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Article

Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and Their Potential Use in Biocontrol of Coffee Wilt Disease

by
Afrasa Mulatu
1,
Negussie Megersa
2,
Tariku Abena
3,
Selvaraju Kanagarajan
4,
Qinsong Liu
5,
Tesfaye Alemu Tenkegna
1,* and
Ramesh R. Vetukuri
4,*
1
Department of Microbial, Cellular and Molecular Biology, Addis Ababa University, Addis Ababa P.O. Box 1176, Ethiopia
2
Department of Chemistry, Addis Ababa University, Addis Ababa P.O. Box 1176, Ethiopia
3
National Agricultural Biotechnology Research Center, Ethiopian Institute of Agricultural Research, Addis Ababa P.O. Box 249, Ethiopia
4
Department of Plant Breeding, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden
5
Key Laboratory of Southwest China Wildlife Resources Conservation, Ministry of Education, College of Life Science, China West Normal University, Nanchong 637009, China
*
Authors to whom correspondence should be addressed.
Crops 2022, 2(2), 120-141; https://doi.org/10.3390/crops2020010
Submission received: 1 April 2022 / Revised: 12 April 2022 / Accepted: 22 April 2022 / Published: 26 April 2022
(This article belongs to the Special Issue Molecular Variability of Crop Pathogens)
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Abstract

:
The present study investigated the distribution status and biodiversity of Trichoderma species surveyed from coffee rhizosphere soil samples from Ethiopia and their potential for biocontrol of coffee wilt disease (CWD) caused by Fusarium xylarioides. Trichoderma isolates were identified based on molecular approaches and morphological characteristics followed by biodiversity analysis using different biodiversity indices. The antagonistic potential of Trichoderma isolates was evaluated against F. xylarioides using the dual confrontation technique and agar diffusion bioassays. A relatively high diversity of species was observed, including 16 taxa and 11 undescribed isolates. Trichoderma asperellum, T. asperelloides and T. longibrachiatum were classified as abundant species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. Trichoderma asperellum was the most abundant species (comprising 39.6% of all isolates) in all investigated coffee ecosystems. Shannon’s biodiversity index (H), the evenness (E), Simpson’s biodiversity index (D) and the abundance index (J) were calculated for each coffee ecosystem, revealing that species diversity and evenness were highest in the Jimma zone (H = 1.97, E = 0.76, D = 0.91, J = 2.73). The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. In vitro confrontation experiments revealed that T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The potential use of these Trichoderma species for disease management of F. xylarioides and to reduce its impact on coffee cultivation is discussed in relation to Ethiopia’s ongoing coffee wilt disease crisis.

1. Introduction

Trichoderma species are widely found in different soil types, ecosystems and climatic zones and categorized based on their metabolic, physiological and genetic diversity features [1]. They are economically significant because of their functions as primary decomposers, producers of antimicrobial compounds and enzymes, and are used as biocontrol agents against diverse phytopathogens [2,3,4,5]. Many research studies have revealed that Trichoderma species inhibit the growth of phytopathogens through mycoparasitism, antibiosis and competition for niches and nutrients [6]. In addition, some Trichoderma species have beneficial effects on plants resulting from plant growth promotion, solubilization of soil micro- and macronutrients [7] and activation of plant systemic resistance [8,9]. To date, studies on Trichoderma diversity have mainly been conducted in Asia, Europe and America [10]; there have been few investigations into the diversity and distribution of Trichoderma in Africa, with the exception of some studies targeting specific ecological niches [11,12]. In particular, there has been only one published study on Trichoderma species inhabiting coffee plants, which focused on species isolated from the rhizosphere of C. arabica in Ethiopia [13]. In contrast to the previous report, the present study covers a broad range of geographical regions with three coffee production systems, viz., garden coffee, semi-forest coffee and forest coffee.
Morphological characterization and distinction were first used by Rifai [14] and later by Bisset [15,16,17,18] to investigate the diversity and evolution of Trichoderma species. However, species identification and delimination based on morphology alone are very difficult, making such approaches unreliable and subjective [19]. A more reliable approach is molecular phylogenetic analysis based on DNA sequencing data; over 375 Trichoderma species have been validly described and characterized in this way [20]. Reliable phylogenetic information is also important for studying the diversity of secondary metabolites of Trichoderma species. Consequently, molecular biological analysis is essential for the accurate identification of Trichoderma [21]. The internal transcribed spacer (ITS) is a widely used “universal” fungal molecular barcode [22,23]. However, it has low species resolution in the genus Trichoderma [24]. Therefore, the sequence of translation elongation factor 1-alpha (TEF1-α) was recommended as an alternative molecular barcode for the phylogenetic analysis of this genus [24].
Trichoderma species stand out among rhizospheric microorganisms due to their high biocontrol potential and their ability to facilitate nutrient uptake by plants while also protecting phytopathogens [25]. To maximize their beneficial effects on crop plants, it is essential to evaluate the functional and structural diversity of Trichoderma species found in specific agro-climatic conditions. The rhizosphere of coffee exhibits particularly high diversity with a wide range of putative Trichoderma species and is a hotspot for the evolution of this genus [13]. Trichoderma species have been extensively studied and used as biocontrol agents against diverse plant pathogens, including bacteria [26,27], fungi [28], oomycetes [29] and nematodes [30] for many different crops and agro-climatic conditions [31].
Ethiopia is the center of origin for Arabica coffee (Coffea arabica L.) and hosts a large germplasm diversity. It is also Africa’s largest coffee producer and the world’s fifth-largest coffee exporter, with a forecasted production of 457,200 metric tons (MT) in 2021/2022, having a value in excess of USD 900 million [32,33]. Coffee cultivation provides a livelihood for around 25 million people [34,35], accounting for 25–30% of total export incomes [36]. In addition to the worldwide reputation of Ethiopia’s genetic resources, coffee plays a major role in the national economy and the livelihoods of approximately 4.5 million coffee farmers [37,38]. Despite its leading position in coffee cultivation in Africa, the Ethiopian coffee sector is underachieving due to the rise of various fungal and bacterial diseases, and these pressures are predicted to increase with climate change [39,40]. During the last decade of the 20th century, coffee wilt disease (CWD) caused by Fusarium xylarioides became the principal production constraint for Arabica coffee in Ethiopia, Uganda, the Democratic Republic of the Congo (DRC) and Tanzania [40]. The yearly coffee yield loss due to CWD in Ethiopia is estimated to be 30–40% [40,41,42,43]. CWD incidence is greatly affected by the farming system, with much higher rates in garden and plantation coffee. CWD has been conventionally managed by uprooting and burning the affected coffee plant and using resistant varieties [44].
The potential use of Trichoderma species for plant pathogen control is now well-documented, although this approach is largely unexploited for many diseases of tropical perennial crops. Therefore, given the importance of coffee in Ethiopia’s national economy, the damaging nature of CWD, the limited availability of resistant crop lines and the lack of information on the biocontrol of CWD, a study on the potential of Trichoderma species to suppress the growth of F. xylarioides is needed to identify new genomic resources for management of this pathogen. Screening the biodiversity of different coffee ecosystems and the ecophysiology of Trichoderma species from a genomic perspective and analyzing their diversity will provide important insights into the potential value of Trichoderma for controlling CWD in the future.
The prospect of influencing coffee rhizospheres by inoculating potential Trichoderma species to control CWD and enhancing coffee growth and health was studied substantially under laboratory, greenhouse and field conditions (Mulatu A., unpublished data). However, the reduced efficiency of biocontrol agents under field conditions is hindered due to their ability to adapt to local biotic and abiotic environmental conditions. To understand this phenomenon, it is necessary to study the biocontrol agents’ geographical distribution and habitat preference in the rhizosphere. Hence, the present investigation was undertaken to study the distribution and biodiversity patterns of Trichoderma species in major coffee-growing regions of Ethiopia to assess their potential as biocontrol agents of CWD.

2. Materials and Methods

2.1. Collection of Soil Samples and Isolation of Trichoderma Species

Trichoderma isolates were collected from ten major Ethiopian coffee-growing areas (Jimma, Kaffa, Benchi Maji, Sheka, Bunno Bedele, Bale, Sidama, Gedio, West Wollega and West Guji) in different agro-climatic zones. Trichoderma isolates were obtained from coffee rhizosphere soil gathered during surveys conducted between May 2016 and August 2017. The surveys covered all major coffee-growing areas of Ethiopia’s southern, western and southwestern regions. The upper surface soil litter (4–6 cm) was discarded during soil collection, and 200 g soil samples were collected from a depth of approximately 10–15 cm. Over 184 soil samples were obtained from 28 districts (categorized under 10 zones) along the main roads (Figure 1 and Table S1). The soil samples were placed in sterile polyethylene bags, transported to the laboratory and processed immediately. The strains were isolated using Trichoderma Specific Medium (TSM) according to previously reported methods by Gil et al. [45] and Saravanakumar et al. [46] and purified by subculturing on potato dextrose agar (PDA). Fusarium xylarioides (DSM No. 62457, strain: IMB 11646), the causative agent of coffee wilt disease [9,47,48], was used as a test pathogen to evaluate the biocontrol potential of Trichoderma species.

2.2. Morphological Characteristics

The Trichoderma isolates were characterized based on their morphology by growing them on PDA at 28 ± 2 °C for 5 days following the protocol described by Samuels and Hebbar [49]. The Trichoderma colonies were visually observed to determine their color (obverse and reverse), texture, margin and sporulation. All Trichoderma isolates were classified and identified at the species level using morphological characteristics as suggested by Rifai [31] and Leahy and Colwell [50]. For further experiments and long-term storage, Trichoderma isolates were subcultured, and slants were prepared in cryovials overlaid with 20% glycerol and stored at −80 °C, respectively.

