Characterization and Pathogenicity of Colletotrichum truncatum Causing Stem Anthracnose of Red‐Fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During August 2010 and January 2011, 10 isolates of Colletotrichum were recovered from stem anthracnose lesions of Hylocereus polyrhizus in the states of Kedah and Penang, Malaysia. Based on the morphological characteristics of colony colour and appearance, and shapes of conidia as well as sequences of internal transcribed spacer regions (ITS), β‐tubulin, actin (ACT) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), the fungus was identified as Colletotrichum truncatum. Pathogenicity test showed that C. truncatum isolates were pathogenic to the artificially inoculated H. polyrhizus stem. This is the first report of C. truncatum causing anthracnose on H. polyrhizus stems in Malaysia.     

Identification and characterization of Colletotrichum species associated with anthracnose on persimmon in Brazil

Persimmon (Diospyros kaki) anthracnose is a major threat in production areas worldwide. Most of the studies are focused on Colletotrichum horii, but other species have been reported as well. The association of distinct Colletotrichum species present in Brazilian persimmon production regions as well as their host ranges are yet elusive. The aims of this work were to identify and characterize Colletotrichum species associated with the persimmon anthracnose. In a survey performed in four production regions of Brazil, 88.7% and 11.3% out of 231 isolates were identified as members of Colletotrichum gloeosporioides species complex (Cgc) or Colletotrichum acutatum species complex (Cac), respectively. A subset of 18 isolates were identified through multilocus phylogenetic analysis, using ITS-rDNA region and two loci, namely GAPDH and TUB2. This study revealed the presence of four species: C. horii (38.8%) and Colletotrichum fructicola (27.7%) from the Cgc and Colletotrichum nymphaeae (27.7%) and Colletotrichum melonis (5.8%), from the Cac. Additionally, 13 isolates were selected for morphological, physiological, and pathogenic analyses. Contrasting characteristics were observed among species of the Cgc and Cac complexes. The optimal temperature for mycelial growth and germination were higher for Cgc species. The percentage of appressoria melanisation also varied across complexes. All the identified species were able to cause anthracnose-like symptoms on persimmon fruit, leaves, shoots, and sepals. Colletotrichum species from persimmon were also able to infect apple and pear. The findings will support decisions to manage anthracnose of persimmon under high infection risk due to multiple host susceptibility.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Genetic Diversity and Pathogenic Variability of Colletotrichum truncatum Causing Anthracnose of Pepper in Malaysia

Colletotrichum truncatum was initially described from pepper and has been reported to infect 180 host genera in 55 plant families worldwide. Samples were collected from pepper plants showing typical anthracnose symptoms. Diseased samples after isolation were identified as C. truncatum based on morphological characters and ITS‐rDNA and β‐tubulin sequence data. Intersimple sequence repeat (ISSR) markers were used to estimate genetic diversity in C. truncatum from Malaysia. A set of 3 ISSR primers revealed a total 26 allele from the amplified products. Cluster analysis with UPGMA method clustered C. truncatum isolates into two main groups, which differed with a distance of 0.64. However, the genetic diversity of C. truncatum isolates showed correlation between genetic and geographical distribution, but it failed to reveal a relationship between clustering and pathogenic variability. Phylogenetic analyses discriminated the C. truncatum isolates from other reference Colletotrichum species derived from GenBank. Among the morphological characters, shape, colour of colony and growth rate in culture were partially correlated with the ISSR and phylogenetic grouping. Pathogenicity tests revealed that C. truncatum isolates were causal agents for pepper anthracnose. In the cross‐inoculation assays, C. truncatum isolates were able to produce anthracnose symptoms on tomato, eggplant, onion, lettuce and cabbage. A pathogenicity and cross‐inoculation studies indicated the potential of C. truncatum for virulence and dominancy on plant resistance.     

Genetic differentiation of Colletotrichum gloeosporioides and C. truncatum associated with Anthracnose disease of papaya (Carica papaya L.) and bell pepper (Capsium annuum L.) based on ITS PCR-RFLP fingerprinting

Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1–5.8S–ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment length polymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1–5.8S–ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.     

