Waléria Guerreiro Lima

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of São Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in São Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of São Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.

Species of Lasiodiplodia associated with papaya stem-end rot in Brazil

This study aims to identify and characterize species of Lasiodiplodia associated with stem-end rot of papaya in six different populations in the Northeast of Brazil. Fungal identifications were made using a combination of morphology together with a phylogenetic analysis based on partial translation elongation factor 1-α sequence (EF-1α) and internal transcribed spacers (ITS). Five species of Lasiodiplodia were identified: Lasiodiplodia brasiliense sp. nov., L. hormozganensis, L. marypalme sp. nov., L. pseudotheobromae and L. theobromae. Only L. theobromae had previously been reported in papaya, while all the other species are reported for the first time in association with this host in Brazil and worldwide. Lasiodiplodia theobromae was the most prevalent species. All species of Lasiodiplodia were pathogenic on papaya fruit, with L. hormozganensis being the most virulent.

The impact of phenotypic and molecular data on the inference of Colletotrichum diversity associated with Musa

ABSTRACT Developing a comprehensive and reliable taxonomy for the Colletotrichum gloeosporioides species complex will require adopting data standards on the basis of an understanding of how methodological choices impact morphological evaluations and phylogenetic inference. We explored the impact of methodological choices in a morphological and molecular evaluation of Colletotrichum species associated with banana in Brazil. The choice of alignment filtering algorithm has a significant impact on topological inference and the retention of phylogenetically informative sites. Similarly, the choice of phylogenetic marker affects the delimitation of species boundaries, particularly if low phylogenetic signal is confounded with strong discordance, and inference of the species tree from multiple-gene trees. According to both phylogenetic informativeness profiling and Bayesian concordance analyses, the most informative loci are DNA lyase (APN2), intergenic spacer (IGS) between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), β-tubulin (TUB2), and a new marker, the intergenic spacer between GAPDH and an hypothetical protein (GAP2-IGS). Cornmeal agar minimizes the variance in conidial dimensions compared with potato dextrose agar and synthetic nutrient-poor agar, such that species are more readily distinguishable based on phenotypic differences. We apply these insights to investigate the diversity of Colletotrichum species associated with banana anthracnose in Brazil and report C. musae, C. tropicale, C. theobromicola, and C. siamense in association with banana anthracnose. One lineage did not cluster with any previously described species and is described here as C. chrysophilum.

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 β-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.

DIVERSIDADE GENOTÍPICA E PATOGÊNICA DE Colletotrichum musae NO ESTADO DE PERNAMBUCO

Sixty Colletotrichum isolates originating from fields producing banana in the municipalities of Vicencia, Sao Vicente Ferrer and Machados in the State of Pernambuco were evaluated for physiological, morphological, molecular, cultural, virulence and genetic diversity characteristics. The isolates were identified as C. musae, with most conidia, straight, oblong with rounded summits. The mycelial growth rate ranged from 1.36 to 1.91 cm / day. Three groups of staining for colonies were found: white, cream and salmon, while the presence of sectors ranged from 0 to 8 per isolate, and in most isolates (73.3%) there were the presence of microsclerotia. The difference in virulence was significant for the area under the disease progress curve, indicating variability among isolates. The dendrogram generated by UPGMA analysis of ISSR-PCR markers revealed the formation of three groups by Dice similarity coefficient which correspond mostly to the three sampled areas.

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.

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.