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Article

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide

1
Departamento de Agronomía (DAUCO María de Maeztu Unit of Excellence 2021–2023), Campus de Rabanales, Universidad de Córdoba, Edif. C4, 14071 Córdoba, Spain
2
Laboratory of Improvement and Protection of Olive Genetic Resources, Olive Tree Institute, BP 208 Cité Mahrajene, Tunis 1082, Tunisia
3
Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, University of Bari Aldo Moro, 70126 Bari, Italy
4
School of Science and Health, Western Sydney University, Penrith 2747, Australia
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this article.
Academic Editor: Lei Cai
J. Fungi 2021, 7(9), 741; https://doi.org/10.3390/jof7090741
Received: 16 August 2021 / Revised: 3 September 2021 / Accepted: 6 September 2021 / Published: 9 September 2021
(This article belongs to the Special Issue Fungal Biodiversity and Ecology 2.0)

Abstract

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.
Keywords: anthracnose; Colletotrichum spp.; diversity; Olea europaea; pathogenicity; phenotype; phylogenetic analysis anthracnose; Colletotrichum spp.; diversity; Olea europaea; pathogenicity; phenotype; phylogenetic analysis

1. Introduction

The olive (Olea europaea L. subsp. europaea) is the most important tree crop worldwide, covering over 11 million hectares, more than the whole of stone fruit species [1]. Most olives are grown near the Mediterranean Sea, especially Spain, Italy, Greece, Tunisia, and Portugal. The excellent adaptation of the olive plant to different conditions has prompted a spread of olive farming to countries where it is not a traditional crop, such as Australia, Brazil, or China [2,3]. Due to this expansion through different areas, the olive plant has been gradually exposed to new pathogens. This situation is particularly striking in olive anthracnose, the most important disease of the fruit.
Olive anthracnose caused by numerous Colletotrichum species causes dramatic losses of fruit yield and oil quality during epidemic years [4,5,6,7]. The pathogen infects through the seasons, but disease symptoms appear at the beginning of ripening when the color of the fruit changes from green to black [6,8]. Typical symptoms are depressed, round, and ochre or brown lesions leading to fruit rot with great orange conidial masses (Figure 1a,b), the “soapy olive” syndrome that gives its name to this disease in Spanish [9]. Subsequently, fruit are mummified (Figure 1c,d) when the temperature falls, relative humidity increases in late autumn-winter, and most of them fall to the soil [6,10]. The pathogen also causes the dieback of olive branches via phytotoxins (Aspergillomarasmine A) produced by the fungus in the rotten fruit (Figure 1e,f) [8,11,12]. Likewise, the pathogen can cause the blight of olive inflorescences, mainly when mummies remain attached to the tree canopy during the flowering [8,12,13]. In addition, the pathogen may act as a secondary invader of injured tissue and can also survive as endophyte or saprophyte. The ability to survive and multiply in the absence of symptoms may explain why anthracnose fungi often cause unexpected crop losses in olives [12,14].
The causal agent of olive anthracnose was described for the first time in Portugal by de Almeida [15] as Gloeosporium olivarum. Subsequently, this species was reclassified as Colletotrichum gloeosporioides (anamorph of Glomerella cingulata) after reviewing the Gloeosporium genus by von Arx [16]. Later, the species C. gloeosporioides was considered a heterogeneous species complex affecting about 300 plant species [17]. Currently, over 1000 epithets are listed in Mycobank [18] under Colletotrichum, which comprises 248 accepted species, most of them grouped into 14 species complexes [19].
As with many other crops, olives can be affected by a wide range of Colletotrichum species [20,21,22]. To date, a total of 14 Colletotrichum spp. have been associated with olive anthracnose over the world. These species belong to three Colletotrichum complexes: C. acutatum, C. boninense, and C. gloeosporioides [5,13,23,24]. Among them, the species C. acutatum sensu stricto (from now on C. acutatum), C. fioriniae, C. godetiae, C. nymphaeae, C. rhombiforme, and C. simmondsii, all of them belonging to the C. acutatum species complex, are currently considered the major pathogens of this genus [5,13,24]. While several Colletotrichum species can be found in an olive-growing area, there is usually one dominant one and some secondary [7]. For example, the species C. nymphaeae is dominant in the olive orchards of Portugal [4,25], while C. godetiae is the prevalent species in several Mediterranean countries such as Greece, Montenegro, and Spain [6,7,24,26].
In southern Italy, Faedda et al. [26] found that the Colletotrichum population of olive trees consisted of mainly those dominated by C. clavatum. However, Damm et al. [27] considered C. clavatum as a synonym of the older species C. godetiae. Later, different studies revealed a wide distribution of C. acutatum and C. godetiae together with four species—C. aenigma, C. gloeosporioides, C. cigarro (C. gloeosporioides especies complex), and C. karstii (C. boninense complex)—associated with olive anthracnose in southern Italy [23,24,28,29]. Consequently, Mosca et al. [28] and Schena et al. [24] have hypothesized that C. acutatum is an emerging olive pathogen in Italy. This latter species has also been reported to cause olive anthracnose in Australia, Brazil, Greece, Portugal, South Africa, Tunisia, and Uruguay [4,5,13,30,31,32].
Furthermore, other species belonging to the C. gloeosporioides species complex have been described in Australia (C. siamense and C. theobromicola), Montenegro (C. queenslandicum), and Uruguay (C. alienum and C. theobromicola) as associated with olive fruit [13,23]. Nevertheless, the pathogenicity of several of these species (C. aenigma, C. cigarro, C. karstii, C. queenslandicum, and C. siamense) on olive fruit is still uncertain, suggesting that their role in the fruit infection could be secondary [5].
From the first descriptions of C. acutatum sensu lato and C. gloeosporioides s. l. as the causal agents of the olive anthracnose in 1999 [33], many studies focused on aetiology have been conducted mainly in Italy and Portugal, generating relevant knowledge about the diversity of Colletotrichum species associated with the disease [4,23,24,28,34]. In the case of Spain, the etiological knowledge about olive anthracnose is much more limited and suggests that there are two prevalent species, with C. godetiae being dominant [7]. Therefore, more etiological studies are necessary to elucidate the diversity of Colletotrichum species involved in the olive anthracnose. Furthermore, differences in virulence among Colletotrichum species, and isolates of the same species, have also been described in different woody crops [5,35,36,37,38]. However, the number of species tested is still slight, and broader studies on the pathogenicity of Colletotrichum spp. causing olive anthracnose are necessary.
During these last two decades, many Colletotrichum isolates from olive fruit showing anthracnose symptoms in orchards located in the Iberian Peninsula, Spain, and Portugal were studied in our laboratory. Both Spain and Portugal produce around 65% of the global supply of olive oil [1]. In parallel, many Colletotrichum isolates from olives or other susceptible hosts to anthracnose from Australia, Brazil, California, Greece, Portugal, Italy, Tunisia, and Uruguay have been studied in close collaboration with different international research groups. Thus, the main goal of the present study was to characterize a vast collection of Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries. To this end, in the present study, we combined different techniques for characterization of the Colletotrichum population affecting olive fruit around the world, including morphological characteristics [4,5,16,26,31], physiological traits including tolerance to fungicides and enzymatic activity [4,5,7,33], and molecular tools [4,19,23,27]. Therefore, the specific objectives of this study were (i) to obtain a wide collection of Colletotrichum isolates representative of the geographic origin described above and from different hosts showing anthracnose symptoms; (ii) to characterize them based on phenotypic (colony and conidial morphology; and benomyl-sensitive and casein-hydrolysis tests) and molecular characters (multilocus alignments and phylogenetic analyses); (iii) to determine the pathogenicity of Colletotrichum isolates to olive fruit; and (iv) to evaluate cross-pathogenicity on different Colletotrichum hosts. Elucidating the biodiversity of Colletotrichum species causing olive anthracnose is essential for a better understanding of the aetiology and epidemiology of the most critical fruit disease of this legendary crop.

2. Materials and Methods

2.1. Collection of Fungal Isolates

Olive fruit samples showing symptoms of anthracnose were collected from many commercial orchards from 1998 to 2016. Symptomatic fruit were collected from different provinces across Spain, emphasizing the orchards located in the Andalusia region of southern Spain, the world’s leading olive-producing region. Many other samples were collected from commercial orchards situated in southern Portugal, where olive anthracnose is endemic [34,39]. Isolations were made from affected fruit with the typical anthracnose lesions. Diseased fruit were surface disinfested with commercial bleach (Cl at 50 g L–1) at 10% (v/v) in sterile water for 1 min, and air-dried on sterile filter paper for 30 min. Affected tissues were cut with a sterile scalpel and plated on potato dextrose agar (PDA) (Difco Laboratories®, Detroit, MI, USA) acidified with lactic acid (2.5 mL of 25% [v/v] per liter of medium) to minimize bacterial growth (APDA). When the affected fruit tissues showed abundant pathogen sporulation, masses of conidia were removed using a sterile needle and cultured in Petri dishes on APDA. Petri dishes were incubated at 23 ± 2 °C under a 12-h daily photoperiod of cool fluorescent light (350 μmol m–2 s–1) for 5 days. Single-spore isolates were prepared before use in further experiments using serial dilutions [40]. Moreover, Colletotrichum isolates recovered from olive fruit showing anthracnose symptoms in Australia, Brazil, California (the USA), Greece, Italy, Tunisia, and Uruguay were also included in this study as collaboration with several international research groups from those major olive-growing regions of the world (Table 1). All the isolates were maintained on colonized PDA into sterile plastic tubes with sterile paraffin oil (Panreac Química SA, Barcelona, Spain) at 4 °C in darkness for long-term storage in the fungal collection of the Department of Agronomy at the University of Cordoba (UCO; Spain).

2.2. Phenotypic Characterization

2.2.1. Colonies and Conidial Morphology

Thirty-eight representative isolates belonging to C. acutatum (27 isolates), C. boninense (two isolates), and C. gloeosporioides (nine isolates) species complexes (Table 1) were used to study mycelium colony and conidium morphology. To this end, all the isolates were grown on PDA (Difco® Laboratories, Detroit, MI, USA) for two weeks at the same incubation conditions described above. There were three replicated Petri dishes per isolate.
Characteristics of mycelia (texture, density, color, and zonation) were recorded by visual observations on 7-day-old colonies [41]. For all isolates, color was determined using a color scale [42]. For conidial measures, conidial masses removed from the margin of 10-day-old colonies were placed on slides with a drop of 0.005% acid fuchsine in lactoglycerol (1:1:1 lactic acid, glycerol, and water) and covered with a coverslip. For each isolate and replicate Petri dish, the size, and the shape of 50 conidia were measured utilizing a Nikon Eclipse 80i microscope (Nikon Corp., Tokyo, Japan) at 400× magnification. The conidial size was determined by measuring its length and width, and the length/width ratio was calculated. According to their shape, conidia were classified into three categories: (0) Conidia with two rounded ends (ellipsoid); (1) Conidia with one rounded end and the other acute (clavate); and (2), Conidia with two acute (sharp) ends (fusiform). Data were expressed as a percentage (%) of each type of conidium.

2.2.2. Benomyl-Sensitive Assay

Twenty-seven representative isolates belonging to C. acutatum (16 isolates), C. boninense (two isolates), and C. gloeosporioides (nine isolates) species complexes were used to evaluate their sensitivity to benomyl by in vitro sensitivity assay (Table 1). Based on previous studies [4,43], and our preliminary trials using isolates of C. acutatum and C. gloeosporioides species complexes, we determined a threshold of 5 µg of benomyl per milliliter to differentiate sensitive and tolerant isolates to this fungicide. Thus, mycelial plugs (5 mm in diameter) obtained from the margins of 7-day old actively growing colonies on PDA were transferred to Petri dishes with PDA amended with 5 µg mL−1 of benomyl (benomyl 50%, WP, Adama Agriculture, Madrid, Spain). Mycelial plugs of each isolate were plated on non-amended PDA as control. All Petri dishes were incubated under the described conditions. There were three replicated Petri dishes per isolate and treatment (benomyl and control), and the experiment was conducted twice.
The evaluation was performed at 7 days, measuring the largest and smallest diameters of each colony. For each isolate, the inhibition percentage (%) was calculated by comparing the growth on PDA and on PDA amended with benomyl. Data from repetitions of the experiment were combined after checking for homogeneity of variances of the experimental error of the two replicated experiments by the F test. Subsequently, analysis of variance (ANOVA) was conducted using a randomized complete block design with the two repetitions of the experiment as blocks, fungal isolate as the independent variable, and inhibition percentage as the dependent variable. Mean comparisons were made using Tukey’s honestly significant difference (HSD) test [44]. Data were analyzed using Statistix 10 [45].

2.2.3. Casein-Hydrolysis Assay

The 27 Colletotrichum isolates, previously studied according to their sensitive/tolerance benomyl fungicide, were also characterized according to their ability to hydrolyse the casein. Thus, the 27 Colletotrichum isolates were transferred, as described previously, to hydrolyse casein medium (CHM). We formulated the CHM media using a 15% milk powder solution (Sveltesse Nestle®, Esplugues de Llobregat, Barcelona, Sapin) in deionized water sterilized at 120 °C for 15 min, and 20 mL of the sterile milk solution was added to 980 mL of sterilized Water Agar (WA; Biokar-Diagnostics, Allonne, France) before solidification (around 50 °C) and homogenized for 2 min using a magnetic rotor (Agimatic-N, JP-Selecta, Barcelona, Spain).
A 5-mm diameter plug of each Colletotrichum isolate was plated to Petri dishes with CHM and incubation for 5 days as described for the benomyl sensitivity assay. For each isolate, mycelial plugs were plated on non-amended PDA as control. We visually determined the presence or absence of the hydrolysis halo surrounding the Colletotrichum colony growing on CHM media. There were three replicated Petri dishes per isolate and treatment (milk powder suspension and control), and the experiment was conducted twice.

