Antifungal effects of Conyza bonariensis (L.) Cronquist essential oil against pathogenic Colletotrichum musae and its incorporation in gum Arabic coating to reduce anthracnose development in banana during storage.

AIM: This study evaluated the inhibitory effects on mycelial growth and damage on membrane integrity and enzymatic activity caused by Conyza bonariensis essential oil (CBEO) on distinct pathogenic Colletotrichum musae isolates, as well as the preventive and curative effects of coatings with gum Arabic (GA) and CBEO to reduce anthracnose development in banana during room temperature storage. The effects of GA-CBEO coatings on some physicochemical parameters of banana were investigated during room temperature storage. METHOD AND RESULTS: CBEO (0.4-1 µl ml-1 ) inhibited the mycelial growth of C. musae isolates in laboratory media. The exposure of C. musae conidia to CBEO (0.6 µl ml-1 ) for 3 and 5 days resulted in high percentages of conidia with damaged cytoplasmic membrane and without enzymatic activity. Coatings with GA (0.1 mg ml-1 ) and CBEO (0.4-1 µl ml-1 ) reduced the anthracnose development in banana artificially contaminated with C. musae during storage. In most cases, the disease severity indexes found for GA-CBEO-coated banana were lower than or similar to those for banana treated with commercial fungicide. GA-CBEO-coated banana had reduced alterations in physicochemical parameters during storage, indicating more prolonged storability. CONCLUSION: The application of GA-CBEO coatings is effective to delay the anthracnose development in banana during storage, which should help to reduce the amount of fungicides used to control postharvest diseases in this fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing the efficacy of coatings formulated with GA and CBEO to delay the development of anthracnose in banana, as well as to decrease alterations in physicochemical parameters indicative of postharvest quality of this fruit during storage. In a practical point of view, GA-CBEO coatings could be innovative strategies to delay the anthracnose development and postharvest losses in banana.

Production and partial purification by PEG/citrate ATPS of a ß-galactosidase from the new promising isolate Cladosporium tenuissimum URM 7803.

ß-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and ß-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. ß-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 ß-galactosidase showed a profile suitable for prebiotics production.

Optimal markers for the identification of Colletotrichum species.

Colletotrichum is among the most important genera of fungal plant pathogens. Molecular phylogenetic studies over the last decade have resulted in a much better understanding of the evolutionary relationships and species boundaries within the genus. There are now approximately 200 species accepted, most of which are distributed among 13 species complexes. Given their prominence on agricultural crops around the world, rapid identification of a large collection of Colletotrichum isolates is routinely needed by plant pathologists, regulatory officials, and fungal biologists. However, there is no agreement on the best molecular markers to discriminate species in each species complex. Here we calculate the barcode gap distance and intra/inter-specific distance overlap to evaluate each of the most commonly applied molecular markers for their utility as a barcode for species identification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone-3 (HIS3), DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), and intergenic spacer between GAPDH and a hypothetical protein (GAP2-IGS) have the properties of good barcodes, whereas sequences of actin (ACT), chitin synthase (CHS-1) and nuclear rDNA internal transcribed spacers (nrITS) are not able to distinguish most species. Finally, we assessed the utility of these markers for phylogenetic studies using phylogenetic informativeness profiling, the genealogical sorting index (GSI), and Bayesian concordance analyses (BCA). Although GAPDH, HIS3 and ß-tubulin (TUB2) were frequently among the best markers, there was not a single set of markers that were best for all species complexes. Eliminating markers with low phylogenetic signal tends to decrease uncertainty in the topology, regardless of species complex, and leads to a larger proportion of markers that support each lineage in the Bayesian concordance analyses. Finally, we reconstruct the phylogeny of each species complex using a minimal set of phylogenetic markers with the strongest phylogenetic signal and find the majority of species are strongly supported as monophyletic.

Thiophanate-Methyl Resistance and Fitness Components of Colletotrichum musae Isolates from Banana in Brazil.

Anthracnose, caused by Colletotrichum musae, is the most important postharvest disease of banana and is widely distributed among the banana production regions in Brazil. Although thiophanate-methyl is a fungicide frequently used in Brazilian banana orchards to control Sigatoka leaf spot, Collettotrichum populations are also exposed, resulting in the evolution of fungicide resistance and the inability to manage banana anthracnose. We investigated 139 Brazilian isolates of C. musae for thiophanate-methyl sensitivity in vitro. The 50% mycelial growth inhibition (EC50) values varied between 0.003 and 48.73 µg/ml. One-hundred and thirty isolates were classified as sensitive, with EC50 values ranging from 0.003 to 4.84 µg/ml, while the remaining nine isolates were considered moderately resistant, with EC50 values ranging between 10.43 and 48.73 µg/ml. Resistant or highly resistant isolates (EC50 > 100 µg/ml) were not found. A substitution of TAC for TTC at codon 200 in a coding region of the ß-tubulin gene was associated with the moderately resistant phenotype. Applications of thiophanate-methyl formulation to detached banana fruit at the label rate (500 µg/ml) showed low efficacy in controlling the moderately resistant isolates on banana fruits. However, there is no indication of a reduction in fitness associated with fungicide resistance as sensitive and moderately resistant isolates do not differ with respect to mycelial growth rate (P = 0.098), spore production (P = 0.066), spore germination (P = 0.366), osmotic sensitivity (P = 0.051), and virulence (P = 0.057). Our results revealed absence of adaptability cost for the moderately resistant isolates, suggesting that they can be dominant in population if the fungicide continue to be applied.

