Genetic and Phytopathogenic Characterization of Endemic Colletotrichum Isolates in Major Olive Cultivars of Greece

Abstract

Olive anthracnose outbreaks caused by the Colletotrichum species complex in the Mediterranean region decrease both fruit yield and olive oil production while also drastically degrading olive oil quality. The presence of various Colletotrichum species able to produce disease symptoms in olive fruits significantly deteriorates the efforts for an efficient crop protection strategy. In this report, the major olive productive area of Peloponnese was screened for Colletotrichum species capable of generating anthracnose symptoms. Olive fruits of 12 different olive cultivars were collected from 60 groves distributed analogously in the Peloponnese. Thirty-two fungal strains isolated from asymptomatic olive drupes were identified morphologically as Colletotrichum spp. and were multilocus genetically analyzed. The 32 isolates were grouped into two primary lineages resembling the previously characterized Colletotrichum acutatum and Colletotrichum nymphaeae based on the conducted genetic analysis for five genetic loci. The virulence of 16 Colletotrichum spp. strains were evaluated in a detached fruit assay of 10 Greek olive cultivars. The results clearly suggested that fungal isolates belonging to both C. acutatum and C. nymphaeae exhibited different levels of pathogenicity in a cultivar-dependent manner. Thus, cultivars examined in terms of the % Disease Index (%DI) were divided into highly tolerant, tolerant, and susceptible, and those analyzed regarding the % Disease Severity Index (%DSI) were divided into tolerant and susceptible. Our results suggest that the Greek cultivars of Athinolia and Megaritiki are highly tolerant to the vast majority of Colletotrichum strains isolated from Peloponnesian groves and consist of a significant genetic material for the future design of crop protection programs against anthracnose breakouts.

1. Introduction

The olive tree (Olea europaea L.) is one of the most commonly cultivated species in the Mediterranean basin and a vital source of revenue for local societies. Greece is the third olive oil-producing country in the world, after Spain and Italy [1]. The prefecture of Peloponnese represents approximately 25% of olive oil and olive production in Greece [2]. Olive anthracnose, caused by the Colletotrichum species complex, is the most important fungal disease in Mediterranean olive-producing countries [3]. However, Colletotrichum caused anthracnose has been also reported in all continents, where olive trees are cultivated [3]. The first report of Colletotrichum in Greece dates back to 1920, at Corfu Island in the Ionian Sea [4], and until recently, it was considered a less significant disease with limited economic importance. A notable outbreak of anthracnose has recently occurred, mainly in West-Central Greece, Peloponnese, and the island of Crete, which collectively represent 80% of Greek production [2] (ELSTAT, 2021), resulting in a significant deterioration of both olive fruits and olive oil yield [5,6]. This outbreak of anthracnose was caused by the extensive rainfalls in autumn and winter [5]. Moreover, other studies reported anthracnose outbreaks simultaneously in Italy and Spain [7].

While Colletotrichum spp. mainly infect drupes, they also produce considerable symptoms in various plant structures of the host [8]. In olive trees, Colletotrichum spp. typically impact flowers and olive fruit as a primary target, with olive leaves and branches being less frequently affected. Plant organs such as flowers, green shoots, stems, pedicels, and petioles may also be affected to a lesser degree [8]. Therefore, these various infected structures serve as the main potential source of infection for future disease outbreaks [3,9].

The autumn season in Greece as well as in other Mediterranean countries is typically characterized by elevated humidity and increased rainfall levels, which provides ideal conditions for anthracnose development, as it facilitates the germination of fungal conidia [10]. Indeed, symptoms of olive anthracnose are mainly manifested in autumn, which coincides with the time of fruit ripening processes [4]. Moral et al. [11] pointed out that the olive fruit maturity index plays an important role in the progression of the fungal infection. Mature drupes are preferentially affected by the pathogen, although green immature drupes are also susceptible under favorable environmental conditions [11,12]. Moreover, it has been suggested that the persistence of the infection may vary across seasons, which is mainly influenced by factors such as the type of olive cultivar, environmental conditions, agricultural practices, and the virulence of the pathogen [13].

