First report of Gnomoniopsis castaneae associated with branch dieback on chestnut tree (Castanea sativa) in Southern Chile.

ABSTRACT

European chestnut (Castanea sativa Mill.) currently reaches 1,470 ha, distributed from the Maule region to the Los Rios region in Chile. Almost 3000 tons of fruit have been exported in the last three years. A survey was carried out in January 2023 in an eight-year-old orchard located in Vilcún (38°34'46.22"S 72° 9'58.61"O), Araucanía Region. Chestnut trees with branch die back and reduced growth and vigor were detected. The incidence in the orchard was 3% (6 out of 200 trees) estimated by visual observation. Cross and longitudinal sections of the woody trunk of two trees were collected and examined, and an internal dark-brown discoloration to partial necrosis lesion was observed. To identify the causal agent, small pieces of wood from the edge of the symptomatic area were surface sterilized with 70% ethanol, rinsed twice with sterile distilled water, blotted on dry sterile filter paper, plated on potato dextrose agar (PDA) and incubated at 22°C. Fungal colonies were consistently isolated, and after 5 days, pure cultures were obtained by transferring mycelium to new PDA plates, preliminarily identified as Gnomoniopsis sp. (Visentin et al. 2012, Shuttleworth 2012). All cultures exhibited characteristics consistent with the description of G. castaneae (Syn. G. smithogilvyi), such as concentric development of greyish-brown mycelium, abundant stroma, hyaline conidia of 7.2 ±0.54 (6.1-8.1) X 2.3 ±0.26 (1.5-2.9) µm (n= 30), mainly biguttulate and fusoid. Total DNA was extracted, rDNA amplified using ITS1/ITS4 primers (White et al. 1990), and the fragment was Sanger sequenced and the sequence was deposited in GenBank (OR665735). BLAST analysis revealed a 99% identity to G. castaneae (MH384925). In addition, the DNA of the isolate was evaluated in a species-specific multiplex PCR (Silva-Campos et al. 2022), and the amplicons were electrophoretically separated, giving a similar band profile to G. smithogilvyi RGM 2903 and RGM 2904 strain from Chilean Collection of Microbial Genetic Resources. Pathogenicity of G. castaneae isolate (CV-11) was tested on ten replicates of 3-year-old C. sativa plants. Two wounds were made on the same season growing shoot and two on the previous season shoot. Longitudinal wounds (5 mm long, 4 mm wide and 2 mm depth) were made using a scalpel without removing the outer bark to inoculate the plants. Each wound was inoculated with a 5-mm mycelium plug, covered with the outer bark, and wrapped with Parafilm. Plugs of PDA were placed onto the wounds of two plants as control. The plants were kept in a growth chamber (22 ±1 0C and 90± 5% RH). All plants showed dark brown cankers measuring 20 to 40 mm long two weeks after inoculation. Also, most plants inoculated in the same season shoot presented wilted and chlorotic foliage. Mature conidiomata with cirri developed in most of the cankers. No symptoms were observed in the control. Fungal colonies of G. castaneae were reisolated on PDA from all inoculated chestnut plants and were not recovered from the controls. Recently, G. smithogilvyi has been identified as the causal agent of brown rot on chestnut nuts in Chile (Cisterna Oyarce et al. 2022); however, in several countries, it has also been associated as the causal agent of cankers in branch and stem of chestnut, as well as an endophyte in different hardwood species. Future studies on the incidence of this pathogen and its impact on chestnut yield should be carried out in the producing regions because it represents an emerging threat to Chilean chestnut production.