2.3. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted according to Gontia-Mishra et al. (2014). Polymerase chain reaction (PCR) amplification of the TEF1-α region was performed using EF2-EF1728M primer following the conditions given by White et al. [51]. PCR amplifications were carried out in a total reaction volume of 12.5 μL, including 0.25 μL of each primer, 1.25 µL of BSA, 6.25 of Taq polymerase (including dNTPs), 0.25 µL of genomic DNA (30 ng/µL); 0.25 µL DMSO and 4 µL of sterile ultrapure water. PCR conditions for TEF1-α were 94 °C/2 min, followed by nine cycles at 94 °C/35 s, 66 °C/55 s and 35 cycles at 94 °C/35 s, 56 °C/55 s and 72 °C/1 min 30 s. PCR products were visualized by Gelred (Thermo Fisher Scientific, Bremen, Germany) staining following electrophoresis of 4 μL of each product in 1% agarose gel. The PCR products were sequenced by the Eurofins Sanger sequencing facility, Germany.

2.4. Phylogenetic Analysis

Consensus sequences were assembled from forward and reverse sequencing chromatograms using the CLC Main Workbench 8.1 software packages. TEF1-α contigs of all isolates were compared to homologous sequences deposited in the NCBI GenBank database. Sequences generated and used in the current study were deposited in this database (Table 1). Sequences utilized from other studies were retrieved from the NCBI GenBank database for use in our phylogenetic analyses. Sequence alignments were carried out using MUSCLE as implemented in MEGA 10 [52]. Before phylogenetic analyses, the most appropriate nucleotide substitution model for each locus was chosen using MRMODELTEST v.2126. Finally, the maximum likelihood phylogenetic tree was constructed using MEGA 10 software [53]. Maximum likelihood phylogenetic inference was used in this study, since it is consistent on gapped multiple sequence alignments (MSAs), as long as substitution rates across each edge were greater than zero. Maximum likelihood analyses were estimated with nucleotide substitution of HKY + I + G model. Trichoderma species matching the isolates obtained in this work were retrieved and used to construct the phylogenetic tree, including two Nectria species as the outgroup. Nodal robustness was checked using the bootstrap method, and phylogenetic robustness was evaluated using 1000 replicates. Only sequences that matched published results identified through BLASTN searches with >97% sequence identity and an e-value of zero were used.

2.5. Diversity Analysis of Trichoderma Species

The degree of dominance index (Y) was used to quantitatively categorize the habitat preference of Trichoderma isolates in the coffee rhizosphere. The dominance values were computed using the following equation:
Y = ni ∗ fi N
Here, “N” is the total number of Trichoderma isolates, “ni” is the number of the genus (species) i, and “fi” is the frequency with which genus (species) i appears in the samples. The species i is dominant when Y > 0.02 [54]. Species richness (the total number of species), abundance (the sum of the number of isolates of each species) and diversity were evaluated using the Simpson biodiversity index (D) [55], Shannon’s biodiversity index (H) [56], Pielou species evenness index (E) [57] and Margalef’s abundance index (J) [58]. These ecological indices were used to quantitatively describe the diversity and habitat preference of Trichoderma species in different coffee ecosystems and major coffee-growing zones of Ethiopia.
Trichoderma species diversity, defined as the product of the evenness and the number of species, was evaluated using the Shannon biodiversity index (H) [56,59]. Simpson’s diversity index was calculated to assess the dominance of individual species [55,60]. This index shows the probability that two species selected randomly from a given ecosystem will belong to different species categories. Margalef’s abundance index was used to evaluate the species richness while the Pielou index was used to determine the evenness of the Trichoderma population. The biological diversity indices were calculated using the following equations:
D = 1 Σ i − 1 s P i 2 ,   P i 2 = ni ( ni − 1 ) N ( N − 1 )
H = ∑ i = 1 N Pi ln Pi , Pi = ni N
E = H H max , H max = lnS
J = S − 1 ln N
Here, “S” is the total number of Trichoderma species, “N” is the sum of all Trichoderma species isolates, “Pi” is the relative quantity of Trichoderma species “i’, and “ni” is the number of isolates of Trichoderma species “i’.

2.6. In Vitro Bioassay

In the present study, a total of 175 Trichoderma isolates were tested against F. xylarioides according to the method of Dennis and Webster [61]. Briefly, mycelial disks (5 mm in diameter) from seven-day-old growing edges of Trichoderma and F. xylarioides were put on opposite sides of a PDA Petri dish (3 cm away from each other). Control plates were also prepared without a Trichoderma disk. The culture plates were incubated at 25 °C with a 12 h photoperiod for 7 days. Following the methodology of [62], the percentage of colonization (%C) of each Trichoderma isolate was computed using the formula:
% C = ( DT − DE DE ) ∗ 100
Here, DT is the distance between colonies after mycelial growth stabilizes and DE is the initial distance between the two mycelial discs.
In brief, Trichoderma species (1 × 107 spores/mL) were inoculated into 1 L of PDB at pH 7.2 and cultured for 21 days at 28 °C. After the incubation, the liquid culture was subjected to ethyl acetate extraction and the crude extract was concentrated using a rotary evaporator. Finally, the concentrated extracts were dissolved in methanol for further partial purification using Sephadex LH-20. A total of 25 fractions were collected from the chromatographic column and subjected to agar diffusion assay against F. xylarioides on King B medium.

2.7. Statistical Data Analysis

Experimental results were analyzed using one-way analysis of variance (ANOVA) with SPSS, version 25. All statistical analyses of ecological indices used to evaluate the biodiversity of Trichoderma species were performed using Microsoft Excel 2019 and R software. The significance of differences between the mean results for treatments was evaluated using the Highest Significant Difference (HSD) based on the Tukey test with a significance threshold of p ≤ 0.05.

3. Results

3.1. Isolation and Morphological Characterization of Trichoderma Isolates

Trichoderma isolates were collected from the coffee rhizosphere conducted in southern, western and southwestern parts of Ethiopia. A total of 175 Trichoderma isolates were obtained from 184 rhizospheric soil samples collected from 28 districts distributed across different agroclimatic zones with soil pH ranging from 4.3 to 8.2. They were morphologically characterized by culturing on PDA plates to capture a full-scale Trichoderma diversity and distribution profile. Macroscopic morphological analysis revealed colonies with fast mycelial growth, concentric halos and floccose or compact surfaces on the culture medium (Figure 2). They were found to form colonies with white mycelia, becoming green when forming conidia and conidiophores. The mycelium, initially a white color, acquired green or yellow shades, or remained white, due to the abundant production of conidia and secondary metabolites. Concentric rings on culture media were observed for some isolates. Morphological variants, including phialides, conidial arrangements and conidial structures, were also observed among the Trichoderma isolates. Microscopic analysis revealed plentiful sporulation of conidia originating from verticillate conidiophores. The conidia of most Trichoderma isolates were ellipsoidal, globose and subglobose, with the apex broadly rounded and the base more narrowly rounded (Figure 2). However, morphological characteristics were insufficient to distinguish between different Trichoderma isolates. Therefore, molecular identification was needed to differentiate the complex and overlapping Trichoderma isolates.

3.2. Molecular Identification of Trichoderma Isolates

In total, 164 isolates of Trichoderma were identified at the species level based on their TEF1-α sequences and morphological analysis. The isolates were assigned to 16 putative species of Trichoderma, namely T. asperellum (64 isolates), T. asperelloides (32), T. longibrachiatum (20), T. harzianum (8), T. aethiopicum (6), T. hamatum (6), T. viride (4), T. reesei (4), T. koningiopsis (3), T. brevicompactum (3), T. citrinoviride (3), T. gamsii (3), T. erinaceum (2), T. orientale (2), T. bissettii (3) and T. paratroviride (1) (Figure 3 and Supplementary Table S1). These results represent the first observations for the following nine Trichoderma species in Ethiopia: T. asperellum, T. bissettii, T. brevicompactum, T. citrinoviride, T. erinaceum, T. orientale, T. paratroviride, T. reesei and T. viride. In addition, 11 undescribed and different isolates could not be matched to any other sequence in Genbank, demonstrating the considerable unresolved biodiversity of Trichoderma in the coffee ecosystem.

3.3. Phylogenetic Analysis

The TEF1-α phylogenetic analysis indicated that the 164 Trichoderma isolates were grouped into 16 highly supported monophyletic groups on the phylogeny. The TEF1-α phylogenetic analysis and the resulting maximum likelihood tree achieved good resolution for most of the analyzed isolates and effectively discriminated between members of the detected clades. Five basic clades were categorized following the identification manual for Trichoderma, namely Brevicompactum, Longibrachiatum, Hamatum, Harzianum and Viride (Figure 4). One hundred and two isolates were categorized into three known species belonging to the clades Hamatum: T. asperellum, T. asperelloides and T. Hamatum, while fifteen isolates were identified as T. orientale, T. koningiopsis, T. viride, T. erinaceum, T. paratroviride and T. gamsii in the clade Viride. In addition, 36 isolates were identified as T. longibrachiatum, T. aethiopicum, T. citroviride, T. bissettii and T. reesei in the clade Longibrachiatum. Eight isolates were identified as T. harzianum in the clade Harzianum, and three isolates were grouped as T. brevicompactum belonging to the clade Brevicompactum (Figure 4).