2.3. Molecular Characterization

2.3.1. DNA Extraction, PCR, Sequencing, and Nucleotide Alignment

Genomic DNA was extracted from 100 mg of mycelium of the 185 Colletotrichum isolates growing on PDA (Table 1). Mycelial tissues were ground using a FastPrep®-24 grinder machine (MP Biomedicals, Irvine, CA, USA). Subsequently, DNA extractions were carried out using E.Z.N.A.® Fungal DNA Mini Kit (OMEGA bio-tek, Norcross, GA, USA) following the manufacturer’s instructions. The concentration and purity of extracted DNA were determined by means of MaestroNano® spectrophotometer (MaestroGen, Hsinchu City, Taiwan).
Six genomic areas, 5.8S nuclear ribosomal gene with two flanking internal transcribed spacers (ITS), beta-tubulin (TUB2), actin (ACT), partial sequences of the chitin synthase 1 (CHS-1), histone 3 (HIS3), and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were amplified and sequenced. For that, the following primer pairs were correspondingly used: ITS4 and ITS5 [46], Bt-2a and Bt-2b [47], ACT-512F and ACT-783R [48], CHS-354R and CHS-79F [48], CYLH3F and CYLH3R [49], and GDF1 and GDR1 [50]. Additionally, to infer the identity of fungal isolates belonging to the C. gloeosporioides complex, the intergenic region between Apn2 and Mat1-2 genes (ApMat) was also amplified and sequenced with the primer pair AMF1 and AMR1 [51].
PCR amplifications were performed in a MyCyclerTM Thermal Cycler (BIO-RAD) in a total volume of 25 µL. All PCRs mixture contained 5 µL of 5×MyTaq reaction buffer, 0.13 µL of MyTaq DNA polymerase (Bioline), and 20 ng of genomic DNA template. Additionally, 0.2 µM of each primer was added for the ACT, CHS-1, HIS3, and GADPH PCRs, and 0.4 µM of each primer for ITS, TUB2, and ApMat PCRs. Negative control was included in all PCRs using ultrapure water instead of DNA. The PCRs cycling programs were conducted as follows: an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, annealing for 15 s and 72 °C for 10 s, and a final extension at 72 °C for 7 min. The annealing temperatures used were: 48 °C for ITS, 52 °C for GAPDH, CHS-1, HIS3, TUB2, and ACT, and 55 °C for ApMat. All PCRs were stopped at 4 °C.
Amplification products were checked by electrophoresis in 1.7% (wt/vol) agarose gel stained with RedSafe (Intron Biotechnology, Sagimakgol-ro Joongwon-gu Seongnam-Si Korea, Republic of South) and visualized under ultraviolet light. DNA gTP-Ladder (gTPbio) was used for electrophoresis as DNA size markers. Single-band products were purified using MEGAquick-spinTM Total Fragment DNA Purification kit (INTRON Biotechnology), following the manufacturer’s instructions. Subsequently, purified PCR products were sequenced in both forward and reverse directions by the Central Service Support Research (SCAI) at the University of Córdoba (Spain).
Generated sequences were assembled and edited using the software SeqMan® v. 7.0.0. (DNASTART LaserGen, Madison, WI, USA). Consensus sequences for all isolates were compiled into a single file (Fasta format) and were deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/ accessed on 1 Augest 2021) (Table 1).

2.3.2. Phylogenetic Analyses and Species Delimitation

All consensus sequences were blasted against the NCBIs GenBank nucleotide database to determine the closest relative species of Colletotrichum to our isolates. In total, 185 isolates of Colletotrichum were included in the molecular phylogenetic analyses. Additionally, sequences from 70 species of Colletotrichum (90 isolates in total) were downloaded from GenBank and included in the analysis as reference sequences or outgroups (Table 1). Reference sequences were selected based on their high similarity with our query sequences using MegaBLAST and were added to the data set and aligned using CLUSTAL W v. 2.0.11 [52].
A Neighbour-Joining (NJ) analysis was performed individually for each genomic area using the Maximum Composite Likelihood method and 2000 bootstrap replications to determine whether the sequence datasets were congruent and combinable. Tree topologies of 70% reciprocal bootstrap generated individually for each locus were compared visually. Because no supported nodes were in conflict, the data of different loci were combined into single concatenated datasets. Three different datasets were analyzed to compare and identify our Colletotrichum isolates correctly. For a first identity approach, one phylogeny was constructed using a combination of ITS and TUB2 sequences (dataset I). This phylogeny consisted of 40 taxa of the Colletotrichum genus including species belonging to C. acutatum, C. boninense, and C. gloeosporioides species complexes, among other Colletotrichum spp., with C. dracaenophilum (CBS 118199) as an outgroup. Subsequently, a second phylogeny was performed by multilocus alignment of ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH sequences (dataset II) to identify our isolates [27]. This second phylogeny consisted of 41 taxa of the Colletotrichum genus including species belonging to C. acutatum, C. boninense, and C. gloeosporioides species complexes, with C. dracaenophilum (CBS 118199) again as an outgroup. A third multilocus alignment combining the ITS, TUB2, and ApMat sequences (dataset III) was performed for inferring organismal phylogeny of 14 isolates belonging to the C. gloeosporioides species complex [53,54].
For multilocus alignments, phylogenetic analyses were conducted by Bayesian Inference (BI) and Maximum Parsimony (MP). The MP trees were obtained using the Tree-Bisection-Regrafting (TBR) algorithm with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates). All positions containing gaps and missing data were eliminated. A set of 2000 bootstrap replications evaluated the robustness of the generated trees. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were recorded. BI analyses were performed with MrBayes v.3.2.6 [55], which uses Markov Chain Monte Carlo to approximate the posterior probability of trees. Two analyses with four chains each were run at the same time, for 1 × 107 generations, sampled every 100 generations, and starting from a random tree topology. The “temperature” parameter was set to 0.2. For the consensus tree, the first 25% of the saved trees were discarded as the burn-in phase of the analysis. Each of the individual genes and a combined data set were aligned, adjusted manually, and analyzed by NJ or MP using MEGA v.7 [56]. In BI and NJ analyses, the best evolutionary model for each gene partition was also determined by MEGA v.7. The genes were concatenated in a single nucleotide alignment using Phylogenetic Data Editor (PhyDE-1).

2.4. Pathogenicity Test

2.4.1. Pathogenicity on Olive Fruit

The following Colletotrichum isolates were evaluated according to their pathogenicity on olive fruit: C. acutatum isolates from olive fruit (Col-193 and Col-256) and almond fruit (Col-536); C. fioriniae isolate from olive fruit (Col-172); C. gloeosporioides from olive fruit (Col-41) and sweet orange fruit (Col-69); C. godetiae from olive fruit (Col-30, Col-57, Col-88, Col-508, Col-515, and Col-519) and almond fruit (Col-522); C. karstii from sweet orange fruit (Col-79); and C. nymphaeae isolates from olive fruit (Col-42 and Col-506) and strawberry fruit (Col-84 and Col-86) (Table 1). Violet (color class 3) olive fruit of the highly susceptible cv. Hojiblanca were collected from olives growing in the World Olive Germplasm Bank (WOGB), belonging to the IFAPA located in the Córdoba province [57]. Before inoculation, the olive fruits were washed and surface-disinfested according to Moral et al. [9]. Surface-disinfected olive fruits were placed in moist chambers (plastic containers, 22 × 16 × 10 cm) at 100% relative humidity (RH) and inoculated by spraying them up run-off with a conidial suspension adjusted with a haemocytometer to 105 conidia mL−1. After inoculation, humid chambers were incubated at 23 ± 2 °C with a 12-h photoperiod. Additionally, olive fruit sprayed with sterile distilled water were included as a control. There were three replicated humid chambers per isolate and 20 fruits per humid chamber, and the experiment was conducted twice. A completely randomized design was used with fungal isolates as the independent variable and moist chambers as replications. The pathogens were re-isolated from the olive fruit as described above.

2.4.2. Pathogenicity on Other Hosts

Colletotrichum godetiae isolates from olive fruit (Col-9 and Col-57), C. gloeosporioides from sweet orange fruit (Col-69), C. karstii from sweet orange fruit (Col-79), C. nymphaeae from olive fruit (Col-42), and C. siamense from strawberry fruit (Col-44) were selected to evaluate their pathogenicity on different hosts (Table 1). Fruits of almond (Prunus dulcis (Mill.) D.A.Webb) cv. Guara, apple (Malus domestica Borkh.) cv. Golden Delicious, sweet orange (Citrus sinensis L.) cv. Lanelate, and strawberry (Fragaria × Ananassa L.) cv. Camarosa, as well as leaves of oleander (Nerium oleander L.) were selected for this assay. Plant material was washed, and surface disinfested as described above. The pathogenicity of the six Colletotrichum isolates was evaluated by independent inoculation on the different hosts. Thus, almond, apple, olive, and strawberry fruits were inoculated by surface deposition of one mycelial plug (9 mm in diameter) per fruit pierced with a sterile needle, according to Moral et al. [9]. Oleander leaves were inoculated by the same method, but in this case, three mycelial plugs (7 mm in diameter) were deposited per leaf. Inoculated fruits and leaves were incubated in moisture chambers at 23 ± 2 °C with a 12-h photoperiod. Additionally, non-inoculated fruits or leaves treated with PDA plugs were included as a control. There were three replicated humid chambers per isolate-host combination, 10 fruits or leaves per humid chamber, and the experiment was conducted twice. A completely randomized design was used with fungal isolates and host as the independent variable and moist chambers as replications. The pathogens were re-isolated from the fruits and leaves as described above.

2.4.3. Disease Assessment and Data Analysis

Disease severity (DS) in inoculated olive and almond fruits was evaluated weekly until most of the fruit achieved the maximum value (approx. 14 and 21 days for olive and almond fruits, respectively). DS was assessed using a 0–5 rating scale: (0) no symptoms; (1) 1–25% of the fruit surface affected; (2) 26–50%; (3) 51–75%; (4) >75%; and (5) 100% [9]. A disease severity index (DSI) was calculated in each replication using the following formula: DSI = [(Σni × i)/(N × 5)] × 100, where i represents a severity (zero to five), ni is the number of fruits with severity i, N is the total number of fruits, and five is the highest value of the severity rating scale. For the rest of the hosts, the largest and smallest diameters of lesions were measured weekly, and mean data were converted to the radial growth rate (mm day−1). DS of the inoculated fruits of apple, sweet orange, and strawberry, and in leaves of oleander was evaluated weekly until most of the fruits or leaves reached 90–100% of their surface affected (approx. 21, 41, 12, and 18 days for apple, sweet orange, strawberry, and oleander, respectively). In all cases, relative areas under the disease progress curve (RAUDPC) were calculated using the trapezoidal integration of DSI values over time. RAUDPC data from the two runs of the experiment were subjected to analysis of variance (ANOVA). The non-pathogenic isolates were excluded from the statistical analysis. The RAUDPC data were logarithmically transformed when necessary to the homogeneity of variances or normality. When ANOVA showed significant differences for each host, means were compared according to Tukey’s honestly significant difference (HSD) test at p = 0.05 [44]. Data were analyzed using Statistix 10 [45].

3. Results

3.1. Collection of Fungal Isolates

In total, 137 Colletotrichum isolates were obtained from different hosts across the Iberian Peninsula: 83 of them isolated from olive trees in Portugal, and 54 of them isolated from olives and other hosts in Spain. Forty-six of the Spanish isolates were obtained from olives located in the four major olive-producing regions (Andalusia, Extremadura, Catalonia, and Valencia; located at Southern, South-western, North-eastern, and Eastern Spain, respectively). The other eight Spanish isolates were recovered from almond (two isolates), Citrus (two isolates), Pistacia terebinthus (one isolate), and strawberry (three isolates). In addition to Iberian isolates, we included 16 isolates from Australia, 1 isolate from Brazil, 5 isolates from California, 6 isolates from Greece, 12 isolates from Italy, 5 isolates from Tunisia, and 3 isolates from Uruguay obtained from affected olive fruit (Table 1).

3.2. Phenotypic Characterization

3.2.1. Colonies and Conidial Morphology

Most Colletotrichum colonies were similar regarding texture and density characteristics with abundant aerial mycelium with regular margins. However, the Australian isolates Col-166, Col-200, Col-152, and Col-214 had colonies with lobulated margins. The growth pattern of all colonies was radial with concentric circles. Nevertheless, the colonies showed a broad variation in color, mainly white, whitish to dark gray, and pinkish-orange being the most common colors observed. Thus, colony color was helpful to discriminate color sub-groups. In general, colonies of all C. godetiae isolates were gray (from dark to light gray), colonies of C. acutatum isolates showed pinkish-orange tones, and C. fioriniae and C. gloeosporioides isolates were light gray. In particular, the isolate C. siamense Col-44 from strawberry showed a distinctive greenish-gray colony color. However, the rest of the Colletotrichum spp. isolates showed colonies with many variations in color within the same species, so it was impossible to establish a relationship between species and the color of their colonies (Table 2; Figure 2).
The average length of the conidia ranged between 8.3 and 14.8 μm for C. acutatum isolate Col-175 and C. gloeosporioides Col-41, respectively. The average width varied from 2.7 to 5.1 μm for C. acutatum isolate Col-175 and for the isolates C. godetiae Col-30 and C. gloeosporioides Col-69, respectively. In general, the conidia were hyaline, varying in type (ellipsoid, clavate, and fusiform) between isolates within species complex or even within the same fungal species. Isolates belonging to C. acutatum species complex had the three types of conidia. Isolates of C. fioriniae and C. nymphaeae showed fusiform and clavate conidia, respectively. Most isolates identified as C. godetiae showed clavate conidia, except isolates Col-88 and Col-522, which showed ellipsoid and fusiform conidia, respectively. Colletotrichum simmondsii isolate Col-169 showed fusiform conidia. Concerning the isolates belonging to the C. boninense complex, differences in the type of conidia were also observed between species. For example, C. boninense isolate Col-178 showed clavate conidia, while C. karstii isolate Col-79 showed ellipsoid conidia. Finally, most isolates belonging to the C. gloeosporioides complex showed ellipsoid conidia, except two isolates identified as C. siamense (isolates Col-44 and Col-184), which showed clavate conidia (Table 2).

