Corynespora cassiicola causing leaf spots on Boehmeria nivea (rami) in Brazil
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A new leaf spot disease caused by necrotrophic fungus Corynespora cassiicola is reported on Boehmeria nivea for the first time in Brazil. Identity was clarified based on morphological and molecular evidence and pathogenicity to ramie was demonstrated.
KeywordsAscomycota Eye-spot Dematiaceae Fiber crop Leaf disease Necrotroph Pathogenicity
A dematiaceus fungus was regularly found growing on the center of the ramie leaf spots. Homogeneous pure cultures were obtained by transferring single conidia from sporulating lesions onto potato dextrose-agar (PDA) plates with a sterile fine pointed needle. One representative isolate was deposited in the local culture collection of the Universidade Federal de Viçosa (Accession No COAD 1964).
Seven-day-old colonies grown at 25 °C under a 12 h light regime in PDA were described following the terminology of Crous et al. (2009) and the color terminology of Rayner (1970). Fungal structures were scraped from lesions with a scalpel and slides were mounted in lactoglycerol and observed with a Olympus BX 51 light microscope fitted with differential interference contrast (DIC) illumination and connected to a Olympus Qcolor 3™ camera. The fungus had the following morphology: Conidiophores amphigenous, cylindrical, straight to slighlty curved, 132 ̶ 384 × 3 ̶ 5 μm, 6–13 septate, pale brown, smooth; conidia solitary or in short acropetal chains, cylindrical to subcylindrical, 92–154 × 3–6 μm, 10 ± 20 pseudoseptate, pale brown, smooth (Fig. 1e and f). In culture: Fast-growing (7.8 cm diameter after 7 days in PDA), edges entire, umbonate, felty, pale vinaceous, bay reverse, not sporulating (Fig. 1d). Fungal morphology was equivalent to that of Corynespora cassiicola as described by Dixon et al. (2009) and Ellis and Holliday (1971).
To further confirm fungus identity, genomic DNA was extracted from 7- day old colonies using the Wizard Genomic DNA Purification Kit (Promega) by following the manufacturer’s instructions. The ITS region of rRNA of the fungus was PCR amplified with ITS4 (TCC TCC GCT TAT TGA TAT GC) and ITS5 (GGA AGT AAA AGT CGT AAC AAG G) primers (White et al. 1990) and sequenced by Macrogen (Korea). The resulting sequence was deposited in GenBank (accession no. MH536840). This sequence had 99% identity with a previously deposited ITS sequence of C. cassiicola on Plectranthus barbatus (accession No. FJ852592) (Dixon et al. 2009).
A pathogenicity test was performed by inoculating five healthy six-month-old potted B. nivea with a 1 × 104 conidia/mL suspension of COAD 1964. Sporulating cultures were obtained on PDA after 10 days growth under black light (40 W) for 48–72 h. Sporulating cultures were flooded with sterile water and scraped with a paintbrush to produce the conidial suspension, which was then adjusted to the correct concentration with the help of a haemocytometer. The conidial suspension was then sprayed on the abaxial surface of leaves until runoff. Plants were kept in a humid chamber, for 48 hs after inoculation, and then transferred to a greenhouse bench. Six days after inoculation leaf spot symptoms identical to those originally seen on the field appeared on inoculated plants, but not on controls – two healthy plants treated similarly but sprayed with distilled water. Corynespora cassiicola was isolated from necrotic tissues of inoculated plants and pure cultures of equivalent morphology to that COAD 1964 were obtained, and fungal structures mounted in lactoglycerol were observed confirming its identity as C. cassiicola, fulfilling Koch’s postulates.
Corynespora cassiicola is a common and cosmopolitan species, especially abundant in the tropics, with a wide range of host plants (Ellis and Holliday 1971; Dixon et al. 2009; Lopez et al. 2018). Farr and Rossman (2019) listed records of C. cassiicola in association with B. nivea from China and Malaysia but ramie is reported here for the first time as a host of C. cassiicola in Brazil.
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