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Diversity 2010, 2(4), 527-549; https://doi.org/10.3390/d2040527

Article
The Rhizosphere of Coffea Arabica in Its Native Highland Forests of Ethiopia Provides a Niche for a Distinguished Diversity of Trichoderma
Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9-1665, A-1060 Vienna, Austria
*
Author to whom correspondence should be addressed.
Received: 25 February 2010; in revised form: 24 March 2010 / Accepted: 24 March 2010 / Published: 5 April 2010

Abstract

:
The southwestern highlands forests of Ethiopia are the origin of the coffee plant Coffea arabica. The production of coffee in this area is affected by tracheomycosis caused by a soil-born fungus Gibberella xylarioides. The use of endemic antagonistic strains of mycoparasitic Trichoderma species would be a nature conserving means to combat this disease. We have used molecular methods to reveal that the community of Trichoderma in the rhizosphere of C. arabica in its native forests is highly diverse and includes many putatively endemic species. Among others, the putative new species were particularly efficient to inhibit growth of G. xylarioides.
Keywords:
biocontrol; coffee; diversity; DNA barcode; Ethiopia; Hypocrea; molecular phylogeny; rhizosphere

1. Introduction

Coffee (Coffea arabica L.) is a tropical crop, which today contributes 70% of the world’s commercial coffee market, and is currently grown in 75 countries with a total production close to 108 million ton per year [1,2]. For the African country of Ethiopia, coffee is the major export crop, accounting for over 60% of total value of exports [3]. It originated in the former Province of Kaffa in the southwest of the country [4,5,6] rendering Ethiopia the origin and center of infraspecific genetic diversity for C. arabica.
The production of coffee is today severely affected by fungal wilt diseases including tracheomycosis (coffee wilt disease) caused by Fusarium xylarioides Steyaert (teleomorph: Gibberella xylarioides Heim and Saccas) [7,8,9,10]. The disease is responsible for a reduction in the production of coffee beans and is also accompanied by severe damage and death of millions of coffee bushes [7].
Currently, attempts to control coffee wilt disease are fundamentally based on the breeding of resistant plants, plant and environmental management, and synthetic fungicides [11]. The high cost of pesticides, the emergence of fungicide-resistant pathogen biotypes and other social and health-related impacts of conventional agriculture on the environment have however recently led to an increased interest in agricultural sustainability and biodiversity conservation [12]. Thus, there is a need for new solutions of plant disease problems that provide effective control while minimizing cost and negative consequences for human health and the environment [13].
Biological control—i.e., the antagonism and eventual killing of plant pathogens by other living organisms, which are themselves not harmful to the plants—could present an attractive alternative for combating wilt disease. Species of the anamorphic genus Trichoderma (teleomorph: Hypocrea, Ascomycota) have been proven as effective biocontrol agents of soil-borne plant diseases [14,15]. Trichoderma would be especially suitable for combating coffee wilt disease because many of its species are rhizosphere competent [16,17], and the coffee roots are the first target for the attack by pathogens [18,19]. In support of this hypothesis, Trichoderma spp. have already been applied successfully to suppress Fusarium spp. causing Asparagus root rot [20], bean root rot [21], and carnation wilt [22].
Yet the antagonistic efficacy of a biocontrol agent in the field will also depend on the environment where it will be applied [23], and therefore—ideally—screening for appropriate biocontrol agents should be done in the same environment. This would also avoid the introduction of invasive microorganisms and conserve the microbial composition of the respective biotopes. To date, information about the diversity and abundance of Trichoderma in natural for coffee forest of Ethiopia is unknown.
The objective of this study was to survey the diversity of Trichoderma inhabiting the rhizosphere of C. arabica; and to compare two different coffee habitats, native populations and plantations in major coffee growing regions of Ethiopia with respect to their abundance and distribution of Trichoderma species.

2. Results

2.1. Habitats of C. arabica and the Sampling Strategy

C. arabica is a relatively small evergreen shrub (normally up to 5 m tall), which grows in shady places in subtropical forests. It usually inhabits the highland areas around 1500 m above the mean sea level, but can be found up to 2800 m. C. arabica can grow best on deep, free-draining, loamy soils, with a good water holding capacity and a slightly acid soil.
The four major coffee growing regions of Ethiopia lie in the south, south-western and south-eastern parts of the country corresponding to Wellega, Jimma, Hararghe and SNNP (Southern Nations, Nationalities and Peoples) regions, respectively (Figure 1). The samples were taken during the rainy season of 2006 (August) on the basis of a road survey. More than 160 soil samples were collected along the main roads every several hundred meters in a way that (with the exception of Hararghe) both undisturbed native forests and disturbed semi-forests were equally represented. The latter ecosystem is common in Ethiopia. It is formed by coffee growers, when they intentionally thin out the forest in a way that the coffee plants get more sunlight but at the same time still have enough of large trees to provide adequate shade. In addition in semi-forests farmers also regularly slash the weeds to facilitate harvesting of the coffee beans. In extreme cases the artificial semi-forest may form after the primary burning of the native forest and subsequent introduction of fast growing large trees, which provide shade to coffee shrubs. The native forest is not disturbed by any anthropogenic pressure. Neither coffee plantations nor gardens were sampled.
As wild coffee inhabits a specific ecological niche it is not surprising that the soils sampled from the rhizosphere of C. arabica are similar in their physicochemical properties (Table 1). The representative samples from four studied regions were similar in carbon (approximately 2.3% in average) and nitrogen (approximately 0.3%) content. The pH of soil solutions was around 5 reaching a maximum of 6.5 in samples from the native forest in Hararghe.
Table 1. Description of sampled areas and physical and chemical properties of soils.
Table 1. Description of sampled areas and physical and chemical properties of soils.
RegionEcosystemCodeAlt. range (m)Soil propertiesNo. of samplesNo. of Trichoderma isolates
ColorTextureC%N%pH
Jimmanative forestJF1320–2150Reddish brownSandy loam2.70.274.803026
semi-forestJC1370–2400Reddish brownSilt loam2.70.64.722927
Welleganative forestWF1500–2300Reddish brownSilt loam2.40.344.971513
semi-forestWC1570–2400Reddish brownSandy loam1.60.244.675311
SNNPnative forestSF1650–2050Reddish brownSandy loam2.30.266.011314
semi-forestSC1670–2080Reddish brownSilt loam2.30.324.811237
Hararghenative forestHF2150Reddish brownSandy loam2.50.236.5322
semi-forestHC1580–2350Reddish brownSandy loam20.194.0164

