains as inocula. Molecular methods are increasingly useful for assessing fungal diversity associated with root segments, as are staining methods used to document and quantify root infection. Mycorrhizal studies are by nature bioassays, involving living plant tissues as the theater for examining diversity, infection, and the importance of these fungi to growth and health of the host.

Endophyte cultures are kept in a herbarium.

In contrast, many fungal endophytes can be grown in axenic culture on plates in the laboratory. These fungi are isolated from fragments cut from surface sterilized leaves, stems, or fruits, such that the endophyte community associated with any given host part can be easily quantified. Once in isolation, the endophytes are organized into morphospecies according to a suite of hyphal, whole colony, and spore characters. The overall collection is maintained as a living herbarium, with preserved vouchers made of the many new species we encounter. Each morphospecies is recorded with a living and freeze dried tissue sample, a digital photograph of hyphae and of the whole colony, and a microscope slide bearing fruiting bodies. In order to integrate our many collections and to properly organize our samples, these data are included in an Internet accessible diversity database. At the University of Arizona, we now maintain several hundred fungal morphospecies isolated from cocoa, with hundreds more coming from co-occurring tree species in forest and agroforest settings.

Endophytic hypha next to a leaf vein.

Subsets of the endophytes are tested in vitro against witches’ broom, black pod (isolated in a similar manner from infected tissues), and monilia. We have found that roughly 30% of the morphospecies isolated and tested to date inhibit one or more of these pathogenic fungi. Our next steps include expanding the molecular methods we now use to examine Oomycete DNA to the endophytes, in order to assess our morphospecies groupings and the genetic diversity present within morphospecies; and beginning within plant bioassays, in which the actual costs and benefits to cocoa and other hosts are assessed, both in the presence and absence of pathogens.

Monilia infected cacao.

Collections and Study Sites

For both the pathogen and endophyte surveys, the critical first step is to make adequate collections from cocoa at a variety of sites. To date, we have collected several thousand fungal strains representing pathogens, soil fungi, and fruit/leaf endophytes from cocoa in five Panamanian locations, which include a range of habitats, disease regimes, and agricultural settings. Concurrently, we have established long term monitoring of tree health and production at four of those sites (Table I). On Barro Colorado Island, in central Panamá, cocoa grows in a feral state beneath a canopy of forest trees that has been untouched by humans for over five hundred years. At Plantation Loop, also in central Panamá, an abandoned, 80 year old plantation grows beneath dense secondary forest cover. In Bocas del Toro, on the Atlantic coast of western Panamá, cocoa is grown commercially in monoculture and in mixed species stands, but production has been severely limited by monilia infection. At the Instituto Nacional, in Herrera province, we are monitoring relatively healthy trees, while on the Atlantic coast, near Nombre de Dios, we have found evidence of infection by monilia, black pod, and witches’ broom. Notably, our record of witches’ broom appears to be the northernmost record to date, and it is clear that previous attempts to eradicate the disease from eastern Panamá have been unsuccessful.

Our research sites in Panamá.

Our collections represent a rich diversity of fungal strains. To date, our endophyte group has isolated >500 morphospecies of endophytes representing >800 endophyte strains, with an average of >12 fungal species occurring in each cocoa leaf sampled. This richness is typical for tropical trees, but is unexpected relative to temperate zone fungal diversity, and we will continue to explore the implications of this entirely unexpected biodiversity in the months to come. Table 1 lists a few of our sampling sites and gives an overview of the endophytes collected at each one; these data are preliminary, however, and represent the endophytes taken from only a very few (usually <10) leaves at each site. As our sampling becomes both broader and deeper, we will be able to compare the number of endophytes per leaf found in each site, or to examine taxonomic differences among the endophytes from one agricultural setting to another. Finally, we hope to relate these endophyte data to disease incidence and severity as our study progresses.

Table 1: A summary of endophyte samples taken to data, and an overview of sampling sites. Note that this overview represents only preliminary samples; we expect the endophyte richness at each site to be orders of magnitude greater than these values represent.

Site Diseases* Cultivation Type Sample Fungal Type Strains Morphospecies
Barro Colorado P Intact forest Healthy leaves Endophytes >240 >120
Bocas del Toro M, P All types Healthy leaves Endophytes >100 >60
Nombre de Dios M, P, WB Monoculture Healthy leaves Endophytes 272 221
Instituto Nacional TBA TBA Healthy leaves Endophytes 176 69
Plantation Loop M, P Forest canopy Healthy leaves Endophytes 39 20
Nombre de Dios M, P, WB Monoculture Fruits Mixed 68 32
Total         895 >522
*: M=monilia, P=Phytophthora, WB=witches’ broom.

Synthesis and Potential

Our collections of endophytes and pathogens, and our ability to manipulate mycorrhizal communities, set the stage for novel exploration of the ecological importance of each of these fungal types to cocoa grown in forest, agroforest, and monoculture sites. Concentrating on the spectrum of cocoa cultivation methods employed in Panamá, we are poised to examine how these major components of the fungal community associated with cocoa influence growth, productivity, and other interrelated indicators of plant health. In the coming year we will concentrate on equalizing our sampling among our main research sites, continuing our monitoring of cocoa tree health at each site, integrating our endophyte collections, and continuing our in vitro and in vivo assays examining the potential for these naturally occurring, diverse fungi to influence cocoa defense against disease. It is our hope that our understanding of cocoa symbionts, and the relationship of these symbionts to the agricultural setting in which cocoa occurs, will not only enhance our understanding of plant-fungus interactions in general, but directly influence the development of cocoa as both a conservation tool, and a sustainable crop.

Collaborative Team

Appreciations

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