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.

First Report of Anthracnose Caused by Colletotrichum theobromicola on Barbados Cherry (Malpighia emarginata) in Brazil.

Barbados cherry, also called acerola, is a fruit originated from tropical America that is well-known for its high content of vitamin C and nutritional value. Anthracnose is one of the most common diseases on Barbados cherry. In Brazil, this disease is associated with Colletotrichum gloeosporioides sensu lato (2). In 2012, necrotic and sunken spots were observed on Barbados cherry fruit (cv. Rubra) in Sao Paulo State, Brazil, from which a Colletotrichum species was isolated on potato dextrose agar (PDA). The isolate was grown on PDA at 25°C and 12-h photoperiod under fluorescent light. The colony was gray on the upper surface and the reverse part was dark gray. Conidia (n = 50) were cylindrical to subcylindrical, hyaline, and 12 to 15 (avg. 12.7) × 3.8 to 5.9 (avg. 4.3) µm. Conidia length/width ratio was 2 to 3.6. Pathogenicity was confirmed on Barbados cherry fruit. Inoculation was carried out by depositing 40-µl droplets of a conidial suspension (1 × 105 conidia ml-1) on fruit wounded with a sterilized needle and on non-wounded fruit. Fruit were incubated in a moist chamber at 25°C. First symptoms appeared 3 and 5 days after inoculation on wounded and non-wounded fruit, respectively. No symptoms were observed on control fruit inoculated with water. Six isolates recovered from inoculated fruit showed the same morphological characteristics of the previous isolate. The DNA of the fungus was extracted by a CTAB protocol (1) and the sequences of ITS, GAPDH, ACT, CHS-1, TUB, and CAL genes (4) were generated. Sequences were used in BLAST searches in GenBank and were 100% similar to C. theobromicola, except for GAPDH. The ITS (KC566724) and CAL (KC566437) sequences matched strain ICMP 17099 (JX010285 and JX009588, respectively) with 100% identity. The BTUB (KC566148), GAPDH (KC566578), ACT (KC566870), and CHS-1(KC566292) sequences matched with the strains ICMP 18649 (JX010447, 100% identity), ICMP 17099 (JX009957, 99% identity, 1 pb), ICMP 18567 (JX009457, 100% identity), and ICMP 18613 (JX009771, 100% identity), respectively. The sequences were also compared with authentic culture of C. gloeosporioides (IMI 356878) and the identities were: ITS 99% (JX010148), CAL 91% (JX009729), BTUB 90% (JX010445), GAPDH 83% (GU174561), ACT 93% (JX009494), and CHS-1 98% (JX009747). Based on the multi-gene sequencing, the isolate was identified as C. theobromicola. C. theobromicola was described in 2010 (3) and it is considered as a widely distributed species occurring on different hosts in tropical and subtropical regions (4). This report shows the necessity of the identification of Colletotrichum species from tropical fruits to elucidate the etiology of anthracnose diseases of which C. gloeosporioides sensu lato is considered to be the causal agent. To our knowledge, this is the first report of C. theobromicola on Barbados cherry. References: (1) M. G. Murray and W. F. Thompson. Nucleic Acids Res. 8:4321, 1980. (2) R. Ritzinger et al. Acerola em Foco 13:1, 2007. (3) E. I. Rojas et al. Mycologia 102:1318, 2010. (4) B. S. Weir et al. Stud. Mycol. 73:115, 2012.