3.4. Biodiversity and Distribution of Trichoderma Isolates

3.4.1. Diversity Analysis of Trichoderma Species

The dominance value (Y) was 0.048 (>0.02), indicating that the genus Trichoderma was dominant in coffee rhizosphere soil. T. asperellum, T. asperelloides and T. longibrachiatum were classified as the principal species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. The analyzed data were used to compute Simpson’s biodiversity index (D), Shannon’s biodiversity index (H), evenness (E), and the abundance index (J) for each coffee ecosystem and coffee-growing zone, as shown in Supplementary Table S1. The highest species diversity and evenness (H = 1.97, E = 0.79, D = 0.81) were recorded for the forest and semi-forest coffee ecosystems of Kaffa, Jimma and Bale. The Shannon and Simpson diversity indices estimated for the West Guji and Bunno Bedele zones garden coffee ecosystem showed that they had lower species diversity (H = 1.57, D = 0.7). The calculated species abundance values were E = 2.71 for forest coffee, E = 2.64 for semi-forest coffee and E = 2.14 for garden coffee. The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4 (Table 2). Simpson’s index and the evenness index were close to 1 except in the West Guji zone, indicating a very high diversity of Trichoderma species in major coffee-growing areas of Ethiopia. The numbers of species and isolates, and the dominant species of Trichoderma, varied geographically (Table S2). These results reveal that the forest, semi-forest and garden ecosystems had a high diversity of Trichoderma species. The rhizosphere of C. arabica in Ethiopia thus hosts a large and highly diverse population of Trichoderma species.

3.4.2. Distribution of Trichoderma Species in Different Coffee-Growing Zones

Distribution and habitat preference analysis showed that Trichoderma species were widely dispersed throughout different coffee production systems. The proportion and composition of Trichoderma species varied among the sampled coffee-growing districts and zones. The proportion of Trichoderma species obtained from the Jimma zone was the highest (27%), followed by the Sheka zone (16%) and Bench Maji zone (13%); the lowest proportion was obtained from the Bunno Bedele zone (3%) (Figure 5). Species richness was highest in the Jimma zone (25 soil samples), for which 11 Trichoderma species were identified, followed by the Sheka zone (9 species, 18 soil samples), whereas Bunno Bedele had only 3 Trichoderma species. Among the identified isolates, T. asperellum (39.6%) and T. asperelloides (28%) were the most abundant species, being found in all major coffee-growing zones and districts of Ethiopia (Figure 5). Conversely, T. paratroviride was noted only in soil samples collected from the Jimma zone. The number of Trichoderma species declined going from the southwest to the south. The 11 known species identified in the Jimma zone were T. asperellum, T. asperelloides, T. longibrachiatum, T. harzianum, T. aethiopicum, T. citrinoviride, T. viride, T. reesei, T. koningiopsis, T. erinaceum and T. paratroviride. On the other hand, Trichoderma species obtained from the Sheka zone were T. asperellum, T. asperelloides, T. longibrachiatum, T. viride, T. hamatum, T. brevicompactum, T. koningiopsis, T. citrinoviride and T. bissettii. T. asperellum and T. asperelloides were found in all major coffee-growing areas and were the most widely dispersed species. Another widely distributed species was T. longibrachiatum, which was scattered in all zones except Kaffa. However, some species were unique to one zone; for instance, T. paratroviride was isolated only from the Jimma zone (Figure 5).

3.4.3. Distribution of Trichoderma Species in a Coffee Ecosystem

There were slight differences in the communities of Trichoderma species observed in the coffee rhizosphere soils of the different coffee ecosystems. Their high biodiversity was apparent in the distribution of Trichoderma species (Table S1). In total, 72 soil samples were collected from the native forest ecosystem, yielding 68 isolates representing 12 species of Trichoderma. Fifty-nine soil samples were collected from disturbed semi-forests, yielding 62 isolates representing 13 species. Fewer samples were collected from garden coffee ecosystems (53 soil samples), yielding only nine different Trichoderma species. The isolation frequency of Trichoderma in the native forest ecosystem was 39%, which was substantially higher than that for garden coffee ecosystems (29%; Figure 6). Except for species represented by single isolates, all species were found in multiple areas, showing that they may be regularly distributed within the coffee rhizosphere. However, there were some notable exceptions; T. erinaceum and T. brevicompactum were mostly isolated from the forest rhizosphere, T. paratroviride and T. citrinoviride were only found in semi-forest zones, and T. orientale was only observed in the garden coffee ecosystem (Figure 6).

3.5. Screening of Biocontrol Trichoderma

All isolates were capable of significantly inhibiting the mycelial growth of F. xylarioides. Twelve isolates exhibited the highest defined level of in vitro antagonistic activity. ANOVA analysis revealed statistically significant (p ≤ 0.05) differences in the mycelial growth inhibition profiles of the Trichoderma isolates against F. xylarioides, with inhibition percentages ranging from 44.5% to 84.8% (Table 3). T. asperellum AU71, T. longibrachiatum AU158 and T. asperellum AU131 were the most effective, causing 79.3%, 82.4% and 84.8% inhibition, respectively (Table 3 and Figure 7A1–C1). The mean inhibitory effect of these isolates against F. xylarioides was such that the pathogen’s growth was suppressed almost completely, whereas it grew rapidly on control plates lacking Trichoderma isolates (Figure 7D1). The inhibition of F. xylarioides radial growth in the dual-culture confrontation assay was attributed to inhibitory secondary metabolites released by one or both organisms as well as competition, mycoparasitism and production of cell-wall-degrading enzymes. The potential Trichoderma species exhibited an average growth rate of 0.45 mm/h in dual-culture bioassays.
Based on the in vitro bioassay results, three potent isolates (T. asperellum AU71, T. asperellum AU131 and T. longibrachiatum AU158) were subjected to secondary metabolite extraction. The agar well diffusion method was used to quantify the antifungal activities of crude metabolites extracted from these species (Table 3 and Figure 7A2–C2). All crude metabolites from these microorganisms inhibited the mycelial growth of F. xylarioides at the point of application around the agar wells; inhibition percentages of 83.5%, 86.7% and 88.2% were observed for the extracts of T. asperellum AU71, T. asperellum AU131 and T. longibrachiatum AU158, respectively, (Figure 7A2–C2) (p ≤ 0.05).

