Amorphophallus konjac anthracnose caused by Colletotrichum siamense in China.

AIMS: Amorphophallus konjac is an important commercial crop grown in China because it is the only plant species which is rich in glucomannan concentration. Recently, an outbreak of anthracnose (incidence ranging from 10-15%) was observed in a field survey conducted from June to August 2018. This study aims to identify the causal agent of A. konjac anthracnose. METHODS AND RESULTS: The pathogen was isolated on potato dextrose agar (PDA) medium. The fungal colony on PDA was greyish to dark grey. Conidia were falcate, one-celled and hyaline. Based on the micro-morphological and cultural characteristics, the pathogen was identified as Colletotrichum sp. blast search and phylogenetic analysis of the ITS, GAPDH, CHS1, ACT, CAL and TUB2 genes revealed the pathogen as Colletotrichum siamense. Koch's postulates were conducted on 2-month konjac leaves with conidial suspension. Development of typical anthracnose disease was recorded 5 days after inoculation and the pathogen's identity was confirmed by re-isolation and molecular identification. CONCLUSIONS: Amorphophallus konjac anthracnose was caused by C. siamense in China. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of causal agent of A. konjac anthracnose will be helpful in designing effective disease control strategies.

Plant pathogenic fungi Colletotrichum and Magnaporthe share a common G1 phase monitoring strategy for proper appressorium development.

To breach the plant cuticle, many plant pathogenic fungi differentiate specialized infection structures (appressoria). In Colletotrichum orbiculare (cucumber anthracnose fungus), this differentiation requires unique proper G1 /S phase progression, regulated by two-component GTPase activating protein CoBub2/CoBfa1 and GTPase CoTem1. Since their homologues regulate mitotic exit, cytokinesis, or septum formation from yeasts to mammals, we asked whether the BUB2 function in G1 /S progression is specific to plant pathogenic fungi. Colletotrichum higginsianum and Magnaporthe oryzae were genetically analyzed to investigate conservation of BUB2 roles in cell cycle regulation, septum formation, and virulence. Expression profile of cobub2Δ was analyzed using a custom microarray. In bub2 mutants of both fungi, S phase initiation was earlier, and septum formation coordinated with a septation initiation network protein and contractile actin ring was impaired. Earlier G1 /S transition in cobub2Δ results in especially high expression of DNA replication genes and differing regulation of virulence-associated genes that encode proteins such as carbohydrate-active enzymes and small secreted proteins. The virulence of chbub2Δ and mobub2Δ was significantly reduced. Our evidence shows that BUB2 regulation of G1 /S transition and septum formation supports its specific requirement for appressorium development in plant pathogenic fungi.

Identification and Characterization of Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Cut branches of deciduous holly (Ilex spp. L.) harboring colorful berries are traditionally used as ornaments in holiday decorations. Since 2012, a fruit rot of unspecified cause has resulted in significant yield reduction and economic losses across Midwestern and Eastern U.S. nurseries. In this study, symptomatic fruit samples collected from nine different locations over five years were analyzed, and several fungal species were isolated. A combination of morphological characterization, multilocus phylogenetic analyses, and pathogenicity assays revealed that Alternaria alternata and Diaporthe ilicicola sp. nov. were the primary pathogens associated with symptomatic fruit. Other fungi including A. arborescens, Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum, and species in the D. eres species complex appeared to be minor pathogens in this disease complex. In detached fruit pathogenicity assays testing the role of wounding and inoculum concentration on disease development, disease incidence and severity increased when fruit was wounded and inoculated with a higher inoculum concentration. These findings indicate that management strategies that can protect fruit from injury or reduce inoculum may lower disease levels in the field. This research established the basis for further studies on this emerging disease and the design of research-based management strategies. To our knowledge, it also represents the first report of species of Alternaria, Colletotrichum, Diaporthe, and Epicoccum causing fruit rot of deciduous holly.

ABC protein CgABCF2 is required for asexual and sexual development, appressorial formation and plant infection in Colletotrichum gloeosporioides.