2.2. Occurrence of Known Trichoderma Species

There were 134 isolates of Trichoderma recovered from the four major coffee growing regions of Ethiopia (Appendix 1; Figure 1). Among them, 54 came from the rhizosphere of coffee plants in forest soil, whereas 80 were obtained from C. arabica rhizosphere from semi-forests. Among these, 11 and 15 of the native (forest) and disturbed (semi-forest) soil samples respectively, were from plants infected with wilt disease (the presence of the disease was not visible at the time of sampling but later discovered in the laboratory).
Figure 1. Location of the main coffee growing areas (green areas) and the corresponding diversity of Hypocrea/Trichoderma in the rhizosphere of C. arabica. The purple line corresponds to Wellega, blue to Jimma, red for the SNNP region and yellow for the Hararghe region. The orange circles indicate the main commercial coffee plantation sites in Ethiopia (Jimma). The diversity of Hypocrea/Trichoderma is shown by separate pie plots (disturbed semi-forest and native forest, respectively) for all coffee growing areas except Hararghe, which was undersampled. The number in the center of each plot indicates the total number of Trichoderma strains isolated from each area. Species are abbreviated as T. harzianum sensu stricto (hz), ‘pseudoharzianum matrix’ (phz), T. hamatum (hm), Hypocrea atroviridis clade E (at E), H. atroviridis clade D (at D), T. asperelloides (as), T. spirale (sp), T. longibrachiatum (lg), T. gamsii (ga), and H. koningiopsis (kp), all potentially new species are numbered as “T. sp.” followed by the corresponding strain number. The world map inset on the upper right side of the image shows the location of Ethiopia in the “coffee belt” where the light green indicates C. arabica production areas, and the dark green shows C. robusta.
Figure 1. Location of the main coffee growing areas (green areas) and the corresponding diversity of Hypocrea/Trichoderma in the rhizosphere of C. arabica. The purple line corresponds to Wellega, blue to Jimma, red for the SNNP region and yellow for the Hararghe region. The orange circles indicate the main commercial coffee plantation sites in Ethiopia (Jimma). The diversity of Hypocrea/Trichoderma is shown by separate pie plots (disturbed semi-forest and native forest, respectively) for all coffee growing areas except Hararghe, which was undersampled. The number in the center of each plot indicates the total number of Trichoderma strains isolated from each area. Species are abbreviated as T. harzianum sensu stricto (hz), ‘pseudoharzianum matrix’ (phz), T. hamatum (hm), Hypocrea atroviridis clade E (at E), H. atroviridis clade D (at D), T. asperelloides (as), T. spirale (sp), T. longibrachiatum (lg), T. gamsii (ga), and H. koningiopsis (kp), all potentially new species are numbered as “T. sp.” followed by the corresponding strain number. The world map inset on the upper right side of the image shows the location of Ethiopia in the “coffee belt” where the light green indicates C. arabica production areas, and the dark green shows C. robusta.
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The ITS1 and 2 oligonucleotide barcode program TrichOKey [24] identified 104 of the 134 isolates at the species level. Eight known species were found: T. harzianum sensu lato and T. hamatum were most abundant (30 and 35 isolates respectively), followed by T. asperelloides (11 isolates), T. spirale (eight isolates), H. atroviridis/T. atroviride (six isolates) and H. koningiopsis/T. koningiopsis (five isolates). T. gamsii and T. longibrachiatum, were represented by four and three specimens respectively. While ITS1 and 2 sequences are sufficient for distinguishing the majority of Hypocrea/Trichoderma species [24], they have only limited power to differentiate at the infraspecific level. As all identified species are very common and known to be cosmopolitan, it is interesting to learn whether the isolates from Ethiopia represent also common haplotypes of these species, which are already known from other geographic regions. In order to learn this, we sequenced the hypervariable 4th long intron of the elongation factor 1-alpha (tef1) of the corresponding strains. Comparison of the resulting sequences with those of the Database of Industrially Important Fungi of Vienna University of Technology and the NCBI GenBank databases showed that the tef1 sequences of T. longibrachiatum, H. koningiopsis/T. koningiopsis and T. gamsii strains were identical to those of strains known from Europe or South America (data not shown). Isolates of T. harzianum sensu lato also covered a large part of the genetic diversity known for this complex taxon, although most strains were identical or highly similar to strains from Africa (Cameroon, Egypt). The majority of strains belonged to the network of recombining holomorphic strains known as the ‘pseudoharzianum matrix’ [25], while at least four isolates were attributed to the new putative agamospecies T. sp. ‘afroharzianum’ [25] and a single isolate C.P.K. 1818 resembled the ex-type strain of T. harzianum CBS 226.95 (i.e., T. harzianum sensu stricto), which is usually found in a temperate climate [26].
Eleven isolates were initially identified as T. asperellum. However this species has been recently reconsidered as an aggregate of two morphological cryptic species T. asperellum s. s. and T. asperelloides [27]. Phylogenetic analysis of tef1 intron and the detection of the diagnostic SNP of ITS2 of the rRNA gene cluster resulted in the attribution of Ethiopian isolates to T. asperelloides (Figure 2), which is frequent in Africa.
A different situation was encountered with the other three species: strains of T. hamatum exhibited three tef1 alleles, which formed a clade separate from all other known T. hamatum strains in the phylogenetic analysis (data not shown). Most of the strains identified as H. atroviridis/T. atroviride clustered in H. atroviridis clade D [28], forming a terminal branch shared by an isolate from Nepal (C.P.K. 300). One isolate (C.P.K. 2622), although belonging to the H. atroviridis/T. atroviride clade, formed a basal single branch, which cannot be attributed to any clades specified Dodd et al. [28] (Figure 2).
A high, as yet unnoticed genetic diversity was found for T. spirale: a phylogenetic tree of tef1 sequences resulted in the formation of several statistically supported clades with the ex-type strain CBS 346.93 forming a separate lineage (Figure 3). Six of the eight Ethiopian T. spirale isolates formed a so far unique terminal clade remote from the ex-type strain, whereas one (C.P.K. 2606) was located within a closely related subclade and one formed a single lineage which occupies an unresolved position on the T. spirale s. l. cladogram.
Figure 2. Identification phylogram of Trichoderma diversity from rhizosphere of C. arabica in a vicinity of H. atroviridis/T. atroviride and T. asperellum inferred from a Bayesian analysis of tef1 intron alignments. The position of the potentially new species is shown in red, while hypothetically endemic populations of known species are marked by blue. Nodes with black circles indicate posterior probabilities >0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and/or strain number. Infraspecific groups and species names are given above branches leading to the corresponding node.
Figure 2. Identification phylogram of Trichoderma diversity from rhizosphere of C. arabica in a vicinity of H. atroviridis/T. atroviride and T. asperellum inferred from a Bayesian analysis of tef1 intron alignments. The position of the potentially new species is shown in red, while hypothetically endemic populations of known species are marked by blue. Nodes with black circles indicate posterior probabilities >0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and/or strain number. Infraspecific groups and species names are given above branches leading to the corresponding node.
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Figure 3. Identification phylogram of Ethiopian tef1 alleles (in blue) of T. spirale based on a Bayesian analysis. Nodes marked by black circles indicate posterior probabilities >0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and the strain number.
Figure 3. Identification phylogram of Ethiopian tef1 alleles (in blue) of T. spirale based on a Bayesian analysis. Nodes marked by black circles indicate posterior probabilities >0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and the strain number.
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2.3. Occurrence of Potentially Endemic Trichoderma Taxa