3.2.2. Benomyl Sensitive Assay

Wide variability in mycelial growth rate was observed among the Colletotrichum isolates grown on PDA amended with 5 µg mL−1 of benomyl. In general, Colletotrichum isolates developed lower aerial mycelium and greater conidial production than those in the presence of the fungicide. There were significant differences (p < 0.001) for mycelial growth inhibition between isolates. According to their sensitivity to benomyl, the Colletotrichum isolates could be grouped into two groups (moderately and highly sensitive). The moderately sensitive group only included isolates belonging to C. acutatum species complex, whose percentages of inhibition ranged from 33.5% to 71.1% for C. acutatum isolate Col-166 and C. fioriniae isolate Col-172, respectively, both from olive trees in Australia (Table 2). The highly sensitive group was formed by isolates belonging to C. boninense and C. gloeosporioides species complexes, whose percentages of mycelial growth inhibition ranged from 93.8% to 100% for C. siamense isolate Col-160 (from olive fruit, Australia) and C. gloeosporioides isolate Col-41 (from olive fruit, Spain), respectively. However, no benomyl-resistant Colletotrichum isolates were observed in any case.

3.2.3. Hydrolysis-Casein Assay

Seventeen out of the 26 tested Colletotrichum isolates caused a casein hydrolysis halo surrounding their colonies in CHM that was observable at 1 day of incubation. At 5 days of incubation, Colletotrichum isolates were classified as able (+ or ++ for the width of halo ≤ 2 or > 2 mm, respectively) or not able (-) to hydrolyze casein. This phenotypic characteristic was also helpful to discriminate isolates between Colletotrichum species complexes, but with some exceptions. Thus, all the isolates belonging to the C. acutatum species complex could hydrolyze casein, except C. fioriniae isolate Col-172 from olive trees in Australia. Most isolates belonging to C. boninense and C. gloeosporioides species complexes could not hydrolyze casein, except three isolates within the C. gloeosporioides species complex (C. alienum isolate Col-211, C. siamense isolates Col-184, and Col-187, all of them from olive trees in Australia) (Table 2).

3.3. Molecular Characterization. Phylogenetic Analyses

Our Colletotrichum isolates were initially identified based on the combined data of ITS and TUB2 sequences alignment. This first analysis (dataset I) included 186 taxa from which 109 were sequences of our isolates, and 77 were reference sequences from GenBank including the outgroup C. dracaenophylum isolate CBS 118199. A total of 867 characters, including gaps, were analyzed (ITS from 1 to 500, and TUB2 from 501 to 867 position). For BI analysis, a K2 + G model was used to combine both regions, and the phylogenetic tree is shown in Figure 3. In the MP analysis of the ITS and TUB2 regions, there were 811 positions in the final dataset, from which 220 characters were parsimony-informative and 591 conserved sites. The five most parsimonious trees were retained (TL = 555 steps, CI = 0.520, RI = 0.956, RC = 0.539, and HI = 0.480). The consensus tree obtained by MP analysis confirmed the topology obtained with BI, and bootstrap supports agreed with Bayesian probability values. Our isolates were grouped into three well-supported clades in this first phylogenetic tree according to the three Colletotrichum species complexes. Likewise, 93 isolates (from different countries and hosts) were grouped into the C. acutatum species complex, two isolates (Col-178 and Col-79, from Australia and Spain, and olive and sweet orange fruits, respectively) were grouped into the C. boninense species complex, and 14 isolates (from Australia, Spain and Tunisia, and most of them from olive trees) were grouped into the C. gloeosporioides species complex. These three different clades were well supported with a Bayesian posterior probability (PP) value of 1.0 for all of them, and with bootstrap support (MP (BS); %) values of 99%, 98%, and 100% for C. acutatum, C. boninense, and C. gloeosporioides species complexes, respectively. Most of the isolates belonging to the C. acutatum species complex clustered in four clades: (i) 45 isolates clustered together with reference isolates of C. godetiae (PP/BS(%):1/99), (ii) 11 isolates clustered with reference isolates of C. acutatum (1/99), (iii) 29 isolates clustered with reference isolates of C. nymphaeae (<0.90/70), and (iv) 7 isolates clustered with reference isolates of C. fioriniae (1/99). The isolate Col-169 from olive fruit (Australia) could not be well-identified with this phylogenetic analysis due to the fact that it clustered between reference sequences of C. paxtonii (IMI 165753) and C. simmondsii (CBS 122122) within the C. acutatum species complex.
Concerning the C. boninense species complex, the isolate Col-178 (from olive fruit, Australia) clustered together with reference isolates of C. boninense (1/99), and the isolate Col-79 (from sweet orange, Spain) clustered together with reference sequences of C. karstii (0.99/93). ITS and TUB2 multilocus alignment was not helpful to distinguish between species belonging to the C. gloeosporioides species complex, which formed a unique clade (1/100) (Figure 3).
A second multigene analysis (dataset II) was performed based on ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions with a total of 188 taxa from which 126 were sequences of our isolates, and 62 were reference sequences from GenBank, including the outgroup C. dracaenophylum isolate CBS 118199. A total of 2143 characters, including gaps, were analyzed. The gene boundaries in the multialignment were ITS (from 1 to 518 positions), TUB2 (519–902), ACT (903–1186), CHS-1 (1187–1464), HIS-3 (1465–1853), and GADPH (1854–2143). For Bayesian analysis, a K2 + G model was selected for ITS, a K2 + I model for TUB2 and ACT, a TN93 + G model for CHS-1 and HIS-3, and a K2 + G+I model for GADPH, and they were incorporated in the analysis. The tree obtained with Bayesian PP values is shown in Figure 4.
Regarding MP analysis, there were 1845 positions in the final dataset, from which 693 characters were parsimony-informative, 1266 conserved sites, and 114 parsimony-uninformative. Two most parsimonious trees were retained (TL = 1673 steps, CI = 0.525, RI = 0.944, RC = 0.529, HI = 0.475). The consensus tree obtained by MP analysis confirmed the topology obtained with Bayesian inference, and BS values agreed with Bayesian probability values. This second phylogenetic improved the identification of isolates belonging to the C. acutatum species complex. Regarding our isolates, 119 were grouped as C. acutatum species complex, one isolate (Col-79) was grouped as C. boninense complex, and 14 isolates were grouped as C. gloeosporioides species complex. These three clades were well supported with a PP value of 1.0 and BS values of ≥ 99%. The isolates belonging to the C. acutatum species complex clustered in five well-supported clades: (i) 84 isolates (one from Italy, 73 from Portugal, and 10 from Spain, most of them from olive trees) clustered together with three reference isolates of C. nymphaeae (1/98), (ii) 25 isolates (20 from Spain, and 5 from Portugal, all of them from olive trees except Col-522 from almond trees) clustered with seven reference isolates of C. godetiae (1/99), (iii) 6 isolates from olive trees (five from California and one from Australia) clustered with six reference isolates of C. fioriniae (1/100), and (iv) 3 isolates with different origins (Australia, Tunisia, and Spain) clustered with the reference isolates of C. acutatum (1/100). In this case, the isolate Col-169 from olive trees (Australia), which could not be identified before based on ITS and TUB2-combined alignment, clustered consistently (1/94) with the reference ex-type isolate of C. simmondsii CBS 122122. The combined alignment of ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions was insufficient to distinguish between species belonging to the C. gloeosporioides species complex (Figure 4).
Finally, an additional multilocus alignment combining ITS, TUB2, and ApMat gene sequences (dataset III) was performed for inferring organismal phylogeny of the isolates belonging to the C. gloeosporioides species complex. It included 42 taxa, from which 14 were sequences of our isolates, and 29 were reference sequences from GenBank including the outgroup C. xanthorrhoeae ICMP17903. A total of 1749 characters, including gaps, were processed. The gene boundaries in the multialignment were ITS (1–484), TUB2 (485–842), and ApMat (843–1749). For Bayesian analysis, a K2 + G model was selected for ITS and ApMat, while a K2 model was used for TUB2. The tree obtained with Bayesian posterior probability values is shown in Figure 5. Regarding MP analysis, there were a total of 1576 positions in the final dataset, from which 363 characters were parsimony-informative, 1097 conserved sites, and 116 parsimony-uninformative. The four most parsimonious trees were retained (TL = 869 steps, CI = 0.764, RI = 0.894, RC = 0.683, and HI = 0.236). MP analysis confirmed the tree obtained by BI and BS agreed with PP values. The 14 isolates from this study classified within the C. gloeosporioides complex were identified as C. alienum (two isolates from olive trees, Australia; 1/99), C. fructicola (one isolate from olive trees, Spain; 1/99), C. gloeosporioides (four isolates, two of them from Tunisia (Col-251 and Col-295); and the other two from Spanish olive (Col-41) and sweet orange trees (Col-69); 1/100), C. perseae (one isolate from olive trees, Australia; 1/100), C. siamense (five isolates, four of them from olive trees Australia, and one from strawberry, Spain (Col-44; 1/83), and C. theobromicola syn. C. fragariae (one isolate from olive trees, Australia; 1/99) (Figure 5).
Considering just the new olive tree isolates in this study, we molecularly identified 177 isolates belonging to 12 species of Colletotrichum. Most of these were Portuguese and Spanish isolates, 83 and 45 isolates, respectively. The species C. nymphaeae and C. godetiae were the most frequent (87 and 59 isolates, respectively) followed by C. acutatum, C. fioriniae, and C. gloeosporioides with 10, 7, and 3 isolates, respectively. The rest of the species (C. boninense, C. fructicola, C. perseae, C. simmondsii, and C. theobromicola) were represented by just one. At the same time, two and four Australian isolates were identified as C. alienum and C. siamense, respectively. Overall, C. godetiae was the dominant species in all the European countries (Greece, Italy, and Spain) except in Portugal, where the C. nymphaeae (89%) was the predominant species followed by C. godetiae (11%) (Figure 6).
Besides the isolates from olive trees, we have included eight Colletotrichum isolates obtained from other hosts in Spain, such as almonds, sweet orange trees, strawberries, and terebinth (Pistacia terebinthus). Among these isolates, six species were identified: C. acutatum from an almond tree, C. godetiae from almond and terebinth trees, C. nymphaeae (two isolates) and C. siamense from strawberries, and C. gloeosporioides and C. karstii from a sweet orange tree (Table 1).

3.4. Pathogenicity Tests

3.4.1. Pathogenicity to Olive Fruit

Significant differences in virulence (p ≤ 0.001) were observed between isolates depending on the Colletotrichum species and the original host. Most of the isolates tested were pathogenic in olive fruit except for C. nymphaeae isolates Col-84 and Col-86, and both originated from strawberries. Among the pathogenic isolates, RAUDPC values varied from 4.7% to 83.1% for C. karstii isolate Col-79 and C. nymphaeae isolate Col-506. The olive tree isolates Col-508 and Col-515 of C. godetiae, together with the isolates Col-506 of C. nymphaeae, were the most virulent olive fruit (RAUDPC > 64%) (Figure 7 and Figure 8). Overall, olive tree isolates belonging to C. godetiae and C. nymphaeae caused the typical “soapy rot”, i.e., rot covering the totality of the fruit surface with abundant conidia in mucilaginous orange masses.

3.4.2. Pathogenicity on Other Hosts

Most of the Colletotrichum isolates tested in this experiment were pathogenic in all the hosts evaluated except for C. siamense isolate Col-44 from strawberries, which was non-pathogenic to apple and sweet orange fruits, and C. godetiae isolate Col-9, which was non-pathogenic to oleander leaves. There was a significant interaction between isolate and host. In almond fruit, for example, all the Colletotrichum isolates were pathogenic with significant (p = 0.017) differences in virulence among them. In this host, C. siamense isolate Col-44 from strawberries was the less virulent (RAUDPC = 22.6%), while the isolates Col-9 and Col-57 (C. godetiae), and Col-42 (C. nymphaeae) caused RAUDPCs around 50%. In apple and sweet orange fruit, C. gloeosporioides isolate Col-69 from sweet oranges was the most virulent (RAUDPC > 70%) with marked differences in virulence concerning the other isolates tested. In both apples and sweet oranges, C. siamense isolate Col-44 was not pathogenic. Concerning pathogenicity on oleander leaves, C. karstii Col-79 was the most virulent isolate (RAUDPC = 82.3%) followed by C. siamense isolate Col-44 (RAUDPC = 66.1%). Conversely, C. godetiae isolate Col-57 and C. nymphaeae isolate Col-42 were weakly pathogenic (RAUDPC < 5.0%). Finally, in strawberry fruit, C. gloeosporioides Col-69 was the most virulent isolate (RAUDPC = 61.1%), while C. godetiae isolate Col-57, C. nymphaeae isolate Col-42, and C. siamense isolate Col-44 showed moderate levels of virulence (RAUDPC = 37.9%, 31.1%, and 19.4%, respectively). The isolates C. godetiae Col-9 and C. karstii Col-79 showed the lowest levels of virulence in strawberry fruit (RAUDPC < 5%) (Figure 9 and Figure 10).

