Elucidating the impact of environmental factors on the growth of Colletotrichum coccodes strains isolated from potato tubers in Great Britain.

Black dot and silver scurf caused by Colletotrichum coccodes and Helminthosporium solani, respectively, are tuber blemish diseases affecting quality in the fresh and pre-pack potato industry. In the last 20 years, the importance of high-quality tuber appearance has increased considerably due to the growing demand for washed and pre-packed potatoes in the UK. Changing climate characterised by rising temperatures and wetter summers is a threat as this will favour the development of pathogens such as C. coccodes in the soil increasing the risk of food spoilage. Moreover, both diseases can develop not only in the field but also after harvest, with postharvest storage temperatures being a crucial factor in controlling fungal growth. Furthermore, anecdotal evidence showed differences on the aggressiveness of black dot depending on its origin (i.e. England and Scotland) on potato tubers. Silver scurf and black dot are difficult to differentiate as they present similar phenotypes characterised by silvery lesions making it challenging for managers to take the necessary corrective action during storage. Hence, the aim of this study was to give a general insight into the ecological conditions affecting the establishment of the causal agent of potato black dot in the field, and black dot and silver scurf during the supply chain. Therefore, invitro experiments were designed to study the growth rate and lag times simulating both scenarios respectively: on soil extract agar (SEA) media at different temperatures (4, 11, 15 °C) and matric potentials (control [unmodified] and - 1.4 MPa [modified]); and on natural potato dextrose agar (NPDA) for different temperatures (4, 11, 15 and 20 °C) at 99 % relative humidity (RH) for 25 days. When simulating the field environment, drier conditions (matric potential = -1.4 MPa) reduced fungal growth for both isolates by 0.1 cm day-1 at the temperature of 15 °C, suggesting temperature as the main limiting factor for the growth of C. coccodes in the soil. The causal agent of black dot exhibited a faster growth rate under retailer-like conditions (i.e., 15 °C) compared to H. solani. Understanding the environmental influence on both the pathogen and the crop is vital for proper disease management to help reduce food loss and waste.

Exhaustive identification and characterization of Colletotrichum siamense and Colletotrichum fructicola as causative agents of circular leaf spot disease of rubber tree (Hevea brasiliensis) in India.

The rubber tree (Hevea brasiliensis) is one of the major domesticated crops planted commercially for the production of natural rubber (NR) worldwide. In recent years, rubber trees in the Southern states of India and other rubber-producing countries have experienced a severe leaf spot disease, characterized by the appearance of several brown circular spots in the initial stage, which later spread all over the lamina of fully matured leaves, leading to yellowing and defoliation. The causal organism of this Circular Leaf Spot (CLS) disease has not been conclusively identified in any previous studies. In this study, we collected infected leaf samples from various locations in the South Indian states. We aimed to identify the actual fungal pathogen that causes the CLS disease on rubber trees. Based on the morphological and molecular analysis of the most frequently isolated fungi from infected leaf samples were identified as Colletotrichum siamense and Colletotrichum fructicola. Pathogenicity tests also confirmed the involvement of isolated Colletotrichum spp. in the development of CLS disease. These findings provide valuable insights into understanding the CLS disease and its impact on rubber cultivation. To our knowledge, it is the first report of C. siamense and C. fructicola associated with CLS disease of rubber trees in India.

Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight, and premature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin, and Lipu in Guangxi Province and Meishan, Abe Tibetan, and Qiang Autonomous Prefecture of Sichuan Province. Characteristics of mycelia on potato dextrose agar, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], ß-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%), and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.

Endophytic species of Colletotrichum associated with cashew tree in northeastern Brazil.