Thirty (22.6%) of the isolates were identified as eight new alleles of ITS1 and 2 by TrichOKey and thus may constitute new, undescribed species. One of them (T. sp. C.P.K. 1833) accounted for the majority of isolates (16), whereas five of the others putative taxa were encountered only as single specimens. Five, three and two isolates were found for T. sp. C.P.K. 2707, T. sp. C.P.K. 1837 [29] and T. sp. C.P.K. 2612, respectively. Trichoderma sp. C.P.K. 1837 was recently shown to be a new phylogenetic species closely related to H. orientalis and T. longibrachiatum [29] of Trichoderma section Longibrachiatum. For the other putative new species hallmark sequences suggested that they are new members of Trichoderma section Pachybasium and section Trichoderma.
In order to test the hypothesis that these are new phylogenetic species, we also sequenced the tef1 fragment from these 30 isolates and used them for an analysis together with sequences of the most closely related reference species. Figure 4A shows that T. sp. C.P.K. 1828 is a member of the Brevicompactum clade [30], forming a separate branch between T. arundinaceum and H. rodmanii, and the four isolates of T. sp. C.P.K. 2707 form a sister branch to T. helicum (Figure 4B).
Figure 4. Identification phylogram of a potentially new species within the Brevicompactum clade (a) and a potentially new species related to T. helicum (b) based on the Bayesian analysis of tef1 intron. Nodes marked by black circles indicate posterior probabilities > 0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and strain number.
Figure 4. Identification phylogram of a potentially new species within the Brevicompactum clade (a) and a potentially new species related to T. helicum (b) based on the Bayesian analysis of tef1 intron. Nodes marked by black circles indicate posterior probabilities > 0.94. All reference strains are given by the accession number of the corresponding tef1 sequence in GenBank and strain number.
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The remaining five putative new species were located within the Harzianum clade (Figure 5): one branch leading to the most abundant T. sp. C.P.K. 1833 and the single lineage corresponding to T. sp. C.P.K. 2670 formed a sister clade to the T. cerinum–T. tomentosum species pair. The two isolates of T. sp. C.P.K. 2612 formed an isolated branch basal to all other species. And the remaining two hypothetical new species—T. sp. C.P.K. 1812 and C.P.K. 1807 were located on basal branches to the clades containing e.g., Trichoderma species causing green mould disease of Pleurotus (T. pleuroticola and T. pleurotum) and Agaricus (T. aggressivum) respectively (Figure 5). The single putative new species of section Trichoderma (T. sp. C.P.K. 2725) formed a basal branch to H. atroviridis as shown in Figure 2.