4. Discussion

Fungal species belonging to the Colletotrichum genus are characterized by a global distribution associated with anthracnose diseases affecting a wide range of hosts, including many tree crops [5,58,59,60]. Although numerous species of Colletotrichum have been associated with the olive crop, these studies have focused on specific producing regions and lack an overall view [4,5,23,24,31]. Interestingly, the diversity of Colletotrichum species affecting olive trees in Spain, the leading olive oil producer globally, is very little known since the main study was conducted before dividing the Colletotrichum species complex into species to molecular profiles [33]. The present work focused on elucidating the biodiversity of the Colletotrichum species, causing olive anthracnose worldwide, emphasizing the fungi population from the Iberian Peninsula, i.e., Spain and Portugal. To this end, a vast collection of Colletotrichum isolates obtained mainly from olives in the main olive-growing regions of the world (Australia, Brazil, California, Greece, Italy, Portugal, Spain, Tunisia, and Uruguay) were characterized based on morphological, molecular, and pathogenic characters.
Several Colletotrichum species can produce infections in a single host, showing high pathogenic specialization; much more frequent, however, are the Colletotrichum species with the ability to infect multiple hosts [58,60]. These antecedents suggest that correct taxonomic identification of Colletotrichum species is essential to avoid etiological ambiguities. Therefore, determining the aetiology of Colletotrichum diseases will be crucial to develop studies on the epidemiology and control of the disease in the future [10].
By tradition, the taxonomic identification of the species of Colletotrichum genus has been mainly based on phenotypic differences of colony morphology and conidium shape and size [59,61,62]. Several authors considered the curvature of the ends of the conidia as the essential morphological character to distinguish between Colletotrichum species [16,62,63]. This conidial character has been traditionally used to discriminate between C. acutatum (sharp conidium ends, fusiform) and C. gloeosporioides (rounded conidium ends, ellipsoid) [58,64]. Nevertheless, conidia of the isolates characterized morphologically in this study varied in form (ellipsoid, clavate, or fusiform) between fungal isolates within the same species complexes and, even, the same fungal species. For example, within the C. acutatum species complex, the three shapes of conidia were observed for C. acutatum isolates, whereas C. godetiae isolates showed clavate conidia except for the isolates Col-88 and Col-522, which showed ellipsoid and fusiform conidia, respectively. Because of this morphological characteristic (clavate conidia), Faedda et al. [26] described the new species C. clavatum as the most common associated with olive anthracnose in Italy. However, this new species did not show molecular and morphological differences with the previous one, C. godetiae [27]. Likewise, in this study, and previous ones, different types of conidia were also observed between species belonging to C. boninense species complex (clavate or ellipsoid conidia) as well as within the C. gloeosporioides species complex (ellipsoid or clavate conidia).
Regarding the colony color, there were no differences that allowed their specific identification. Usually, colonies of C. godetiae isolates were gray, C. acutatum isolates showed pink tones, and C. fioriniae and C. gloeosporioides isolates showed light gray ones. The only exception was C. siamense from strawberries (Col-44), which showed a distinctive greenish-gray colony color. Similar results about differences between Colletotrichum species affecting almond trees depending on colony color-subpopulations were described by López-Moral et al. [36], who indicated that C. acutatum, C. godetiae, and C. nymphaeae isolates were associated with pinkish-orange, dark gray, and light gray subpopulations, respectively. Despite these specific differences in colony color between Colletotrichum species, it does not correctly identify Colletotrichum species since environmental factors could significantly influence the stability of morphological traits becoming in intermediate forms [58,65,66].
Regarding the sensitivity to the benomyl, all the isolates included within C. boninense and C. gloeosporioides species complexes were highly sensitive to the fungicide with inhibition percentages of mycelial growth higher than 93%. Conversely, the isolates belonging to the C. acutatum species complex were more tolerant (from 33.5% to 71.1%) to the fungicide than those of the C. gloeosporioides species complex. However, there was an unclear association between fungicide tolerance and pathogen species or geographic or host origin. Our results are concordant with those obtained by several authors who indicated that C. gloeosporioides isolates are highly sensitive to benomyl while C. acutatum isolates are moderately tolerant independent of the host origin [4,43,58,67,68]. Species belonging to the C. acutatum complex are predominant in the olive-growing areas in the Andalusia region [7]. Thus, Andalusian C. acutatum isolates show higher tolerance to benomyl than those of C. gloeosporioides and C. boninense. These differences could be a consequence of the use of this fungicide in olive orchards. However, benomyl had not been traditionally used by olive growers in Andalusia to prevent olive diseases and is currently not registered for use [7].
The ability of Colletotrichum isolates to hydrolyze casein was also helpful to discriminate isolates between Colletotrichum species complexes, but with some exceptions. Thus, most isolates belonging to the C. acutatum species complex hydrolyzed the casein, whereas those belonging to C. boninense and C. gloeosporioides species complexes did not. These results are in concordance with those obtained by Martín et al. [69].
Molecular techniques, such as phylogenetic analyses of ribosomal genes (i.e., ITS, 28S, etc.) and functional protein regions (i.e., actin, β-tubulin, calmodulin, etc.) have been set up during these last few years, improving the identification of Colletotrichum species within this genus [27,50,54,66,70]. The combined alignment of ITS and TUB2 helped identify the isolates into the three Colletotrichum species complexes: C. acutatum, C. boninense, and C. gloeosporioides. In general, ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes except for the isolate Col-169, which was identified as C. simmondsii based on the ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions.
In corroboration with previous studies [54,70,71,72], we used an additional alignment combining ITS, TUB2, and ApMat gene sequences for inferring the phylogeny of the isolates previously grouped as C. gloeosporioides species complex. Phylogenetic studies conducted to determine the provided information by ApMat and glutamine synthetase (GS) showed that regions offer similar information, but ApMat discriminates more species in the C. gloeosporioides species complex [70,72].
All the aspects discussed above are in agreement with the ideal polyphasic approach for Colletotrichum systematics described by Cai et al. [73], who suggested that the identification of Colletotrichum species should be based on multi-gene phylogenetic analysis together with recognizable phenotypic characters, such as morphology, physiology, pathogenicity, or cultural characteristics, among others.
Concerning the global distribution of our Colletotrichum isolates, most of those collected from olive trees in Spain were classified within the C. acutatum species complex, with C. godetiae being the most common species, followed by C. nymphaeae. The Spanish isolates of C. godetiae were collected in the Andalusia region, whereas C. nymphaeae isolates showed more diversity regarding the country’s geographic origin. We previously observed that olive anthracnose is caused by the C. acutatum species complex in the olive-growing areas of southern Spain [7]. Still, the molecular identification of these isolates has not been conducted until the present study. The species C. gloeosporioides and C. fructicola were also isolated from olive trees in Valencia (Eastern Spain) and Catalonia (North-Eastern Spain). Remarkably, the species C. fructicola (Col-82) was isolated from olive leaves showing necrotic lesions (an unusual symptom for anthracnose) from plants in a nursery, probably due to cross-contamination with citrus plants. The infection/contamination of olive stock with Colletotrichum could influence the long-distance spread of these pathogens.
In our study, most olive isolates from Greece, Italy, and Tunisia were identified as C. godetiae. These results are in concordance with those obtained by several authors who indicate that the species belonging to the C. acutatum complex are considered the most important ones associated with olive anthracnose in European countries [6,24,26,32]. Conversely, and in concordance to the previous studies [4,25], our study confirmed that the most prevalent species associated with olive anthracnose in Portugal is C. nymphaeae. In initial studies, we observed that the Spanish C. godetiae isolates, coming from olive-growing regions where copper-based fungicides are frequently used by farmers, are more tolerant to copper than C. nymphaeae isolates, while in Portugal, the opposite is true. However, the adaptation to weather and agronomic conditions, including the potential specialization in the local olive cultivars, could explain these differences [7,74]. In addition, in previous studies we occasionally detected interactions between olive cultivar-Colletotrichum spp., but none so important so as to explain such a different species distribution [7,8,9,57].
Although we identified eight Colletotrichum species among the Australian isolates, neither C. godetiae nor C. nymphaeae were found. This substantial variability of species associated with olive anthracnose in Australia was influenced by the fact that the 16 studied isolates were previously selected from a higher search to maximize the variability. Besides, previous studies hypothesized that the center of origin of Colletotrichum could be in Oceania since the highest level of variability and strains of the species complexes occurred mainly in Australia and New Zealand [59]. The species C. siamense and C. theobromicola have been previously described in olive trees in Australia [23]. However, the species C. perseae (Col-205) was identified for the first time as associated with olive anthracnose. The species C. alienum has been identified in a broad diversity of hosts, including olive trees [13,53,75], while C. perseae has been described as novel species associated with avocado anthracnose in Israel [21].
All of the isolates from olive trees from California were identified as C. fioriniae. Nevertheless, the etiological studies of olive anthracnose in this state have not been conducted yet. However, C. fioriniae is a common pathogen of nut trees in California [27,76].
Finally, among all our isolates, species belonging to the C. gloeosporioides complex were only identified for the isolates collected from Australia, Tunisia, and Eastern Spain. These results agree with those described by Talhinhas et al. [5], who indicated that the C. gloeosporioides complex occurs in several countries presenting lower frequency than other species. On the other hand, these authors described C. acutatum as the prevalent species complex associated with olive anthracnose in the Southern Hemisphere.
Regarding the six Colletotrichum species obtained from hosts other than the olive tree (i.e., C. acutatum, C. gloeosporioides, C. godetiae, C. karstii, C. nymphaeae and C. siamense), it is interesting to note that all of them are new reports from the respective hosts in Spain, except C. gloeosporioides from sweet orange and C. acutatum from almond trees [36].
Among the Colletotrichum isolates from almonds, olives, sweet oranges, and strawberries tested for pathogenicity on olive fruit, only the isolates from strawberries were not pathogenic. Overall, the isolates from olive trees were more virulent in olive fruit than those from other hosts [35]. These results agree with López-Moral et al. [36], who observed that Colletotrichum isolates from olives (Col-506 and Col-508) were more virulent than ones from almonds (Col-522 and Col-536) on olive fruit. Overall, the pathogenicity in olive fruit has been confirmed in eight species, which differ in their virulence [5,35]. These pathogenicity tests have demonstrated that C. acutatum and C. nymphaeae are the most virulent species, C. godeatiae and C. fioriniae resulted in an intermediate virulence, and C. gloeosporioides is less virulent [4,23,24]. When cross inoculations were conducted using different isolates and hosts, a notable pathogenic specialization was observed in some cases. For example, C. siamense isolate Col-44 from strawberries resulted as non-pathogenic to apple and sweet orange fruit [7]. Although we can find many differences in virulence between isolates and host combinations, our results demonstrated the pathogenic specialization of Colletotrichum isolates on their host. This characteristic has been used to identify specific or intraspecific taxa in this genus [16,61]. However, further research is needed to determine the pathogenic specialization of Colletotrichum isolates on olive trees.
In conclusion, in the present study, the largest so far, we recorded 12 species of the pathogen affecting the olive tree, C. acutatum, C. alienum, C. boninense, C. fioriniae, C. fructicola, C. gloeosporioides, C. godetiae, C. nymphaeae, C. perseae, C. siamense, C. simmondsii, and C. theobromicola. According to our knowledge, this study is the first report of C. boninense, C. fructicola, and C. perseae affecting olive trees. Other studies have described another six Colletotrichum species associated with this crop, C. aenigma, C. cigarro, C. karstii, C. queenslandicum, C. lupini, and C. rhombiborme [4,5,23,77,78]. Although many other woody crops are affected by numerous species of Colletotrichum [21,54,79,80], the olive tree is one of the plant species affected by the most remarkable diversity of taxa of this fungal genus with 18 species. This fact may be associated with the enormous expansion capacity of the olive tree in the last 30 years, which has led it to be the main woody crop in the world [1]. Our results also showed that the dominant species in Spain, Italy, and Greece is C. godetiae, while C. nymphaeae is the dominant species in Portugal. Interestingly, neither of these two species have been described in Australia, where we have found the highest diversity with eight Colletotrichum spp. These results reinforce the hypothesis that native species of Colletotrichum to each place jumped from other hosts to the olive tree when it colonized new growing areas, rather than the pathogen having moved with the crop.

5. Conclusions

This study aimed to elucidate the biodiversity of Colletotrichum species causing olive anthracnose worldwide. Our results demonstrated that the phenotypic characters (colony and conidium morphology, benomyl-sensitivity, and casein-hydrolyse ability) are not helpful enough to identify Colletotrichum species, although they allow for the separation of some species complexes. For instance, conidia of the Colletotrichum isolates characterized morphologically in this study varied in form (ellipsoid, clavate, or fusiform) among fungal isolates within the same species complexes and even the same fungal species. Thus, molecular tools are essential to infer phylogenetic species within the Colletotrichum genus. In this respect, ITS and TUB2 are enough to distinguish Colletotrichum species within the C. acutatum and C. boninense species complexes. In contrast, ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloeosporioides complex. Consequently, our results reinforce the hypothesis based on the ideal polyphasic approach for Colletotrichum systematics, suggesting that the identification of Colletotrichum species should be based on multi-gene phylogenetic analysis together with recognizable phenotypic characters. Pathogenicity tests in olive showed significant differences in virulence to this host between isolates depending on the Colletotrichum species and host origin. When cross-pathogenicity was conducted, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves. Finally, regarding the diversity of Colletotrichum species causing olive anthracnose worldwide, among the 177 Colletotrichum isolates from olive included in this study, 12 Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified. The species C. godetiae was dominant in Spain, Italy, and Greece. The highest diversity was in Australia, where eight Colletotrichum species were identified. Altogether, this study also reinforces the hypothesis that native species of Colletotrichum to each place jumped from other hosts to the olive tree when it colonized new growing areas, rather than the pathogen having moved with the crop.

