Silverleaf caused by the basidiomycete Chondrostereum purpureum affects numerous woody species, including fruit tree crops like apple, resulting in wood necrosis and foliar silvering. There are no curative alternatives for this disease, and its management is by prevention methods. Therefore, the aim of this study was to develop a rapid diagnostic tool for the detection and identification of C. purpureum directly from woody tissues to help distinguish the pathogen from other basidiomycetes that are commonly found on apple. The silverleaf pathogen was isolated from different hosts and locations, and Koch’s postulates were performed by inoculating the isolates on apple cuttings and measuring internal necrosis. A previously described APN 1 pair of primers specificity was also tested against 25 C. purpureum isolates in this study, using other wood rotting species as negative controls. Seven virulent isolates were inoculated on apple cuttings, and DNA was extracted from the cuttings’ sawdust and amplified using APN 1, after 22 days of incubation. To prove the efficiency of the method in the field, DNA from healthy nursery plants inoculated with two virulent isolates, and naturally infected plants showing different levels of foliar symptoms, were tested. Presence of the fungus was verified by reisolation on APDA in all assays. Koch’s postulates indicated that all C. purpureum isolates were pathogenic, showing different virulence levels, and APN 1 primers were able to discriminate them from other basidiomycetes. The method was also able to detect C. purpureum from artificially inoculated plants as well as naturally infected ones, demonstrating that the protocol may become a rapid minimally destructive diagnostic tool to detect the pathogen without the need to isolate it from tissues, and thus taking measures to prevent its dissemination.

Cichorium intybus var. sativum is an Astereacae plant grown for its fleshy roots, which are rich in inulin, oligofructose and other fructans, and are used as functional food. It was introduced to Chile in 2006, where climate conditions allowed high yields. However, during crop adaptation period a severe crown rot was observed, with yield losses of up to 31% and reduced efficiency in inulin extraction process. Field collections of rotted plants from all cultivated areas, were between 2009 and 2013. Root symptoms started as dark, small, sunken lesions on the crown, turning to firm, black colored cavities as the crop developed. Signs were not visible. Sections of tissue were aseptically excised from the margins of necrotic lesions and placed into Petri plates, containing either water agar or acidified 25% potato dextrose agar. Pure cultures were obtained through hyphal tips. Isolates were identified on the basis of their morphological characteristics on oatmeal agar (OA) and malt agar (MEA), the metabolite ‘E’ test (Aveskamp et al. 2010) and molecular features. DNA was extracted from pure cultures and multilocus sequence typing was performed (Aveskamp et al. 2009). The internal transcribed spacer (ITS) sequence, actin and β-tubulin genes were amplified and sequenced. Primers ITS1 and ITS4 were used for ITS region, Act-512F and Act-783R for actin, and Bt2a and Bt2b for β-tubulin. Acquired sequences were subjected to NCBI BLAST search. Bootstrap analyses were performed on each dataset using neighbour-joining with 1000 replicates. After seven days, colonies grew 39 to 70 mm in diameter on OA and 47 to 82 mm on MEA. Colonies on MEA were olivaceous gray with irregularly scalloped margins, floccose, white to pale olivaceous-gray aerial mycelium and olivaceous-gray to black reverse. Pycnidia were dark, submerse, globose to subglobose, glabrous, solitary or confluent with a yellowish conidial matrix and a non-papillated ostiole. Conidia were hyaline and unicellular, ellipsoidal to oblong (n=100), ranging from 1.9 - 3.2 µm wide (2.8 ± 0.9 µm) x 4.2 - 6.8 µm long (5.7 ± 4.2 μm), with bipolar guttules. Swollen cells were olivaceous and constricted at the septa. Chlamydospores were absent (Boerema et al. 2004). All isolates were E+. Multilocus sequence alignment and tree construction assigned the isolates to Boeremia exigua var. exigua. Pathogenicity tests were performed with four different inoculation methods. Mycelium plugs 70 mm diameter, from colonies on PDA, were inoculated on surface sterilized root discs 50 - 70 mm diameter and incubated in humid chamber, at 22°C and dark. Plugs were also inoculated on surface sterilized entire roots and incubated both in humid chamber and in pasteurized soil, in the greenhouse for 20 days. Controls were inoculated with sterile agar plugs. Subsequently, the fungus was massified in sterile millet grains and mixed with pasteurized soil (0.1 % v/v), where healthy plants were transplanted. The pathogen was able to colonize the root, either with or without injuries to the crown, reproducing black root rot symptoms in all the inoculations. For all the tests, the pathogen was reisolated on PDA, fulfilling Koch’s postulates. Consequently, this is the first report of B. exigua var. exigua on industrial chicory in Chile.