The initial signs of disease on olive fruit appear as tiny brown spots or indentations, which develop rapidly and eventually cover a significant area of the fruit [10]. This progression often leads to premature fruit shedding or under certain environmental conditions to fruit mummification, with the fruit occasionally remaining attached to the trees and serving as the inocula for the next cycle coinciding with spring [4,14]. These outcomes lead to compromised fruit quality and subsequently reduced yield of both fruit and olive oil, as the olive oil of the infected fruit exhibits a reddish color and contains a high concentration of free fatty acids [15,16,17].

Managing anthracnose in plants has become increasingly challenging due to the existence of various and cosmopolitan Colletotrichum species, namely Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum simmondsii, Colletotrichum fioriniae, Colletotrichum clavatum, and Colletotrichum theobromicola, which in many plant species are considered to be severe pathogens [18]. Similarly, at least three Colletotrichum complex species that are able to produce severe pathogenic symptoms in olives have been characterized so far. These are C. acutatum sensu lato and C. gloeosporioides sensu lato, whereas Colletotrichum boninence sensu lato is less capable of producing phytopathological symptoms (e.g., C. kastri) [7,17,19]. Considerable progress has been achieved in comprehending the diversity within the Colletotrichum genus by examining an extensive global assortment of isolates, focusing on phylogenetic, morphological, and pathological characteristics [7,20]. Several Colletotrichum species have been classified under the C. acutatum species complex, including C. acutatum sensu stricto, C. fioriniae, C godetiae, C nymphaeae, C rhombiforme, and C simmpndsii. C. acutatum sensu lato is generally considered the most aggressive, which is linked to recent outbreaks in the Mediterranean basin [7]. Accordingly, several studies indicated that C. nymphaeae and C. acutatum sensu stricto consist of the most aggressive species, thus playing a pivotal role in spreading olive anthracnose disease [3,21]. However, C. acutatum species exhibited altered levels of virulence in olives in a cultivar-dependent manner [3,22,23]. In the Mediterranean region, C. godetiae is commonly found, although there are indications that it is being supplanted by the more aggressive C. acutatum sensu stricto [3,20]. C. gloeosporioides sensu lato, on the other hand, comprises two genera able to develop anthracnose symptoms in olives, namely C. gloeosporioides and C. theobromicola [3]. The recent identification of various species within the species complex has shed light on their role in olive anthracnose disease and the latest outbreak in Greece [8]. Several lines of evidence suggest that the precise fungal species identification is crucial for the analysis of each pair of pathogen–cultivar interactions [7,23]. Taking into account the available data, it is suggested that fungal species characterization is essential for predicting losses and effective disease management [17,24].

The aim of the present study was the isolation and identification of Colletotrichum spp. strains from the Peloponnese region based on morphologically and molecular technics and further detect the adaptability and pathogenicity of the endemic Colletotrichum spp. isolates. A final goal was to evaluate ten olive cultivars from the Peloponnese region for their susceptibility against the two dominant Colletotrichum species, referred as C. acutatum and C. nymphaea.

2. Materials and Methods

2.1. Selection of Experimental Olives Groves and Plant Material

The olive drupes were collected from a total of 60 groves from the Peloponnese region, where 12 different cultivars were systematically cultivated as previously reported [25]. The 12 collected cultivars, namely Asprolia, Arbequina, Athinolia, Kalamon, Koroneiki, Koutsoyrelia, Manaki, Mavrolia, Megaritiki, Myrtolia, Nemoutiana, and Picual, were proportionally divided into in 60 groves, which depended upon the total cultivated area of each cultivar in the Peloponnese region. From the 60 groves, 27 olive groves were selected from the regional unit of Messinia, 18 from the regional unit of Lakonia (Sparta), 7 from the regional unit of Argolis, 5 from the regional unit of Korinthos, and 3 from the regional unit of Arcadia (Figure 1). The experimental olive groves were selected based on their geographical location, variety, and the criteria associated with cultivation practices. A detailed profile of cultural practices for each producer had been created to facilitate the selection of the groves. Additionally, highly productive regions were represented proportionally in the experiment. From each olive grove, 6 representative olive trees were marked and olive fruit was collected at the stage of commercial ripening according to farmers’ practices, which depended on the cultivar, microclimatic conditions, and agricultural practices. From each marked tree, at least 300 gr of olive fruit was collected. Then, the samples were placed in paper bags and transported to the laboratory on the same day for further experiments.

