First Report of Colletotrichum chrysophilum Causing Anthracnose on Blueberry in Brazil.

Highbush (Vaccinium corymbosum L.) and rabbiteye (V. ashei R.) blueberry are the most important export small fruit crops in southern Brazil. Anthracnose has been considered one of the most destructive disease and exclusively associated with C. karstii in Brazil (Rios et al. 2014). In November 2019, severe anthracnose symptoms including leaf spots but particularly twig blights and fruit rots were observed on all blueberry plants (V. ashei) in one organic orchard in Santa Catarina state, Brazil (27º43'48.96"S, 49º0'57.79"W). Four isolates were obtained from necrotic lesions and monosporic cultures were grown on potato dextrose agar at 25°C and with a 12 h photoperiod under near ultra violet light. After 15 days, colonies showed upper surface color varying from grayish-white to pale-orange and the reverse side pale-orange. Conidia were hyaline, cylindrical with rounded ends, and their length and width ranged from 9.5 to 15.5 µm (x ̅=11.8) and 6.5 to 3.5 µm (x ̅=4.9), respectively. The isolates were identified by multilocus phylogenetic analyses using nucleotide sequences of actin (ACT), ß-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), glutamine synthetase (GS), internal transcribed spacer (ITS) and the intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (ApMAT). Nucleotide sequences exhibited from 95 to 100% sequence identity to Colletotrichum chrysophilum ex-type (CMM4268) and were deposited in GenBank database (MW868219 to MW868222, MW868211 to MW868214, MW868215 to MW868218, MW868223 to MW868226, MW868202 to MW868205, MW793353 to MW793356, and MW868207 to MW868210). C. chrysophilum belongs to the C. gloeosporioides species complex and was previously described as C. ignotum in banana and other tropical fruits in Brazil (Vieira et al. 2017; Veloso et al. 2018). In addition, this species was recently reported on apple fruit in New York, USA (Khodadadi et al. 2020). To confirm pathogenicity, one-year-old blueberry plants were inoculated by spraying a suspension of 1×106 conidia/ml, incubated in a moist chamber in the dark for 48 h and then kept in the greenhouse. Plants sprayed with sterile distilled water served as control. Additionally, fruits were immersed for 2 min in a conidial suspension (1×106 conidia/ml) and incubated at 25°C and 12 h photoperiod for 20 days. Inoculated plants exhibited first symptoms in twigs at 10 days after inoculation (dai). Infected twigs showed initially dark brown spots that coalesced and became necrotic. On leaves, reddish-brown lesions with less than 2 mm appeared at low intensity at 15 dai. On fruits, sunken areas associated with an abundant orange mucilaginous mass of acervuli and conidia were seen at 7 dai. Symptoms on plants were identical to those observed under field conditions, and the pathogen was re-isolated from lesions fulfilling Koch's postulates. To the best knowledge, this is the first report of C. chrysophilum causing anthracnose on blueberries in Brazil. The identification of this species causing blueberry anthracnose is crucial to improve the disease control strategies and resistance breeding.

First Report of Colletotrichum karstii Causing Anthracnose on Strawberry in Brazil.

