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Colletotrichum truncatum, the most relevant fungal species associated with soybean anthracnose, is responsible for major losses in the crop. Chemical control via fungicide application is still the most effective strategy for the control of soybean foliar diseases. However, the increase in anthracnose incidence in some regions of Brazil indicates that current chemical control has not been effective against anthracnose. In this study, we assessed the fungicide sensitivity of C. truncatum lineages using isolates representing two important regions of soybean production in Brazil to the fungicides azoxystrobin, thiophanate-methyl, difenoconazole, and fludioxonil. We characterized the molecular resistance to quinone-outside inhibitors (QoI), methyl benzimidazole carbamates (MBC) and demethylation inhibitors (DMI) fungicide groups based on amino acid sequences of the cytochrome b (cytb), β-tubulin gene (β-tub), and P450 sterol 14a-demethylases (CYP51) genes. Multiple resistance of C. truncatum isolates to QoI and MBC was observed associated with mutation points in the β-tub (E198A and F200Y) and cytb (G143A). Alternatively, low EC50 values were found for fludioxonil and difenoconazole indicating high efficacy. Analysis of C. truncatum genomes revealed two potential DMI targets, CYP51A and CYP51B, and higher genetic variability in the CYP51A gene. A slight correlation between genetic differentiation of C. truncatum populations and fungicide sensibility was found (Student’s t-test C. truncatum in Brazil.Highlights-Multiple resistance of C. truncatum to azoxystrobin and thiophanate-methyl-C. truncatum isolates are sensitive to difenoconazole and fludioxonil-Presence of E198A and F200Y β-tubulin mutations and G143A cytochrome b mutation-Presence of CYP51A and CYP51B paralogues and higher genetic variability in the CYP51A

The genus

Soybean (Glycine max L.) is one of the most important crops worldwide as a source of protein-rich foods and animal feeds. Anthracnose, one of the major limiting factors to soybean production (Dias et al. 2016), is caused by species such as Colletotrichum truncatum, C. sojae, and C. plurivorum (Damm et al. 2009, 2019). In December 2016 and 2017, soybean plants of cultivars Monsoy 8768 and Pioneer y-70 with typical symptoms of anthracnose (necrotic and irregular brown lesions on stems, leaves, and pods) were collected in Mato Grosso, Brazil. Commercial fields sampled showed 10 to 15% incidence of anthracnose in 1 ha in each sampled area. In total, 10 different geographic locations were sampled. Colletotrichum strains were isolated and cultured on potato dextrose agar at 25°C with a 12-h light photoperiod from surface-disinfected (70% alcohol followed by 0.5% sodium hypochlorite) plant tissues. Among others, three single-spore isolates (LFN0048 from Sinop, LFN0074 and LFN0090 from Lucas do Rio Verde) showed different morphology; isolates LFN0048 and LFN0074 were selected for further characterization. Total DNA was extracted and partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), and β-tubulin (TUB2) genes were amplified and sequenced. The sequences were deposited in GenBank (accession numbers MN604249 and MK163893 for HIS3, MN604248 and MK142674 for GAPDH, and MN604250 and MK142675 for TUB) and were compared with most similar reference sequences of Colletotrichum (Damm et al. 2019). Both isolates clustered with Colletotrichum musicola epitype (CBS 132885), showing 100 and 98.5% similarity in GAPDH, 99.5 and 98.9% in HIS3, and 99.2% in TUB2. On PDA, colonies showed dark-gray aerial mycelium with entire margins, reverse violaceous-black. Conidia and ascospore size and shape match those previously described by Damm et al. (2019): 12.12 to 15.86 × 4.93 to 6.95 µm and 15.5 to 19.34 × 5 to 7.84 µm, respectively (n = 100). Appressoria (n = 50) were single or in loose groups, violaceous-black with predominant obovoid, truncated, and cylindrical shapes, with smooth, undulate, or lobate margin, and 9.25 to 29.79 × 7.22 to 21.06 µm. Perithecia, paraphyses; and unitunicate eight-spored asci were also observed. Asci were cylindrical to clavate, smooth-walled, and 48.12 to 68.78 × 9.59 to 14.47 µm (n = 50). Soybean anthracnose is seed-borne (Dias et al. 2018; Rogério et al. 2017); therefore, pathogenicity tests were carried out on pregerminated seeds. Five seeds of Brasmax 8579 cultivar were inoculated with 10 µl of a conidial suspension (106 conidia/ml) that was placed in the emerging radicle, and five mock-inoculated seeds were used as a control. Seedlings were planted in vermiculite and incubated at 25°C with a 12-h photoperiod. After 7 days, inoculated plants showed necrotic lesions on the cotyledons, leaflets, and hypocotyl, whereas control plants remained asymptomatic. The experiment was repeated three times. C. musicola was reisolated from the symptomatic tissues, and the identity was confirmed by morphology and multilocus phylogeny. Until now, C. musicola has been reported to be associated with Musa sp. (Damm et al. 2019) and Colocasia esculenta (Vásquez-López et al. 2019) in Mexico, and with Phaseolus lunatus in Brazil (Cavalcante et al. 2018). To our knowledge, this is the first report of C. musicola joining a group of new and emergent species of Colletotrichum causing anthracnose in soybean-producing regions around the world.