Previous studies have investigated the diversity of endophytic fungi associated with coffee88,89,90,91,92, but these were based on surveys restricted to the Americas and Hawaii, where coffee is an exotic introduced species. The endophytic mycobiota found in these studies is dominated by genera such as Colletotrichum, Fusarium, Penicillium, Pestalotia and Xylaria. Such assemblages consist mainly of opportunistic endophytes—seemingly, of little biological significance to their hosts93—with Trichoderma appearing only infrequently. Only one study involved sampling of all the coffee tissues (leaf, berry, stem, root system)89 and, of the 843 isolates obtained, only four were identified as belonging to the genus Trichoderma. Conversely, and in sharp contrast, the Coffea samples from Africa in this study yielded 76 endophytic isolates of Trichoderma from the aerial plant tissues of a relatively small sample size, with a highly diverse taxonomic range, including four new species. At this stage, it is not possible to determine whether the new taxa described herein are geographically restricted to Africa or even to coffee. Nevertheless, we find it significant that a far richer diversity of Trichoderma was found in association with coffee in its African centre of origin compared to that elsewhere, especially in the Neotropics. We also find particularly relevant the complete absence of endophitic Trichoderma species isolates amongst the plethora of fungal isolates obtained from our sampling in semi-wild situations in Brazil. This was entirely unexpected and may indicate the existence of a ‘Trichoderma void’ in the coffee endophyte mycobiota outside of Africa.
The occurrence of Trichoderma in association with C. arabica has been reported previously in Ethiopia15,94, but these studies focused on strains isolated from the rhizosphere and root tissues. The isolates included: T. harzianum sensu lato, T. hamatum, T. asperelloides, T. spirale, T. atroviride, T. koningiopsis, T. gamsii and T. longibrachiatum. Only three of these taxa were isolated during our study of stems, leaves and berries: namely, T. hamatum, T. spirale and T. koningiopsis. These are cosmopolitan species that are frequently isolated from tropical habitats, especially from soil6,7,60,95. Certain Trichoderma species were isolated from more than one plant tissue type: T. koningiopsis and T. spirale from the leaves and stems of C. canephora; T. hamatum from the stems and berries of C. arabica. T. hamatum, T. koningiopsis and T. spirale have also been reported as endophytes in other tropical woody plants, notably cacao and rubber5. Nevertheless, only T. hamatum had previously been reported as endophytic in C. arabica; occurring in the root system15,94. Apart from the four novel species described here, other Trichoderma species were found for the first time as endophytes in coffee: T. atroviride, T. guizhouense, T. breve and T. theobromicola. These species were known from other habitats, such as: tropical soils; decaying wood and bark; as mycoparasites; on mushroom compost; in leaf-cutting ant colonies; and as endophytes in Theobroma spp. (Malvaceae)4,5,20,96,97. Trichoderma guizhouense has a worldwide distribution, mainly in soil, and had only been reported previously as an endophyte in the endemic woody liana, Ancistrocladus korupensis, and in the stems of Cola trees in primary forest in south-west Cameroon4. Previously, T. theobromicola was known only from South America, and reported to be a common endophyte in sapwood of cacao4,22,79 whilst T. breve, a recently described species isolated from soil, was previously known only from northern China12. These two species are new geographical and host records for Africa, but this may simply reflect the poor sampling of Trichoderma in the region, particularly for endophytes. The results of the surveys also suggest that many species of Trichoderma are either cosmopolitan or pantropical.