4. Discussion

A total of 164 isolates belonging to five clades were obtained from coffee rhizosphere soil samples. Trichoderma species were primarily identified based on morphological characteristics, including green coloration interleaved with a white mycelium, which is consistent with the morphological features reported previously for this fungus [31,63]. The identification keys of Samuels et al. [63] and Rifai [31] state that T. longibrachiatum holds subglobous to ovoid conidia and lageniform phialides. Additionally, Moat et al. [35] describes the presence of yellowish-green pigment on the backside of plates of T. longibrachiatum, which was also observed in this work. However, phenotypic characters are varied and depend partly on culture conditions [64] and secondary metabolite production [65]. This plasticity of characteristics means that analyses based solely on phenotypic traits cannot provide conclusive taxonomic identification of Trichoderma species [66,67].
Phylogenetic grouping revealed that the Trichoderma isolates recovered in this study formed a reliable maximum likelihood tree with acceptable taxonomic assumptions [68,69]. Modern methodologies for Trichoderma identification and classification into phylogenetic clades are based on analyses of sequence data [41,67,69]. Five clades were identified in this study, namely; Hamatum, Harzianum, Longibrachiatum, Brevicompactum and Viride (Figure 4). The Hamatum clade contains economically important species such as T. asperellum and T. asperelloides, which are used in agriculture as biological control agents [70,71]. The Longibrachiatum clade has high optimal and maximum growth temperatures and yellow reverse pigmentation due to the production of secondary metabolites such as pyrone. Trichoderma longibrachiatum has been used to produce various antimicrobial substances with important agricultural, health and environmental benefits [19].
The diversity of Trichoderma species in Africa in general [43,72] and Ethiopia in particular [24,73] is somewhat understudied compared to other parts of the world. Nine Trichoderma species were identified for the first time in Ethiopia in this work. It is notable that these species were previously described in America [74], Asia [46,54,75] and in European Mediterranean countries [42,76]; their presence in coffee rhizosphere soils in Ethiopia can be attributed to the diverse ecological substrata and climate conditions of the country’s coffee-growing areas and reflects the high Trichoderma biodiversity present in coffee ecosystems. The only previous study comparable to this one in terms of sampling size and studied area was conducted in the neotropical forests of South America, mainly in Colombia [77]. In that study, a high diversity of Trichoderma (29 species among 183 isolates) was detected, with a high proportion of putative new species among the isolates (11 species, corresponding to 6% of the sample). The main difference between their findings and ours is that we investigated a well-defined microecological niche, namely the rhizosphere of C. arabica.
The biodiversity of Trichoderma species is difficult to evaluate comparatively due to the range of indices suggested for this purpose [78]. In the present study, several widely used diversity indices were tested using a range of simple and multifaceted statistical analyses to evaluate whether some were better for certain analyses than others. The Shannon index values calculated for native forest and semi-forest ecosystem samples were almost twice those obtained for soils in Sardinia at H = 1.97 versus 1.59, respectively, even though the number of samples investigated in the latter case was almost three times that collected in this work. However, the Shannon indices of the Sardinian ecosystems and the garden coffee zones were quite similar (H = 1.59 versus 1.57), possibly reflecting the extensive disturbance of both ecosystems by human activities [79]. These results show that Trichoderma diversity and habitat preference can be used as a natural indicator of rhizosphere soil health. Forest and semi-forest coffee regions had richly varied Trichoderma populations with relatively high diversity and very similar biodiversity indices and evenness values.
The number of Trichoderma species detected in this work was almost twice that reported in earlier studies on biodiversity in Ethiopia [24] and other countries, including Poland [80], Central Europe [76] and China’s Northern Xinjiang region [75]. In addition, significant differences were observed between the Trichoderma populations of different coffee-growing zones; this variation may reflect differences in the zones’ ecological environments. The populations of Trichoderma species in the southwestern Ethiopia forest and semi-forest coffee ecosystems were diverse, and their composition varied between ecosystems. The Jimma zone had 11 Trichoderma species and the largest number of Trichoderma isolates (48), followed by the Sheka (9 species, 27 isolates), Benchi Maji (7 species, 22 isolates) and Bunno Bedele (3 species, 6 isolates) zones (Table 1). Our results suggest that forest and semi-forest ecosystems are particularly favorable for the survival and colonization of Trichoderma, indicating that this genus has a clear environmental preference, in keeping with previous reports [54,75,76].
T. asperellum (39%) was found to be the most widely distributed and abundant fungal species in this work (Figure 3). The occurrence of Trichoderma species is modulated by several factors, including metabolic variety, reproductive ability, substrate availability and the competitive abilities of Trichoderma isolates in nature [76,81,82]. Trichoderma isolates were obtained from different coffee ecosystems, with T. asperellum, T. asperelloides and T. longibrachiatum being the most widely distributed species. T. asperellum is the most dominant and cosmopolitan species, such as T. harzianum [83], whereas T. asperelloides and T. longibrachiatum were found mostly in forest ecosystems of South America and Asia [77,84]. Conversely, previous studies have found T. harzianum, T. hamatum, T. spirale and T. asperelloides to be the most widely distributed species of this genus in coffee ecosystems in Ethiopia [24]. Except for species that were only found as single isolates, all species were obtained in multiple districts, suggesting that they are quite evenly distributed within the coffee rhizosphere. T. erinaceum and T. brevicompactum were only isolated from the native forest; T. paratroviride and T. citrinoviride were only obtained from semi-forest areas, and T. orientale was only isolated from garden coffee ecosystem samples. Studies conducted by Hoyos-Carvajal and Bissett [77] indicated the dominant Trichoderma species in the neotropics are T. asperellum, followed by T. harzianum. Our results confirm the predominance of T. asperellum, followed by T. asperelloides. Conversely, Belayneh et al. [24] reported that T. hamatum was the most dominant species in the rhizosphere of coffee plants. The large number and wide distribution of Trichoderma species identified within Ethiopia’s coffee ecosystem demonstrate the presence of significant genetic diversity, suggesting that further study of these species may offer opportunities to improve the sustainable management of coffee cultivation and discover effective biocontrol agents for managing CWD.
This work represents the first investigation of the biodiversity of Trichoderma species in the rhizospheres of Ethiopia’s coffee ecosystem and their suitability as biological control agents (BCA) against CWD (F. xylarioides). The results presented herein mainly concern the taxonomy of the Trichoderma isolates with some observations on their ecology, and will support the selection of candidate biocontrol agents for the management of CWD in Ethiopia. This work is part of a larger project seeking to control CWD using a classical biological control strategy involving sourcing and releasing potential BCAs from the center of origin of coffea arabica to minimize the incidence and severity of the disease. Such approaches using fungal natural enemies have been used successfully to control various soil-borne plant pathogens [9,24,73,85,86]. Our results indicate that there is a significant number of Trichoderma species that are substantially antagonistic to F. xylarioides and which could be exploited for the biocontrol of CWD in this way. In the previous study, we formulated a biofungicide from T. asperellum AU131 and T. longibrachiatum AU158 under solid-state fermentation (SSF) to control CWD [87].
All Trichoderma strains isolated in this work effectively inhibited the mycelial growth of F. xylarioides colonies. However, there were notable differences between Trichoderma strains in controlling the mycelial growth of F. xylarioides in dual-culture experiments. For example, Filizola et al. [88] state that some isolates of certain species suppress the growth of phytopathogens via hyper-parasitism, whereas others achieve growth suppression via mechanisms such as antibiosis or competition. It has also been reported that Trichoderma species grow faster than competing phytopathogens because they use food sources more efficiently. Another important mechanism involves the secretion of metabolites and hydrolytic enzymes that reduce or hinder the growth of plant pathogens in the area; this mechanism has been suggested to contribute to the success of Trichoderma species against phytopathogenic fungi [89]. The potential of T. asperellum and T. longibrachiatum as effective biocontrol agents of fungi and bacterial strains of both annual and perennial crops was clearly stated by many research reports [90,91]. For instance, T. asperellum exhibits strong control effects on F. graminearum, F. oxysporum and Verticillium wilt of olive [92,93]. On the other hand, T. longibrachiatum is also used as a potential biocontrol agent, being most effective against P. grisea, F. verticillioides, H. oryzae, F. moniliforme and A. alternate with inhibition percentages of 98.9, 96.4, 95.1, 93.6 and 93.0%, respectively [94]. Here, we should point out that the three T. asperellum strains assessed in this work were isolated from coffee rhizosphere in production fields from southwestern Ethiopia. This aspect should be considered a valuable asset for biocontrol applications, as native isolates are better adapted to their local climate conditions and pathogenic targets than foreign isolates.
Various secondary metabolites produced by Trichoderma species, including harzianolide, peptaibols, pyrones and other secondary metabolites, have been suggested to have antimicrobial potential and to act as plant growth promoters (Mulatu, unpublished data). In addition to achieving higher growth rates than F. xylarioides in competition experiments, the Trichoderma strains isolated in this work achieved growth rates significantly exceeding the value of 0.33 mm/h reported by Moretto et al. [95]. Moreover, field and greenhouse experiments using Geisha coffee varieties of C. arabica (the most susceptible to CWD) gave similar results (Afrasa Mulatu, unpublished data). The results obtained indicate that understanding the genetic variation within the genus Trichoderma is essential for selecting novel indigenous Trichoderma species that can be used as biocontrol agents against CWD. In addition, our findings display the distribution and diversity profile of Trichoderma species and provide insights into their potential usefulness as microbial fungicides to safeguard coffee cultivation across different agroclimatic zones in Ethiopia.

5. Conclusions

A total of 175 isolates of Trichoderma were identified at the species level based on TEF1-α sequence analysis, yielding 16 putative species. T. asperellum, T. asperelloides and T. longibrachiatum were classified as the abundant species, and the average diversity values for Trichoderma species originating from coffee ecosystems were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. The results obtained suggest that T. asperellum and T. longibrachiatum are promising suppressors of F. xylarioides’ growth and promoters of plant growth, suggesting that they could be valuable biocontrol agents for the management of CWD. Additionally, our results demonstrate the existence of a guild of Trichoderma species that are potentially antagonistic to F. xylarioides, which could be exploited to develop more effective biological control of CWD. In addition, future research should focus on assessing any toxicity or risks associated with potential Trichoderma species (T. asperellum and T. longibrachiatum) in animal models. Secondary metabolites (volatile and nonvolatile metabolites) must also be characterized and elucidated utilizing various chromatographic methods.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/crops2020010/s1, Table S1: Trichoderma species isolated and identified from major coffee growing zones of Ethiopia, Table S2: Identification, origin, and isolation details of Trichoderma isolates.