The European hazelnut (Corylus avellana) is an important fruit crop cultivated in Chile, with over 17,000 ha planted (46%) in the Maule region, central Chile. During a routine orchard survey in seasons 2020-2021 and 2021-2022, in the Maule region, canker and dieback symptoms were observed in two commercial orchards of European hazelnut cv. Tonda Di Giffoni in San Rafael (8-year-olds) and Linares (15-year-olds), with an incidence between 10% and 36%, respectively, based on external symptoms. Twenty symptomatic branches exhibiting cankers, reduced vigor, wilting, twig death, and dieback, were collected. A cross-section of diseased branches revealed mostly brown V or U-shaped cankers of hard consistency. Branches were cut, and pieces of cankers were surface sterilized in 96% ethanol for 3 s and briefly flamed. Small pieces of wood (5 mm2) from the edge of cankered tissues were placed on Potato Dextrose Agar (2% PDA) amended with 0.1% Igepal CO-630 and incubated at 25°C for five days in the dark (Díaz and Latorre 2014). Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Eight pure cultures (NP-Haz01 to NP-Haz08) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA under near-UV light. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown globose pycnidia after 21 days at 25°C. Conidia were hyaline, aseptate, ellipsoidal, densely granulate, externally smooth, and thin-walled dark, that measured (9.5-) 15.5 ±1.2 (-17.3) x (5.1-) 7.2 ± 0.6 (-9.1) µm (n = 30), with a length/width ratio of 2.15. These isolates were tentatively identified morphologically as Neofusicoccum sp. Molecular identification was performed using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R primers of the internal transcribed spacer (ITS1-5.8S-ITS2) region, a portion of the beta-tubulin (BT) and part of the translation elongation factor (EF1-) genes, respectively (Dissanayake et al. 2015). A MegaBlast search in GenBank showed a 99% similarity to isolate CMW9081, the ex-type of Neofusicocum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. The sequences were added to GenBank (OR393855 to OR393857 for ITS; OR400688 to OR400690 for BT; OR400691 to OR400693 for EF1-). Pathogenicity of three isolates (NP-Haz02, NP-Haz04, NP-Haz09) was studied on freshly made pruning wounds on attached branches of 3-year-old and one-year-old of European hazelnut cv. Tonda Di Giffoni in the San Rafael field. Fifteen pruning wounds were inoculated with 40 µL conidial suspension (105 conidia/mL) of each isolate of N. parvum. Sterile distilled water was used as a control treatment (n=15 branches) for branches of 3-year-olds and one-year-olds. Both pathogenicity tests were repeated once. Attached branches of 3-year-olds (6 months of incubation) and one-year-olds (4 months of incubation), developed necrotic streaks and cankers with a mean length of 33 to 82 mm and 25 to 51 mm, respectively. No necrotic streaks were observed in the branches treated with water. Neofusicoccum parvum was reisolated only from symptomatic tissues of inoculated branches, and morphological and molecularly (EF1-) identified, thus fulfilling Koch's postulates. Previously, other Botryosphaeriaceae spp. as Diplodia coryli (Guerrero and Pérez 2012) and D. mutila (Moya-Elizondo et al. 2023) have been obtained from canker and dieback of hazelnut in Chile. Recently, N. parvum was reported causing nut rot in hazelnuts in Italy (Wagas et al. 2022). To our knowledge, this is the first report of N. parvum causing canker and branch dieback of hazelnut trees in Chile and worldwide.

Silverleaf is an important fungal trunk disease of fruit crops, such as Japanese plum (Prunus salicina). It is known that infection by Chondrostereum purpureum results in discolored wood, “silvered” foliage, and tree decline. However, effects on fruit yield and quality have not been assessed. Therefore, the objectives of this study were to determine C. purpureum pathogenicity on P. salicina and the effects on physiology, fruit yield, and quality, in Chile, in 2019 and 2020. Wood samples from affected plum trees were collected in the Chilean plum productive area. Fungi were isolated by plating wood sections from the necrosis margin on culture media. Morphological and molecular characteristics of the isolates corresponded to C. purpureum (98%). Representative isolates were inoculated from healthy plum plants and after 65-d incubation, wood necrotic lesions and silver leaves were visible. Fungi were reisolated, fulfilling Koch’s postulates. To determine Silverleaf effects, xylem water potential and fruit yield and quality were measured in healthy and Silverleaf-diseased plum trees ‘Angeleno’. Water potential was altered in diseased trees, and fruit yield was reduced by 51% (2019) and by 41% (2020) compared to fruit from healthy trees. Moreover, cover-colour, equatorial-diameter, and weight were reduced, and fruit were softer, failing to meet the criteria to be properly commercialized and exported to demanding markets.

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 disc...

Ugni molinae Turcz. (Myrtaceae), commonly known as murta or ‘‘Chilean guava’’, is a small evergreen shrub, native from the south of Chile. It is grown for the flavor of its fruit and leaf extracts, rich in antioxidant polyphenols and triterpenoids, used in pharmaceutical and cosmetic industries. Symptoms of silverleaf, a fruit and forest crops wood disease, were observed on wild murta plants of the rainforest of Valdivia (39°51'59"S 73°13'26"W). Leaves showed silver color and internal necrosis of the stems, similar to the symptoms on Vaccinium corymbosum and V. asheii (France et al. 2009; 2017). To isolate the causal agent, stems from affected plants were collected and their bark removed. Subsequently, wooden discs were cut and disinfected using bleach 3%, ethanol 70% and rinsed with sterile distilled water. Discs were plated on Petri plates containing acidified one-quarter-strength potato dextrose agar (PDA, Difco) and incubated at 25°C and darkness, until mycelial development (7-14 days). Later, fungal ...