Anthracnose caused by Colletotrichum is the most severe and widely occurring cashew disease in Brazil. Colletotrichum species are commonly found as pathogens, endophytes and occasionally as saprophytes in a wide range of hosts. The endophytic species associated with cashew trees are poorly studied. In this study, we report the Colletotrichum endophytic species associated with cashew trees in two locations in the state of Pernambuco, their prevalence in different plant organs (leaves, veins, branches and inflorescences), and compare the species in terms of pathogenicity and aggressiveness using different inoculation methods (wounded × unwounded). Six species of Colletotrichum were identified according to multilocus phylogenetic analyses, including Colletotrichum asianum, Colletotrichum chrysophilum, Colletotrichum karsti, Colletotrichum siamense, Colletotrichum theobromicola, and Colletotrichum tropicale. There were differences in the percentage of isolation in relation to the prevalence of colonized tissues and collection locations. C. tropicale was the prevalent species in both geographic areas and plant tissues collected, with no pattern of distribution of species between areas and plant tissues. All isolates were pathogenic in injured tissues of cashew plants. The best method to test the pathogenicity of Colletotrichum species was utilizing the combination of leaves + presence of wounds + conidial suspension, as it better represents the natural infection process. C. siamense was the most aggressive species.

PCR-Based Detection for the Quarantine Fungus Colletotrichum kahawae, a Biosecurity Threat to the Coffee (Coffea arabica) Industry Worldwide.

Coffee berry disease is caused by Colletotrichum kahawae, a quarantine fungus still absent from most coffee-producing countries. Given the potential adverse effects on coffee berry production, it is a severe worldwide threat to farmers and industry. Current biosecurity management focuses on exclusion by applying quarantine measures, including the certification of coffee plants and their products. However, methods for detecting C. kahawae by National Plant Protection Organization (NPPO) laboratories still need approval. This research aims to functionally demonstrate, standardize, and validate a method for detecting and discriminating C. kahawae from other Colletotrichum species that may be present in coffee plant samples. The method proposes to use an end-point PCR marker for the mating type gene (MAT1-2-1) and a confirmatory test with a real-time quantitative PCR (qPCR) marker developed on the glutamine synthetase gene. The C. kahawae amplicons for the Cen-CkM10 qPCR marker exhibited specific melting temperature values and high-resolution melt profiles that could be readily differentiated from other tested species, including their relatives. Given the fungus's quarantine status, specificity was tested using artificial mixtures of DNA of C. kahawae with other Colletotrichum species and coffee plant DNA. The described method will enable NPPOs in coffee-producing and exporting countries, especially Colombia, to prevent this pathogen's entry, establishment, and spread.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Chitosan Sensitivity of Fungi Isolated from Mango (Mangifera indica L.) with Anthracnose.

In Mexico, the mango crop is affected by anthracnose caused by Colletotrichum species. In the search for environmentally friendly fungicides, chitosan has shown antifungal activity. Therefore, fungal isolates were obtained from plant tissue with anthracnose symptoms from the state of Guerrero in Mexico and identified with the ITS and ß-Tub2 genetic markers. Isolates of the Colletotrichum gloeosporioides complex were again identified with the markers ITS, Act, ß-Tub2, GADPH, CHS-1, CaM, and ApMat. Commercial chitosan (Aldrich, lot # STBF3282V) was characterized, and its antifungal activity was evaluated on the radial growth of the fungal isolates. The isolated anthracnose-causing species were C. chrysophilum, C. fructicola, C. siamense, and C. musae. Other fungi found were Alternaria sp., Alternaria tenuissima, Fusarium sp., Pestalotiopsis sp., Curvularia lunata, Diaporthe pseudomangiferae, and Epicoccum nigrum. Chitosan showed 78% deacetylation degree and a molecular weight of 32 kDa. Most of the Colletotrichum species and the other identified fungi were susceptible to 1 g L-1 chitosan. However, two C. fructicola isolates were less susceptible to chitosan. Although chitosan has antifungal activity, the interactions between species of the Colletotrichum gloeosporioides complex and their effect on chitosan susceptibility should be studied based on genomic changes with molecular evidence.

VOCs profile of Colletotrichum spp. as a potential tool for quality control of açaí pulp.

Colletotrichum has been identified as responsible for the "dried fruit" disease in açaí (Euterpe oleracea). Besides concern for açaí pulp quality control, the characterization of Colletotrichum has been difficult, which has motivated the search for chemical markers in the Volatile Organic Compounds (VOCs) profile for use as a tool in the identification of açaí pulp contaminated by the fungus. Extracted VOCs by Headspace - Solid Phase Micro Extraction (HS-SPME) were identified through gas chromatography mass spectrometry (GC-MS). From GC-MS analyzes 26 VOCs were identified, with a predominance of the terpenoids. Chemometrically, menthol and menthone were assigned as potential markers of the genus. The analysis of VOCs in açaí pulps contaminated by Colletotrichum under different cultivation conditions enabled the detection of menthone. This result illustrated the selectivity of the culture medium and the potential of this tool for use in the quality control of açaí pulp.