Author Contributions

Field sampling, fungal collection, laboratory tasks, review and editing J.M.; laboratory tasks, data analyses, wrote and edit the manuscript, review C.A.-B.; molecular characterization, writing, and editing M.C.R.; morphological and pathogenic characterization J.J.-B.; morphological and pathogenic characterization, writing, and editing A.L.-M.; field sampling and fungal collection L.F.R.; characterization of fungal isolates from Tunisia M.C.; revised the manuscript A.R.; characterization of fungal isolates from Italy, revised the manuscript F.N.; characterization of fungal isolates from Australia, revised the manuscript V.S.; conceived and designed the study, field sampling and fungal collection, funding acquisition, supervision, revised the manuscript A.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by projects AGL2004-7495 and AGL2008-01683 from the Spanish Ministry of Science and Technology, PCI-A/026301/09 and AP/037045/11 from Spanish Agency for International Development Cooperation (AECID), and P08-AGR-03635 and N027464 from Andalusian Regional Government and FEDER funds. J. Moral is holder of a ‘Ramón y Cajal’ postdoctoral fellowship (contract nº RYC2019-028404-I) from the Spanish Ministry of Science, Innovation and Universities (MICINN). We acknowledge financial support from the Spanish Ministry of Science and Innovation, the Spanish State Research Agency, through the Severo Ochoa and María de Maeztu Program for Centres and Units of Excellence in R&D (Ref. CEX2019-000968-M).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors thank F. Luque for her skilful technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Typical symptoms of olive anthracnose. (a) Disease progression on naturally infected olive fruit (I: incipient symptoms; II: depressed, round, and ochre-brown lesions; III: rotted fruit with orange conidial masses produced by Colletotrichum spp.); (b) detail of orange conidial masses (red arrows) on infected olive fruit; (c,d) mummified fruit remaining in the tree canopy causing flower and leaf blight; (e,f) dieback of shoots and branches caused by Colletotrichum spp. in olive trees.
Figure 1. Typical symptoms of olive anthracnose. (a) Disease progression on naturally infected olive fruit (I: incipient symptoms; II: depressed, round, and ochre-brown lesions; III: rotted fruit with orange conidial masses produced by Colletotrichum spp.); (b) detail of orange conidial masses (red arrows) on infected olive fruit; (c,d) mummified fruit remaining in the tree canopy causing flower and leaf blight; (e,f) dieback of shoots and branches caused by Colletotrichum spp. in olive trees.
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Figure 2. Variability in the colonies of representative Colletotrichum isolates belonging to the following species complexes: (al) Colletotrichum acutatum; (m,n) C. boninense and (ot) C. gloeosporioides. Colonies were grown on PDA for 14 days at 25 ± 2 °C under a 12-h daily photoperiod of cool fluorescent light (350 μmol m–2 s–1). (ad) C. acutatum ((a) Col-166, (b) Col-175, (c) Col-208, (d) Col-536); (eh) C. fioriniae ((e) Col-172, (f) Col-693, (g) Col-695, (h) Col-696); (i,j) C. godetiae ((i) Col-88, (j) Col-522); (k) C. nymphaeae Col-42); (l) C. simmondsii Col-169 (m) C. boninense Col-178; (n) C. karstii Col-79; (o,p) C. alienum ((o) Col-211, (p) Col-214); (q) C. gloeosporioides Col-69; (r,s) C. siamense ((r) Col-44, (s) Col-160); (t) C. theobromicola Col-200.
Figure 2. Variability in the colonies of representative Colletotrichum isolates belonging to the following species complexes: (al) Colletotrichum acutatum; (m,n) C. boninense and (ot) C. gloeosporioides. Colonies were grown on PDA for 14 days at 25 ± 2 °C under a 12-h daily photoperiod of cool fluorescent light (350 μmol m–2 s–1). (ad) C. acutatum ((a) Col-166, (b) Col-175, (c) Col-208, (d) Col-536); (eh) C. fioriniae ((e) Col-172, (f) Col-693, (g) Col-695, (h) Col-696); (i,j) C. godetiae ((i) Col-88, (j) Col-522); (k) C. nymphaeae Col-42); (l) C. simmondsii Col-169 (m) C. boninense Col-178; (n) C. karstii Col-79; (o,p) C. alienum ((o) Col-211, (p) Col-214); (q) C. gloeosporioides Col-69; (r,s) C. siamense ((r) Col-44, (s) Col-160); (t) C. theobromicola Col-200.
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Figure 3. Phylogenetic tree resulting from Bayesian analysis using the combined ITS and TUB2 sequence alignments of Colletotrichum acutatum, C. boninense, and C. gloeosporioides species complexes. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of maximum parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum dracaenophilum (CBS 118199) was used as outgroup.
Figure 3. Phylogenetic tree resulting from Bayesian analysis using the combined ITS and TUB2 sequence alignments of Colletotrichum acutatum, C. boninense, and C. gloeosporioides species complexes. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of maximum parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum dracaenophilum (CBS 118199) was used as outgroup.
Jof 07 00741 g003aJof 07 00741 g003b
Figure 4. Phylogenetic tree obtained by Bayesian analysis using the combined ITS, TUB2, ACT, CHS-1, HIS3, and GAPDH sequence alignments of Colletotrichum acutatum, C. boninense, and C. gloeosporioides species complexes. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of maximum parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum dracaenophilum (CBS 118199) was used as outgroup.
Figure 4. Phylogenetic tree obtained by Bayesian analysis using the combined ITS, TUB2, ACT, CHS-1, HIS3, and GAPDH sequence alignments of Colletotrichum acutatum, C. boninense, and C. gloeosporioides species complexes. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of maximum parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum dracaenophilum (CBS 118199) was used as outgroup.
Jof 07 00741 g004aJof 07 00741 g004b
Figure 5. Phylogenetic tree obtained by Bayesian analysis using the combined ITS, TUB2, and ApMat sequence alignments of the Colletotrichum gloeosporioides species complex. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of Maximum Parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum xanthorrhoeae (ICMP 17903) was used as outgroup.
Figure 5. Phylogenetic tree obtained by Bayesian analysis using the combined ITS, TUB2, and ApMat sequence alignments of the Colletotrichum gloeosporioides species complex. Bayesian posterior probabilities (PP, > 0.9) and bootstrap support values (MP, (BS) > 70%) of Maximum Parsimony analysis are shown in the nodes (PP/MP). The asterisk (*) indicates full support (1/100). Colletotrichum xanthorrhoeae (ICMP 17903) was used as outgroup.
Jof 07 00741 g005
Figure 6. Mosaic Plot representing the relative percentage of twelve Colletotrichum species in the five countries where six or more fungal isolates were studied. n = number of Colletotrichum isolates analyzed.
Figure 6. Mosaic Plot representing the relative percentage of twelve Colletotrichum species in the five countries where six or more fungal isolates were studied. n = number of Colletotrichum isolates analyzed.
Jof 07 00741 g006
Figure 7. Relative area under the disease progression curve (RAUDPC) on the fruits of olive cv. Hojiblanca inoculated with the following isolates: C. acutatum (dark-blue columns) from olives (Col-193 and Col-256) and almonds (Col-536); C. fioriniae (green column) from olives (Col-172); C. gloeosporioides (yellow columns) from olives (Col-41) and sweet oranges (Col-69); C. godetiae (light-blue columns) from olives (Col-30, Col-57, Col-88, Col-508, Col-515, and Col-519) and almonds (Col-522); C. kasrstii (dark-gray column) from sweet oranges (Col-79), C. nymphaeae (red column) from olives (Col-42 and Col-506) and strawberries (Col-84 and Col-86), and C. siamense (light-gray column) from olives (Col-181). Columns are the means of two independent sets (experiments) of three replicated (humid chambers) with 20 fruits per humid chamber. Vertical bars are the standard error of the means. Columns with the same letter do not differ significantly according to Tukey’s HSD test at p = 0.05. * Isolates non-pathogenic to olives.
Figure 7. Relative area under the disease progression curve (RAUDPC) on the fruits of olive cv. Hojiblanca inoculated with the following isolates: C. acutatum (dark-blue columns) from olives (Col-193 and Col-256) and almonds (Col-536); C. fioriniae (green column) from olives (Col-172); C. gloeosporioides (yellow columns) from olives (Col-41) and sweet oranges (Col-69); C. godetiae (light-blue columns) from olives (Col-30, Col-57, Col-88, Col-508, Col-515, and Col-519) and almonds (Col-522); C. kasrstii (dark-gray column) from sweet oranges (Col-79), C. nymphaeae (red column) from olives (Col-42 and Col-506) and strawberries (Col-84 and Col-86), and C. siamense (light-gray column) from olives (Col-181). Columns are the means of two independent sets (experiments) of three replicated (humid chambers) with 20 fruits per humid chamber. Vertical bars are the standard error of the means. Columns with the same letter do not differ significantly according to Tukey’s HSD test at p = 0.05. * Isolates non-pathogenic to olives.
Jof 07 00741 g007
Figure 8. Anthracnose symptoms developed on non-wounded violet (color class 3) olive fruit of cv. Hojiblanca 14 days after inoculation with conidial suspension of the following isolates: (a), Colletotrichum nymphaeae from olives (Col-42); (b), C. godetiae from olives (Col-57); (c), C. karstii from sweet oranges (Col-79); (d), C. gloeosporioides from sweet oranges (Col-69); (e), C. nymphaeae from strawberries (Col-84) and (f), non-inoculated control olive fruit.
Figure 8. Anthracnose symptoms developed on non-wounded violet (color class 3) olive fruit of cv. Hojiblanca 14 days after inoculation with conidial suspension of the following isolates: (a), Colletotrichum nymphaeae from olives (Col-42); (b), C. godetiae from olives (Col-57); (c), C. karstii from sweet oranges (Col-79); (d), C. gloeosporioides from sweet oranges (Col-69); (e), C. nymphaeae from strawberries (Col-84) and (f), non-inoculated control olive fruit.
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Figure 9. Relative area under the disease progression curve (RAUDPC) on fruits of almond cv. Guara, apple cv. Golden Delicious, sweet orange cv. Lane Late, strawberry cv. Camarosa, and on oleander leaves inoculated with the following isolates: Colletotrichum godetiae from olive (Col-9 and Col-57), C. gloeosporioides from sweet orange (Col-69), C. karstii from sweet oranges (Col-79), C. nymphaeae from olives (Col-42), and C. siamense from strawberries (Col-44). Columns are the means of two independent sets (experiments) of three replicated (humid chambers) in each host inoculation, with 10 fruit or leaves per host and per humid chamber. Vertical bars are the standard error of the means. For each host, columns with the same letter are not significantly different according to the Tukey’s HSD test at p = 0.05. * Isolates not pathogenic to these hosts.
Figure 9. Relative area under the disease progression curve (RAUDPC) on fruits of almond cv. Guara, apple cv. Golden Delicious, sweet orange cv. Lane Late, strawberry cv. Camarosa, and on oleander leaves inoculated with the following isolates: Colletotrichum godetiae from olive (Col-9 and Col-57), C. gloeosporioides from sweet orange (Col-69), C. karstii from sweet oranges (Col-79), C. nymphaeae from olives (Col-42), and C. siamense from strawberries (Col-44). Columns are the means of two independent sets (experiments) of three replicated (humid chambers) in each host inoculation, with 10 fruit or leaves per host and per humid chamber. Vertical bars are the standard error of the means. For each host, columns with the same letter are not significantly different according to the Tukey’s HSD test at p = 0.05. * Isolates not pathogenic to these hosts.