2.2. Isolation of Colletotrichum spp. from Olive Drupes

Colletotrichum spp. strains were isolated from ostensibly healthy olive drupes. The collected olive fruits were initially washed thoroughly with tap water and then surface-sterilized using 70% (w/v) ethanol for 5 min, 0.5% (w/v) NaOCl containing 0.2% (w/v) Tween20 for 5 min, and 70% (w/v) ethanol for 30 s. The drupes were finally rinsed at least four times with sterilized double distilled water [26]. The surface-sterilized olive drupes were then cut into small pieces using a sterile scalpel and placed in Petri dishes containing a PDA medium. Plates were incubated at 25 °C and macroscopically observed for fungal colony growth for 7 days. The isolated pure strains were classified based on their conidial microscopic observation and mycelium morphology and color (white–grey and pinkish) prior to further experimentation.

2.3. DNA Extraction of Colletotrichum spp. Isolates

DNA extractions from pure fungal cultures of Colletotrichum spp. strains were prepared using a Pure Link Plant Total DNA Purification Kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNA concentration and the quality of the samples were estimated using NanoDrop One (Thermo Scientific, Carlsbad, CA, USA). Then, the samples were normalized and used for PCR amplification.

2.4. Genetic Characterization of Colletotrichum spp. Isolates

Primers were selected against five genetic loci previously used for Colletotrichum spp. genetic characterization [27,28,29,30,31] (Table S1). In more detail, primers targeted KLAP1 (Key Lime Anthracnose Pathogenicity1), 5.8S-ITS, B-TUBULIN2 (TUB2), HISTONE-H3, and Internal Transcribed Spacer (ITS) genes, as described in Table S1. The PCR mixture used for each reaction contained 1 μL of each primer (Table S1), 4 μL 5× PCR buffer, 0.4 μL dNTPs, 0.2 μL Phusion DNA polymerase (Thermo Fisher Scientific), and 5 μg DNA template. PCR was carried out in a Verity Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using the following cycle: initial denaturation at 98 °C for 30 sec, followed by 35 cycles of 98 °C/10 s (denaturation), 45–72 °C/30 s (annealing), 72 °C/30 s (extension), and a final step of 72 °C for 10 min. A total volume of 5 μL of each PCR product was loaded and visualized after electrophoresis in 1.5% (w/v) agarose gel. The PCR products were purified using a GeneJet PCR Purification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) following the instructions provided by the manufacturer.

2.5. Analyses of Sequences and Species Identification

The phylogenetic analysis of the Colletotrichum spp. strains was performed based on the nucleotide sequences of the five genetic loci that were compared to previously characterized sequences of Colletotrichum spp. available at NCBI (US National Center for Biotechnological Information) (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 August 2024), which are summarized in Table S2, as well as between the different isolates using NCBI BLAST (Basic Local Alignment Tool) and blastn algorithms. A multiple alignment of the sequenced amplicons was then performed using the ClustalW multiple alignment algorithm with the MEGA 11.0 (Molecular Evolutionary Genetics Analysis) program. Phylogenetic trees were constructed using the neighbor-joining method, using the MEGA 11.0 program. The reliability and stability of the dendrogram relationships were assessed through 1000 bootstrap samples.

2.6. Ex Vivo Virulence Evaluation

Sixteen selected fungal strains were inoculated on small-sized detached olive drupes to evaluate their virulence under ex vivo conditions, as previously described in Tsalgatidou et al. [32], with the appropriate modifications. Briefly, sterilized olive drupes were first artificially wounded and then inoculated with an aliquot of 10 μL of conidia suspension (10⁵ spores/mL), using a hemocytometer. Inoculated olive drupes from ten different cultivars (Asprolia, Athinolia, Kalamon, Koroneiki, Koutsoyrelia, Manaki, Mavrolia, Megaritiki, Myrtolia, and Nemoutiana) were transferred to plastic boxes, maintaining high humidity, and were incubated for 7 days in a growth chamber at 25 °C in the dark.