Quantitative Detection and Monitoring of Colletotrichum siamense in Rubber Trees Using Real-Time PCR.

Colletotrichum siamense is one of the most important pathogens of rubber trees in Asia. The proper detection and quantification of C. siamense populations in rubber trees are of importance for monitoring the epidemics of the disease. In this study, we developed an internal transcribed spacer-based real-time PCR method to efficiently detect C. siamense infecting rubber trees, which reliably detected as little as 100 fg of genomic DNA, 100 copies of target DNA, and 20 conidia. The real-time PCR protocol recognized all C. siamense isolates collected from three provinces in China, whereas no amplification was observed with the rubber tree and its other pathogens. Detection and quantification of C. siamense were performed in artificially and naturally infected rubber leaves. We could still detect C. siamense in plant mixes, of which only 0.0001% of the tissue was infected. An accumulation of C. siamense DNA was observed during the whole infection process at all three leaf phenological stages, suggesting that the real-time PCR method can be used to monitor C. siamense development in rubber trees. Finally, the method allowed the detection of C. siamense in naturally infected and symptomless leaves of rubber trees in the fields. Compared with earlier detection methods, the real-time PCR method is more specific and more sensitive, and it will be of great use for studies aiming to gain a better understanding of the epidemiology of Colletotrichum leaf disease, as well as the prediction of disease risk and proposals to control it.

Twig and Shoot Dieback of Citrus, a New Disease Caused by Colletotrichum Species.

(1) Background: This study was aimed at identifying the Colletotrichum species associated with twig and shoot dieback of citrus, a new syndrome occurring in the Mediterranean region and also reported as emerging in California. (2) Methods: Overall, 119 Colletotrichum isolates were characterized. They were recovered from symptomatic trees of sweet orange, mandarin and mandarin-like fruits during a survey of citrus groves in Albania and Sicily (southern Italy). (3) Results: The isolates were grouped into two distinct morphotypes. The grouping of isolates was supported by phylogenetic sequence analysis of two genetic markers, the internal transcribed spacer regions of rDNA (ITS) and ß-tubulin (TUB2). The groups were identified as Colletotrichum gloeosporioides and C. karstii, respectively. The former accounted for more than 91% of isolates, while the latter was retrieved only occasionally in Sicily. Both species induced symptoms on artificially wound inoculated twigs. C. gloeosporioides was more aggressive than of C. karstii. Winds and prolonged drought were the factor predisposing to Colletotrichum twig and shoot dieback. (4) Conclusions: This is the first report of C. gloeosporioides and C. karstii as causal agents of twig and shoot dieback disease in the Mediterranean region and the first report of C. gloeosporioides as a citrus pathogen in Albania.

Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia.

BACKGROUND AND OBJECTIVE: Postharvest disease caused by Colletotrichum spp. caused major losses. The species of Colletotrichum are difficult to distinguish if only seen from their morphology. This study investigated Colletotrichum isolates associated with tropical fruits anthracnose using multi-genetic analysis and the cross-infection potency of each isolate among tropical fruits. MATERIALS AND METHODS: The fruit samples were collected from markets in the Special Region of Yogyakarta, Indonesia and its surrounding area. The fruits affected by anthracnose subjected to isolation, resulting in 15 isolates. Morphology of colony and conidia then characterized and clustered with UPGMA. The seven representative isolates were selected for molecular identification. The multi-genetic analysis was used by combining ITS, Glyceraldehyde 3-phosphate dehydrogenase (gapdh) and tub2 sequence genes. A cross-infection test was conducted by using selected species from the multi-genetic analysis. RESULTS: Multi-genetic analysis clustered the selected isolates into four species. Isolates from banana, avocado, papaya and citrus belonged to gloeosporioides species complex, including C. siamense, C. asianum and C. gloeosporioides. Isolates from apple, guava, mango and citrus belonged to acutatum species complex, including C. sloanei. The cross-infection test in this study showed that C. siamense could cause anthracnose on banana, apple, citrus and avocado, C. asianum on avocado, papaya, apple and citrus, C. gloeosporioides on citrus and apple, C. sloanei on apple, guava, citrus and papaya. CONCLUSION: The C. siamense, C. asianum, C. gloeosporioides and C. sloanei found associated with tropical fruits anthracnose. The potency of the cross-infection test revealed the board range in the pathogenicity of the Colletotrichum isolates.

