Occurrence of Neofusicoccum parvum Causing Canker and Branch Dieback of European Hazelnut in Maule Region, 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.

First report of Calosphaeria pulchella causing cankers in peach (Prunus persica) in Chile.

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.

Silverleaf (Chondrostereum purpureum) Effects on Japanese Plum (Prunus salicina).

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.

First report of Eutypa lata causing cankers and dieback in sweet cherry in Chile.

Sweet cherry (Prunus avium) is one the most important fruit crops in Chile. Its production has significantly grown in recent years, reaching 228,448 tons exported in 2019/2020, to 47 countries. One of the main threats for this expanding crop are fungal pathogens, especially those that cause wood diseases. Cherry orchards (n=35) located in the central area of Chile, from Curicó (34°58'58''S 71°14.366'W) to Angol (37°47'42.7''S 72°42.982'W), were surveyed during 2020. Wood samples were collected (n= 72) from living branches and trunks showing dieback, cankers and dark necrosis, mostly wedge shaped. Small wood sections (0.5-cm) were cut off from the margin of the necrosis and surface disinfected using 0.5% v/v sodium hypochlorite. Sections were plated on a quarter-strength potato dextrose agar amended with 1mg/L tetracycline (PDA-tet). Plates were incubated at 25°C until mycelial development and subsequently the isolates were purified transferring excised fungal tips to PDA. Colonies (n=21) developed white cottony mycelia, which turned slightly greyish and flatter after 10-days at 25°C. Isolates developed black pycnidia which released beige conidial matrixes after subsequent 15-days at 25 +/-2°C and 12-h photoperiod. Conidia were hyaline, curved and filiform, measuring 19.8-(27.9)-36.7 µm length (lineal) x 1.2-(1.7)-1.9 µm width (n=70), according to Eutypa lata (Rappaz, 1984). DNA was extracted from mycelia of the representative isolates HMCe30a, HMCe41a, HMCe109c and HMCe110a. The partial ß-tubulin gene was amplified using bt2A/bt2B primers (Glass & Donaldson 1995) and the internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al. 1990). Sequences were BLAST analyzed, finding that ITS shared 99% and ßTUB 100% identity with E. lata strain CBS 208.87 (Rolshausen et al. 2006). Sequences were accessioned to GenBank (MW363035, MW363034, MW363033 and MW363032 [ITS], and MW366820, MW366819, MW366818and MW366817 [ßTUB]). The isolates were inoculated on sweet cherry healthy plants cv. Kordia, produced by rooting scions in tap water amended with 500 ppm of indole-butyric acid, for 30 days. An injury was made in the upper third of the shoot using a sterile 0.5-cm diameter corkborer. Mycelial plugs were placed on the injuries and covered with plastic film, using sterile agar for controls (n=25). Plants were incubated in aerated tap water for 60 days at 23 +/-3 °C. After incubation, plants were cut exposing dark-brown necrotic lesions, while control plants remained asymptomatic. Moreover, 2-year old potted plants cv. Lapins were inoculated (n=3 per isolate) with mycelial plugs, on fresh cuts of their main lateral branches, in January 20th, and remained under partial shade for 72-days. After incubation, bark was removed from inoculated branches and the necrotic lesions length was measured. HMCe109c was the most virulent isolate (3.6 cm), followed by HMCe30a (2.1 cm), HMCe41a (1.9 cm) and HMCe110a (1.1 cm), while symptoms were not reproduced in controls. Fulfilling Koch's postulates, fungi were reisolated from all inoculated plants in both pathogenicity tests and no fungus was recovered from controls. To our knowledge this is the first report of Eutypa lata causing wood decay in sweet cherry in Chile. The pathogen was recently reported causing dieback of grapevines in Chile (Lolas et al. 2020). These are significant findings due to the frequent proximity of sweet cherry orchards and vineyards, which facilitates cross infections.

First report of Seimatosporium vitifusiforme causing trunk disease in Chilean grapevines (Vitis vinifera).

Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial ß-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and ßtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.