ATP-binding cassette (ABC) proteins are exclusively found in both prokaryotes and eukaryotes. In this study, we have characterized a gene from Glomerella leaf spot pathogen Colletotrichum gloeosporioides that encodes an ABC protein, whose function to date remains unknown. We designated this gene as CgABCF2. Deletion of CgABCF2 showed drastic reduction both growing rate and conidial production in C. gloeosporioides. The Δcgabcf2 mutant did not form the appressoria, lost the capability to infect apple and failed to form lesions on the wounded leaves and fruits. The C. gloeosporioides native CgABCF2 fully recovered defect of the Δcgabcf2 mutant. These data indicated that CgABCF2 was required for fungal development and invasion. The transcriptions of six pectolytic enzymes genes (CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB) significantly reduced in the Δcgabcf2 mutant, indicating that deletion of CgABCF2 impaired the fungal necrotrophic growth. In addition, CgABCF2 mediated sexual development through the positive regulation of the gene MAT1-2-1 expression. These results indicated that CgABCF2 underlies the complex process governing morphogenesis, sexual and asexual reproduction, appressorial formation and pathogenicity in C. gloeosporioides.

Phylogeny and variability of Colletotrichum truncatum associated with soybean anthracnose in Brazil.

AIMS: Fungal diseases are among the main factors limiting high yields of soybean crop. Colletotrichum isolates from soybean plants with anthracnose symptoms were studied from different regions and time periods in Brazil using molecular, morphological and pathogenic analyses. METHODS AND RESULTS: Bayesian phylogenetic inference of GAPDH, HIS3 and ITS-5.8S rDNA sequences, the morphologies of colony and conidia, and inoculation tests on seeds and seedlings were performed. All isolates clustered only with Colletotrichum truncatum species in three well-separated clusters. Intraspecific genetic diversity revealed 27 distinct haplotypes in 51 fungal isolates; some of which were identical to C. truncatum sequences from other regions around the world, while others were related to alternative hosts. Conidia were falcate, hyaline, unicellular and aseptate, formed in acervuli, with variable dimensions. Despite being pathogenic to seedlings by both inoculation methods, variation was observed in the aggressiveness of the tested isolates, which was not correlated with genetic variation. CONCLUSION: The identification of C. truncatum in the sampled isolates was evidenced as being the only causal agent of soybean anthracnose in Brazil until 2007, with relevant genetic, morphological and pathogenic variability as well as a broad geographical origin. The wide distribution of the predominant C. truncatum haplotype indicated the existence of a highly efficient mechanism of pathogen dispersal over long distances, reinforcing the role of seeds as the primary source of disease inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and distribution of Colletotrichum species in soybean-producing regions in Brazil is fundamental for understanding the disease epidemiology and for ensuring effective control strategies against anthracnose.

Morphological, Pathogenic, and Molecular Characterization of Colletotrichum acutatum Isolates Causing Almond Anthracnose in Spain.

Almond anthracnose is a serious and emerging disease in several countries. All isolates causing almond anthracnose have been assigned to the Colletotrichum acutatum species complex, of which only C. fioriniae and C. godetiae have been associated with the disease to date. Here, we characterized Colletotrichum isolates from almond fruit affected by anthracnose in the Andalusia region. Two Colletotrichum isolates causing olive anthracnose were included for comparison. Morphological characteristics were useful for separating the isolates into groups based on colony morphology. Pathogenicity tests in almond, olive, and apple fruit showed differences in virulence and some degree of pathogenic specialization among isolates. Molecular characterization allowed clear identification of the Colletotrichum isolates tested. The olive isolates were identified as C. godetiae and C. nymphaeae, both previously identified in Andalusian olive orchards. Two phylogenetic species were identified among the almond isolates: C. godetiae, with gray colonies, which is well known in other countries, and C. acutatum, with pink-orange colonies. This species identification differs from those of pink-colony subpopulations described in other countries, which are C. fioriniae. Therefore, this study is also the first report of a new species of Colletotrichum causing almond anthracnose within the C. acutatum species complex.

Species boundaries in plant pathogenic fungi: a Colletotrichum case study.