Author Contributions

Conceptualization and designing the experiment, A.M., R.R.V. and T.A.T.; methodology, A.M., T.A.T. and N.M.; DNA extraction, T.A. and A.M., PCR optimization and sequencing, R.R.V., S.K. and Q.L.; software, A.M., and Q.L.; Data validation, T.A.T. and R.R.V.; formal analysis, A.M.; investigation, A.M. and T.A.T.; resources, T.A.T., N.M. and R.R.V.; data curation, A.M. and T.A.T.; writing—original draft preparation, A.M.; writing—review and editing, T.A.T., N.M. and R.R.V.; supervision and project administration, T.A.T., N.M. and R.R.V.; funding acquisition, T.A.T. and R.R.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ethiopian Biotechnology Institute (EBTi), Ethiopia, under the Biotechnology Product Development Research Grant. Open access and sequencing funding was provided by Swedish University of Agricultural Sciences (SLU).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank EBTi for funding this research project under the theme “Bio-fungicide production for CWD control in Ethiopia” and the Department of Microbial, Cellular and Molecular Biology at Addis Ababa University (AAU), Ethiopia, for the laboratory facilities. Special thanks goes to the Department of Plant Breeding at the SLU, Sweden, for the valuable support they provided for the project. RV was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (grant number 2019-01316) and the Swedish Research Council (grant number 2019-04270).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Srinivasa, N.; Devi, T.P.; Sudhirkumar, S.; Kamil, D.; Borah, J.L.; Prabhakaran, N. Bioefficacy of Trichoderma isolates against soil-borne pathogens. Afr. J. Microbiol. Res. 2014, 8, 2710–2723. [Google Scholar] [CrossRef] [Green Version]
  2. Parulekar-Berde, C.V.; Joshi, S.A.; Berde, V.B. Fungal Communities as Biological Control Agents for Different Phytopathogenic Organisms. In Recent Trends in Mycological Research; Springer: Berlin/Heidelberg, Germany, 2021; pp. 189–201. [Google Scholar]
  3. Zamanizadeh, H.R.; Hatami, N.; Aminaee, M.M.; Rakhshandehroo, F. Application of biofungicides in control of damping disease off in greenhouse crops as a possible substitute to synthetic fungicides. Int. J. Environ. Sci. Technol. 2011, 8, 129–136. [Google Scholar] [CrossRef] [Green Version]
  4. Woo, S.L.; Ruocco, M.; Vinale, F.; Nigro, M.; Marra, R.; Lombardi, N.; Pascale, A.; Lanzuise, S.; Manganiello, G.; Lorito, M. Trichoderma-based products and their widespread use in agriculture. Open Mycol. J. 2014, 8, 71–126. [Google Scholar] [CrossRef] [Green Version]
  5. Sood, M.; Kapoor, D.; Kumar, V.; Sheteiwy, M.S.; Ramakrishnan, M.; Landi, M.; Araniti, F.; Sharma, A. Trichoderma: The “Secrets” of a Multitalented Biocontrol Agent. Plants 2020, 9, 762. [Google Scholar] [CrossRef]
  6. López-Bucio, J.; Pelagio-Flores, R.; Herrera-Estrella, A. Trichoderma as biostimulant: Exploiting the multilevel properties of a plant beneficial fungus. Sci. Hortic. 2015, 196, 109–123. [Google Scholar] [CrossRef]
  7. Altomare, C.; Norvell, W.; Bjorkman, T.; Harman, G. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl. Environ. Microbiol. 1999, 65, 2926–2933. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Shoresh, M.; Harman, G.E.; Mastouri, F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu. Rev. Phytopathol. 2010, 48, 21–43. [Google Scholar] [CrossRef] [Green Version]
  9. Kubicek, C.P.; Herrera-Estrella, A.; Seidl-Seiboth, V.; Martinez, D.A.; Druzhinina, I.S.; Thon, M.; Zeilinger, S.; Casas-Flores, S.; Horwitz, B.A.; Mukherjee, P.K. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol. 2011, 12, R40. [Google Scholar] [CrossRef] [Green Version]
  10. Jaklitsch, W.; Voglmayr, H. Biodiversity of Trichoderma (Hypocreaceae) in Southern Europe and Macaronesia. Stud. Mycol. 2015, 80, 1–87. [Google Scholar] [CrossRef] [Green Version]
  11. du Plessis, I.L.; Druzhinina, I.S.; Atanasova, L.; Yarden, O.; Jacobs, K. The diversity of Trichoderma species from soil in South Africa, with five new additions. Mycologia 2018, 110, 559–583. [Google Scholar] [CrossRef]
  12. Haouhach, S.; Karkachi, N.; Oguiba, B.; Sidaoui, A.; Chamorro, I.; Kihal, M.; Monte, E. Three New Reports of Trichoderma in Algeria: T. atrobrunneum (South), T. longibrachiatum (South), and T. afroharzianum (Northwest). Microorganisms 2020, 8, 1455. [Google Scholar] [CrossRef] [PubMed]
  13. Belayneh, T.; Kubicek, C.P.; Druzhinina, I.S. The rhizosphere of Coffea arabica in its native highland forests of Ethiopia provides a niche for a distinguished diversity of Trichoderma. Diversity 2010, 2, 527–549. [Google Scholar] [CrossRef]
  14. Rifai, M.A. Revision of the genus Trichoderma. Mycol. Pap. 1969, 116, 1–56. [Google Scholar]
  15. Bissett, J. A revision of the genus Trichoderma. III. Section Pachybasium. Can. J. Bot. 1991, 69, 2373–2417. [Google Scholar] [CrossRef]
  16. Bissett, J. A revision of the genus Trichoderma. I. Section Longibrachiatum sect. nov. Can. J. Bot. 1984, 62, 924–931. [Google Scholar] [CrossRef]
  17. Bissett, J. A revision of the genus Trichoderma. II. Infrageneric classification. Can. J. Bot. 1991, 69, 2357–2372. [Google Scholar] [CrossRef]
  18. Bissett, J. A revision of the genus Trichoderma. IV. Additional notes on section Longibrachiatum. Can. J. Bot. 1991, 69, 2418–2420. [Google Scholar] [CrossRef]
  19. Spring, O.; Gomez-Zeledon, J.; Hadziabdic, D.; Trigiano, R.N.; Thines, M.; Lebeda, A. Biological characteristics and assessment of virulence diversity in pathosystems of economically important biotrophic oomycetes. Crit. Rev. Plant Sci. 2018, 37, 439–495. [Google Scholar] [CrossRef]
  20. Cai, F.; Druzhinina, I.S. In honor of John Bissett: Authoritative guidelines on molecular identification of Trichoderma. Fungal Divers. 2021, 107, 1–69. [Google Scholar] [CrossRef]
  21. Samuels, G.J.; Dodd, S.L.; Gams, W.; Castlebury, L.A.; Petrini, O. Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 2002, 94, 146–170. [Google Scholar] [CrossRef]
  22. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. Guide Meth. Appl. 1990, 18, 315–322. [Google Scholar]
  23. Schoch, C.L.; Seifert, K.A.; Huhndorf, S.; Robert, V.; Spouge, J.L.; Levesque, C.A.; Chen, W. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. USA 2012, 109, 6241–6246. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Chaverri, P.; Branco-Rocha, F.; Jaklitsch, W.; Gazis, R.; Degenkolb, T.; Samuels, G.J. Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains. Mycologia 2015, 107, 558–590. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Schmidt, J.E.; Vannette, R.L.; Igwe, A.; Blundell, R.; Casteel, C.L.; Gaudin, A.C. Effects of agricultural management on rhizosphere microbial structure and function in processing tomato plants. Appl. Environ. Microbiol. 2019, 85, e01064-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Leelavathi, M.; Vani, L.; Reena, P. Antimicrobial activity of Trichoderma harzianum against bacteria and fungi. Int. J. Curr. Microbiol. Appl. Sci. 2014, 3, 96–103. [Google Scholar]
  27. Anwar, J.; Iqbal, Z. Effect of growth conditions on antibacterial activity of Trichoderma harzianum against selected pathogenic bacteria. Sarhad J. Agric. 2017, 33, 501–510. [Google Scholar] [CrossRef]
  28. Motlagh, M.R.S.; Samimi, Z. Evaluation of Trichoderma spp., as biological agents in some of plant pathogens. Ann. Biol. Res. 2013, 4, 173–179. [Google Scholar]
  29. Nandini, B.; Hariprasad, P.; Shankara, H.N.; Prakash, H.S.; Geetha, N. Total crude protein extract of Trichoderma spp. induces systemic resistance in pearl millet against the downy mildew pathogen. 3 Biotec. 2017, 7, 183. [Google Scholar] [CrossRef]
  30. Lamondia, J.A.; Cowles, R.S. Effect of Entomopathogenic Nematodes and Trichoderma harzianum on the Strawberry Black Root Rot Pathogens Pratylenchus penetrans and Rhizoctonia fragariae. J. Nematol. 2002, 34, 351–357. [Google Scholar]
  31. Poveda, J.; Abril-Urias, P.; Escobar, C. Biological control of plant-parasitic nematodes by filamentous fungi inducers of resistance: Trichoderma, mycorrhizal and endophytic fungi. Front. Microbiol. 2020, 11, 992. [Google Scholar] [CrossRef]
  32. Tefera, A.; Tefera, T. Ethiopia Coffee Annual Report ET2020-0004; USDA Foreign Agricultural Service: Addis Ababa, Ethiopia, 2021; pp. 1–6.
  33. Moat, J.; Williams, J.; Baena, S.; Wilkinson, T.; Gole, T.W.; Challa, Z.K.; Demissew, S.; Davis, A.P. Resilience potential of the Ethiopian coffee sector under climate change. Nat. Plants 2017, 3, 17081. [Google Scholar] [CrossRef] [PubMed]
  34. ICO. Total Production by All Exporting Countries; ICO: London, UK, 2016. [Google Scholar]
  35. Panggabean, Y.; Arsyad, M. Coffee farming business development: E-commerce technology utilization. IOP Conf. Ser. Earth Environ. Sci. 2021, 807, 032011. [Google Scholar] [CrossRef]