Soybean anthracnose caused by Colletotrichum species: Current status and future prospects.

Soybean (Glycine max) is one of the most important cultivated plants worldwide as a source of protein-rich foods and animal feeds. Anthracnose, caused by different lineages of the hemibiotrophic fungus Colletotrichum, is one of the main limiting factors to soybean production. Losses due to anthracnose have been neglected, but their impact may threaten up to 50% of the grain production. TAXONOMY: While C. truncatum is considered the main species associated with soybean anthracnose, recently other species have been reported as pathogenic on this host. Until now, it has not been clear whether the association of new Colletotrichum species with the disease is related to emerging species or whether it is due to the undergoing changes in the taxonomy of the genus. DISEASE SYMPTOMS: Typical anthracnose symptoms are pre- and postemergence damping-off; dark, depressed, and irregular spots on cotyledons, stems, petioles, and pods; and necrotic laminar veins on leaves that can result in premature defoliation. Symptoms may evolve to pod rot, immature opening of pods, and premature germination of grains. CHALLENGES: As accurate species identification of the causal agent is decisive for disease control and prevention, in this work we review the taxonomic designation of Colletotrichum isolated from soybean to understand which lineages are pathogenic on this host. We also present a comprehensive literature review of soybean anthracnose, focusing on distribution, symptomatology, epidemiology, disease management, identification, and diagnosis. We consider the knowledge emerging from population studies and comparative genomics of Colletotrichum spp. associated with soybean providing future perspectives in the identification of molecular factors involved in the pathogenicity process. USEFUL WEBSITE: Updates on Colletotrichum can be found at http://www.colletotrichum.org/. All available Colletotrichum genomes on GenBank can be viewed at http://www.colletotrichum.org/genomics/.

Species of Colletotrichum on bamboos from China.

Twenty-seven Colletotrichum isolates associated with asymptomatic tissues of bamboo (Bambusoideae, Gramineae) were isolated from Anhui, Beijing, and Guangxi in China. Based on multilocus (internal transcribed spacer [ITS], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS], actin [ACT], beta-tubulin [TUB2]) phylogenetic analyses and morphological characteristics, three species were distinguished, including two novel species, C. bambusicola and C. guangxiense, and one known species, C. metake, which is a first report for China. These species have hitherto only been discovered on Bambusoideae, indicating that they probably have host preference.

Phospholipid signaling pathway in Capsicum chinense suspension cells as a key response to consortium infection.

BACKGROUND: Mexico is considered the diversification center for chili species, but these crops are susceptible to infection by pathogens such as Colletotrichum spp., which causes anthracnose disease and postharvest decay in general. Studies have been carried out with isolated strains of Colletotrichum in Capsicum plants; however, under growing conditions, microorganisms generally interact with others, resulting in an increase or decrease of their ability to infect the roots of C. chinense seedlings and thus, cause disease. RESULTS: Morphological changes were evident 24 h after inoculation (hai) with the microbial consortium, which consisted primarily of C. ignotum. High levels of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA) were found around 6 hai. These metabolic changes could be correlated with high transcription levels of diacylglycerol-kinase (CchDGK1 and CchDG31) at 3, 6 and 12 hai and also to pathogen gene markers, such as CchPR1 and CchPR5. CONCLUSIONS: Our data constitute the first evidence for the phospholipids signalling events, specifically DGPP and PA participation in the phospholipase C/DGK (PI-PLC/DGK) pathway, in the response of Capsicum to the consortium, offering new insights on chilis' defense responses to damping-off diseases.

Different loci control resistance to different isolates of the same race of Colletotrichum lindemuthianum in common bean.