After seven days of incubation, Disease Incidence (DI%) was calculated as the percentage of infected olive drupes. The infected area of each olive drupe by Colletotrichum was measured using the ImageJ software v.1.8.0 analysis tool, and Disease Severity (DS%) was determined as the percentage of the infected area. The Disease Severity Index (DSI%) was scored on a 0 to 9 rating scale, with 0 = healthy fruits, 1 = 1–10%, 3 = 11–25%, 5 = 26–50%, 7 = 51–75% and 9 = >75% infected fruit area and was calculated based on the following formula:

% Disease Severity Index, (DSI) = [Σ(n × i)/(N × Z)] × 100,

where n stands for the number of fruits in a specific value of the disease rating scale, i stands for the corresponding value of the scale, N stands for the total number of fruits, and Z stands for the highest value of the disease rating scale.

Colletotrichum strains were reisolated from the symptomatic olive tissues, confirming their identity and Koch’s postulates.

2.7. Statistical Analysis

The experiments of detached fruit assay were arranged as a completely randomized design with three replications of at least ten fruits per strain.

In order to assess the effect of cultivars and strains on %DSI and %DI parameters, the data were analyzed as two-way ANOVA with the factors being the cultivar (Asprolia, Athinolia, Kalamon, Koroneiki, Koutsoyrelia, Manaki, Mavrolia, Megaritiki, Myrtolia, and Nemoutiana) and strain (FAth9.1, FAth24.1, FArb3.4, FKor1.4, FKor4.2, FKor5.1, FKor7.2, FKor10.1, FKor18.2, FKor34.1, FKor37.2, FKor44.3, FKor47.1, FKal19.1, FKal46.1, and FMeg61.1). The significant differences were determined by Tukey’s HSD test (α = 0.05).

One-way ANOVA was used to assess significant differences among strains for each cultivar separately. The significant differences were determined by Tukey’s HSD test (α = 0.05).

Statistical analysis was carried out using JMP 10.0 (SAS, USA). A cluster heatmap for cultivars and strains was constructed based on the Euclidean distance using a publicly available online tool (https://www.bioinformatics.com.cn/en, accessed on 6 August 2024) [33].

3. Results

3.1. Morphology and Geographical Distribution of Colletotrichum Isolates

Colletotrichum isolates were detected in 6 out of the 12 cultivars selected for the present study across the Peloponnese region (Figure 1A), whereas the vast majority, 22 out of the 32 isolates in total, were isolated from the Koroneiki cultivar (Figure 1B). Furthermore, 25 out of the 32 Colletotrichum strains were isolated from the Messinia region, whereas only 5 strains were isolated from Lakonia (Figure 1C), despite the fact that the latter exhibited comparable number of olive trees (ELSTAT 2021). In more detail, Colletotrichum strains were isolated from ostensibly healthy olive drupes, which were derived from groves located in four different geographical areas (Messinia, Lakonia, Argolis, and Korinthos) of the Peloponnese region (Figure 1C).

A total of 32 Colletotrichum spp. isolates were morphologically classified into two main categories (Figure 2). The first included isolates showing pale grey-to-white aerial mycelia, while the Petri dish bottom view of the PDA culture was pinkish (Figure 2A). The second category comprised fungal colonies with a grey center and white peripheral aerial mycelia, and the bottom view of the PDA culture was white with grey spore masses (Figure 2B). The first group encompassed fungal strains classified as the C. acutatum species complex and the second as C. nymphaeae. Conidia morphology examination revealed that C. acutatum were cylindrical, fusiform, and unicellular, while the C. nymphaeae samples were hyaline and appeared slightly translucent under the microscope and unicellular, but some became septate as they matured (Figure 2C). The conidium morphology for all isolates is illustrated in Figure S1, while the representative conidia for C. acutatum and C. nymphaeae are presented in Figure 2C.