  36. Tefera, A.; Tefera, T.; Gray, Q. Coffee Annual Report; USDA Foreign Agricultural Service: Addis Ababa, Ethiopia, 2012.
  37. Ethiopian Economic Association. Report on the Ethiopian Economy; Ethiopian Economic Association: Addis Ababa, Ethiopia, 2005; Volume 5. [Google Scholar]
  38. International Coffee Organization. Total Production by All Exporting Countries. 2016. Available online: http://www.ico.org (accessed on 18 June 2021).
  39. Teferi, D.; Belachew, K. Evaluation of released arabica coffee varieties (Coffea arabica L.) for major coffee diseases with especial emphasis to coffee wilt disease (Gibberella xylarioides) at jimma, Ethiopia. Evaluation 2015, 5, 81–87. [Google Scholar]
  40. Adugna, G.; Million, A.; Hindorf, H.; Zeru, A.; Teferi, D.; Jefuka, C. (Eds.) Coffee Wilt Disease in Ethiopia; CAB International: Oxfordshire, UK, 2010; p. 50. [Google Scholar]
  41. Zeru, A.; Assefa, F.; Adugna, G.; Hindorf, H. Occurrence of fungal diseases of Coffea arabica L. in montane rainforests of Ethiopia. J. Appl. Bot. Food Qual. 2012, 82, 148–151. [Google Scholar]
  42. Getachew, S.; Adugna, G.; Lemessa, F.; Hindorf, H. Coffee wilt disease (Gibberella xylarioides Heim and Saccas) in forest coffee systems of southwest and southeast Ethiopia. Plant Pathol. J. 2012, 11, 10–17. [Google Scholar] [CrossRef] [Green Version]
  43. Tiru, M.; Muleta, D.; Bercha, G.; Adugna, G. Antagonistic effect of rhizobacteria against coffee wilt disease caused by Gibberella xylarioides. Asian J. Plant Pathol. 2013, 7, 109–122. [Google Scholar] [CrossRef] [Green Version]
  44. Adugna, G. Diversity in Pathogenicity and Genetics of Gibberella xylarioides (Fusarium xylarioides) Populations and Resistance of Coffea spp. Ph.D. Thesis, University of Bonn, Bonn, Germany, 2004. [Google Scholar]
  45. Gil, S.V.; Pastor, S.; March, G. Quantitative isolation of biocontrol agents Trichoderma spp., Gliocladium spp. and Actinomycetes from soil with culture media. Microbiol. Res. 2009, 164, 196–205. [Google Scholar]
  46. Saravanakumar, K.; Yu, C.; Dou, K.; Wang, M.; Li, Y.; Chen, J. Biodiversity of Trichoderma community in the tidal flats and wetland of southeastern China. PLoS ONE 2016, 11, e0168020. [Google Scholar] [CrossRef]
  47. Adugna, G.; Hindorf, H.; Steiner, U.; Nirenberg, H.; Dehne, H.-W.; Schellander, K. Genetic diversity in the coffee wilt pathogen (Gibberella xylarioides) populations: Differentiation by host specialization and RAPD analysis/Genetische Diversität in der Population des Erregers der Kaffeewelke (Gibberella xylarioides): Differenzierung durch Wirtsspezifität und RAPD Analyse. J. Plant Dis. Protec. 2005, 112, 134–145. [Google Scholar]
  48. Adugna, G.; Hindorf, H. Tracheomycosis (Gibberella xylarioides) on coffee (Coffea arabica). In Proceedings of the Conference on International Agricultural Research for Development, Berlin, Germany, 5–7 October 2004; Deutscher Tropentag: Berlin, Germany; pp. 9–11. [Google Scholar]
  49. Samuels, G.J.; Hebbar, P.K. Trichoderma: Identification and Agricultural Applications; APS Press: Eagan, MN, USA, 2015. [Google Scholar]
  50. Leahy, J.G.; Colwell, R.R. Microbial degradation of hydrocarbons in the environment. Microbiol. Rev. 1990, 54, 305–315. [Google Scholar] [CrossRef]
  51. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Mycologia 2013, 64, 315–322. [Google Scholar]
  52. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  54. Ma, J.; Tsegaye, E.; Li, M.; Wu, B.; Jiang, X. Biodiversity of Trichoderma from grassland and forest ecosystems in Northern Xinjiang, China. 3 Biotech 2020, 10, 362. [Google Scholar] [CrossRef] [PubMed]
  55. Simpson, E.H. Measurement of Diversity. Nature 1949, 163, 688. [Google Scholar] [CrossRef]
  56. Shannon, C.E. A Mathematical theory of communication. Bell Syst. Technol. J. 1948, 27, 379–423. [Google Scholar] [CrossRef] [Green Version]
  57. Pielou, E.C. The measurement of diversity in different types of biological collections. J. Theor. Biol. 1966, 13, 131–144. [Google Scholar] [CrossRef]
  58. Margalef, R. Information theory in biology. Gen. Syst. Yearb. 1958, 3, 36–71. [Google Scholar]
  59. Konopiński, M.K. Shannon diversity index: A call to replace the original Shannon’s formula with unbiased estimator in the population genetics studies. PeerJ 2020, 8, e9391. [Google Scholar] [CrossRef]
  60. Gregorius, H.-R.; Gillet, E.M. Generalized Simpson-diversity. Ecol. Model. 2008, 211, 90–96. [Google Scholar] [CrossRef]
  61. Dennis, C.; Webster, J. Antagonistic properties of species-groups of Trichoderma: II. Production of volatile antibiotics. Trans. Br. Mycol. Soc. 1971, 57, 41–48. [Google Scholar] [CrossRef]
  62. Szekeres, A.; Leitgeb, B.; Kredics, L.; Antal, Z.; Hatvani, L.; Manczinger, L.; Vágvölgyi, C. Peptaibols and related peptaibiotics of Trichoderma. Acta Microbiol. Immunol. Hung. 2005, 52, 137–168. [Google Scholar] [CrossRef] [PubMed]
  63. Samuels, G.J.; Ismaiel, A.; Bon, M.-C.; De Respinis, S.; Petrini, O. Trichoderma asperellum sensu lato consists of two cryptic species. Mycologia 2010, 102, 944–966. [Google Scholar] [CrossRef]
  64. Mazrou, Y.S.; Makhlouf, A.H.; Elseehy, M.M.; Awad, M.F.; Hassan, M.M. Antagonistic activity and molecular characterization of biological control agent Trichoderma harzianum from Saudi Arabia. Egypt J. Biol. Pest Contr. 2020, 30, 4. [Google Scholar] [CrossRef] [Green Version]
  65. Hermosa, R.; Cardoza, R.E.; Rubio, M.B.; Gutiérrez, S.; Monte, E. Secondary metabolism and antimicrobial metabolites of Trichoderma. In Biotechnology and Biology of Trichoderma; Elsevier: Amsterdam, The Netherlands, 2014; pp. 125–137. [Google Scholar]
  66. Bridge, P.; Spooner, B.; Roberts, P. The impact of molecular data in fungal systematics. Adv. Bot. Res. 2005, 42, 33–67. [Google Scholar]
  67. Atanasova, L.; Le Crom, S.; Gruber, S.; Coulpier, F.; Seidl-Seiboth, V.; Kubicek, C.P.; Druzhinina, I.S. Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism. BMC Gen. 2013, 14, 121. [Google Scholar] [CrossRef] [Green Version]
  68. Druzhinina, I.; Kubicek, C.P. Species concepts and biodiversity in Trichoderma and Hypocrea: From aggregate species to species clusters? J. Zhejiang Univ. Sci. B 2005, 6, 100. [Google Scholar] [CrossRef] [Green Version]
  69. Druzhinina, I.S.; Kubicek, C.P. Genetic engineering of Trichoderma reesei cellulases and their production. Microb. Biotechnol. 2017, 10, 1485–1499. [Google Scholar] [CrossRef]
  70. Liu, B.; Ji, S.; Zhang, H.; Wang, Y.; Liu, Z. Isolation of Trichoderma in the rhizosphere soil of Syringa oblata from Harbin and their biocontrol and growth promotion function. Microbiol. Res. 2020, 235, 126445. [Google Scholar] [CrossRef]
  71. Sumida, C.H.; Daniel, J.F.; Araujod, A.P.C.; Peitl, D.C.; Abreu, L.M.; Dekker, R.F.; Canteri, M.G. Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants. Biocontr. Sci. Technol. 2018, 28, 142–156. [Google Scholar] [CrossRef]
  72. Mutawila, C.; Vinale, F.; Halleen, F.; Lorito, M.; Mostert, L. Isolation, production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases. Plant Pathol. 2016, 65, 104–113. [Google Scholar] [CrossRef] [Green Version]
  73. Belayneh, T.; Druzhinina, I.S.; Kubicek, C.P.; Atanasova, L. Novel endophytic Trichoderma spp. isolated from healthy Coffea arabica roots are capable of controlling coffee Tracheomycosis. Diversity 2013, 5, 750–766. [Google Scholar]
  74. Holmes, K.A.; Schroers, H.-J.; Thomas, S.E.; Evans, H.C.; Samuels, G.J. Taxonomy and biocontrol potential of a new species of Trichoderma from the Amazon basin of South America. Mycol. Progr. 2004, 3, 199–210. [Google Scholar] [CrossRef]
  75. Jiang, Y.; Wang, J.-L.; Chen, J.; Mao, L.-J.; Feng, X.-X.; Zhang, C.-L.; Lin, F.-C. Trichoderma biodiversity of agricultural fields in east china reveals a gradient distribution of species. PLoS ONE 2016, 11, e0160613. [Google Scholar] [CrossRef] [PubMed]
  76. Błaszczyk, L.; Strakowska, J.; Chełkowski, J.; Gąbka-Buszek, A.; Kaczmarek, J. Trichoderma species occurring on wood with decay symptoms in mountain forests in Central Europe: Genetic and enzymatic characterization. J. Appl. Gen. 2016, 57, 397–407. [Google Scholar] [CrossRef] [Green Version]
  77. Hoyos-Carvajal, L.; Bissett, J. Biodiversity of Trichoderma in neotropics. In The Dynamical Processes of Biodiversity-Case Studies of Evolution and Spatial Distribution; InTech: London, UK, 2011. [Google Scholar]
  78. Morris, E.K.; Caruso, T.; Buscot, F.; Fischer, M.; Hancock, C.; Maier, T.S.; Meiners, T.; Müller, C.; Obermaier, E.; Prati, D. Choosing and using diversity indices: Insights for ecological applications from the German Biodiversity Exploratories. Ecol. Evol. 2014, 4, 3514–3524. [Google Scholar] [CrossRef] [Green Version]
  79. Migheli, Q.; Balmas, V.; Komoñ-Zelazowska, M.; Scherm, B.; Fiori, S.; Kopchinskiy, A.G.; Kubicek, C.P.; Druzhinina, I.S. Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma. Environ. Microbiol. 2009, 11, 35–46. [Google Scholar] [CrossRef]