Why species delimitation matters for fungal ecology: Colletotrichum diversity on wild and cultivated cashew in Brazil

Anthracnose is one of the most important plant diseases globally, occurring on a wide range of cultivated and wild host species. This study aimed to identify the Colletotrichum species associated with cashew anthracnose in Brazil, determine their phylogenetic relationships and geographical distribution, and provide some insight into the factors that may be influencing community composition. Colletotrichum isolates collected from symptomatic leaves, stems, inflorescences, and fruit of cultivated and wild cashew, across four Brazilian biomes, were identified as Colletotrichum chrysophilum, Colletotrichum fragariae, Colletotrichum fructicola, Colletotrichum gloeosporioides sensu stricto, Colletotrichum queenslandicum, Colletotrichum siamense and Colletotrichum tropicale. Colletotrichum siamense was the most dominant species. The greatest species richness was associated with cultivated cashew; leaves harbored more species than the other organs; the Atlantic Forest encompassed more species than the other biomes; and Pernambuco was the most species-rich location. However, accounting for the relative abundance of Colletotrichum species and differences in sample size across strata, the interpretation of which community is most diverse depends on how species are delimited. The present study provides valuable information about the Colletotrichum/cashew pathosystem, sheds light on the causal agents identification,and highlights the impact that species delimitation can have on ecological studies of fungi.     

Colletotrichum species associated with sugarcane red rot in Brazil

Sugarcane is a widely cultivated crop in Brazil and in many parts of the world. However, the red rot causes huge losses due to the reduction of sucrose and deterioration of the juice. The aim of this study was to identify Colletotrichum species associated with the red rot through polyphasic approaches; which included phylogenetic, morpho-cultural analyzes and pathogenicity tests. Nine isolates from the states of Alagoas and two from São Paulo, Brazil, were preliminary analyzed with the glyceraldehyde-3 phosphate dehydrogenase gene (GAPDH), as an initial measure for species diversity. Later on, the representative isolates of each species were sequenced with the β-tubulin (TUB2) gene, calmodulin (CAL), DNA lyase (APN2/MAT IGS) and the ITS-rDNA region. Morphocultural characterization was performed by evaluating the mycelial growth rate (MGR), colony appearance and the shape and size of 50 conidia and appressoria. For the pathogenicity test asymptomatic leaves and stalks of sugarcane were tested with and without injuries. Phylogenetic analysis associated with morphocultural characteristics and the pathogenicity test of the eleven isolates revealed three Colletotrichum species: Colletotrichum falcatum (8 isolates), Colletotrichum siamense (1 isolate) and Colletotrichum plurivorum (2 isolates) causing the red rot disease in sugar cane. All species were pathogenic in wounded leaves and stalks, being C. falcatum the one causing the largest lesions (1.12 cm) in leaves and C. plurivorum in stalks (0.67 cm). Therefore, this study confirms the association of C. falcatum as a sugarcane pathogen and records for the first time worldwide the occurrence of C. siamense and C. plurivorum associated with this host.     

Genetic variation of <Emphasis Type="Italic">Colletotrichum magnum</Emphasis> isolated from <Emphasis Type="Italic">Carica papaya</Emphasis> as revealed by DNA fingerprinting

Mexico is one of the five largest producers of papaya worldwide, but losses caused by pathogens, mainly fungus, at the pre- and post-harvest stages are often more than 50% of the crop. Papaya anthracnose, caused by three different species of the Colletotrichum genus in Mexico, occupies a preponderant place in this problem. Although two of these species, C. gloeosporiodes and C. truncatum, have been characterized morphologically and genotypically, this has not occurred with C. magnum, the third species involved, about which there is very little information. Because of this, it is vital to know its genetic characterization, much more so considering that the studies carried out on the other two species reveal a wide genetic diversity, differences in pathogenicity and in the response to fungicides of the different strains characterized. In this work, Colletotrichum spp. isolates were collected at different papaya orchards in the south-southeast of Mexico. C. magnum isolates identified by species-specific primers were characterized by morphological and molecular approaches. Differences in colony characteristics resulted in five morphological groups. AP-PCR, DAMD and ISSR markers were found to be very efficient for revealing the interspecific variability of this species. The high genetic variability found in the accessions of C. magnum was linked to the geographical area where they were collected. Isolates from Chiapas State were the most variable, showing point mutations in the ITS1-ITS2 region. These results will enable a better phytosanitary management of anthracnose in papaya in this region of Mexico.     