3.2. Phylogenetic Analysis

Thirty-two isolates exhibiting morphological similarities to previously characterized Colletotrichum spp. were subjected to multilocus analysis for five genetic loci, namely KLAP1, 5.8S-ITS, TUB2, HISTONE-H3, and ITS genes. The genetic investigation of the sequences revealed that the 32 isolates were categorized into Colletotrichum spp. and were subjected to further phylogenetic analysis. The concatenated complete sequences of the 32 strains for the five genetic loci examined in this study were aligned with previously characterized Colletotrichum species (Table S2). All the studied isolates belong to the C. acutatum species complex, forming two major lineages and exhibiting extensive similarity to the characterized C. acutatum and C. nymphaeae (Figure 3). Interestingly, isolates belonging to the C. gloeosporioides species complex were not detected among the studied samples. The majority of the isolates (22 samples) were grouped under the C. acutatum species complex, while 10 strains were similar to C. nymphaeae. Intriguingly, FKor7.1 and FAth24.1 exhibited less genetic similarity to the other isolates grouped in the same lineage. The accession numbers of all isolates are presented in Table S3.

3.3. The Pathogenicity of Ex Vivo Colletotrichum spp. Isolates

After the initial screening of the 32 isolated Colletotrichum spp. strains, 16 isolates that exhibited high virulence were systematically evaluated for their ability to cause anthracnose in olive fruit of the 10 cultivars mainly cultured in the Peloponnese region, specifically Athinolia, Koroneiki, Asprolia, Kalamon, Koutsourelia, Manaki, Mavrolia, Megaritiki, Myrtolia, and Nemoutiana. Among the representative strains selected for analysis, 10 were classified as C. acutatum (FArb3.4, FAth9.1, FKal19.1, FKor1.4, FKor4.2, FKor5.1, FKor7.1, FKor10.1, FKor18.2, and FKal46.1), and 6 (FAth24.1, FKor34.1, FKor37.2, FKor44.3, FKor47.1, and FMeg61.1) were identified as C. nymphaeae (Figure 4). The pathogenicity of the fungal strains regardless of the species showed significant fluctuation among the olive varieties (Figure 4). Additionally, for strains that exhibited high %DSI, the %DI was mitigated and vice versa (e.g., FKor5.1 and Fkal19.1 both belonging to C. acutatum and FAth24.1 belonging to C. nymphaeae). Indeed, strains with low rates of fruit infection were capable of producing severe symptoms, while strains exhibiting a high number of infected fruits generated mild symptoms (Table S5). These results are more obvious in Figure 5A,B, where the heatmap for the %DI value of classified olive cultivars is presented for (i) susceptible cvs (Koroneiki, Mavrolia, Nemoutiana, Asprolia, and Kalamon), (ii) tolerant cvs (Manaki and Koutsourelia), and (iii) highly tolerant cvs (Athinolia, Myrtolia, and Megaritiki) On the other hand, the heatmap for the %DSI value is shown for cultivars categorized as tolerant (Athinolia, Myrtolia and Megaritiki, Manaki, and Koutsourelia) and susceptible (Koroneiki, Mavrolia, Nemoutiana, Asprolia, and Kalamon). However, both heatmaps (Figure 5) clearly suggest that Koroneiki, Mavrolia, Nemoutiana, Asprolia, and Kalamon are susceptible varieties for the majority of the examined strains, excluding FArb3.4 and FKor1.4. These particular strains were less efficient in infecting olive fruit and simultaneously produced less severe symptoms in all the tested cultivars. The most vulnerable cultivar to anthracnose, regarding both %DI and %DSI values, was Kalamon; conversely, the most tolerant olive cultivar was Athinolia (Figure 5C,D). Interestingly, the strains FKor7.1 and FAth24.1 showed limited similarity to other congener C. acutatum isolates and were extremely virulent since they demonstrated high values for both %DI and %DSI (Tables S4 and S5). Paradoxically, Koutsourelia cv. was tolerant to the most virulent strains and susceptible to isolates that are generally non-virulent even for the susceptible cultivars. Among the most virulent strains of Colletotrichum spp. was FAth24.1, isolated from Athinolia, which according to the results was the most tolerant cultivar.