  80. Błaszczyk, L.; Popiel, D.; Chełkowski, J.; Koczyk, G.; Samuels, G.J.; Sobieralski, K.; Siwulski, M. Species diversity of Trichoderma in Poland. J. Appl. Genet. 2011, 52, 233–243. [Google Scholar] [CrossRef] [Green Version]
  81. Contreras-Cornejo, H.A.; Macías-Rodríguez, L.; Del-Val, E.; Larsen, J. Ecological functions of Trichoderma spp. and their secondary metabolites in the rhizosphere: Interactions with plants. FEMS Microbiol. Ecol. 2016, 92, fiw036. [Google Scholar] [CrossRef] [Green Version]
  82. Kumar, G.; Maharshi, A.; Patel, J.; Mukherjee, A.; Singh, H. Trichoderma: A potential fungal antagonist to control plant diseases. ATSA Mukhapatra-Ann. Technol. Issue 2017, 21, 206–218. [Google Scholar]
  83. Kredics, L.; Hatvani, L.; Naeimi, S.; Körmöczi, P.; Manczinger, L.; Vágvölgyi, C.; Druzhinina, I. Biodiversity of the genus Hypocrea/Trichoderma in different habitats. In Biotechnology and Biology of Trichoderma; Elsevier: Amsterdam, The Netherlands, 2014; pp. 3–24. [Google Scholar]
  84. Cummings, N.; Ambrose, A.; Braithwaite, M.; Bissett, J.; Roslan, H.; Abdullah, J.; Stewart, A.; Agbayani, F.; Steyaert, J.; Hill, R.A. Diversity of root-endophytic Trichoderma from Malaysian Borneo. Mycol. Progr. 2016, 15, 50. [Google Scholar] [CrossRef]
  85. Flood, J. Coffee Wilt Disease; CABI: London, UK, 2010. [Google Scholar]
  86. Mulatu, A.; Megersa, N.; Alemu, T. Characterization of fungal extracts from Trichoderma isolates: Their effects against coffee wilt pathogen (Gibberella xylarioides). SINET Ethiop. J. Sci. 2013, 36, 81–92. [Google Scholar]
  87. Mulatu, A.; Alemu, T.; Megersa, N.; Vetukuri, R.R. Optimization of Culture Conditions and Production of Bio-Fungicides from Trichoderma Species under Solid-State Fermentation using Mathematical Modeling. Microorganisms 2021, 9, 1675. [Google Scholar] [CrossRef] [PubMed]
  88. Filizola, P.R.B.; Luna, M.A.C.; de Souza, A.F.; Coelho, I.L.; Laranjeira, D.; Campos-Takaki, G.M. Biodiversity and phylogeny of novel Trichoderma isolates from mangrove sediments and potential of biocontrol against Fusarium strains. J. Microb. Cell Factor. 2019, 18, 89. [Google Scholar] [CrossRef]
  89. Waghunde, R.R.; Shelake, R.M.; Sabalpara, A.N. Trichoderma: A significant fungus for agriculture and environment. Afr. J. Agri. Res. 2016, 11, 1952–1965. [Google Scholar]
  90. Zhang, S.; Xu, B.; Zhang, J.; Gan, Y. Identification of the antifungal activity of Trichoderma longibrachiatum T6 and assessment of bioactive substances in controlling phytopathgens. Pestic. Biochem. Phys. 2018, 147, 59–66. [Google Scholar] [CrossRef]
  91. Degani, O.; Khatib, S.; Becher, P.; Gordani, A.; Harris, R. Trichoderma asperellum Secreted 6-Pentyl-α-Pyrone to Control Magnaporthiopsis maydis, the Maize Late Wilt Disease Agent. Biology 2021, 10, 897. [Google Scholar] [CrossRef]
  92. Carrero-Carrón, I.; Trapero-Casas, J.L.; Olivares-García, C.; Monte, E.; Hermosa, R.; Jiménez-Díaz, R.M. Trichoderma asperellum is effective for biocontrol of Verticillium wilt in olive caused by the defoliating pathotype of Verticillium dahliae. Crop Prot. 2016, 88, 45–52. [Google Scholar] [CrossRef]
  93. Wu, Q.; Sun, R.; Ni, M.; Yu, J.; Li, Y.; Yu, C.; Dou, K.; Ren, J.; Chen, J. Identification of a novel fungus, Trichoderma asperellum and comprehensive evaluation of its biocontrol efficacy. PLoS ONE 2017, 12, e0179957. [Google Scholar] [CrossRef]
  94. Elamawi, R.M.; Al-Harbi, R.E.; Hendi, A.A. Biosynthesis and characterization of silver nanoparticles using Trichoderma longibrachiatum and their effect on phytopathogenic fungi. Egy. J. Biol. Pest Contr. 2018, 28, 28. [Google Scholar] [CrossRef] [Green Version]
  95. Moretto, K.C.; Gimenes-Fernandes, N.; dos Santos, J.M. Influence of Trichoderma spp. on Colletotrichum acutatum mycelial growth and morphology and on infection of “Tahiti” lime detached flowers. Summa Phytopathol. 2001, 27, 357–364. [Google Scholar]
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Figure 1. Map of study areas and illustration of the geographical locations of districts from which rhizospheric soil samples were collected, Ethiopia. SNNP = South Nations and Nationalities Peoples region.
Figure 1. Map of study areas and illustration of the geographical locations of districts from which rhizospheric soil samples were collected, Ethiopia. SNNP = South Nations and Nationalities Peoples region.
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Figure 2. Morphological characteristics of Trichoderma species colony grown on PDA: T. asperellum (1), T. asperelloides (2), T. longibrachiatum (3), T. harzianum (4), T. aethiopicum (5), T. citrinoviride (6), T. hamatum (7), T. reesei (8), T. viride (9), T. bissettii (10), T. brevicompactum (11), T. erinaceum (12), T. gamsii (13), T. koningiopsis (14), T. orientale (15) and T. paratroviride (16); x = structure of conidiophores. Conidiophores were observed at 400× magnification.
Figure 2. Morphological characteristics of Trichoderma species colony grown on PDA: T. asperellum (1), T. asperelloides (2), T. longibrachiatum (3), T. harzianum (4), T. aethiopicum (5), T. citrinoviride (6), T. hamatum (7), T. reesei (8), T. viride (9), T. bissettii (10), T. brevicompactum (11), T. erinaceum (12), T. gamsii (13), T. koningiopsis (14), T. orientale (15) and T. paratroviride (16); x = structure of conidiophores. Conidiophores were observed at 400× magnification.
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Figure 3. Trichoderma species isolated and identified from coffee rhizospheric soil samples; the numbers in parentheses was the percentage of each Trichoderma species.
Figure 3. Trichoderma species isolated and identified from coffee rhizospheric soil samples; the numbers in parentheses was the percentage of each Trichoderma species.
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Figure 4. Phylogenetic tree constructed from maximum likelihood analysis of TEF1-α genes of Trichoderma. The TEF1-α nucleotide sequences were aligned with similar sequences from taxa of Trichoderma species available in the GenBank. The bootstrap scores are based on 1000 iterations. The scale bar represents 50 substitutions per nucleotide position. Sequences from this study were designated with isolate ID: AU.
Figure 4. Phylogenetic tree constructed from maximum likelihood analysis of TEF1-α genes of Trichoderma. The TEF1-α nucleotide sequences were aligned with similar sequences from taxa of Trichoderma species available in the GenBank. The bootstrap scores are based on 1000 iterations. The scale bar represents 50 substitutions per nucleotide position. Sequences from this study were designated with isolate ID: AU.
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Figure 5. Distribution of Trichoderma species in major coffee-growing zones of Ethiopia.
Figure 5. Distribution of Trichoderma species in major coffee-growing zones of Ethiopia.
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Figure 6. Distribution of Trichoderma species in different coffee production ecosystems.
Figure 6. Distribution of Trichoderma species in different coffee production ecosystems.
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Figure 7. Antagonistic effects of Trichoderma species against F. xylarioides: (A1–C1) dual-culture bioassay and (A2–C2) agar diffusion bioassay. T. asperellum AU71 (A), T. asperellum AU131 (B), T. longibrachiatum AU158 (C) and F. xylarioides (D1) alone as a control. Red arrows indicate the growth of the test pathogen.
Figure 7. Antagonistic effects of Trichoderma species against F. xylarioides: (A1–C1) dual-culture bioassay and (A2–C2) agar diffusion bioassay. T. asperellum AU71 (A), T. asperellum AU131 (B), T. longibrachiatum AU158 (C) and F. xylarioides (D1) alone as a control. Red arrows indicate the growth of the test pathogen.
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Table
Table 1. Identification, origin, NCBI Genbank accession numbers and isolation details of Trichoderma species from coffee rhizospheric soil of Ethiopia.
Table 1. Identification, origin, NCBI Genbank accession numbers and isolation details of Trichoderma species from coffee rhizospheric soil of Ethiopia.
Trichoderma SpeciesIsolate IDAccession Number (TEF1-α)District/LocationZoneCoffee Ecosystem
Trichoderma hamatumAU2MZ361591GeraJimmaSemi-forest
Trichoderma asperellumAU3MZ361592GeraJimmaSemi-forest
Trichoderma asperellumAU6MZ361593MelkoJimmaSemi-forest
Trichoderma asperellumAU8MZ361594GeraJimmaSemi-forest
Trichoderma virideAU9MZ361595YekiJimmaSemi-forest
Trichoderma asperelloidesAU10MZ361596GeraJimmaSemi-forest
Trichoderma asperelloidesAU11MZ361597GeraJimmaSemi-forest
Trichoderma asperellumAU13MZ361598GeraJimmaSemi-forest
Trichoderma longibrachiatumAU14MZ361599GeraJimmaSemi-forest
Trichoderma asperellumAU15MZ361600YekiShekaSemi-forest