KEY MESSAGE: Linkage and genome-wide association analyses using high-throughput SNP genotyping revealed different loci controlling resistance to different isolates of race 65 of Colletotrichum lindemuthianum in common bean. Development of varieties with durable resistance to anthracnose is a major challenge in common bean breeding programs because of the extensive virulence diversity of Colletotrichum lindemuthianum fungus. We used linkage and genome-wide association analyses to tap the genomic regions associated with resistance to different isolates of race 65. Linkage mapping was done using an F2 population derived from the cross between the Mesoamerican common beans BRS Estilo x Ouro Vermelho, inoculated with two different isolates of race 65. Association genetics relied on a diversity common bean panel containing 189 common bean accessions inoculated with five different isolates of race 65 as an attempt to validate the linkage analysis findings and, eventually, identify other genomic regions associated with resistance to race 65. The F2 population and diversity panel were genotyped with the BARCBean6K_3 Illumina BeadChip containing 5398 SNP markers. Both linkage and genome-wide association analyses identified different loci controlling resistance to different isolates of race 65 on linkage group Pv04. Genome-wide association analysis also detected loci on Pv05, Pv10 and Pv11 associated with resistance to race 65. These findings indicate that resistance to race 65 can be overcome by the virulence diversity among different isolates of the same race and could lead to the loss of resistance after cultivar release. We identified 25 resistant common bean cultivars to all five isolates of race 65 in the diversity panel. The accessions should be useful to develop cultivars combining different resistance genes that favor durable resistance to anthracnose in common bean.

Real-time PCR assay for Colletotrichum acutatum sensu stricto quantification in olive fruit samples.

Olive anthracnose is caused by fungal species within the Colletotrichum acutatum, C. gloeosporioides and C. boninense complexes. Anthracnose causes severe pre- and post-harvest olive drupe fall. This study aimed to design a species-specific qPCR assay, based on klap1 gene, suitable for C. acutatum s.s. quantification in cv. Galega Vulgar fruit samples. The developed qPCR assay presented a detection limit of 10.14 fg/reaction, and a linear cycle threshold of R2 = 0.996. C. acutatum inoculum was detected in pulverized olive fruits, and in early infection stages, before symptom appearance, 16 h after inoculation (Ct values = 28.29 ± 1.1). In olive samples, the derived melting curve was specific presenting a single dissociation peak (Tmelting = 88.7 °C). The designed assay was effectively applied in C. acutatum detection and quantification using infected olive samples, with a LOD of 0.59 ng and a LOQ of 1.8 ng, allowing its application to orchard management.

Conyza bonariensis as an alternative host for Colletotrichum species in Argentina.

AIMS: This study investigated the diversity of Colletotrichum isolates recovered from Conyza bonariensis leaves through the use of morphological characteristics, growth rate, carbon sources utilization and phylogenetic analysis. METHODS AND RESULTS: In all, 30 Colletotrichum isolates recovered from C. bonariensis leaves showing symptoms of disease were included in the present study. Based on the analysis of morphology and sequences, the isolates were distributed into six Colletotrichum species complexes. The concatenated alignment of GAPDH and ITS sequences showed that 20 out of 30 isolates were included in four species complexes which comprise the most important pathogens causing anthracnose in soybean or anthracnose and stalk rot in maize: C. truncatum, C. orchidearum, C. gloeosporioides and C. graminicola. The remaining 10 isolates were included in the C. boninense and C. destructivum species complexes or could not be assigned to any complex with the available information. CONCLUSION: Weeds belonging to genus Conyza are host to soybean and maize potential pathogenic species of Colletotrichum and could have a role as inoculum reservoir for cross contamination in the agroecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of morphological, kinetics and physiological parameters of growth and phylogenetic analysis in Colletotrichum isolates from Conyza leaves allowed the detection of species complexes previously not identified in Argentina.

A rare case of Colletotrichum truncatum keratitis in a young boy with complete healing after medical treatment.

Colletotrichum is a rare fungal pathogen, which is known to cause anthracnose in plants and keratitis or subcutaneous infections in humans. Among the seven Colletotrichum species reported in eye infections, truncatum species is usually virulent with poor visual prognosis even after surgical treatment. Here we report a case of Colletotrichum truncatum keratitis in a young boy with thorn injury that completely resolved with topical natamycin and voriconazole.

Effectiveness of Cymbopogon citratus Oil Encapsulated in Chitosan on Colletotrichum gloeosporioides Isolated from Capsicum annuum.