Diversity of Colletotrichum species associated with torch ginger anthracnose.

Anthracnose caused by Colletotrichum species is one of the most important diseases of torch ginger. The disease leads to loss of aesthetic and commercial value of torch ginger stems. This study aimed to characterize Colletotrichum species associated with torch ginger anthracnose in the production areas of Pernambuco and Ceará. A total of 48 Colletotrichum isolates were identified using molecular techniques. Pathogenicity tests were performed on torch ginger with representative isolates. Phylogenetic analyses based on seven loci-DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), calmodulin (CAL), intergenic spacer between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a hypothetical protein (GAP2-IGS), glutamine synthetase (GS), and ß-tubulin (TUB2)-revealed that they belong to five known Colletotrichum species, namely, C. chrysophilum, C. fructicola, C. siamense, C. theobromicola, and C. tropicale, and three newly discovered species, described here as C. atlanticum, C. floscerae, and C. zingibericola. Of these, C. atlanticum was the most dominant. Pathogenicity assays showed that all isolates were pathogenic to torch ginger bracts. All species are reported for the first time associated with torch ginger in Brazil. The present study contributes to the current understanding of the diversity of Colletotrichum species associated with anthracnose on torch ginger and demonstrates the importance of accurate species identification for effective disease management strategies.

Elucidating the Colletotrichum spp. diversity responsible for papaya anthracnose in Brazil.

Papaya (Carica papaya L.) is among the most important tropical fruits produced in Brazil and is grown in nearly every state. However, several diseases can affect papaya production. Anthracnose stands out among these diseases due to high postharvest yield losses. Previous studies identified Colletotrichum magna (invalid name) and Colletotrichum gloeosporioides causing anthracnose of papaya in Brazil, but species identification was inadequate due to reliance on nuclear ribosomal internal transcribed space (nrITS) and glutamine synthetase (GS) sequences. Thus, the diversity of Colletotrichum spp. causing papaya anthracnose in Brazil may be underestimated. The present study aims to identify the Colletotrichum species associated with papaya anthracnose in Brazil based on broad geographical sampling and multilocus phylogenetic analysis, as well as to assess the prevalence and aggressiveness of the species found. Here, we report C. chrysophilum, C. fructicola, C. gloeosporioides, C. karsti, C. okinawense, C. plurivorum, C. queenslandicum, C. siamense, C. theobromicola, Colletotrichum truncatum causing papaya anthracnose in Brazil. We are also synonymizing Colletotrichum corchorum-capsularis under C. truncatum. Colletotrichum okinawense was the most prevalent species in general and in most sampled locations, and with C. truncatum represents the most aggressive species.

Control of anthracnose caused by Colletotrichum species in guava, mango and papaya using synergistic combinations of chitosan and Cymbopogon citratus (D.C. ex Nees) Stapf. essential oil.

This study assessed the efficacy of chitosan (Chi) and Cymbopogon citratus (D.C. ex Nees) Stapf. essential oil (CCEO) combinations to control the mycelial growth of five pathogenic Colletotrichum species (C. asianum, C. siamense, C. fructicola, C. tropicale and C. karstii) in vitro, as well as the anthracnose development in guava (Psidium guajava L.) cv. Paluma, mango (Mangifera indica L.) cv. Tommy Atkins and papaya (Carica papaya L.) cv. Papaya artificially inoculated with these species. Combinations of Chi (2.5, 5 or 7.5mg/mL) and CCEO (0.15, 0.3, 0.6 or 1.25µL/mL) inhibited the mycelial growth of all tested fungal species in vitro. Examined Chi-CCEO combinations showed additive or synergistic interactions to inhibit the target Colletotrichum species based on the Abbott index. Coatings formed by synergistic Chi (5mg/mL) and CCEO (0.15, 0.3 or 0.6µL/mL) combinations decreased anthracnose lesion development in guava, mango and papaya inoculated with any of the tested Colleotrichum species during storage. Overall, anthracnose lesion development inhibition in fruit coated with synergistic Chi-CCEO combinations was higher than that observed in fruit treated with synthetic fungicides. These results show that the application of coatings formed by Chi-CCEO synergistic combinations could be effective to control postharvest anthracnose development in fruit.

Draft genome assembly of Colletotrichum musae, the pathogen of banana fruit.

Colletotrichum musae is an important cosmopolitan pathogenic fungus that causes anthracnose in banana fruit. The entire genome of C. musae isolate GM20 (CMM 4420), originally isolated from infected banana fruit from Alagoas State, Brazil, was sequenced and annotated. The pathogen genomic DNA was sequenced on HiSeq Illumina platform. The C. musae GM20 genome has 50,635,197 bp with G + C content of 53.74% and in its present assembly has 2763 scaffolds, harboring 13,451 putative genes with an average length of 1626 bp. Gene prediction and annotation was performed by Funannotate pipeline, using a pattern for gene identification based on BUSCO.