Mycoparasitism—the ecological relationship where one fungus parasitizes another98—has now been reported for a number of species of Trichoderma, notably: T. atroviride, T. hamatum, T. longibrachiatum, T. reesei and T. virens, and it has recently been established that mycoparasitism is an ancestral trait of the genus19,99 Mycoparasitic Trichoderma spp. have a wide range of hosts, including true fungi, such as Botrytis cinerea, Rhizoctonia solani, Alternaria alternata and Fusarium spp., as well as Oomycetes, such as Pythium ultimum49,98. However, the species found as mycoparasites of H. vastatrix reported here—T. aggressivum, T. andinense, T. parareesei, T. petersenii and T. pseudopyramidale—are the first in the genus to be reported attacking the Hemileia rusts associated with coffee. Three of the species of Trichoderma obtained during the surveys are well-known mycoparasites, but were found here only growing as endophytes in coffee, namely: T. atroviride, T. hamatum and T. virens. Trichoderma pseudopyramidale may deserve special attention as a potential biocontrol agent of CLR, since it was the most common mycoparasitic species obtained from both Cameroon and Ethiopia (77.8% of total mycoparasites). In Ethiopia this species was commonly associated with a purported new species of Hemileia (cf. H. coffeicola) on wild C. arabica in cloud forest (ca. 2000 m). It was also frequently isolated as an endophyte from the leaves and stems of both semi-wild and wild C. arabica in Ethiopia (see Table 1). It may encompass dual roles as an endophytic bodyguard of coffee and also as a contact mycoparasite of CLR.
Mycoparasitic fungi associated with coffee rust have been studied n regions of the world where coffee is not a native species, such as in Mexico100. It is interesting to note that this Mexican survey identified six purported mycoparasites: Acremonium byssoides, Calcarisporium ovalisporum, C. arbuscula, Fusarium pallidoroseum, Sporothrix guttuliformis and Verticillium (= Akanthomyces lecanii). A more recent publication reporting the results of an investigation in Mexico and Puerto Rico, involving the use of single-molecule DNA sequencing of fungal rRNA gene barcodes to identify putative mycoparasites in pustules of H. vastatrix, yielded 15 fungal taxa associated with CLR, none of which belonged to Trichoderma101. Information on the ecology of the new Trichoderma species described here, and their role in nature, is limited because relatively few strains of each species were isolated during the survey; the exceptions being T. botryosum and T. pseudopyramidale, which constituted over 60% of the total isolations and seem to have a close association with their Coffea hosts, in both Cameroon and Ethiopia, in wild, semi-wild and cultivated situations.
The aim of the present study was to collect and catalogue endophytes of Coffea species—as well as the mycoparasites of the associated Hemileia rusts—in their African centres of origin, as part of a project to screen and assess these isolates as potential biological control agents of CLR. The target area is Central America where the rust has become a critical constraint to coffee production, as well as causing a socio-economic crisis, over the past decade53,102,103. The work presented here covers only the taxonomy with some observations on the ecology of the Trichoderma isolates resulting from the surveys in Africa, but these data will be pivotal for selecting candidate biocontrol agents for the potential management of H. vastatrix in the Americas.
The philosophy behind the overall project is based on the concepts of classical biological control and, in the case of CLR, on the Enemy Release Hypothesis which posits that exotic species become invasive and achieve pest status because of increased fitness in the absence of their coevolved natural enemies104. One solution to address the problem of invasive alien pests is to source, import and release coevolved natural enemies from the centres or regions of origin of the target species in order to reduce ‘pest’ fitness: the classical biological control strategy. This approach using fungal natural enemies, such as entomopathogens and plant pathogens, has been employed successfully to control invasive alien arthropod pests and weeds105,106, but never against alien plant diseases using mycoparasites. The evidence from our study indicates that there is a guild of Trichoderma species, potentially antagonistic to H. vastatrix in Africa, which could be exploited for biological control of CLR in Central America following the classical approach. There are claims that non-specific, indigenous mycoparasites; notably, Lecanicillium lecanii—now Akanthomyces lecanii107—can reduce the impact of CLR in the Americas108, but this is not evident based on the continuing rust outbreaks.
Another scenario has been suggested to further explain the invasiveness of alien plant species: the Endophyte-Enemy Release Hypothesis109, which posits that alien plants arrive not only without their coevolved natural enemies but also deficient in, or completely lacking, coevolved endophytes, some of which may be acting as symbionts (‘bodyguards’); protecting their hosts against adverse abiotic and biotic factors. Thus, in their absence, exotic crops thrive and alien weeds invade, with no natural enemies reducing plant fitness and fecundity and no bodyguards to ‘pay’ for protection. In crop species, the consequences can be catastrophic when coevolved natural enemies—lacking their own natural enemies, such as mycoparasites (in the case of fungal pathogens)—eventually catch up with their endophyte-deficient plant hosts. Such may be the case with H. vastatrix in Central America—and, of course, this may explain the devastating rust epiphytotics that destroyed coffee cultivation in Sri Lanka (Ceylon) in the nineteenth century, as well as in all the global regions where the rust has invaded110.