2.4. Habitat Preference of Trichoderma

In order to assess the richness of Trichoderma in the rhizosphere of C. arabica, we calculated Shannon’s biodiversity index H and evenness E, as well as the Simpson’s biodiversity index (Table 2). Simpson’s index and the evenness index were close to 1, indicating very high diversity and lack of dominance of any species. When the indexes are calculated separately for cultivated and uncultivated areas it becomes apparent that the diversity and evenness is equal in both ecosystems.
The high diversity was also reflected in the distribution of Trichoderma species. With the exception of species which were only obtained as single isolates, all other species were encountered from more than one area indicating that they may be evenly distributed within the coffee rhizosphere. Yet some remarkable exceptions could be seen: T. sp. C.P.K. 1833 was mostly isolated from semi-forest soils and T. sp. C.P.K. 2707 was only observed in the native forest. We also note that T. hamatum was particularly abundant in Jimma province, both in semi-forest as well as native forest rhizosphere (Figure 1).
Figure 5. Distribution of Ethiopian Trichoderma strains within Harzianum clade as inferred from a Bayesian analysis of tef1 intron showing the position of potentially new species (red) and hypothetically endemic populations (blue). Nodes marked by black circles indicate posterior probabilities >0.94. GenBank accession numbers of the corresponding tef1 sequence of the reference strains are given in [25].
Figure 5. Distribution of Ethiopian Trichoderma strains within Harzianum clade as inferred from a Bayesian analysis of tef1 intron showing the position of potentially new species (red) and hypothetically endemic populations (blue). Nodes marked by black circles indicate posterior probabilities >0.94. GenBank accession numbers of the corresponding tef1 sequence of the reference strains are given in [25].
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In order to learn whether there would be any ecological preference of individual species, we performed a correlation analysis of the occurrence of strains with habitat specific properties such as altitude, pH, and the carbon and nitrogen content of the soils. Undisturbed forest soils in SNNP and Hararghe areas had a higher pH values (6.0–6.5) compared to other locations. Nevertheless, results from these analyses showed that there was no correlation between occurrence of any Trichoderma species and any of factors monitored in this study. Only exception was the observation that cosmopolitan species which were also known from other geographic regions and continents appeared as better adapted to grow on soils poor in nitrogen and carbon. For example, occurrence of T. asperelloides was significantly higher on such soils (ANOVA, P < 0.05).
Table 2. Diversity of Trichoderma in major Ethiopian coffee growing regions.
Table 2. Diversity of Trichoderma in major Ethiopian coffee growing regions.
Index AverageEcosystemSampled regions
ForestSemi-forestWellegaJimmaSNNPHararghe
Shannon diversityH2.472.342.292.142.192.151.56
Shannon evennessEH'0.810.830.830.930.790.840.97
SimpsonD0.880.870.870.90.840.860.93

2.5. Antagonistic Activity of Trichoderma Isolates against Gibberella xylarioides

In order to test whether the isolates of this study would be applicable as agents of biological control against plant pathogenic fungi (biocontrol agents), we tested a subset of randomly selected strains against an indigenous strain of G. xylarioides causing the coffee wilt in Ethiopia. Table 3 shows that all 10 isolates of Trichoderma tested were in fact able to inhibit the growth of G. xylarioides in vitro (PIRG between 55% and 76%). The hypothetically new taxon T sp. C.P.K. 2612 showed the highest antagonistic activity in this test. Interestingly, three strains (T. sp. C.P.K. 1817, H. atroviridis/T. atroviride C.P.K. 2622, and T. sp. C.P.K. 1834) also produced a zone of inhibition indicative of the formation of secondary metabolite(s) inhibiting G. xylarioides.