Strawberry (Fragaria x ananassa Duch.) is one of the most important fruit crops worldwide. With increasing cultivated area in the last decades, Brazil has become the largest strawberry producer in South America. Anthracnose caused by Colletotrichum spp. has been considered one of the most destructive diseases in Brazil. In May 2019, irregular and circular dark brown leaf spots sometimes associated with chlorosis and petiole necrosis were observed on strawberry plants (cv. Pircinque) organically cultivated in Santa Catarina state, Brazil (27°45'40"S, 49°59'06''W). The Colletotrichum isolate was obtained from leaf, and monosporic culture was grown on potato dextrose agar at 25°C and 12-h photoperiod under near ultraviolet light. Colonies at the age of 15 days showed upper surface color varying from white to orange and the reverse side grayish to orange. Conidia were hyaline, cylindrical with rounded ends, 13.9 to 9.2 × 4.2 to 6.7 µm ((x ) ̅= 11.3 × 5.2, n = 100). Perithecia were produced in vitro and their diameter ranged from 265.2 to 142.5 µm ((x ) ̅= 198.4). Asci were 47.3 to 39.9 × 5.2 to 7.2 µm ((x ) ̅= 42.8 × 5.9, n = 50), and ascospores 12.6 to 8.1 × 4.3 to 2.1 ((x ) ̅= 10.3 × 2.9, n = 100). To confirm pathogenicity, 90-day-old plants of strawberry (cv. Pircinque) were inoculated by spraying a suspension of 1×106 conidia/ml, incubated in a moist chamber in the dark for 48 h and then kept in a greenhouse for further 30 days. Plants sprayed with sterile distilled water served as control. Additionally, detached leaves were inoculated with six drops of 10 µl (1×106 conidia/ml) onto abaxial surface and incubated in a moist chamber at 25°C and 12 h photoperiod for 15 days. Inoculated plants exhibited first symptoms in both leaves and petioles at 15 days after inoculation (dai). On leaf, irregular and circular dark brown spots evolved to necrotic lesions and were frequently surrounded by chlorotic halos. In petioles, lesions were reddish-brown, elongated, and depressed. Typical anthracnose symptoms on fruits at 6 dai showed as circular, slightly sunken lesions that enlarged over time and produced an abundant orange mucilaginous mass of acervuli and conidia, and after 20 days, fruits became mummified. In the detached-leaf-assay, symptoms appeared at 7 dai, with presence of circular dark brown lesions measuring 1 to 15 mm and then evolved to necrosis. The same pathogen was consistently re-isolated from the inoculated leaves, petioles, and fruits, and confirmed by morphological characterization and molecular assays as described in this note. A representative isolate (MANE189) was molecularly identified using genomic regions of actin (ACT), ß-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), glutamine synthetase (GS), and internal transcribed spacer (ITS). Nucleotide sequences exhibited 100% homology to the typical Colletotrichum karstii strains (CBS:127535, CBS:128500 and ML1792) and were deposited in GenBank database (MW396420, MW396430, MW396460, MW396440, MW396450, and MW331606). This species belongs to the C. boninense species complex (Damm et al. 2012) and was previoStrawberry (Fragaria x ananassa Duch.) is one of the most important fruit crops worldwide. With increasing cultivated area in the last decades, Brazil has become the largest strawberry producer in South America. Anthracnose caused by Colletotrichum spp. has been considered one of the most destructive diseases in Brazil. In May 2019, irregular and circular dark brown leaf spots sometimes associated with chlorosis and petiole necrosis were observed on strawberry plants (cv. Pircinque) organically cultivated in Santa Catarina state, Brazil (27°45'40"S, 49°59'06''W). The Colletotrichum isolate was obtained from leaf, and monosporic culture was grown on potato dextrose agar at 25°C and 12-h photoperiod under near ultraviolet light. Colonies at the age of 15 days showed upper surface color varying from white to orange and the reverse side grayish to orange. Conidia were hyaline, cylindrical with rounded ends, 13.9 to 9.2 × 4.2 to 6.7 µm ((x ) ̅= 11.3 × 5.2, n = 100). Perithecia were produced in vitro and their diameter ranged from 265.2 to 142.5 µm ((x ) ̅= 198.4). Asci were 47.3 to 39.9 × 5.2 to 7.2 µm ((x ) ̅= 42.8 × 5.9, n = 50), and ascospores 12.6 to 8.1 × 4.3 to 2.1 ((x ) ̅= 10.3 × 2.9, n = 100). To confirm pathogenicity, 90-day-old plants of strawberry (cv. Pircinque) were inoculated by spraying a suspension of 1×106 conidia/ml, incubated in a moist chamber in the dark for 48 h and then kept in a greenhouse for further 30 days. Plants sprayed with sterile distilled water served as control. Additionally, detached leaves were inoculated with six drops of 10 µl (1×106 conidia/ml) onto abaxial surface and incubated in a moist chamber at 25°C and 12 h photoperiod for 15 days. Inoculated plants exhibited first symptoms in both leaves and petioles at 15 days after inoculation (dai). On leaf, irregular and circular dark brown spots evolved to necrotic lesions and were frequently surrounded by chlorotic halos. In petioles, lesions were reddish-brown, elongated, and depressed. Typical anthracnose symptoms on fruits at 6 dai showed as circular, slightly sunken lesions that enlarged over time and produced an abundant orange mucilaginous mass of acervuli and conidia, and after 20 days, fruits became mummified. In the detached-leaf-assay, symptoms appeared at 7 dai, with presence of circular dark brown lesions measuring 1 to 15 mm and then evolved to necrosis. The same pathogen was consistently re-isolated from the inoculated leaves, petioles, and fruits, and confirmed by morphological characterization and molecular assays as described in this note. A representative isolate (MANE189) was molecularly identified using genomic regions of actin (ACT), ß-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), glutamine synthetase (GS), and internal transcribed spacer (ITS). Nucleotide sequences exhibited 100% homology to the typical Colletotrichum karstii strains (CBS:127535, CBS:128500 and ML1792) and were deposited in GenBank database (MW396420, MW396430, MW396460, MW396440, MW396450, and MW331606). This species belongs to the C. boninense species complex (Damm et al. 2012) and was previously reported causing anthracnose on strawberry leaves in Taiwan (Chung et al. 2020). To our knowledge, this is the first report of C. karstii causing anthracnose on strawberry in Brazil. The accurate identification of the pathogen will assist in the disease management and resistance breeding. usly reported causing anthracnose on strawberry leaves in Taiwan (Chung et al. 2020). To our knowledge, this is the first report of C. karstii causing anthracnose on strawberry in Brazil. The accurate identification of the pathogen will assist in the disease management and resistance breeding.