BACKGROUND: Accurate delimitation of plant pathogenic fungi is critical for the establishment of quarantine regulations, screening for genetic resistance to plant pathogens, and the study of ecosystem function. Concatenation analysis of multi-locus DNA sequence data represents a powerful and commonly used approach to recognizing evolutionary independent lineages in fungi. It is however possible to mask the discordance between individual gene trees, thus the speciation events might be erroneously estimated if one simply recognizes well supported clades as distinct species without implementing a careful examination of species boundary. To investigate this phenomenon, we studied Colletotrichum siamense s. lat., which is a cosmopolitan pathogen causing serious diseases on many economically important plant hosts. Presently there are significant disagreements among mycologists as to what constitutes a species in C. siamense s. lat., with the number of accepted species ranging from one to seven. RESULTS: In this study, multiple approaches were used to test the null hypothesis "C. siamense is a species complex", using a global strain collection. Results of molecular analyses based on the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) and coalescent methods (e.g. Generalized Mixed Yule-coalescent and Poisson Tree Processes) do not support the recognition of any independent evolutionary lineages within C. siamense s. lat. as distinct species, thus rejecting the null hypothesis. This conclusion is reinforced by the recognition of genetic recombination, cross fertility, and the comparison of ecological and morphological characters. Our results indicate that reproductive isolation, geographic and host plant barriers to gene flow are absent in C. siamense s. lat. CONCLUSIONS: This discovery emphasized the importance of a polyphasic approach when describing novel species in morphologically conserved genera of plant pathogenic fungi.

Bypassing both surface attachment and surface recognition requirements for appressorium formation by overactive ras signaling in Magnaporthe oryzae.

Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2(G18V) allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2(G18V) transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2(G18V) had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens.

A modular plasmid system for protein co-localization and bimolecular fluorescence complementation in filamentous fungi.

To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.

Molecular, phenotypic and pathogenic variability in Colletotrichum isolates of subtropical region in north-eastern India, causing fruit rot of chillies.

AIM: To study the genetic variability and population structure of Colletotrichum species found to be associated with anthracnose of chilli in the north-eastern region of India. METHODS AND RESULTS: Twenty-three Colletotrichum isolates were obtained from infected chilli fruits and leaves from the chilli growing regions of north-eastern Uttar Pradesh, India, identified as Colletotrichum capsici and Colletotrichum acutatum using species-specific primers. Genetic variability among the isolates was analysed using RAPD and ISSR markers. The RAPD marker efficiently grouped the isolates at species level, while ISSR marker was effective in separating the isolates based on geographical origin. In vitro pathogenic test revealed the inability of C. acutatum isolates to infect unripe fruits, while C. capsici isolates were found to infect both ripe and unripe fruits at disease severity scale 7-9. Growth rate on different media was recorded to cross-confirm the classification of isolates, which clearly grouped the two species into distinct group on PCA plot. CONCLUSIONS: Two species, viz. C. capsici and C. acutatum, prevalent in the region were found to infect the fruits at postripening stage. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study proposes the implementation of management strategies at postripening stages of the crop to control the spread of disease, thereby leading to increment in production of chilli in the given region.

Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes.

AIM: To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. METHODS AND RESULTS: We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. CONCLUSION: This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process.

Colletotrichum lindemuthianum exhibits different patterns of nuclear division at different stages in its vegetative life cycle.

Live-cell imaging with fluorescent protein labeling is providing major new insights into nuclear dynamics in filamentous fungi. With this approach we provide a detailed report of nuclear organization and behavior during mitosis in the bean pathogen Colletotrichum lindemuthianum. Nuclear division and nuclear migration were analyzed in ungerminated conidia, conidial germlings and the mature colony. Ungerminated conidia were uninucleate and completion of mitosis was found not to be essential for germ tube formation, conidial anastomosis tube (CAT) formation or fusion. Nuclei in fused conidial germlings exhibited asynchronous mitoses, and nuclear migration through fused CATs occurred after the nuclei had divided. Different patterns of nuclear division were found in vegetative hyphae of the mature colony. Synchronous, parasynchronous and asynchronous patterns of mitosis were observed in apical hyphal compartments at the colony border, while only synchronous and asynchronous mitoses occurred in subapical hyphal compartments. These findings have revealed unexpected diversity in the patterns of mitosis in different cells of C. lindemuthianum.

Colletotrichum truncatum: an unusual pathogen causing mycotic keratitis and endophthalmitis.

In recent years, the well-known plant pathogens of the Colletotrichum genus were increasingly reported to cause ophthalmic infections in humans. Among 66 species in the Colletotrichum genus, only a few are known to be pathogenic for humans. We report here five cases of ophthalmic infections due to Colletotrichum truncatum, a species never reported earlier to cause human infection. The isolates were identified by morphological characteristics and the sequencing of internal spacer regions of ribosomal DNA. The progress of lesions in those patients was slow compared to that of lesions caused by Aspergillus or Fusarium infections. The surgical management included total penetrating keratoplasty in patients with keratitis and pars plana vitrectomy in endophthalmitis. Two patients were treated additionally with intravitreal amphotericin B deoxycholate, one patient with oral itraconazole, and another patient with oral and topical fluconazole therapy. The present series therefore highlights the expanding spectrum of agents causing eye infections and the inclusion of C. truncatum as a human pathogen.