Utility of internally transcribed spacer region of rDNA (ITS) and β‐tubulin gene sequences to infer genetic diversity and migration patterns of Colletotrichum truncatum infecting Capsicum spp.

Anthracnose is among the most economically important diseases affecting pepper (Capsicum spp.) production in the tropics and subtropics. Of the three species of Colletotrichum implicated as causal agents of pepper anthracnose, C. truncatum is considered to be the most destructive in agro‐ecosystems worldwide. However, the genetic variation and the migration potential of C. truncatum infecting pepper are not known. Five populations were selected for study and a two‐locus (internally transcribed spacer region, ITS1‐5.8S‐ITS2, and β‐tubulin, β‐TUB) sequence data set was generated and used in the analyses. Sequences of the ITS region were less informative than β ‐ tubulin gene sequences based on comparisons of DNA polymorphism indices. Trinidad had the highest genetic diversity and also had the largest effective population size in pairwise comparisons with the other populations. The Trinidad population also demonstrated significant genetic differentiation from the other populations. AMOVA and STRUCTURE analyses both suggested significant genetic variation within populations more so than among populations. A consensus Maximum Likelihood tree based on β‐TUB gene sequences revealed very little intraspecific diversity for all isolates except for Trinidad. Two clades consisting solely of Trinidad isolates may have diverged earlier than the other isolates. There was also evidence of directional migration among the five populations. These findings may have a direct impact on the development of integrated disease management strategies to control C. truncatum infection in pepper.     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

Molecular and Phenotypic Analyses Reveal Association of Diverse Colletotrichum acutatum Groups and a Low Level of C. gloeosporioides with Olive Anthracnose

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and β-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from β-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.     

Pectinolytic enzyme production by Colletotrichum truncatum,causal agent of soybean anthracnose

BackgroundColletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi.ObjectivesTo explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.–soybean system, yet unknown.MethodsTen strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source.ResultsAll isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08 U/ml PG, 1.05 U/ml PMG, 156 U/ml PL), after a short incubation period (7–10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected.ConclusionsThe disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated.     

Differential Organ Distribution,Pathogenicity and Benomyl Sensitivity of Colletotrichum spp. from Blackberry Plants in Northern Colombia

Blackberry anthracnose, caused by Colletotrichum spp., is an important disease of cultivated blackberry in the world. In Colombia, it is the number one limiting factor for commercial production. This study was conducted to determine the species of Colletotrichum infecting blackberry plants as well as the organ distribution, pathogenicity and response to benomyl of the isolated strains. Sixty isolates from stems (n = 20), thorns (n = 20) and inflorescences (n = 20) were identified as Colletotrichum acutatum and Colletotrichum gloeosporioides by a species‐specific polymerase chain reaction (PCR). Both Colletotrichum species were found in the same plant but on different organs. Colletotrichum gloeosporioides species predominated in thorn lesions (n = 16) and C. acutatum in stems (n = 15) and inflorescence (n = 15). Pathogenicity assays on detached blackberry organs demonstrated differences between the two species with an average period of lesion development of 8.7 days for C. gloeosporioides and 10.3 days for C. acutatum. Wound inoculated organs had 90% disease development compared to 17.5% in non‐wounded. All C. acutatum isolates (n = 34) were benomyl tolerant, whereas C. gloeosporioides isolates (n = 26) were 30.7% sensitive and 69.2% moderately tolerant. Phylogenetic analysis with ITS sequences of a subset of 18 strains showed that strains classified as C. gloeosporioides had 100% identity to Colletotrichum kahawae, which belongs to the C. gloeosporioides species complex, whereas C. acutatum strains clustered into two different groups, with high similarity to the A2 and the A4 molecular groups. These data demonstrate for the first time the differential distribution of both species complexes in blackberry plant organs and further clarifies the taxonomy of the strains.     

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.TaxonomyWhile 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 symptomsTypical 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.ChallengesAs 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 websiteUpdates 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/.     