4. Discussion

A plethora of Colletotrichum species exhibiting high morphological and genetic diversity is capable of causing anthracnose symptoms in olive trees, which renders the strategic planning for effective disease management extremely challenging [3,13,23]. Our knowledge of the specific Colletotrichum species involved in a field can aid in understanding the epidemiology of the disease, including its spread, survival mechanisms, and infection cycles. This information is crucial for predicting disease outbreaks and implementing timely interventions. Furthermore, identifying the species of Colletotrichum can assist in breeding programs aimed at developing olive cultivars with resistance to specific pathogens. This is essential for long-term, sustainable control of the disease. Some Colletotrichum species might be subjected to regulatory and quarantine measures to prevent their spread to new regions. Accurate identification helps in complying with these regulations and protecting other olive-producing areas from potential outbreaks.

In the present study, the multilocus genetic analysis of Colletotrichum isolates linked the initial morphological classification to the strains’ genetic background. Indeed, endemic Peloponnesian Colletotrichum strains were grouped into two major lineages very similar to C. acutatum and C. nymphaeae species. ITS single-locus genetic analysis is not a reliable marker for this fungal genus due to its failure to phylogenetically resolve cryptic species [34]. Thus, five genetic loci were analyzed, namely KLAP1, 5.8S-ITS, TUB2, HISTONE-H3 and ITS genes to effectively clarify the isolates’ genetic diversity. Both lineages of isolated fungi were categorized into the C. acutatum sensu lato group, which is in accordance with previously published reports suggesting that the dominant phytopathogenic species is C. acutatum, whereas the important phytopathogen C. gloeosporioides, very common in the neighboring country Italy, has not been detected in Greece thus far [3,5,7,8]. Similarly, C. godetiae responsible for anthracnose outbreaks in several European countries was absent from our samples, confirming previously published reports from other Greek research groups [5,6,7,8]. Among the genetic loci examined in this study, the most significant was KLAP1, which is recognized as an important pathogenicity factor in Colletotrichum acutatum [35,36]. Specifically, its function in pathogenicity involves acting as an effector that manipulates host cellular processes to suppress plant immune responses and facilitate infection. KLAP1 is often associated with the formation of appressoria, which are specialized structures that generate turgor pressure to breach the host epidermis. The formation and function of appressoria are critical for the successful invasion of the plant by Colletotrichum species. KLAP1 is involved in the early stages of infection, helping the fungus to colonize the host plant tissues. Also, KLAP1 might contribute to host specificity and the degree of virulence of C. acutatum [35]. In this study, single-locus phylogenetic analysis of KLAP1 effectively classified the fungal isolates to C. acutatum and C. nymphaeae species, which renders this marker extremely important for C. acutatum genetic analysis.

The most crucial species for olive anthracnose are C. acutatum and C. gloeosporioides species complexes [10]. C. acutatum is characterized by its fusiform (spindle-shaped) conidia, while other similar species like C. nymphaeae are recognized by their distinct conidia shape and pathogenicity in olives [37]. By contrast, C. gloeosporioides produces more cylindrical conidia and has a wide distribution [23]. Several reports suggest that the most virulent Colletotrichum species belong to the C. acutatum species complex, including C. acutatum, C. godetiae, and C. nymphaeae [3,23]. Our results suggested that certain strains of C. acutatum and C. nymphaeae were comparably aggressive, exhibiting high %DI and %DSI values.

Identifying Colletotrichum species in an olive field is crucial as they impact agricultural productivity, disease management, and economic stability, and all these factors translate into economic benefits with the corresponding environmental sustainability. It is well documented that different species of Colletotrichum can vary in their pathogenicity, virulence, and the symptoms they cause [7,8,23]. Thus, species identification helps in developing targeted and effective disease management strategies, including the use of appropriate fungicides and cultural practices [38]. The 16 strains of Colletotrichum isolated in the present study exhibited different levels of virulence in a cultivar-dependent manner, which is in accordance with previous studies with Greek, Italian, Spanish, and Portuguese cultivars [3,7,8,23]. These data suggest that cultivar-specific metabolic regulation is implicated in plant–pathogen interactions, possibly via the biosynthesis of certain metabolites such as amino acids, signaling-related sugars, and secondary metabolism compounds such as phenolics [4,8].