One of the principal etiological agents associated with losses in horticultural crops is the fungus Colletotrichum sp. This study aimed to evaluate the in vitro effectiveness of the essential oil (EO) from Cymbopogon citratus in chitosan supports for the control of Colletotrichum gloeosporioides isolated from sweet pepper plants. METHODS: The extraction and phytochemical analysis of the EO of C. citratus were performed along with its encapsulation in chitosan-agar in order to compare it with other techniques and determine its effect on C. gloeosporioides. RESULTS: The EO from the citral chemotype (58%) encapsulated in the chitosan-agar, with an 83% encapsulation efficiency in mass percentage, resulted in the total inhibition of mycelial growth at a minimum inhibitory concentration of 1370 ppm. This concentration was effective in controlling the disease under greenhouse conditions. The effectivity of the capsules containing EO was superior to that of other controls using EO evaluated in vitro. The capsules demonstrated an effective period of 51 days, with an additional 30 days of effectiveness after a reinfection cycle, thus providing similar results to the control with Trichoderma sp. CONCLUSIONS: Chitosan capsules present a promising strategy in the use of C. citratus EO on C. gloeosporioides, and they are highly effective and stable under in vitro and field conditions.

Detection of quiescent fungi in harvested fruit using CMOS biosensor: A proof of concept study.

Postharvest fruit decay is caused by fungal pathogens and leads to major losses. In this study, specific mRNA sequences that are upregulated in the fungus Colletotrichum gloeosporioides during its quiescent stage in fruits, were identified using a CMOS sensor. The identification process was based on sandwich approach, where strands complementary to the C. gloeosporioides mRNA sequences (quiescent stage-specific) were immobilized on the CMOS surface, and exposed to the target complementary reporter strands. In the presence of a target sequence, the reporter strand (linked to the enzyme horseradish peroxidase (HRP)) was left in the system and a measurable light signal was produced. The complementary strands specifically anneal to the mRNA in the sample. The sensitivity of the technology was assessed by mRNA sequences isolated from C. gloeosporioides, and identified as 10 nM RNA. The effect of the pathogenicity state on the sensor performance was also evaluated. The CMOS sensor could detect quiescent fungi, which are barely detectable by other means. The unique capability of the proposed system to detect and recognize the fungus during both pathogenic and quiescent stages, will allow the development of new sensors that can monitor the amount of undetectable quiescent fungi in harvested fruit, enabling improved food management.

An integrated approach to improve plant protection against olive anthracnose caused by the Colletotrichum acutatum species complex.

The olive tree (Olea europaea L.) is the most important oil-producing crop of the Mediterranean basin. However, although plant protection measures are regularly applied, disease outbreaks represent an obstacle towards the further development of the sector. Therefore, there is an urge for the improvement of plant protection strategies based on information acquired by the implementation of advanced methodologies. Recently, heavy fungal infections of olive fruits have been recorded in major olive-producing areas of Greece causing devastating yield losses. Thus, initially, we have undertaken the task to identify their causal agent(s) and assess their pathogenicity and sensitivity to fungicides. The disease was identified as the olive anthracnose, and although Colletotrichum gloeosporioides and Colletotrichum acutatum species complexes are the two major causes, the obtained results confirmed that in Southern Greece the latter is the main causal agent. The obtained isolates were grouped into eight morphotypes based on their phenotypes, which differ in their sensitivities to fungicides and pathogenicity. The triazoles difenoconazole and tebuconazole were more toxic than the strobilurins being tested. Furthermore, a GC/EI/MS metabolomics model was developed for the robust chemotaxonomy of the isolates and the dissection of differences between their endo-metabolomes, which could explain the obtained phenotypes. The corresponding metabolites-biomarkers for the discrimination between morphotypes were discovered, with the most important ones being the amino acids L-tyrosine, L-phenylalanine, and L-proline, the disaccharide α,α-trehalose, and the phytotoxic pathogenesis-related metabolite hydroxyphenylacetate. These metabolites play important roles in fungal metabolism, pathogenesis, and stress responses. The study adds critical information that could be further exploited to combat olive anthracnose through its monitoring and the design of improved, customized plant protection strategies. Also, results suggest the necessity for the comprehensive mapping of the C. acutatum species complex morphotypes in order to avoid issues such as the development of fungicide-resistant genotypes.