3. Discussion

Rhizosphere competence—i.e., the ability to competitively colonize plant roots—has often been emphasized as a prerequisite for strains of genus Trichoderma to act as biocontrol agents (cf. [14]). However, to the best of our knowledge, the issue whether Trichoderma spp. prefer the rhizosphere as an ecological niche in soil has never been systematically addressed. In this study, we show that the rhizosphere of a C. arabica exhibits a notably high diversity of Trichoderma taxa. In fact, a correlation analysis failed to detect any other factor which appeared to influence species richness and distribution, and we thus conclude that the ecological-physiological constraint put by C. arabica ecological niche is the main parameter determining the presence of Trichoderma.
The major coffee growing regions of Ethiopia constitute one of the 34 biodiversity hotspot areas of the world [31]. In total they comprise 80% of the landmass of the Afromontane biodiversity hotspot that is inhabited by more than 700 plant species. The rhizosphere of C. arabica exhibited one of the highest richness of Trichoderma species detected so far (cf. [32,33,34]). The only study which may be comparable in sampling size relative to the studied area was performed in neotropical forests of South America, mainly in Colombia [34]. In that study the exclusively high diversity of Trichoderma (29 species detected among 183 isolates) was also accompanied by a high fraction of putatively new species (11 species corresponding to 6% of the sample). However the study of Hoyos-Carvajal et al. [34] included strains isolated from a great diversity of ecological niches and substrata and therefore the observed species richness likely reflects the overall microbial biodiversity potential for neotropical regions. The principal difference of our findings is that we studied a limited and well defined microecological niche of rhizosphere of C. arabica. In another investigation of Trichoderma diversity based on a cultivation-dependent approach on Sardinia [32], around 500 Trichoderma strains have been isolated from different non-rhizosphere soil samples. The Shannon index calculated for all samples from the rhizosphere of C. arabica in Ethiopia is nearly two times higher compared to the same statistics calculated for soils in Sardinia (2.47 versus 1.59 respectively), although that sample was three times larger as compared to the present study. As Sardinian ecosystems were found to be seriously disturbed by various human activities and while in Ethiopia we sampled the largely undisturbed areas, our current results demonstrate that Trichoderma diversity may be used as an ecological indicator of ‘soil health’.
Table 3. Ability of selected Trichoderma strains isolated from rhizosphere of C. arabica to inhibit G. xylarioides.
Table 3. Ability of selected Trichoderma strains isolated from rhizosphere of C. arabica to inhibit G. xylarioides.
Isolate No.Species PIRG1Clear Zone
C.P.K. 2612T. sp. C.P.K.261276a-
C.P.K. 2614T. sp. C.P.K.261272b-
C.P.K. 1808T. sp. C.P.K.183364c-
C.P.K. 2619T. sp. C.P.K.183362c-
C.P.K. 1817T. sp. C.P.K.183762c1.5
C.P.K. 2698T. hamatum60c-
C.P.K. 2622H. atroviride56d2
C.P.K. 1819T. asperelloides56d-
C.P.K. 1888T. spirale 55d-
C.P.K. 1834T .sp. C.P.K.270755d1.5
F. xylarioides (Control)0-
1 See the explanations in the Experimental Section.
The samples isolated from the rhizosphere of coffee plants also contain more of putatively new taxa than any other previous study (28% of all isolates; eight known species versus nine potentially new taxa; compare with Migheli et al. [32], Zachow et al. [33] Hoyos-Carvajala [34] or Kullnig et al. [35]). No new species have been detected among 500 isolates from soils in Sardinia. Although our identification of new taxa was based only on ITS1 and 2 sequence analysis and tef1 phylogeny, and this claim still needs to be verified by analysis of additional loci, we consider this procedure reliable. In all our previous studies, prediction of new taxa by a combination of ITS1 and 2 and tef1 polymorphisms has later on always been confirmed by multiloci phylogeny (e.g., [35,36]).
One of these potentially new species (T. sp. C.P.K. 1833) was remarkable as it was the third most frequent taxon sampled in this study (16 isolates), while at the same time mainly being recovered from C. arabica rhizosphere growing in semi-forests. Comparison with our database revealed that strains of this putative species have been isolated previously from soil in Siberia ([35], erroneously identified as T. oblongisporum), Guatemala [24], Slovakia and Kenya (unpublished data), and thus it is likely cosmopolitan. Phylogenetically, this species appears to be closely related to T. tomentosum and T. cerinum. The fact that it was so abundant in Ethiopia and that its phylogenetic clade was even accompanied by a basal branch to a further potentially new taxon (T. sp. C.P.K. 2670) may suggest that T. sp. C.P.K. 1833 has an East African origin. Its high antagonistic ability renders it a potential candidate for biological control trials, because it has been isolated from disturbed forests.
The diversity of known species was dominated by T. harzianum, T. hamatum, T. spirale and T. asperelloides. All of these species are regarded as the most frequent representatives of the genus in soil and known to be cosmopolitan. They all have been also abundantly detected in Sardinian soils. We therefore consider them as ecological opportunists, which have very efficient abilities in conidial dispersal and germination. Their presence could thus be interpreted to be due to invasion. However, T. spirale is more frequently isolated from tropical environments [24,34,35,36], and also T. asperelloides has been reported to be abundant in South America and Cameroon [27,37]. In addition, most strains of T. harzianum sensu lato, for instance, T. sp. ‘afroharzianum’ [25] also belonged to tef1 phylogenetic clades that were either rich in tropical isolates or being exclusively African.
In this study, we also tested the hypothesis that Trichoderma taxa hypothetically endemic to the rhizosphere of C. arabica would exhibit antagonistic activity against the coffee pathogen G. xylarioides. Although we randomly selected only a small subsample of our isolates for these tests and made only in vitro dual confrontation assays, the results performed verify this hypothesis. Moreover, in planta experiments, performed with different varieties of C. arabica, gave similar results (T. B. Mulaw, unpublished data). The fact, that the putative new taxa were particularly efficient antagonists, supports the above hypothesis for enrichment of these taxa in the coffee rhizosphere. Selecting biocontrol strains by looking for isolates associated with the respective plant may therefore be a method best suited for ecosystems with a largely preserved microbial flora.

