First report of silverleaf caused by Chondrostereum purpureum in hazelnut (Corylus avellana) in Chile.

Hazelnut is among the most important nut crops in Chile, currently covering 46,000 ha. In 2023, the country exported 30,000-ton. In recent years the incidence of plants with internal discoloration, cankers and dieback has been increasing. In some cases, the trees died and had to be removed and, after a year, purple resupinate fruiting bodies were observed growing from the stumps. To determine the etiology of the symptoms and signs, wood samples (n=318) were collected since 2020, from 38 symptomatic orchards from Maule to La Araucanía Regions, primarily from the cvs. Tonda di Giffoni and Lewis. Wood sections 0.5 cm diameter were cut from the symptomatic tissues, disinfected using a sodium hypochlorite (10%) solution, and plated on a quarter-strength acidified potato dextrose agar (aPDA1/4). The plates were incubated and purified on PDA. Subsequently, isolates were identified by morphological and molecular means. Almost half of the isolates (47%) were preliminarily identified as basidiomycetes, based on mycelial features such as the presence of clamp connections, with 45% of them exhibiting abundant whitish cottony fast-growth mycelia, resembling Chondrostereum purpureum (Grinbergs et al., 2020). DNA was extracted and the 500-bp fragment, located between 5S and 18S ribosomal regions, was amplified using APN1 specific primers (Becker et al. 1999), identifying the isolates as C. purpureum. In addition, 5.8S gene of RGM1 (35°13'40.9"S 71°25'14.1"W), RGM2 (36°31'27.95"S 71°46'58.31"W), RGM3 (37°10'54.8"S 72°03'39.6"W), RGM4 (35°19'25.2"S 71°19'54.7"W) and RGM5 (36°35'30.8"S 72°05'18.8"W) isolates, representing different locations within the hazelnut growing area, was amplified using ITS1/ITS4 primers (White et al., 1990). The PCR product was sequenced, and the analysis showed 100% homology among isolates (Genebank codes: PP839283, PP839284, PP839285, PP839286 and PP839287, respectively). To determine the pathogenicity of the isolates, 30-cm healthy cuttings cv. Lewis were inoculated with mycelial plugs, while control shoots were inoculated with sterile agar plugs. Cuttings were vertically arranged in pots with 3-cm water and incubated for 60-d at 22°C. In addition, fresh cuts of 3-y potted plants cv. Lewis were inoculated with mycelial plugs and incubated for 137-d in a shadehouse. After incubation, bark was removed from inoculated cuttings and the length of necrotic lesions was measured. Although discoloration was reproduced by all the isolates in both pathogenicity tests, RGM1 isolate was the most aggressive, causing the complete discoloration of the cuttings and the death of the inoculated plants. To our knowledge this is the first report of C. purpureum causing wood disease in hazelnut. These findings are significant because the disease may not only reduce orchard longevity but also decrease fruit yield and quality, as observed in other fruit crops (Grinbergs et al., 2021).

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.

Occurrence of Alternaria alternata and A. tenuissima causing black rot in cherry fruits (Prunus avium) in Central Chile.