Trichoderma hamatumAU19MZ361601GeraJimmaSemi-forest
Trichoderma asperellumAU21MZ361602GeraJimmaSemi-forest
Trichoderma asperellumAU22MZ361603YekiShekaSemi-forest
Trichoderma hamatumAU23MZ361604GeraJimmaSemi-forest
Trichoderma asperellumAU24MZ361605Odo ShakisoWest GujiGarden Coffee
Trichoderma asperellumAU26MZ361606Odo ShakisoWest GujiGarden Coffee
Trichoderma asperelloidesAU28MZ361607GeraJimmaGarden Coffee
Trichoderma asperelloidesAU29MZ361608GeraJimmaGarden Coffee
Trichoderma hamatumAU30MZ361609ShebedinoSidamaGarden Coffee
Trichoderma longibrachiatumAU32MZ361610GeraJimmaGarden Coffee
Trichoderma asperelloidesAU34MZ361611YekiShekaForest
Trichoderma asperellumAU37MZ361612GeraJimmaForest
Trichoderma asperellumAU38MZ361613YekiShekaForest
Trichoderma asperellumAU39MZ361614GimboKaffaForest
Trichoderma longibrachiatumAU40MZ361615GommaJimmaForest
Trichoderma brevicompactumAU41MZ361615YekiShekaForest
Trichoderma asperellumAU42MZ361615GommaJimmaForest
Trichoderma asperellumAU44MZ361618GommaJimmaForest
Trichoderma asperellumAU46MZ361619GommaJimmaForest
Trichoderma asperelloidesAU47MZ361620ChenaKaffaForest
Trichoderma longibrachiatumAU49MZ361621AndarachaShekaForest
Trichoderma hamatumAU50MZ361622AndarachaShekaForest
Trichoderma asperellumAU51MZ361623AndarachaShekaForest
Trichoderma asperellumAU53MZ361624AndarachaShekaForest
Trichoderma asperelloidesAU55MZ361624MenaJimmaSemi-forest
Trichoderma asperellumAU58MZ361626GewataKaffaForest
Trichoderma bissettiiAU59MZ361627Aleta WondoSidamaGarden Coffee
Trichoderma asperelloidesAU61MZ361628GommaKaffaGarden Coffee
Trichoderma asperellumAU69MZ361629WonagoGedeoGarden Coffee
Trichoderma koningiopsisAU70MZ361630WonagoGedeoGarden Coffee
Trichoderma asperellumAU71MZ361631Yirga cheffeSidamaSemi-forest
Trichoderma longibrachiatumAU72MZ361632Yirga cheffeGedeoSemi-forest
Trichoderma asperellumAU73MZ361633GewataKaffaSemi-forest
Trichoderma asperellumAU74MZ361634Aleta WondoSidamaSemi-forest
Trichoderma asperellumAU75MZ361635DaleSidamaGarden
Trichoderma orientaleAU77MZ361636DaleSidamaGarden
Trichoderma harzianumAU78MZ361637GimboKaffaForest
Trichoderma asperellumAU81MZ361638HaruWest WollegaForest
Trichoderma asperellumAU82MZ361639ShekoBenchi MajiForest
Trichoderma harzianumAU84MZ361640ShekoBenchi MajiForest
Trichoderma asperelloidesAU85MZ361641ShekoBenchi MajiForest
Trichoderma gamsiiAU86MZ361642ShekoBenchi MajiForest
Trichoderma harzianumAU87MZ361643GeraJimmaForest
Trichoderma harzianumAU88MZ361644GeraJimmaForest
Trichoderma asperellumAU91MZ361645ChenaKaffaForest
Trichoderma asperelloidesAU93MZ361646ChenaKaffaSemi-forest
Trichoderma asperelloidesAU94MZ361647ChenaKaffaSemi-forest
Trichoderma asperellumAU95MZ361648Limmu SakaJimmaSemi-forest
Trichoderma asperellumAU97MZ361649Limmu SakaJimmaGarden Coffee
Trichoderma asperelloidesAU98MZ361650Limmu SakaJimmaGarden Coffee
Trichoderma asperelloidesAU99MZ361651Limmu SakaJimmaGarden Coffee
Trichoderma asperellumAU100MZ361652Limmu SakaJimmaGarden Coffee
Trichoderma asperelloidesAU103MZ361653Limmu SakaJimmaSemi-forest
Trichoderma asperellumAU104MZ361654GeishaKaffaForest
Trichoderma aethiopicumAU106MZ361655GeishaKaffaForest
Trichoderma asperellumAU108MZ361656YekiShekaSemi-forest
Trichoderma longibrachiatumAU109MZ361657YekiShekaSemi-forest
Trichoderma asperellumAU110MZ361658YekiShekaSemi-forest
Trichoderma virideAU112MZ361659YekiShekaGarden Coffee
Trichoderma longibrachiatumAU114MZ361660YekiShekaGarden Coffee
Trichoderma asperellumAU115MZ361661YekiShekaGarden Coffee
Trichoderma citrovirideAU116MZ361662YekiShekaSemi-forest
Trichoderma asperelloidesAU118MZ361663Limmu SakaJimmaSemi-forest
Trichoderma asperellumAU122MZ361664YekiShekaSemi-forest
Trichoderma paratrovirideAU123MZ361665Limmu SakaJimmaSemi-forest
Trichoderma longibrachiatumAU125MZ361666YayuBuno BedeleForest
Trichoderma asperellumAU126MZ361667YayuBuno BedeleForest
Trichoderma asperellumAU129MZ361668YayuBuno BedeleForest
Trichoderma asperellumAU131MZ361669GeraJimmaForest Coffee
Trichoderma asperellumAU133MZ361670ShekoBenchi MajiSemi-forest
Trichoderma asperellumAU134MZ361671ShekoBenchi MajiSemi-forest
Trichoderma asperellumAU135MZ361672ShekoBenchi MajiSemi-forest
Trichoderma longibrachiatumAU136MZ361673ShekoBenchi MajiForest
Trichoderma longibrachiatumAU138MZ361674Semien BenchiBenchi MajiSemi-forest
Trichoderma asperelloidesAU139MZ361675Semein BenchiBenchi MajiSemi-forest
Trichoderma longibrachiatumAU141MZ361676Semein BenchiBenchi MajiSemi-forest
Trichoderma longibrachiatumAU143MZ361677ShekoBenchi MajiSemi-forest
Trichoderma asperelloidesAU144MZ361678ShekoBenchi MajiForest
Trichoderma reeseiAU145MZ361679ShekoBenchi MajiForest
Trichoderma harzianumAU148MZ361680HaruWest WollegaForest
Trichoderma asperelloidesAU149MZ361681HaruWest WollegaSemi-forest
Trichoderma gamsiiAU150MZ361682HaruWest WollegaSemi-forest
Trichoderma asperelloidesAU155MZ361683Delo MenaBaleForest
Trichoderma asperelloidesAU158MZ361684YekiShekaSemi-forest
Trichoderma longibrachiatumAU161MZ361685BerbereBaleForest
Trichoderma asperelloidesAU162MZ361686KerchaWest GujiGarden Coffee
Trichoderma longibrachiatumAU164MZ361687JarsoWest WollegaSemi-forest
Trichoderma asperellumAU165MZ361688JarsoWest WollegaSemi-forest
Trichoderma erinaceumAU166MZ361689Aira GulisoWest WollegaSemi-forest
Trichoderma asperellumAU167MZ361690Semein BenchiBenchi MajiSemi-forest
Trichoderma asperellumAU169MZ361691KerchaWest GujiGarden Coffee
Trichoderma asperellumAU171MZ361692Aira GulisoWest WollegaGarden Coffee
Trichoderma longibrachiatumAU173MZ361693Bule HoraWest GujiGarden Coffee
Trichoderma asperellumAU174MZ361694Bule HoraWest GujiGarden Coffee
Trichoderma asperellumAU175MZ361695BedeleBuno BedeleForest
Table
Table 2. Univariate diversity indices analysis of Trichoderma isolates in different coffee ecosystems and major coffee-growing zones of Ethiopia.
Table 2. Univariate diversity indices analysis of Trichoderma isolates in different coffee ecosystems and major coffee-growing zones of Ethiopia.
Ecological IndicesCoffee Ecosystem Major Coffee-Growing Zones
Native ForestSemi-ForestGarden CoffeeAverageJimmaKaffaBench MajiShekaBunno BedaleWest WollegaWest GujiGedioSidamaBale
Simpson index (D)0.810.810.70.760.910.940.830.820.70.640.530.90.80.87
Shannon’s index (H)1.971.961.571.771.971.821.71.830.750.941.291.331.541.89
Pielou evenness index (E)0.790.790.710.750.760.950.870.830.860.720.680.960.860.97
Abundance index (J)2.712.642.142.42.732.581.972.491.241.951.171.862.092.6
Table
Table 3. In vitro evaluation of Trichoderma isolates against F. xylarioides by dual confrontation culture technique and agar diffusion assay.
Table 3. In vitro evaluation of Trichoderma isolates against F. xylarioides by dual confrontation culture technique and agar diffusion assay.
Trichoderma SpeciesMycelia Inhibition over Control (%)Scale of Antagonistic Activity
Dual Culture Agar Diffusion Assay
T. hamatum AU2376.9 ab ± 1.0767.32 ab ± 4.06+++
T. longibrachiatum AU3275.2 b ± 0.769.82 ab ± 4.20+++
T. asperellum AU5372.6 b ± 0.366.3 c ± 2.3+++
T. koningiopsis AU7062.59 d ± 0.970.71 ab ± 4.82++
T. asperellum AU7181.8 a ± 3.0383.5 a ± 4.83++++
T. asperellum AU9779.3 b ± 1.076.42 b ± 3.68++++
T. harzianum AU10578.7 c ± 1.2 75.82 b ± 4.81+++
T. aethiopicum AU10679.3 d ± 5.168.50 c ± 5.12++
T. longibrachiatum AU12179.2 b ± 0.963.4 c ± 3.4+++
T. asperellum AU13184.8 a ± 0.986.7 a ± 1.6++++
T. longibrachiatum AU15882.4 a ± 0.588.2 a ± 3.5++++
T. asperellum AU17177.7 ab ± 0.366.4 c ± 2.5+++
Mean ± standard deviation77.54 ± 1.374.25 ± 3.74+++
Scale of antagonistic activity: ++++: very high antagonistic activity (>75%), +++: high antagonistic activity (61–75%), ++: moderate antagonistic activity (51–60%). Different alphabets depicted in superscript in the columns indicate mean treatments that are significantly different according to Tukey’s HSD post hoc test at p < 0.05; each value is an average of 3 replicate samples ± standard error.
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Mulatu, A.; Megersa, N.; Abena, T.; Kanagarajan, S.; Liu, Q.; Tenkegna, T.A.; Vetukuri, R.R. Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and Their Potential Use in Biocontrol of Coffee Wilt Disease. Crops 2022, 2, 120-141. https://doi.org/10.3390/crops2020010

AMA Style

Mulatu A, Megersa N, Abena T, Kanagarajan S, Liu Q, Tenkegna TA, Vetukuri RR. Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and Their Potential Use in Biocontrol of Coffee Wilt Disease. Crops. 2022; 2(2):120-141. https://doi.org/10.3390/crops2020010

Chicago/Turabian Style

Mulatu, Afrasa, Negussie Megersa, Tariku Abena, Selvaraju Kanagarajan, Qinsong Liu, Tesfaye Alemu Tenkegna, and Ramesh R. Vetukuri. 2022. "Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and Their Potential Use in Biocontrol of Coffee Wilt Disease" Crops 2, no. 2: 120-141. https://doi.org/10.3390/crops2020010

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