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

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.

Molecular identification of Botrytis cinerea, Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile.

BACKGROUND: In Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae. AIMS: We carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens. METHODS: Diseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae. RESULTS: Seventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470bp. CONCLUSIONS: Three Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.

Characterization, In Vitro Culture, and Molecular Analysis of Thecaphora solani, the Causal Agent of Potato Smut.

ABSTRACT The fungus Thecaphora solani (syn.: Angiosorus solani), the causal agent of potato smut, was cultivated in vitro for the first time. Teliospores obtained from galls of infected potato plants were used to inoculate commonly used solid and liquid media. The teliospores produced two kinds of vegetative tissue depending on the nutrient status of the media. A very slow radial-growing, hyaline, and septate mycelium, as usually seen in most of the in vitro-cultivated filamentous fungi, was obtained in wateragar medium after 30 to 40 days. On the other hand, a white, sponge-like mycelial mass was obtained in HCM + 1% activated charcoal, and on common potato dextrose agar or malt-yeast-peptone solid or liquid media, after 40 to 50 days under lab conditions. The identity among teliospores and the sponge-like mycelial mass was corroborated by DNA fingerprinting and partial sequencing of the large subunit (LSU) rDNA region. The sexual cycle of the pathogen was completed under lab conditions based on the development of teliospores on the sponge-like mycelial mass. The first attempt to reproduce the disease under controlled conditions was successful, inducing a gall in a cv. Desirée potato explant cultivated in vitro inoculated with radial-growing mycelia. Phylogenetic analysis of LSU rDNA data of the genus Thecaphora and other smut fungi confirmed the initial classification of the pathogen as T. solani.

Molecular identification of Botrytis cinerea, Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall) in Southern Chile / Identificación molecular de Botrytis cinerea, Botrytis paeoniae y Botrytis pseudocinerea asociadas a la pudrición gris de peonías (Paeonia lactiflora Pall) en el sur de Chile

Background. In Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae. Aims. We carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens. Methods. Diseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300 km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae. Results. Seventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470 bp. Conclusions. Three Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae (AU)

Antecedentes. La peonía es la principal flor ornamental de exportación en Chile. La pudrición gris, causada por los hongos Botrytis cinerea y Botrytis paeoniae, es uno de los problemas fitopatológicos más importantes de su cultivo. Objetivos. Realizar una primera estimación de la diversidad de especies del género Botrytis asociadas a la pudrición gris de la peonía en el sur de Chile. Métodos. Se recogieron hojas de peonías con síntomas de pudrición gris en siete lugares del sur de Chile, cubriendo una distancia de 300 km. Se estudiaron morfológica y molecularmente los aislamientos de Botrytis obtenidos. Finalmente, se desarrolló un ensayo de PCR específico para identificar B. paeoniae basado en el gen nep1. Resultados. Se obtuvieron 17 aislamientos del género Botrytis; todos ellos causaron pudrición gris en plantas de peonía en ensayos de invernadero. Los análisis morfológicos mostraron que cuatro aislamientos compartían características comunes que los distinguen del resto. La homología y el análisis filogenético basado en el gen G3PDH y la determinación del alelo Bc-hch permitieron identificar 12 cepas como B. cinerea, 4 como B. paeoniae y un aislamiento como Botrytis pseudocinerea. Finalmente, el ensayo de PCR resultó específico para B. paeoniae al amplificar una sola banda de 470 pb. Conclusiones. Tres especies de Botrytis están presentes en Chile asociadas a la pudrición gris de las peonías, y se describe por primera vez en este país la presencia de B. paeoniae y B. pseudocinerea como especies patógenas de los cultivos de esta planta. Hasta donde conocen los autores, se describe por primera vez un ensayo de PCR para identificar específicamente B. paeoniae (AU)

Differences in the fly-load of Haematobia irritans (Diptera: Muscidae) on cattle is modified by endophyte infection of pastures

Background: The horn fly, Haematobia irritans, is an obligate bloodsucking ectoparasite of pastured cattle and is a major pest of livestock production in North and South America and Europe. In this study, we investigated the potential to use cattle pastures, infected with non-toxic, "friendly" fungal-endophyte-infected (E+) tall fescue, Festuca arundinacea Schreb., as a strategy for reducing horn fly loads in cattle, and to evaluate the possible bioinsecticide effect on horn fly larvae. Results: When cattle grazed in E+ tall fescue, a decrease in fly-load was observed, compared with other pastures (endophyte-free (E-) pastures). The infestation of horn fly load decreased according to an increase in the percentage of endophyte present in the different pastures (0 to 100%). Moreover, two groups of animals with significant differences in the fly-load (high and low fly-load) in the same herd were observed (P < 0.05). Additionally, it was possible to determine a bioinsecticide effect of cattle dung, upon horn fly larvae (80%), from animals fed E+ tall fescue. Conclusions: These results constitute the first report on the potential for exploiting pasture management for controlling 1) horn fly-loads on cattle and 2) the normal development of horn fly larvae. In conclusion, this information provides preliminary understanding of the role of cattle pasture diet management for controlling horn fliesas part of an integrated pest management strategy for this major pest of farmed livestock.