Jof 07 00741 g009
Figure 10. Anthracnose symptoms developed on fruit or leaves of several hosts 14 days after inoculation with a conidial suspension of Colletrotrichum isolates. (a,b) almond cv. Guara inoculated with C. acutatum from almonds (Col-536) and C. godetiae from almonds (Col-522) (c) apple cv. Golden Delicious inoculated with C. gloeosporioides from sweet oranges (Col-69); (d) sweet orange cv. Lane Late inoculated with C. gloeosporioides from sweet oranges (Col-69); (e) strawberry cv. Camarosa inoculated with C. godetiae from strawberries (Col-57); (f) leaves of Nerium oleander inoculated with C. gloeosporioides from sweet oranges (Col-69).
Figure 10. Anthracnose symptoms developed on fruit or leaves of several hosts 14 days after inoculation with a conidial suspension of Colletrotrichum isolates. (a,b) almond cv. Guara inoculated with C. acutatum from almonds (Col-536) and C. godetiae from almonds (Col-522) (c) apple cv. Golden Delicious inoculated with C. gloeosporioides from sweet oranges (Col-69); (d) sweet orange cv. Lane Late inoculated with C. gloeosporioides from sweet oranges (Col-69); (e) strawberry cv. Camarosa inoculated with C. godetiae from strawberries (Col-57); (f) leaves of Nerium oleander inoculated with C. gloeosporioides from sweet oranges (Col-69).
Jof 07 00741 g010
Table 1. Isolates of Colletotrichum spp. used in this study, with collection details and GenBank accessions.
Table 1. Isolates of Colletotrichum spp. used in this study, with collection details and GenBank accessions.
SpeciesIsolate b,cOrigin, YearSubstrate, HostGenBank Accession No. a
ITSTUB2ACTCHS-1HIS3GAPDHApMat
C. abscissumCOAD 1877TBrazil, CafelandiaPsidium guajavaKP843126KP843135KP843141KP843132KP843138KP843129-
C. acerbumCBS 128530T, ICMP 12921, PRJ 1199.3New ZealandMalus domesticaJQ948459JQ950110JQ949780JQ949120JQ949450JQ948790-
C. acutatumCol-166; UWS-65 d,eAustralia; 2009Fruit, Olea europaeaMH685231MH713165MH717594MH801883MH713299MH717458-
Col-175; UWS-79 d,eAustralia; 2009Fruit, Olea europaeaMH685234MH713166-----
Col-190; UWS-101Australia; 2009Fruit, Olea europaeaMH685239MH713167-----
Col-193; UWS-120 fAustralia; 2009Fruit, Olea europaeaMH685240MH713168-----
Col-208;UWS-149 d,eAustralia; 2009Fruit, Olea europaeaMH685243MH713169-----
Col-231Uruguay; 2010Fruit, Olea europaea cv. HojiblancaMH685249MH713170-----
Col-256; IOT COL-04.1 fNabeul, Tunisia; 2010Fruit, Olea europea cv. MeskiKM594095KP197006MH717595MH801884MH713300MH717459-
Col-258; IOT COL-06.5Takelsa, Tunisia; 2010Fruit, Olea europea cv. ArbequinaKM594093KP185116-----
Col-275; IOT COL-15.3Nabeul, Tunisia; 2010Fruit, Olea europea cv. QueslatiKM594101KP197011-----
Col-391Bari, Italy; 2012Fruit, Olea europea cv. ArbequinaMH685260MH713171-----
Col-536 d,fLebrija, Sevilla, Spain; 2014Fruit, Prunus dulcisKY171894KY171902KY171910KY171918KY171926KY171934-
CBS 112996, ATCC 56816, STE-U 5292TAustraliaCarica papayaJQ005776JQ005860JQ005839JQ005797JQ005818JQ948677-
CBS 129952, PT227, RB015PortugalOlea europaeaJQ948364JQ950015JQ949685JQ949025JQ949355JQ948695-
CBS 127598, 223/09South AfricaOlea europaeaJQ948363JQ950014JQ949684JQ949024JQ949354JQ948694-
C. aenigmaICMP 18608TIsraelPersea americanaJX010244JX010389----KM360143
C. aeschynomenesICMP 17673TUSAAeschynomene virginicaJX010176JX010392----KM360145
C. alataeICMP 17919IndiaDioscorea alataJX010190JX010383----KC888932
C. alienumCol-211;UWS-152 d,eAustralia; 2009Fruit, Olea europaeaMH685244MH713162----MH717580
Col-214;UWS-156 d,eAustralia; 2009Fruit, Olea europaeaMH685245MH713163----MH717581
ICMP 12071TNew ZealandMalus domesticaJX010251JX010411----KM360144
C. annellatumCBS 129826TColombiaLeaf, Hevea brasiliensisJQ0055222JQ005656JQ005570JQ005396JQ005483JQ005309-
C. aotearoaICMP 18537TNew ZealandCoprosma sp.JX010205JX010420----KC888930
C. asianumICMP 18580T;CBS 130418ThailandCoffea arabicaFJ972612JX010406----FR718814
C. australeCBS 116478TSouth AfricaTrachycarpus fortuneiJQ948455JQ950106JQ949776JQ949116JQ949446JQ948786
C. boninenseCol-178; UWS-82 d,eAustralia; 2009Fruit, Olea europaea MH685235MH713152-----
CBS 123755T,MAFF 305972JapanCrinum asiaticum cv. sinicumJQ005153JQ005588JQ005501JQ005327JQ005414JQ005240-
CBS 128547, ICMP 10338New ZealandCamellia sp.JQ005159JQ005593JQ005507JQ005333JQ005420JQ005246-
C. brisbanenseCBS 292.67TAustraliaCapsicum annuumJQ948291JQ949942JQ949612JQ948952JQ949282JQ948621
C. cairnsenseBRIP 63642T, CBS 140847AustraliaCapsicum annuumKU923672KU923688KU923716KU923710KU923722KU923704-
C. catinaenseCBS 142417T; CPC 27978Italy, CataniaCitrus reticulataKY856400KY856482KY855971KY856136KY856307KY856224-
C. chrysanthemiIMI 364540, CPC 18930ChinaChrysanthemun coronariumJQ948273JQ949924JQ949594JQ948934JQ949264JQ948603-
C. citriCBS 134233ChinaCitrus aurantiifoliaKC293581KC293661KY855973KY856138KY856309KC293741-
C. citricolaCBS 134228ChinaCitrus unchiuKC293576KC293656KC293616KY856140KY856311KC293736-
C. clidemiaeICMP 18658THawaii, USAClidemia hirtaJX010265JX010438----KC888929
C. coccodesCBS 369.75TThe Netherlands Solanum tuberosumHM171679JX546873-----
C. constrictumCBS 128504New ZealandCitrus limonJQ005238JQ005672JQ005586JQ005412KY856313JQ005325-
C. cordylinicolaICMP 18579TThailandCordyline fruticosaJX010226JX010440----JQ899274
C. cosmiCBS 853.73, PD 73/856TThe NetherlandsCosmos sp.JQ948274JQ949925JQ949595JQ948935JQ949265JQ948604-
C. costaricenseCBS 330.75TCosta RicaCoffea arabicaJQ948180JQ949831JQ949501JQ949120JQ949450JQ948790-
C. cuscutaeIMI 304802, CPC 18873TDominicaCuscuta sp.JQ948195JQ949846JQ949516JQ949025JQ949355JQ948695-
C. dracaenophilumCBS 118199ChinaDracaenaJX519222JX519247JX519238JX519230JX546756JX546707-
C. fioriniaeCol-172;UWS-70 d,e,fAustralia; 2009Fruit, Olea europaeaMH685233MH713172MH717596MH801885MH713301MH717460-
Col-237Uruguay; 2010Fruit, Olea europaea cv. ArbequinaMH685250MH801882-----
Col-693 dCalifornia, USA; 2017Fruit, Olea europaeaMH685372MH713173MH717597MH801886MH713302MH717461-
Col-694 dCalifornia, USA; 2017Fruit, Olea europaeaMH685373MH713174MH717598MH801887MH713303MH717462-
Col-695 dCalifornia, USA; 2017Fruit, Olea europaeaMH685374MH713175MH717599MH801888MH713304MH717463-
Col-696 dCalifornia, USA; 2017Fruit, Olea europaeaMH685375MH713176MH717600MH801889MH713305MH717464-
Col-697 dCalifornia, USA; 2017Fruit, Olea europaeaMH685376MH713177MH717601MH801890MH713306MH717465-
IMI 345583, CPC 18889USAFragaria × ananassaJQ948333JQ949984JQ949654JQ005797JQ005818JQ948677-
IMI 345575, CPC 18888USAFragaria × ananassaJQ948332JQ949983JQ949653JQ949116JQ949446JQ948786-
CBS 125396; GJS 08-140AUSAMalus domesticaJQ948299JQ949950JQ949620JQ948952JQ949282JQ948621-
CBS 129946, PT170, RB021PortugalOlea europaeaJQ948342JQ949993JQ949663JQ949024JQ949354JQ948694-
CBS 293.67, DPI 13120AustraliaPersea americanaJQ948310JQ949961JQ949631JQ948934JQ949264JQ948603-
CBS 127537, STE-U 5289USAVaccinium sp.JQ948318JQ949969JQ949639JQ948935JQ949265JQ948604-
C. fructicolaCol-82Valencia, Spain; 2003Leaf, Olea europaeaMH685214MH713153MH713292MH713285MH713414MH717489MH717582
CBS 130416, ICMP 18581ThailandCoffea arabicaJX010165JX010405----JQ807838
C. gloeosporioidesCol-41 d,e,fMontsia, Tarragona, Spain; 1999Fruit, Olea europaeaMH685203MH713154MH713293MH713286MH713415MH717490MH717583
Col-69 d,e,fFuente la Palomera, Córdoba, Spain; 2001Citrus sinensisMH685212MH713155MH713294MH713287MH713416MH717491MH717584
Col-251; IOT COL-02Nabeul, Tunisia; 2010Fruit, Olea europaeaKM594085KP176441----MH717585
Col-295; IOT COL-25.3Nabeul, Tunisia; 2010Fruit, Olea europea cv. MeskiKM594112KP197021----MH717586
CBS 112999ItalyCitrus sinensisJQ005152JQ005587JQ005500JQ005326JQ005413JQ005239JQ807843
C. godetiaeCol-1 dAlmodóvar, Córdoba, Spain; 1998Fruit, Olea europaea cv. HojiblancaMH685200MH713003-----
Col-9 d,e,fAntequera, Málaga, Spain: 1998Fruit, Olea europea cv. HojiblancaMH685201MH713004MH717602MH713178MH713307MH717466-
Col-30 d,e,fLlanos D. Juan, Córdoba, Spain; 1998Fruit, Olea europaea cv. HojiblancaMH685202MH713005-----
Col-50 d,eLucena, Córdoba, Spain, 1999Fruit, Olea europeaMH685206MH713006MH717603MH713179MH713308MH717467-
Col-51 d,eLucena, Córdoba, Spain, 1999Fruit, Olea europeaMH685207MH713007MH717604MH713180MH713309MH717468-
Col-52 dAntequera, Málaga, Spain; 1999Fruit, Olea europeaMH685208MH713008MH717605MH713181MH713310MH717469-
Col-57 d,e,fArchidona, Málaga, Spain; 2002Fruit, Olea europaeaMH685209MH713009-----
Col-59 d,eArchidona, Málaga, Spain; 2001Fruit, Olea europaea cv. HojiblancaMH685210MH713010MH717606MH713182MH713311MH717470-
Col-60 d,eArchidona, Málaga, Spain; 2001Fruit, Olea europaea cv. HojiblancaMH685211MH713011MH717607MH713183MH713312MH717471-
Col-88 d,eMontilla, Córdoba Spain; 2004Fruit, Olea europaea cv. PicudoMH685217MH713012MH717608MH713184MH713313MH717472-
Col-90CIFA Cabra, Córdoba Spain; 2004Fruit, Olea europaea cv. PicudoMH685218MH713013MH717609MH713185MH713314MH717473-
Col-104Cabra, Córdoba, Spain; 2014Fruit, Olea europaea cv. PicudoMH685219MH713014MH717610MH713186MH713315MH717474-
Col-107La Rambla, Córdoba, Spain; Fruit, Olea europaea cv. HojiblancaMH685220MH713015MH717611MH713187MH713316MH717475-
Col-111Mengíbar, Jaén, Spain; 2014Fruit, Olea europaea cv. OcalMH685221MH713016MH717612MH713188MH713317MH717476-
Col-121Montilla, Córdoba, Spain; 2014Fruit, Olea europaea cv. HojiblancaMH685224MH713017MH717613MH713189MH713318MH717477-
Col-124Puente Genil, Córdoba, Spain; 2014Fruit, Olea europaea MH685225MH713018MH717614MH713190MH713319MH717478-
Col-250El Pedroso, Sevilla, Spain; 2011Fruit, Pistacia terebinthusMH685251MH713019-----
Col-332Parga, Greece; 2012Fruit, Olea europaeaMH685252MH713020-----
Col-338Parga, Greece; 2012Fruit, Olea europaeaMH685253MH713021-----
Col-347Parga, Greece; 2012Fruit, Olea europaeaMH685254MH713022-----
Col-350Parga, Greece; 2012Fruit, Olea europaeaMH685255MH713023-----
Col-378Parga, Greece; 2012Fruit, Olea europaeaMH685256MH713024-----
Col-384Parga, Greece; 2012Fruit, Olea europaeaMH685257MH713025-----
Col-388Bari, Italy; 2012Fruit, Olea europea cv. ArbosanaMH685258MH713026-----
Col-389Bari, Italy; 2012Fruit, Olea europea cv. ArbequinaMH685259MH713027-----
Col-392Bari, Italy; 2012Fruit, Olea europea cv. Cellina di NardòMH685261MH713028-----
Col-393Bari, Italy; 2012Fruit, Olea europea cv. Cellina di NardòMH685262MH713029----
Col-394Bari, Italy; 2012Fruit, Olea europea cv. Cellina di NardòMH685263MH713030-----
Col-395Bari, Italy; 2012Fruit, Olea europea cv. Ogliarola SalentinaMH685264MH713031-----
Col-396Bari, Italy; 2012Fruit, Olea europea cv. Ogliarola SalentinaMH685265MH713032-----
Col-397Bari, Italy; 2012Fruit, Olea europea cv. Cellina di NardòMH685266MH713033-----
Col-398Bari, Italy; 2012Fruit, Olea europea cv. Ogliarola SalentinaMH685267MH713034-----
Col-399Bari, Italy; 2012Fruit, Olea europea cv. Ogliarola SalentinaMH685268MH713035-----
Col-400Bari, Italy; 2012Fruit, Olea europea cv. Cellina di NardòMH685269MH713036-----
Col-454Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. ArbequinaMH685271MH713037-----
Col-457Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685272MH713038------
Col-462Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. ArbequinaMH685275MH713039-----
Col-471Montilla, Córdoba, Spain; 2013Fruit, Olea europaea cv. PicudoMH685277MH713040-----
