Abstract

Peach (Prunus persica) is an important stone fruit crop in Chile, with 7,665 h in 2022. Trunk diseases symptoms, including shoot dieback, longitudinal cankers and internal dark-brown to purple discolorations in cross sections were observed in a commercial orchard, in March of 2021. In severe cases, mostly in old trees, periderm sections were detached from the cankers, showing circinate groups of black long necked perithecia. To isolate the causal agent, wood samples were collected from March 2021 to October 2022, from symptomatic trees (n=23) of commercial orchards (n=12) (34°12'36.47"S 70°46'3.43"O to 34°34'26.48"S 70°58'17.97"O), located in O’Higgins Region, in the center of Chile. Isolations were performed cutting wood sections (0.5 cm) from the necrosis progress area, disinfecting them in sodium hypochlorite (10%), plating on a quarter-strength potato dextrose agar amended with 1 mg/L tetracycline (aPDA-tet) and incubating at 25°C, until mycelial development. Cultures were purified on PDA and identified by morphological means. Colonies on PDA were dark-pink and purple to orange-red, with regular margins, usually white, and produced abundant hyaline ellipsoidal to allantoid conidia (3.8–5.7 × 1.3 μm). In some cases, perithecia developed on disinfected wood on culture media, showing clavate unitunicate asci with hyaline allantoid ascospores 4.5–6.2 × 0.7 μm. The morphological characteristics of anamorph and teleomorph structures of field and culture isolates accorded to those described for Calosphaeria pulchella (1,2). DNA from representative isolates was extracted and the ITS region was amplified by PCR using ITS1/ITS4 primers (3), sequenced and BLAST analyzed. BLAST results revealed that ITS sequences identity of the representative isolates HMDu263 and HMDu271, shared 99 and 100% similarity, respectively, when compared to isolate CBS115999 (EU367451) (1,2,4). Sequences were accessioned to GenBank (OP216663 and OP216664 [ITS]). To determine the pathogenicity of C. pulchella, 17 representative isolates were inoculated on peach canes (25 cm) (n=7 per isolate), that were previously rooted on tap water amended with 500 ppm of indole-butyric acid, for 30 d. Mycelial plugs (0.5 cm) from actively growing colonies on PDA were placed on circular injuries made in the upper third of the shoots using a sterile corkborer and covered with plastic film. Sterile agar was used for controls. After 60 d of incubation in aerated tap water, at 23 +/-3 °C, bark was removed, and the necrosis length was measured and compared. Mean length values of lesions went from 9.5 to 27 cm. The most virulent isolates (n=7) were inoculated on fresh cuts of main shoots of nursery plants (n=3 per isolate) cv. Royal Glory, with 200 µL conidial suspensions (1x10⁵ conidia/mL), on March 18th, 2022. Plants were incubated at shadehouse for 102 d and after the incubation period, shoots were cut (30 cm), bark was removed, and discoloration length was measured. All the isolates were pathogenic, with differences among their virulence (ANOVA, LSD, P < 0.05) from 5.2 cm (HMDu246) to 24.3 cm (HMDu266). Fungus was successfully reisolated from symptomatic canes (100%) and trees (98.7%), but not from the controls in both pathogenicity tests, fulfilling Koch’s postulates. Calosphaeria pulchella was recently reported causing trunk disease in sweet cherry in Chile (4) and these results contribute to the knowledge of trunk diseases of fruit crops in Chile and to the understanding of the pathogen worldwide.