Thus, the ideal classical biological control agent for CLR would combine the best of both worlds in the form of an endophytic mycoparasite, and—as our results indicate—the genus Trichoderma contains such candidates. Potentially, these would not only be used to colonize the coffee leaf and parasitize the external rust pustules—as well as to target the invasive, intercellular mycelium of the rust—but also to bolster host defences through induced resistance66,84,111. There is increasing evidence that, in addition to induced resistance to diseases and pests, endophytic Trichoderma species confer a range of other benefits to their plant host, in particular, drought tolerance, resistance to abiotic factors such as salt stress and growth stimulation5,21,23,27,28,30,32,36,43,71,72,81,85,86,87,112.
Preliminary data, using Trichoderma isolates from the survey, are showing positive results in the laboratory with evidence of reduction in rust disease severity113 (Authors, unpublished). Greenhouse screening of four isolates of Trichoderma (COAD 2418, COAD 2417, COAD 2535 and COAD 2439), belonging to T. hamatum and T. pseudopyramidale sp. nov.., showed their ability to inhibit the germination of H. vastatrix urediniospores above 70% in vitro. Isolate COAD 2396 (T. atroviride) reduced the severity of the disease to less than 50% of the levels observed in the controls when applied before or simultaneously with H. vastatrix on coffee leaf discs. In addition, an isolate of T. parareesei (COAD 2482) promoted the growth and increased the biomass of tomato roots by 33% and 57%, respectively; whilst others are now showing the ability to increase drought tolerance113 (Authors, unpublished).
The methodology employed during the survey for the isolation of endophytes has proven to be robust. It has been emphasized previously that endophyte isolation is a method-dependent process and this will determine the quality and quantity of fungi obtained114. In our experience, isolating in situ—directly in the field from tree stems—or immediately after collection, eliminates or reduces contamination by many of the opportunistic endophytes and favours the slower-growing, potentially obligate endophytes. This has consistently been demonstrated not only during the present coffee survey in Africa, but also from previous surveys of wild species of Theobroma and Hevea in South American rainforests where this approach was pioneered20,36,39 These surveys resulted not only in the discovery of numerous new Trichoderma taxa—which are still being described4,41—but in many other taxonomic novelties, including new endophytic lineages of Tolypocladium and a new class of Pezizomycotina115,116. Moreover, they reveal the paucity of endophytes in cultivated exotic plants—in this instance, cacao and rubber—compared to wild populations of Theobroma and Hevea in natural ecosystems39,41,117,118,119. This has been confirmed during the present study, when a survey of coffee endophytes in four states of Brazil, failed to isolate any species of Trichoderma, providing compelling evidence that centres of origins or diversity of plants harbour unique guilds of endophytic Trichoderma species—as well as other genera—that could be exploited not only for classical biological control but also as potential reservoirs of novel metabolites.
In conclusion, our surveys in Africa for endophytes and mycoparasites associated with the genus Coffea and with its Hemileia rusts have revealed a highly diverse range of fungi, with many novel species; Trichoderma being just one component. Because of the relatively few countries (3) and localities (18) visited, and the restricted number of host plants sampled, this can only be viewed as a snapshot of the actual diversity of endophytes, as well as of mycoparasites, associated with Coffea in Africa, especially in forest ecosystems. Potentially, in Madagascar, where the diversity of the genus is richer with 59 confirmed species120, this still-untapped diversity could be even higher. Loss of forest habitats in Africa and Madagascar means that many of these fungi will go extinct, along with their host plants, before being described. The potential loss of such key antagonists of the CLR fungus—as well as of Coffea germplasm—should be cause for concern to coffee stakeholders.