4. Experimental Section

4.1. Soil Sampling and Characterization

At each sampling site, a visually healthy adult C. arabica plant was located and a sample was taken within its rhizosphere area. The top layer of a soil litter and the upper soil horizon (4–6 cm) was discarded, and 100 g of soil from approximately 10 cm depth was collected, placed in polyethylene bags and labeled. Afterwards, similar samples from one subarea (geographic region, type of growth of C. arabica, latitude and tracheomycosis state of the forest) were merged in several larger samples, which were subsequently sieved and dried on sterile paper for 2–3 days. Thereafter, they were stored at 4 °C until isolation of Trichoderma strains occurred.
The soil color was defined using a standard color scale for soil science (Munsell Soil Color Charts, U.S. Dept. of Agriculture). All chemical analyses were performed using the fine earth fraction. To measure pH, 1 g of soil was suspended in 100 mL of 1M KCl, and after shaking for one hour pH was determined with a glass electrode. The total nitrogen content was determined according to the Kjeldahl method [38] on a Vapodest 30 (Gerhardt, Germany). The total organic carbon content was measured using the Liechtenfelder method [38], which oxidizes carbon with potassium dichromate, and quantifies the generated Cr3+ photometrically (DIN 19684).

4.2. Strain Isolation and Purification

10 g of soil samples (if necessary pulverized by means of a mortar and pestle, and passed through a 0.5 mm soil screen mesh to remove large debris and root fragments; [39] ) were suspended in 90 mL sterile distilled water and thoroughly mixed. A 10 mL aliquot was then used to prepare a series of dilutions in the range of 10–1 to 10–3, and inoculated on to potato dextrose agar (PDA), malt extract agar (MEA) and synthetic low nutrient agar (SNA) [40], each supplemented with streptomycin (50 mg/L) to prevent bacterial growth. Three replicates were done for each medium and soil sample. Plates were incubated at 25 °C for a period of 10 days, and examined daily for colony development. Putative Trichoderma colonies, indicated by their extensive fluffy white mycelium and distinctively green sporulation, were collected, and used to prepare single spore cultures. They were maintained in 50% (w/v) glycerol at −80 °C in the Collection of Industrially Important Microorganisms of Vienna University of Technology, Austria.

4.3. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted from mycelia grown on MEA with the Plant DNeasy Minikit (QIAgen GmbH, Hilden, Germany) according to the manufacturer’s instructions. A region of nuclear DNA containing the ITS1 and two regions of the rRNA gene cluster, was amplified by PCR with the primer combinations SR6R and LR1 [40], and an approximately 1-kb portion of the gene encoding translation elongation factor 1-alpha (tef1) was amplified and sequenced using the primers EF1 (5’-ATGGGTAAGGA(A/G)GACAAGAC-3’) and EF2 (GGA(G/A)GTACCA GT(G/C)ATCATGTT-3) as described by Jaklitsch et al. [40]. PCR products were purified with the QIAquick PCR Purification Kit (QIAgen) and were subjected to automated sequencing at MWG (Martinsried, Germany). The sequences obtained in this study have been deposited at GenBank; their accession numbers are given in Appendix 1.

4.4. Identification of Trichoderma

For species identification, ITS 1 and 2 sequences were subjected to analysis by TrichOKey (http://www.isth.info/tools/molkey/index.php; [24]). In ambiguous cases, the result was re-checked by sequence analysis of the large intron of tef1 using a sequence similarity search against a database of type sequences implemented in TrichoBLAST (www.isth.info/tools/blast, [41]). For analysis of unusual ITS1 and 2 or tef1 alleles, sequences were automatically aligned with ClustalX [42] and visually checked using Genedoc 2.6.002 [43]. Potentially unique alleles were then confirmed by BLAST analysis against NCBI GenBank, and the database of Collection of Industrially Important Microorganisms of Vienna University of Technology, Austria which currently contains more than 3500 Hypocrea/Trichoderma strains with more than 5800 sequences of phylogenetic marker loci.

4.5. Molecular Phylogenetic Analysis

A multiple sequence alignment file was formatted with PAUP*4.0b10, and manually adapted for the MrBayes v 3.0B4 program [44,45,46]. The model of evolution and prior settings for individual loci were used as estimated for different taxa of Hypocrea/Trichoderma [47]. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) sampling was performed with four incrementally heated chains that were simultaneously run for 1,000,000 and 3,000,000 generations. To check for potentially poor mixing of MCMCMC, each analysis was repeated several times. The convergence of MCMCMC was monitored by examining the value of the marginal likelihood through generations. Convergence of substitution rate and rate heterogeneity model parameters were also checked. Bayesian posterior probabilities (PP) were obtained from the 50% majority rule consensus of trees sampled every 100 generations after removing the 500 first trees.