Col-474Montilla, Córdoba, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685278MH713041-----
Col-477Castro del Río, Córdoba, Spain; 2013Fruit, Olea europaea cv. PicudoMH685279MH713042-----
Col-480Castro del Río, Córdoba, Spain; 2013Fruit, Olea europaea cv. PicudoMH685280MH713043MH717615MH713191MH713320MH717479-
Col-490Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685281MH713044-----
Col-493Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685282MH713045-----
Col-499Montilla, Córdoba, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685283MH713046-----
Col-502Fuente la Palomera, Córdoba, Spain; 2013Fruit, Olea europaeaMH685284MH713047-----
Col-508d,fHornachuelos, Córdoba, Spain; 2014Fruit, Olea europaea cv. ArbequinaKY171892KY171900KY171908KY171916KY171924KY171932-
Col-511Hornachuelos, Córdoba, Spain; 2014Fruit, Olea europaea cv. PicualMH685286MH713048MH717616MH713192MH713321MH717480-
Col-512Hornachuelos, Córdoba, Spain; 2014Fruit, Olea europaea cv. PicualMH685287MH713049-----
Col-514Córdoba, Spain; 2014Fruit, Olea europaea cv. PicualMH685288MH713050MH717617MH713193MH713322MH717481-
Col-515 fCórdoba, Spain; 2014Fruit, Olea europaea cv. PicualMH685289MH713051MH717618MH713194MH713323MH717482-
Col-519 fCórdoba, Spain; 2014Fruit, Olea europaea cv. HojiblancaMH685290MH713052MH717619MH713195MH713324MH717483-
Col-522 d,fLebrija, Sevilla, Spain; 2014Fruit, Prunus dulcisKY171893KY171901KY171909KY171917KY171925KY171933-
Col-556Beja, Portugal; 2014Fruit, Olea europaeaMH685291MH713053MH717620MH713196MH713325MH717484-
Col-558Beja, Portugal; 2014Fruit, Olea europaeaMH685292MH713054-----
Col-560Beja, Portugal; 2014Fruit, Olea europaeaMH685293MH713055-----
Col-562Beja, Portugal; 2014Fruit, Olea europaeaMH685294MH713056MH717621MH713197MH713326MH717485-
Col-563Beja, Portugal; 2014Fruit, Olea europaeaMH685295MH713057-----
Col-564Beja, Portugal; 2014Fruit, Olea europaeaMH685296MH713058-----
Col-577Montesandinha, Portugal; 2014Fruit, Olea europaea cv. ArbequinaMH685301MH713059MH717622MH713198MH713327MH717486-
Col-578Capela, Portugal; 2014Fruit, Olea europaea cv. ArbequinaMH685302MH713060MH717623MH713199MH713328MH717487-
Col-581Montesandinha, Portugal; 2014Fruit, Olea europaea cv. ArbequinaMH685305MH713061MH717624MH713200MH713329MH717488-
CBS 133.44TDenmarkClarkia hybridaJQ948402JQ950053JQ949723JQ949063JQ949393JQ948733-
CBS 130251, OL 10, IMI 398854ItalyOlea europaeaJQ948413JQ950064JQ949734JQ949074JQ949404JQ948744-
CBS 193.32GreeceOlea euroapeaJQ948415JQ950066JQ949736JQ949076JQ949406JQ948746-
CBS 130252, IMI 398855, OL 20ItalyOlea europaeaJQ948414JQ950065JQ949735JQ949075JQ949405JQ948745-
CBS 126527, PD 93/1748United KingdomPrunus aviumJQ948408JQ950059JQ949729JQ949069JQ949399JQ948739-
CBS 126522, PD 88/472, BBA 70345The NetherlandsPrunus cerasusJQ948411JQ950062JQ949732JQ949072JQ949402JQ948742-
CBS 129934, ALM-IKS-7QIsraelPrunus dulcisJQ948431JQ950082JQ949752JQ949092.1JQ949422JQ948762-
C. guajavaeIMI 350839, CPC 18893TIndiaPsidium guajavaJQ948270JQ949921JQ949591JQ948931JQ949261JQ948600-
C. henanenseLC3030, CGMCC 3.17354, LF238TChinaCamellia sinensisKJ955109KJ955257----KJ954524
C. horiiICMP 10492TJapanDiospyros kakiGQ329690JX010450----JQ807840
C. indonesienseCBS 127551, CPC 14986TIndonesiaEucalyptus sp.JQ948288JQ949939JQ949609JQ948949JQ949279JQ948618-
C. jiangxienseLC3463, CGMCC 3.17363, LF687TChinaCamellia. sinensisKJ955201KJ955348----KJ954607
C. johnstoniiCBS 128532, ICMP 12926, PRJ 1139.3TNew ZealandSolanum lycopersicumJQ948444JQ950095JQ949435JQ949105JQ949105JQ948775-
C. kahawae subsp. kahawaeIMI 319418, ICMP 17816Kenya Coffea arabicaJX010231JX010444----JQ894579
C. karstiiCol-79 d,eHuelva, SpainCitrus sp.MH685213MH713151MH713295MH713288MH713417MH717492-
CBS 126532South AfricaCitrus sp.JQ005209JQ005643JQ005557JQ005383JQ005470JQ005296-
CBS 128500, ICMP 18585New Zealand Fruit, Annona cherimolaJQ005202JQ005636JQ005550JQ005376JQ005463JQ005289-
CBS 124969, LCM 232PanamaLeaf, Quercus salicifoliaJQ005179JQ005613JQ005527JQ005353JQ005440JQ005266-
CBS 115535, STE-U 5210Portugal, MadeiraProtea obtusifoliaJQ005214JQ005648JQ005562JQ005388JQ005475JQ005301-
C. kinghorniiCBS 198.35TUnited KingdomPhormium sp.JQ948454JQ950105JQ949775JQ949115JQ949445JQ948785-
C. laticiphilumCBS 112989, IMI 383015, STE-U 5303TIndiaHevea basiliensisJQ948289JQ949940JQ949610JQ948950JQ949280JQ948619-
C. limetticolaCBS 114.14TFlorida, USACitrus aurantifoliaJQ948193JQ949844JQ949514JQ948854JQ949184JQ948523-
C. lupiniCBS 109225; BBA 70884TUkraineLupinus albusJQ948155JQ949806JQ949476JQ948816JQ949146JQ948485-
C. melonisCBS 159.84TBrazilCucumis meloJQ948194JQ949845JQ949515JQ948855JQ949185JQ948524-
C. musaeICMP 19119, CBS 116870USAMusa sp.JX010146HQ596280----KC888926
C. nymphaeaeCol-42 d,eTarragona, Spain, 1999Fruit, Olea europaeaMH685204MH713062MH717625MH713201MH713330MH717496-
Col-84 d,e,fSevilla, Spain; 2004Fruit, Fragaria × ananassaMH685215MH713063MH717626MH713202MH713331MH717497-
Col-86 d,e,fSevilla, Spain; 2004Fruit, Fragaria × ananassaMH685216MH713064MH717627MH713203MH713332MH717498-
Col-116Montefalco, Perugia, Italy; 2014Fruit, Olea europaea cv. MoraioloMH685222MH713065MH717628MH713204MH713333MH717499-
Col-120Navalvillar de Pela, Badajoz, Spain; 2014Fruit, Olea europaea cv. Verdial de BadajozMH685223MH713066MH717629MH713205MH713334MH717500-
Col-142Elvas, Portugal; 2008 Fruit, Olea euroapeaMH685226MH713067-----
Col-143Elvas, Portugal; 2008 Fruit, Olea europaeaMH685227MH713068MH717630MH713206MH713335MH717501-
Col-150Puebla de Guzman, Huelva, Spain; 2008Fruit, Olea europaeaMH685228MH713069MH717631MH713207MH713336MH717502-
Col-151Puebla de Guzman, Huelva, Spain; 2008Fruit, Olea europaeaMH685229MH713070-----
Col-222Caçapava, Brasil; 2010Fruit, Olea europaea cv. ArbequinaMH685246MH713071-----
Col-228Uruguay; 2010Fruit, Olea europaeaMH685248MH713072-----
Col-451Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. ArbequinaMH685270MH713073MH717632MH713208MH713337MH717503-
Col-459Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685273MH713074MH717633MH713209MH713338MH717504-
Col-460Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. HojiblancaMH685274MH713075-----
Col-466Jerez, Cádiz, Spain; 2013Fruit, Olea europaea cv. ArbequinaMH685276MH713076MH717634MH713210MH713339MH717505-
Col-506 d,fHornachuelos, Córdoba, Spain; 2014Fruit, Olea europaea cv. ArbequinaKY171891KY171899KY171907KY171915KY171923KY171931-
Col-510Hornachuelos, Córdoba, Spain; 2014Fruit, Olea europaea cv. PicualMH685285MH713077MH717635MH713211MH713340MH717506-
Col-572Montesardinha,Portugal; 2014Fruit, Olea europaea cv. PicualMH685297MH713078MH717636MH713212MH713341MH717507-
Col-573Capela, Portugal; 2014Fruit, Olea europaea cv. PicualMH685298MH713079MH717637MH713213MH713342MH717508-
Col-574Montesardinha,Portugal; 2014Fruit, Olea europaea cv. ArbequinaMH685299MH713080MH717638MH713214MH713343MH717509-
Col-575Montesardinha,Portugal; 2014Fruit, Olea europaea cv. PicualMH685300MH713081MH717639MH713215MH713344MH717510-
Col-579Capela, Portugal; 2014Fruit, Olea europaea cv. PicualMH685303MH713082MH717640MH713216MH713345MH717511-
Col-580Montesardinha,Portugal; 2014Fruit, Olea europaea cv. PicualMH685304MH713083MH717641MH713217MH713346MH717512-
Col-615Portugal; 2016Fruit, Olea europaea MH685306MH713084MH717642MH713218MH713347MH717513-
Col-616Portugal; 2016Fruit, Olea europaea MH685307MH713085MH717643MH713219MH713348MH717514-
Col-617Portugal; 2016Fruit, Olea europaea MH685308MH713086MH717644MH713220MH713349MH717515-
Col-618Portugal; 2016Fruit, Olea europaea MH685309MH713087MH717645MH713221MH713350MH717516-
Col-619Portugal; 2016Fruit, Olea europaea MH685310MH713088MH717646MH713222MH713351MH717517--
Col-620Portugal; 2016Fruit, Olea europaea MH685311MH713089MH717647MH713223MH713352MH717518-
Col-621Portugal; 2016Fruit, Olea europaea MH685312MH713090MH717648MH713224MH713353MH717519-
Col-622Portugal; 2016Fruit, Olea europaea MH685313MH713091MH717649MH713225MH713354MH717520-
Col-623Portugal; 2016Fruit, Olea europaea MH685314MH713092MH717650MH713226MH713355MH717521-
Col-624Portugal; 2016Fruit, Olea europaea MH685315MH713093MH717651MH713227MH713356MH717522-
Col-625Portugal; 2016Fruit, Olea europaea MH685316MH713094MH717652MH713228MH713357MH717523-
Col-626Portugal; 2016Fruit, Olea europaea MH685317MH713095MH717653MH713229MH713358MH717524-
Col-627Portugal; 2016Fruit, Olea europaea MH685318MH713096MH717654MH713230MH713359MH717525-
Col-628Portugal; 2016Fruit, Olea europaea MH685319MH713097MH717655MH713231MH713360MH717526-
Col-629Portugal; 2016Fruit, Olea europaea MH685320MH713098MH717656MH713232MH713361MH717527-
Col-630Portugal; 2016Fruit, Olea europaea MH685321MH713099MH717657MH713233MH713362MH717528-
Col-631Portugal; 2016Fruit, Olea europaea MH685322MH713100MH717658MH713234MH713363MH717529-
Col-632Portugal; 2016Fruit, Olea europaea MH685323MH713101MH717659MH713235MH713364MH717530-
Col-633Portugal; 2016Fruit, Olea europaea MH685324MH713102MH717660MH713236MH713365MH717531-
Col-634Portugal; 2016Fruit, Olea europaea MH685325MH713103MH717661MH713237MH713366MH717532-
Col-635Portugal; 2016Fruit, Olea europaea MH685326MH713104MH717662MH713238MH713367MH717533-
Col-636Portugal; 2016Fruit, Olea europaea MH685327MH713105MH717663MH713239MH713368MH717534--
Col-637Portugal; 2016Fruit, Olea europaea MH685328MH713106MH717664MH713240MH713369MH717535-
Col-638Portugal; 2016Fruit, Olea europaea MH685329MH713107MH717665MH713241MH713370MH717536-
Col-639Portugal; 2016Fruit, Olea europaea MH685330MH713108MH717666MH713242MH713371MH717537-
Col-640Portugal; 2016Fruit, Olea europaea MH685331MH713109MH717667MH713243MH713372MH717538-
Col-641Portugal; 2016Fruit, Olea europaea MH685332MH713110MH717668MH713244MH713373MH717539-
Col-642Portugal; 2016Fruit, Olea europaea MH685333MH713111MH717669MH713245MH713374MH717540-
Col-643Portugal; 2016Fruit, Olea europaea MH685334MH713112MH717670MH713246MH713375MH717541-
Col-644Portugal; 2016Fruit, Olea europaea MH685335MH713113MH717671MH713247MH713376MH717542-
Col-645Portugal; 2016Fruit, Olea europaea MH685336MH713114MH717672MH713248MH713377MH717543-
Col-646Portugal; 2016Fruit, Olea europaea MH685337MH713115MH717673MH713249MH713378MH717544-
Col-647Portugal; 2016Fruit, Olea europaea MH685338MH713116MH717674MH713250MH713379MH717545-
Col-648Portugal; 2016Fruit, Olea europaea MH685339MH713117MH717675MH713251MH713380MH717546-
Col-649Portugal; 2016Fruit, Olea europaea MH685340MH713118MH717676MH713252MH713381MH717547-
Col-650Portugal; 2016Fruit, Olea europaea MH685341MH713119MH717677MH713253MH713382MH717548-
Col-651Portugal; 2016Fruit, Olea europaea MH685342MH713120MH717678MH713254MH713383MH717549-
Col-652Portugal; 2016Fruit, Olea europaea MH685343MH713121MH717679MH713255MH713384MH717550-
Col-653Portugal; 2016Fruit, Olea europaea MH685344MH713122MH717680MH713256MH713385MH717551-
Col-654Portugal; 2016Fruit, Olea europaea MH685345MH713123MH717681MH713257MH713386MH717552-
Col-655Portugal; 2016Fruit, Olea europaea MH685346MH713124MH717682MH713258MH713387MH717553-
Col-656Portugal; 2016Fruit, Olea europaea MH685347MH713125MH717683MH713259MH713388MH717554-
Col-657Portugal; 2016Fruit, Olea europaea MH685348MH713126MH717684MH713260MH713389MH717555-
Col-658Portugal; 2016Fruit, Olea europaea MH685349MH713127MH717685MH713261MH713390MH717556-
Col-659Portugal; 2016Fruit, Olea europaea MH685350MH713128MH717686MH713262MH713391MH717557-
Col-660Portugal; 2016Fruit, Olea europaea MH685351MH713129MH717687MH713263MH713392MH717558-
Col-661Portugal; 2016Fruit, Olea europaea MH685352MH713130MH717688MH713264MH713393MH717559-