The Colletotrichum strains were isolated from asymptomatic olive drupes and healthy olive fields, and they were collected from the main olive-producing areas of the Peloponnese region such as Messinia, Lakonia (Sparta), Argolis, Arcadia, and Korinthia, in which olive has been cultivated since ancient times. In half of the cultivars studied, the fungus was isolated in the laboratory, although the isolated samples were asymptomatic. Interestingly, the most important oil-producing variety in Greece (Koroneiki), which has been generally considered highly resistant to C. acutatum by researchers [4,39], was found to be a very susceptible phenotype in the present study; this was associated with high values for both %DI and %DSI after inoculation with the isolated endemic C. acutatum strains (Figure 5). It is also noteworthy that the vast majority (78.13%) of the Colletotrichum strains were isolated from Koroneiki (Figure 1B). This fact defines the variety Koroneiki as particularly sensitive to attacks by Colletotrichum. This sensitivity, if combined with favorable conditions, such as high humidity and rainfalls in autumn and abnormal temperature fluctuations [24], leads to great destruction and may partly explain the recent outbreaks in the Peloponnese region, where, until recently, anthracnose was considered an insignificant disease.

Two of our fungal isolates, FAth24.1 and FKor7.1, which were phylogenetically classified as C. acutatum, showed limited genetic similarity to other C. acutatum isolates. Additionally, FAth24.1 phenotypically resembled the C. nymphaeae strains, while FKor7.1 exhibited a typical pinkish C. acutatum phenotype. It is well known that the same genus of fungi can have colonies of different phenotypes. Phenotypic switching in fungi is an in vitro reversible phenomenon that is defined as the spontaneous emergence of colonies with altered colony morphology [40]. Furthermore, changing environmental conditions can affect cellular morphology and lead to the formation of new morphotypes, with each morphotype having a potential impact on both pathogen survival and disease epidemiology [41]. More experiments for the precise genetic classification of FAth24.1 are needed since this strain was characterized as highly aggressive in ex vivo experiments. Interestingly, this strain was isolated from Athinolia, which included the most tolerant of the 10 varieties examined, suggesting an active plant–pathogen crosstalk.

The analysis of %DSI and %DI clarified the categorization of the studied cultivars into susceptible, tolerant, and highly tolerant. The most susceptible proved to be Koroneiki, Mavrolia, Nemoutiana, Asprolia, and Kalamon. The Koroneiki olive cv. is the cornerstone of Greek olive cultivation, celebrated for its high oil yield, superior oil quality, and adaptability to the Mediterranean climate. The cultivar’s resilience and economic importance make it a favorite among Greek olive growers and a vital part of Greece’s agricultural heritage [42,43,44]. Furthermore, the Kalamon olive cultivar is highly desirable for its excellent table olives [45]. This cultivar is well adapted to the Mediterranean climate and soils, demonstrating good drought tolerance and moderate resistance to pests and diseases. It is a cherished component of Greek cuisine and a valuable agricultural export. The susceptibility to Colletotrichum infections, for both Koroneiki and Kalamon, must be taken into account and dealt with in a timely manner since these are very important Greek products. Mavrolia, Nemoutiana, and Asprolia account for a smaller area of cultivation and are therefore less marketable products; nevertheless, it is important to take timely measures against anthracnose for cultivation to be effective. Understanding the relationship between these factors and disease development is crucial for effective management. By implementing integrated disease management practices, including cultural methods, chemical treatments, and the use of resistant varieties, the impact of this disease can be mitigated, ensuring healthier olive crops and better yields.

This study sheds light on the gradation of the susceptibility of Greek olive cultivars and categorizes isolated species to the Colletotrichum species complex according to the cultivation area, findings that may be useful in strategic management programs against anthracnose.