4.6. Antagonism of Trichoderma Isolates against Gibberella xylarioides

Ten different isolates of Trichoderma were individually tested for their antagonistic property against G. xylarioides using the dual culture technique. Agar pieces of 6 mm diameter of G. xylarioides and Trichoderma isolates were taken from pure cultures 7 days old, and placed on plates of potato dextrose agar (3%, Merck, Germany) in a distance 6 cm between G. xylarioides and the Trichoderma strain. Plates were incubated at room temperature for 5 days. Five plates were prepared for each isolate. Plates inoculated with G. xylarioides alone served as control. Clear zone of inhibition (CZI) was also determined by measuring the clearance between the colony margins of the G. xylarioides and Trichoderma. Radial growth of both G. xylarioides and Trichoderma were measured after 5 days after inoculation.
The percentage of inhibition of radial growth (PIRG) was calculated as ([R1-R2]x100)/R1, where R1 is the colony diameter of the pathogen in the control and R2 is the diameter of the pathogen during antagonistic interaction. Antagonism was assessed in semi-quantitative means [48]: >75 PIRG indicating very high antagonistic activity, 61–75 PIRG indicating high antagonistic activity, 51–61 PIRG, indicating moderate antagonistic activity, <50 PIRG, indicating low antagonistic activity, and 0 indicating no activity.
The data were analyzed using SPSS version 17.0 software.

4.7. Statistical Analysis

The Shannon biodiversity index H was used to evaluate the species diversity, which appears as the product of evenness E and the number of species [49]. It measures the likelihood that the next individual will be the same species as the previous sample. Given a sample size with many (more than 5) species, a value near 0 would indicate that every species in the sample is the same, whereas a value near or 4.6 would indicate that the number of individuals is evenly distributed between the 5 species. Dominance of individual species was also measured by Simpson's diversity index [50], using formula D= 1 − ∑ini (ni − 1)i / N(N − 1), where ni represents specimens of a species and N represents the total number of species. This index reflects the probability that two individuals randomly selected from a sample will belong to different species.

5. Conclusions

Our data strongly support the speculation that the C. arabica rhizosphere in Ethiopia is a hotspot for speciation of several Trichoderma spp. The putative Ethiopian endemic nature of several of the new taxa and populations encountered in this study would be consistent with an allopatric speciation scenario. Yet this usually occurs by geographic isolation [51], and although it is possible that the East African highland could present a barrier causing such isolation, the cosmopolitan nature of many of the taxa found in this study argues against this possibility. Therefore, we rather suppose that the association with rhizosphere of C. arabica, which is native for the region, considerably contributed to this speciation. C. arabica is known to display a high genetic diversity in Ethiopia [52] and Tanzania [53]. We consider it possible that this genetic diversity has given rise to new Trichoderma populations and taxa capable of establishing themselves in the rhizosphere of a genetically variable plant. It will thus be interesting to look for Trichoderma populations outside the rhizosphere of C. arabica or/and to compare rhizosphere samples from Tanzania, as well as in areas growing other coffee species (for instance, C. robusta) and/or genetically less variable cultivars of Arabica coffee such as Indonesia or Central America [54].
Given that this investigation is the first of its kind in coffee growing areas of Ethiopia, and that studies on the wild C. arabica-associated microbial community are generally lacking, there is high demand for further research in this field. It is also recommended that further studies be conducted to determine microbial communities using both culture-dependent and culture-independent techniques to reveal the true picture of Trichoderma diversity in such specific microbial communities.

Acknowledgements

This work was partly supported by the Austrian Science Fund grants FWF P-17859 to I.S.D and by OeAD 894/07 North-South Dialogue Program, doctorate scholarship to T.B. Mulaw. The authors are grateful to Monika Komon-Zelazowska (Vienna University of Technology, Austria) for her laboratory assistance and to Vera Terekhova (Moscow State University, Soil Science Faculty, Russia) for her help with the soil analyses.