Col-662Portugal; 2016Fruit, Olea europaea MH685353MH713131MH717689MH713265MH713394MH717560-
Col-663Portugal; 2016Fruit, Olea europaea MH685354MH713132MH717690MH713266MH713395MH717561-
Col-664Portugal; 2016Fruit, Olea europaea MH685355MH713133MH717691MH713267MH713396MH717562-
Col-665Portugal; 2016Fruit, Olea europaea MH685356MH713134MH717692MH713268MH713397MH717563-
Col-666Portugal; 2016Fruit, Olea europaea MH685357MH713135MH717693MH713269MH713398MH717564-
Col-667Portugal; 2016Fruit, Olea europaea MH685358MH713136MH717694MH713270MH713399MH717565-
Col-668Portugal; 2016Fruit, Olea europaea MH685359MH713137MH717695MH713271MH713400MH717566-
Col-669Portugal; 2016Fruit, Olea europaea MH685360MH713138MH717696MH713272MH713401MH717567-
Col-670Portugal; 2016Fruit, Olea europaea MH685361MH713139MH717697MH713273MH713402MH717568-
Col-671Portugal; 2016Fruit, Olea europaea MH685362MH713140MH717698MH713274MH713403MH717569-
Col-672Portugal; 2016Fruit, Olea europaea MH685363MH713141MH717699MH713275MH713404MH717570-
Col-673Portugal; 2016Fruit, Olea europaea MH685364MH713142MH717700MH713276MH713405MH717571-
Col-674Portugal; 2016Fruit, Olea europaea MH685365MH713143MH717701MH713277MH713406MH717572-
Col-675Portugal; 2016Fruit, Olea europaea MH685366MH713144MH717702MH713278MH713407MH717573-
Col-676Portugal; 2016Fruit, Olea europaea MH685367MH713145MH717703MH713279MH713408MH717574-
Col-677Portugal; 2016Fruit, Olea europaea MH685368MH713146MH717704MH713280MH713409MH717575-
Col-678Portugal; 2016Fruit, Olea europaea MH685369MH713147MH717705MH713281MH713410MH717576-
Col-679Portugal; 2016Fruit, Olea europaea MH685370MH713148MH717706MH713282MH713411MH717577-
Col-680Portugal; 2016Fruit, Olea europaea MH685371MH713149MH717707MH713283MH713412MH717578-
CBS 515.78TThe NetherlandsNymphaea albaJQ948197JQ949848JQ949518JQ948858JQ949188JQ948527-
CBS 231.49PortugalOlea europaeaJQ948202JQ949853JQ949523JQ948863JQ949193JQ948532-
CBS 129945, PT135, RB012PortugalOlea europaeaJQ948201JQ949852JQ949522JQ948862JQ949192JQ948531-
C. orchidophilumCBS 119291, MEP 1545PanamaCycnoches aureumJQ948154JQ949805JQ949475JQ948815JQ949145JQ948484-
CBS 632.80TUSADendrobium sp.JQ948151JQ949802JQ949472JQ948812JQ949142JQ948481-
C. paranaenseCBS 134729TBrazil, ParanaMalus domesticaKC204992KC205060KC205077KC205043KC205004KC205026-
C. paxtoniiIMI 165753, CPC 18868TSaint LuciaMusa sp.JQ948285JQ949936JQ949606JQ948946JQ949276JQ948615-
C. perseaeCol-205; UWS-139Australia; 2009Fruit, Olea europaeaMH685242MH713156----MH717588
CBS141365IsraelPersea americanaKX620308KX620341----KX620177
C. phormiiCBS 118194, AR 3546TGermanyPhormium sp.JQ948446JQ950097JQ949767JQ949107JQ949437JQ948777-
C. psidiiICMP 19120TItalyPsidium sp.JX010219JX010443----KC888931
C. pyricolaCBS 128531, ICMP 12924, PRJ 977.1TNew ZealandPyrus communisJQ948445JQ950096JQ949766JQ949106JQ949436JQ948776-
C. queenslandicumICMP 1778TAustraliaCarica papayaJX010276JX010414----KC888928
C. rhombiformeCBS 129953, PT250, RB011TPortugalOlea europaeaJQ948457JQ950108JQ949778JQ949115JQ949448JQ948788-
C. salicisCBS 607.94TThe NetherlandsSalix sp.JQ948460JQ950111JQ949781JQ949121JQ949451JQ948791-
C. salsolaeICMP 19051THungarySalsola tragusJX010242JX010403----KC888925
C. scovilleiCBS 126529, PD 94/921-3, BBA 70349TIndonesiaCapsicum sp.JQ978267JQ949918JQ949588JQ948928JQ948928JQ948597-
C. siamenseCol-44; IMI-345047 d,e,fSpain 1999Fragaria vescaMH685205MH713157MH713296MH713289MH713418MH717493MH717589
Col-160-UWS-13 d,eAustralia 2009Fruit, Olea europaeaMH685230MH713158MH713297MH713290MH713419MH717494MH717590
Col-181; UWS-90 fAustralia; 2009Fruit, Olea europaeaMH685236MH713159----MH717591
Col-184; UWS-92 d,eAustralia; 2009Fruit, Olea europaeaMH685237MH713160----MH717592
Col-187; UWS-94 d,eAustralia; 2009Fruit, Olea europaeaMH685238MH713161----MH717587
ICMP 18578, CBS-130417ThailandCoffea arabicaJX010171JX010404----JQ899289
C. siamense (syn. C. jasminisambac)CBS 130420, ICMP 19118VietnamJasminum sambacHM131511JX010415----JQ807841
C. siamense (syn. C. hymenocallidis)CBS 125378, ICMP 18642, LC0043ChinaHymenocallis americanaJX010278JX010410----JQ899283
C. simmondsiiCol-169-UWS-68 d,eAustralia; 2009Fruit, Olea europaeaMH685232MH713150MH717708MH713284MH713413MH717579-
CBS 122122, BRIP 28519TAustraliaCarica papayaJQ948276JQ949927JQ949597JQ948937JQ949267JQ948606-
C. sloaneiIMI 364297, CPC 18929TMalaysiaTheobroma cacaoJQ948287JQ949938JQ949608JQ948948JQ949278JQ948617-
C. tamarilloiCBS 129814, T.A.6TColombiaSolanum betaceumJQ948184JQ949835JQ949505JQ948845JQ949175JQ948514-
C. theobromicolaCol-200;UWS-131 d,eAustralia; 2009Fruit, Olea europaeaMH685241MH713164MH713298MH713291MH713420MH717495MH717593
CBS 124945T, ICMP 18649PanamaTheobroma cacaoJX010294JX010447----KC790726
C. theobromicola (syn. C. fragariae)CBS 142.31T, ICMP 17927USAFragaria ananassaJX010286JX010373----JQ807844
C. tiICMP 4832New ZealandCordyline sp.JX010269JX010442----KM360146
C. tropicaleCBS 124949, ICMP 18653PanamaTheobroma cacaoJX010264JX010407----KC790728
C. walleriCBS 125472, BMT(HL)19TVietnamCoffea sp.JQ948275JQ949926JQ949596JQ948936JQ949266JQ948605-
C. wuxienseCGMCC 3.17894TChinaCamellia sinensisKU251591KU252200----KU251722
C. xanthorrhoeaeICMP 17903TAustraliaXanthorrhoea preissiiJX010261JX010448----KC790689
a ITS: internal transcribed spacers; TUB2: beta-tubulin gene; ACT: actin gene; CHS-1: partial sequences of the chitin synthase 1; HIS3: histone H3 gene; GAPDH: 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase; ApMat: intergenic region between Apn2 and Mat1-2 genes. b Sequences from Genbank used in the phylogenetic analysis indicated in bold type; T: Isolates are ex-type or from samples that have been linked morphologically to type material of the species. c ATCC: American Type Culture Collection, Virginia, U.S.A.; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; BRIP: Plant Pathology Herbarium, Department of Employment, Economic Development, and Innovation, Queensland, Australia; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; PD: Plantenziektenkundige Dienst Wageningen, Nederland; UWS: University of Western Sydney; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa. Sequences from GenBank used in the phylogenetic analysis indicated in bold type (Damm et al., 2012). d,e,f Representative Colletotrichum spp. isolates selected for morphological characterization with regards on mycelium and conidium characteristics; in vitro sensitivity tests to determine sensitivity against benomyl and ability to hydrolyse casein; and pathogenicity test to olives or to other hosts, respectively.
Table 2. Phenotypical characters of mycelia and conidia of representative Colletotrichum spp. isolates belonging to C. acutatum, C. boninense, and C. gloeosporioides species complexes collected from olive trees and other hosts showing anthracnose symptoms from different geographic origins.
Table 2. Phenotypical characters of mycelia and conidia of representative Colletotrichum spp. isolates belonging to C. acutatum, C. boninense, and C. gloeosporioides species complexes collected from olive trees and other hosts showing anthracnose symptoms from different geographic origins.
Species Complex/Fungal SpeciesIsolateMyceliumConidia d
Color aBenomyl Inhibition (%) bCasein Hydrolysis cLength (µm)Width (µm)Length/WidthType
Colletotrichum acutatum complex
Colletotrichum acutatumCol-166/UWS-65Pink white33.5+11.1 ± 2.183.2 ± 0.803.6 ± 0.86Ellipsoid
Col-175/UWS-79Pink-orange57.4+8.3 ± 1.512.7 ± 0.583.1 ± 0.53Ellipsoid
Col-208/UWS-149Pink gray67.4+13.2 ± 2.284.7 ± 1.453.0 ± 0.71Clavate
Col-536Pink-orangeN/DN/D10.4 ± 1.193.2 ± 0.623.3 ± 0.53Fusiform
Colletotrichum fioriniaeCol-172/UWS-70Light gray71.1-13.4 ± 1.164.4 ± 0.493.1 ± 0.43Fusiform
Col-693WhiteN/DN/D10.4 ± 0.873.3 ± 0.233.3 ± 0.32Fusiform
Col-694WhiteN/DN/D10.4 ± 0.623.4 ± 0.233.2 ± 0.19Fusiform
Col-695Orange grayN/DN/D9.3 ± 0.553.2 ± 0.153.0 ± 0.28Fusiform
Col-696Pink-orangeN/DN/D10.3 ± 0.453.8 ± 0.312.8 ± 0.13Fusiform
Col-697WhiteN/DN/D9.9 ± 0.663.4 ± 0.233.0 ± 0.15Fusiform
Colletotrichum godetiaeCol-1Dark grayN/DN/D14.8 ± 0.855.0 ± 0.003.0 ± 0.17Clavate
Col-9Dark gray55.7++14.8 ± 1.454.9 ± 0.183.0 ± 0.33Clavate
Col-30Dark gray63.7++12.5 ± 1.755.1 ± 0.382.4 ± 0.38Clavate
Col-50Dark gray59.8++13.9 ± 1.235.0 ± 0.122.8 ± 0.25Clavate
Col-51Dark gray67.6++13.5 ± 1.405.0 ± 0.182.7 ± 0.30Clavate
Col-52Dark gray N/DN/D13.1 ± 1.383.8 ± 0.193.5 ± 0.35Clavate
Col-57Dark gray57.2++13.5 ± 1.435.0 ± 0.182.7 ± 0.31Clavate
Col-59Dark gray69.0++13.8 ± 1.515.0 ± 0.182.8 ± 0.37Clavate
Col-60Dark gray67.0++14.0 ± 1.724.9 ± 0.382.9 ± 0.42Clavate
Col-88Dark gray64.5++12.9 ± 1.195.0 ± 0.042.6 ± 0.28Ellipsoid
Col-508Dark grayN/DN/D14.4 ± 1.253.8 ± 0.413.8 ± 0.47Clavate
Col-522Light grayN/DN/D12.8 ± 1.293.7 ± 0.333.5 ± 0.93Fusiform
Colletotrichum nymphaeaeCol-42Light gray41.4++13.9 ± 1.563.5 ± 0.544.2 ± 1.06Fusiform
Col-84Light gray60.5++13.5 ± 1.403.6 ± 0.893.9 ± 0.35Clavate
Col-86Light gray58.7+14.0 ± 1.223.6 ± 0.433.9 ± 0.92Clavate
Col-506Light grayN/DN/D12.1 ± 1.443.4 ± 0.623.6 ± 0.57Clavate
Colletotrichum simmondsiiCol-169/UWS-68Whitish gray64.6+12.4 ± 1.123.9 ± 0.673.2 ± 0.49Fusiform
Colletotrichum boninense complex
Colletotrichum boninenseCol-178/UWS-82Whitish gray95.0-13.2 ± 1.024.8 ± 0.372.9 ± 0.32Clavate
Colletotrichum karstiiCol-79Pink-orange99.4-12.6 ± 1.315.0 ± 0.022.6 ± 0.26Ellipsoid
Colletotrichum gloeosporioides complex
Colletotrichum alienumCol-211/UWS-152White94.0++14.1 ± 1.22 4.6 ± 0.713.2 ± 0.55Ellipsoid
Col-214/UWS-156Pink White95.1-13.9 ± 1.144.6 ± 0.433.1 ± 0.33Ellipsoid
Colletotrichum fructicolaCol-82Light gray95.0-12.0 ± 1.63.7 ± 0.633.3 ± 0.56Ellipsoid
Colletotrichum gloeosporioidesCol-41Whitish gray100-14.8 ± 1.454.4 ± 0.723.5 ± 0.80Ellipsoid
Col-69Light gray99.7-13.2 ± 1.055.1 ± 0.232.6 ± 0.24Ellipsoid
Colletotrichum perseaseCol-205/UWS-139Light gray98.2-14.8 ± 1.384.8 ± 0.863.2 ± 0.55Ellipsoid
Colletotrichum siamenseCol-44Green gray96.5-13.9 ± 1.414.6 ± 0.703.1 ± 0.66Clavate
Col-160/UWS-13Whitish gray93.8-12.3 ± 1.014.5 ± 0.672.8 ± 0.50Ellipsoid
Col-184/UWS-92Whitish gray93.9+11.9 ± 1.383.5 ± 0.543.4 ± 0.61Clavate
Col-187/UWS-94Whitish gray96.0++13.3 ± 1.033.8 ± 0.433.5 ± 0.49Ellipsoid
Colletotrichum theobromicolaCol-200/UWS-131White94.4-13.3 ± 2.554.9 ± 0.42.8 ± 0.48Ellipsoid
HSD0.05 e--5.67-1.70.640.63
a Colony color of single conidial cultures of Colletotrichum spp. isolates was determined on PDA by visual observations after 7 days growing at 25 ± 2 °C with a 12-h diurnal photoperiod of cool fluorescent light (350 μmol m–2 s–1). Color was determined using a color scale [42]. b Inhibition percentage (%) of mycelial growth on PDA amended with benomyl at 5 μg mL−1. Values represent the means of two independent experiments, each with three replicated Petri dishes per isolate. c Levels of proteolytic activity of Colletotichum spp. isolates: ‘-’ non-ability to hydrolyse casein; ‘+’ ability to hydrolyse casein. Presence of one or two plus symbols represents differences of halo size (‘+’: hydrolysis halo ≤ 2 mm in width; ‘++’: hydrolysis halo > 2 mm in width). Data were obtained from the means of two independent experiments, each with three replicated Petri dishes per isolate. d Conidia were obtained from colonies grown on PDA at 25 ± 2 °C with a 12-h photoperiod of fluorescent light (350 mmol m−2 s−1) for 10 days. Length and width measures and the relation between length and width (Length/Width) values represent the mean of 150 conidia ± error standard of the mean. e Critical value for comparison according to the Tukey HSD test at p = 0.05. N/D non-determined.
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