Collacyclumines A-D from the endophytic fungus Colletotrichum salsolae SCSIO 41021 isolated from the mangrove Kandelia candel.

Four undescribed alkaloids, namely collacyclumines A-D, along with a known analogue, agrocybenine, were isolated from the endophytic fungus Colletotrichum salsolae SCSIO 41021 derived from the mangrove plant Kandelia candel (L.) Druce. Collacyclumine A represents the first case of dimeric pyrrolidine alkaloid in nature. The structures of these compounds were elucidated by a combination of NMR spectra, HRESIMS data, and X-ray diffraction experiment. A proposed biosynthetic pathway of these isolated compounds were also discussed. None of compounds showed cytotoxic effects against ten cell lines.

Discovery of pathogenicity genes in the crucifer anthracnose fungus Colletotrichum higginsianum, using random insertional mutagenesis.

Agrobacterium tumefaciens-mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-beta2, ornithine decarboxylase, beta-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.

Morphological, genetic, and pathogenic characterization of Colletotrichum acutatum, the cause of anthracnose of almond in Australia.

Almond anthracnose was reported for the first time in Australia in 1998 and has since been observed in all of the major almond-growing regions. The organism causing anthracnose was confirmed as Colletotrichum acutatum using taxon-specific polymerase chain reaction (PCR). Three main morphotypes of C. acutatum from almond in Australia were identified (namely, pink, orange, and cream colony color) and the optimum temperature for mycelial growth of representative isolates was 25 degrees C. Australian isolates of C. acutatum were more similar morphologically to the pink subpopulation of C. acutatum from California than to the gray Californian subpopulation and the isolates of Colletotrichum from Israel. Inter-simple-sequence-repeat (ISSR) PCR analysis revealed that the majority of Australian isolates shared an identical banding pattern whereas Australian isolates of C. acutatum from almond were distinct from isolates of the pink and gray subpopulations of C. acutatum from almond in California and of Colletotrichum spp. from almond in Israel. Sequence analysis of the internally transcribed spacer (ITS1-2) ribosomal DNA region of representative isolates differed from the results of ISSR-PCR in that polymorphisms were revealed among isolates, indicating that some genetic variation may be present. Pathogenicity experiments on detached leaves and fruit revealed pathogenic variation among representative isolates of C. acutatum from almond in Australia, California, and Israel; however, all isolates tested caused disease. Distinct subgroups among Australian isolates of C. acutatum from almond were not supported on the basis of morphology, mycelial growth rates, ISSR-PCR, and pathogenicity.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species.

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia ("espinheira-santa") in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Col1 and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides.

BACKGROUND: In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria. RESULTS: We found that in the closely related plant pathogen Colletotrichum gloeosporioides, blocking of the cell cycle did not prevent spore germination and appressoria formation. The cell cycle always lagged behind the morphogenetic changes that follow spore germination, including germ tube and appressorium formation, differentiation of the penetrating hypha, and in planta formation of primary hyphae. Nuclear division was arrested following appressorium formation and was resumed in mature appressoria after plant penetration. Unlike in M. grisea, blocking of mitosis had only a marginal effect on appressoria formation; development in hydroxyurea-treated spores continued only for a limited number of cell divisions, but normal numbers of fully developed mature appressoria were formed under conditions that support appressoria formation. Similar results were also observed in other Colletotrichum species. Spores, germ tubes, and appressoria retained intact nuclei and remained viable for several days post plant infection. CONCLUSION: We showed that in C. gloeosporioides the differentiation of infection structures including appressoria precedes mitosis and can occur without nuclear division. This phenomenon was also found to be common in other Colletotrichum species. Spore cell death did not occur during plant infection and the fungus primary infection structures remained viable throughout the infection cycle. Our results show that the control of basic cellular processes such as those coupling cell cycle and morphogenesis during fungal infection can be substantially different between fungal species with similar lifestyles and pathogenic strategies.

Development of Colletotrichum acutatum on tolerant and susceptible Olea europaea L. cultivars: a microscopic analysis.

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192 h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.

Microscopic analysis of colonization of Colletotrichum abscissum in citrus tissues.

Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.