Diversity,structure, and synteny of the cutinase gene of Colletotrichum species

Colletotrichum species complexes are among the top 10 economically important fungal plant pathogens worldwide because they can infect climacteric and nonclimacteric fruit at the pre and/or postharvest stages. C. truncatum is the major pathogen responsible for anthracnose of green and red bell pepper fruit worldwide. C. brevisporum was recently reported to be a minor pathogen of red bell pepper fruit in Trinidad, but has recently been reported as pathogenic to other host species in other countries. The ability of these phytopathogens to produce and secrete cutinase is required for dismantling the cuticle of the host plant and, therefore, crucial to the necrotrophic phase of their infection strategy. In vitro bioassays using different lipid substrates confirmed the ability of C. truncatum and C. brevisporum isolates from green and red bell peppers to secrete cutinase. The diversity, structure and organization and synteny of the cutinase gene were determined among different Colletotrichum species. Cluster analysis indicated a low level of nucleotide variation among C. truncatum sequences. Nucleotide sequences of C. brevisporum were more related to C. truncatum cutinase nucleotide sequences than to C. gloeosporioides. Cluster patterns coincided with haplotype and there was evidence of significant positive selection with no recombination signatures. The structure of the cutinase gene included two exons with one intervening intron and, therefore, one splice variant. Although amino acid sequences were highly conserved among C. truncatum isolates, diversity “hot spots” were revealed when the 66‐amino acid coding region of 200 fungal species was compared. Twenty cutinase orthologues were detected among different fungal species, whose common ancestor is Pezizomycotina and it is purported that these orthologues arose through a single gene duplication event prior to speciation. The cutinase domain was retained both in structure and arrangement among 34 different Colletotrichum species. The order of aligned genomic blocks between species and the arrangement of flanking protein domains were also conserved and shared for those domains immediately located at the N‐ and C‐terminus of the cutinase domain. Among these were an RNA recognition motif, translation elongation factor, signal peptide, pentatricopeptide repeat, and Hsp70 family of chaperone proteins, all of which support the expression of the cutinase gene. The findings of this study are important to understanding the evolution of the cutinase gene in C. truncatum as a key component of the biotrophic–necrotrophic switch which may be useful in developing gene‐targeting strategies to decrease the pathogenic potential of Colletotrichum species.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

Infra-specific diversity of Colletotrichum truncatum associated with chilli anthracnose in India based on microsatellite marker analysis

Colletotrichum truncatum is a fungal species associated with anthracnose disease in many economically important crops within the plant families Fabaceae and Solanaceae. Understanding the degree of genetic diversity within C. truncatum population will provide insights into the ability of this species to evolve in response to environmental conditions, and thus be helpful in designing effective control strategies for this pathogen. In this study, microsatellite markers from 27 loci were used to investigate the genetic diversity and population structure among 99 isolates of C. truncatum from India. All the loci (100%) were polymorphic and a total of 140 different alleles were amplified. Six distinct groups were obtained based on unweighted pair group method with arithmetical average cluster analysis. The isolates belonging to Group V showed the highest level of genetic diversity and a broad host range. Analysis of molecular variance analysis showed that the variation occurs mostly within groups. Microsatellite markers-based genetic diversity estimation revealed high diversity among C. truncatum isolates from India.     

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.     

Genetic diversity of Colletotrichum gloeosporioides species complex associated with Citrus wither‐tip of twigs in Tunisia using microsatellite markers

Anthracnose Citrus disease has been associated with several symptoms worldwide and it is recently compromising Citrus production in the Mediterranean area. Four species complexes are mainly involved: Colletotrichum boninense, C. acutatum, C. gloeosporioides and C. truncatum. In this study, we investigated the genetic diversity of Colletotrichum spp. in Tunisia associated with wither‐tip of twigs on Citrus. Specific primers ITS4‐CgInt allowed the identification of C. gloeosporioides species complex in all the 54 isolates, sampled from three regions and four Citrus species. Overall, our genotypic analysis using 10 SSR markers showed a moderate diversity level in Tunisian C. gloeosporioides population and highlighted that C. gloeosporioides reproduce mainly clonally. In addition, heterothallic isolates were present in our population, suggesting that the pathogen population may undergo parasexual recombinations. The highest genetic diversity in C. gloeosporioides was recorded in Nabeul and on orange, which likely constitutes the area and the host of origin for the Citrus anthracnose disease in Tunisia. In addition, no population subdivision was detected at the geographic, host species or cultivars’ origin levels. However, our study identified two genetic subpopulations and indicated a rapid C. gloeosporioides population change at temporal scale that should be further examined over several consecutive growing seasons in order to understand its population dynamics.