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Appendix

Appendix 1. Strains of Trichoderma isolated in this study and NCBI GenBank accession numbers of their sequences.
Appendix 1. Strains of Trichoderma isolated in this study and NCBI GenBank accession numbers of their sequences.
RegionEcosystemAlt. (m)SpeciesNo. of IsolatesStrain No.GenBank accession No.
C.P.K.ITS 1 and 2tef1
Wellegadisturbed semi-forest2500T. sp. ‘afroharzianum’22618FJ412028FJ436158
2400 2617FJ412059FJ436157
2250T. harzianum sensu lato32620FJ412029
2500 2616FJ412026
2800 2615FJ412025
1900T. asperelloides11839FJ412074FJ436141
2800H. atroviridis/T. atroviride clade E12622FJ412094FJ436160
1570T. hamatum21826FJ411979FJ436130
2400 2613FJ411986
2500T. sp. C.P.K.261212614FJ412013FJ436156
2400T. sp. C.P.K.183312619FJ412060FJ436159
native forest1750T. harzianum sensu lato52610FJ412024FJ436154
1900 1825FJ412017FJ436129
1750 32611FJ412027
1750 2608FJ412023
1750 2609FJ412032
1750T. spirale22606FJ412078FJ436153
1750 2607FJ412079
1560T. asperelloides21893FJ412049FJ436148
1500 1838FJ412047FJ436140
1950H. atroviridis/T. atroviride clade D12605FJ412093FJ436152
2300T. hamatum11827FJ411980
1750T. sp. C.P.K.261212612FJ412011FJ436155
1740T. sp. C.P.K. 183711837FJ412090FJ436139
Hararghedisturbed semi-forest2300T. sp. ‘afroharzianum’11840FJ412018FJ436142
2100T. asperelloides11829FJ412048FJ436132
2080T. sp. C.P.K.182811828FJ412085FJ436131
1580T. sp. C.P.K. 183711841 FJ436143
native forest2150T. harzianum sensu lato22623FJ412030
2150 2624FJ412031
Jimmadisturbed semi-forest1950T. harzianum sensu lato12673FJ412038FJ436175
2200T. harzianum sensu stricto11818FJ412016FJ436123
1750T. harzianum sensu lato52664FJ412034
1800 2506FJ412020
1850 2507FJ412021
1950 2672FJ412037
1590 2684FJ412036
1660T. spirale11888FJ412075FJ436144
1650H. atroviridis/T. atroviride clade D22662FJ412095FJ436170
1700 2663FJ412096FJ436171
1360H. koningiopsis/T. koningiopsis21816FJ412100FJ436121
1850T. gamsii12508FJ412103FJ436151
1600T. hamatum82682FJ411994
1500 2686FJ411996
1500 2687FJ411997FJ436180
2400 1810FJ411977FJ436116
1770 1814FJ411981FJ436119
1500 2685FJ411995FJ436179
1680 1811FJ411978
2000 2505FJ411985FJ436150
1330H. koningiopsis/T. koningiopsis11831FJ412091FJ436133
1950T. longibrachiatum11815FJ412086FJ436120
2300T. sp. C.P.K. 183711817FJ412089FJ436122
2150T. sp. C.P.K.180711807FJ412014FJ436113
2250T. sp. C.P.K.183331808FJ412055FJ436114
2300 1809FJ412056FJ436115
1800 2671FJ412069
native forest2100T. harzianum sensu lato32510FJ412022
1750 2504FJ412019
1700 2668FJ412035
1670T. spirale 32674FJ412071FJ436176
1880 2679FJ412072FJ436178
1800 2502FJ412076FJ436149
1800H. atroviridis/T. atroviride clade D11832FJ412092FJ436134
1950H. koningiopsis/T. koningiopsis11813FJ412099FJ436118
1750T. hamatum112499FJ411982
1880 2678FJ411991
1900 2680FJ411992
1780 2675FJ411989
1600 2500FJ411983
1700 2501FJ411984
1800 2667FJ411987
1640 2669FJ411988
1780 2676FJ411990
1900 2681FJ411993
1950 1835FJ436137
1960T. sp. C.P.K.183311833FJ412058FJ436135
2150T. sp. C.P.K.181211812FJ412015FJ436117
1720T. sp. C.P.K.267012670FJ412107FJ436174
1830T. sp. C.P.K.270732677FJ412083FJ436177
1600 2666FJ412082FJ436173
1800 1834FJ412080FJ436136
SNNPdisturbed semi-forest2070T. sp. ‘afroharzianum’12632FJ412033FJ436162
2000T. harzianum sensu lato12718FJ412039
1750T. spirale 22694FJ412077FJ436184
1670 1822FJ412073FJ436127
1670T. asperelloides71836FJ412046FJ436138
1750 2692FJ412051FJ436183
2000 2722FJ412053FJ436191
1750 2697FJ412052FJ436185
1750 1819FJ412044FJ436124
1700 1820FJ412045FJ436125
1750 2688FJ412050FJ436181
1750H. atroviridis/T. atroviride clade D12690FJ412097FJ436182
1780H. koningiopsis/T. koningiopsis11823FJ412102FJ436128
2000T. gamsii12723FJ412106FJ436192
1750T. hamatum102689FJ411998
1750 2700FJ412004
1750 2703FJ412007
1750 2691FJ411999
1750 2695FJ412000
1750 2696FJ412001
1750 2698FJ412002
1750 2699FJ412003
1750 2702FJ412005
1750 2704FJ412006
1800T. longibrachiatum21889FJ412087FJ436145
1770 1890FJ412088FJ436146
1690T. sp. C.P.K.1833112645FJ412068FJ436169
2000 2719FJ412070FJ436190
2200 2634FJ412063FJ436163
1750 1892FJ412057FJ436147
1850 2626FJ412061
1970 2630FJ412062FJ436161
2200 2636FJ412064FJ436164
2260 2637FJ412065FJ436165
1790 2641FJ412066FJ436166
1690 2642FJ412067FJ436167
1700 2643 FJ436168
native forest1850T. harzianum sensu lato52710FJ412040FJ436189
1900 2727FJ412012FJ436194
1850 2712FJ412042
1850 2711FJ412041
1900 2728FJ412043
2000T. asperelloides12724FJ412054
1750H. koningiopsis/T. koningiopsis11821FJ412101FJ436126
1650T. gamsii22706FJ412104FJ436186
2000 2709FJ412105FJ436188
1650T. hamatum32705FJ412008
2050 2715FJ412009
2050 2717FJ412010
2050T. sp. C.P.K. 272512725FJ412098FJ436193
1950T. sp. C.P.K.270712707FJ412084FJ436187
Bold font indicates potentially new species.
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