During the harvest of 2020 and 2021, sweet cherry (Prunus avium) fruit showed a firm rot with irregular pale to dark brown lesions on the fruit surface, with green to light brown fungal growth resembling Alternaria-like infection (Simmons, 2007). Diseased cherries (n= 80 fruit) were collected at harvest in mature (over 10-year-old) commercial orchards of cherry tree varieties Lapins, Regina, Santina, Skeena, and Sweetheart planted in four localities of the regions O´Higgins (33°59´ S, 70°42´W; San Francisco de Mostazal and Graneros) and Maule (35°00'S, 71°23´W; Curicó and Sagrada Familia), Central Chile. The incidence of black rot was 1.9 and 3.2% in O´Higgins and Maule region, respectively, and it was increased to up to 5% during cold storage. The fruit collected previously, were transported to the lab, and surface disinfected in 75% ethanol for 15 s, and rinsed in sterile water. Internal pieces from the junction of diseased and healthy tissues of fruits were placed on potato dextrose agar (PDA, 2%) for 7 days at 20°C. Forty-two isolates of Alternaria-like (Simmons, 2007) were recovered consistently from pure cultures taking hyphal tips from 7 days old cultures. On PDA, 28 isolates (group A) were characterized by cottony, white-gray to green colonies and conidial chains (4 to 10 conidia) with secondary chains (1 to 5 conidia) branching on the conidiophore. Conidia were ovate to obclavate (mean 22.8 ± 5.1 x 8.8 ± 1.5 µm; n=40) with 3 to 7 transepta and 1 longisepta. The remaining 14 isolates (group B) were characterized by cottony, olive-green to olive-brown colonies following a ring pattern of growth and white margins, with conidial chains (4 to 14 conidia) and uncommon secondary chains (1 to 4 conidia). Conidia were obpyriform to ovate, light brown to brown with a cylindrical short beak at the tip (mean 24.7 ± 5.9 × 11.2 ± 1.3 µm; n=40) with 2 to 4 transepta, and 0 to 2 longisepta. Two representative isolates of group A (Sant-02-2020 and Bing-03-2020) and group B (Sant-26-2021 and Skeen-43-2021) were amplified for the Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), and calmodulin (Cal) loci following the protocols described by Hong et al. (2005) and Lawrence et al. (2013). A MegaBlast search of sequences of group A (GenBank nos. OR267293- OR267294, OR258001- OR258002, and OR267297- OR267298, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains UCD10529 and UCD10539 of A. alternata, and group B (GenBank nos. OR267295- OR267296, OR258003- OR258004, and OR258005- OR258006, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains EGS 34-015 and A30 of A. tenuissima. Combined phylogenetic analysis using MEGA X clustered isolates Sant-02-2020 and Bing-03-2020, and Sant-26-2021 and Skeen-43-2021 with ex-type of A. alternata and A. tenuissima, respectively. Pathogenicity tests were conducted using isolates of A. alternata (Sant-02-2020; Bing-03-2020) and A. tenuissima (Sant-26-2021; Skeen-43-2021). Detached ripe cherry fruit var. Sweetheart (n=40 fruits/isolate) and Regina (n=40 fruits/isolate) were surfaces disinfested (75% ethanol, 30 s), wounded in the middle with a sterile needle (2 mm in depth), and inoculated with 20 µL of conidial suspension (106 conidia/mL). An equal number of healthy cherries (n=40 fruits) treated with sterile water were used as controls. The experiment was repeated once. All inoculated fruit incubated for 7 days at 22°C, developed between 13 ± 2.7 to 23 ± 2.5 mm and 14.1 ± 1.1 to 19 ± 3.6 mm in lesion diameter for A. alternata and A. tenuissima isolates, respectively. Koch´s postulates were fulfilled by 100% reisolation of the causal pathogen from inoculated fruit, and molecular identification of A. alternata and A. tenuissima isolates. Previously, A. alternata has been described as causing rots on cherries in Chile (Acuña 2010), and China (Zhao and Liu, 2012; Ahmad et al., 2020). To our knowledge, this is the first occurrence of cherry black rot caused by A. alternata and A. tenuissima in Central Chile. Epidemiological studies are necessary to develop integrated management of cherry black rot in Central Chile.

Inflammatory biomarkers and pendelluft magnitude in ards patients transitioning from controlled to partial support ventilation.

The transition from controlled to partial support ventilation is a challenge in acute respiratory distress syndrome (ARDS) patients due to the risks of patient-self-inflicted lung injury. The magnitude of tidal volume (VT) and intrapulmonary dyssynchrony (pendelluft) are suggested mechanisms of lung injury. We conducted a prospective, observational, physiological study in a tertiary academic intensive care unit. ARDS patients transitioning from controlled to partial support ventilation were included. On these, we evaluated the association between changes in inflammatory biomarkers and esophageal pressure swing (ΔPes), transpulmonary driving pressure (ΔPL), VT, and pendelluft. Pendelluft was defined as the percentage of the tidal volume that moves from the non-dependent to the dependent lung region during inspiration, and its frequency at different thresholds (- 15, - 20 and - 25%) was also registered. Blood concentrations of inflammatory biomarkers (IL-6, IL-8, TNF-α, ANGPT2, RAGE, IL-18, Caspase-1) were measured before (T0) and after 4-h (T4) of partial support ventilation. Pendelluft, ΔPes, ΔPL and VT were recorded. Nine out of twenty-four patients (37.5%) showed a pendelluft mean ≥ 10%. The mean values of ΔPes, ΔPL, and VT were - 8.4 [- 6.7; - 10.2] cmH2O, 15.2 [12.3-16.5] cmH2O and 8.1 [7.3-8.9] m/kg PBW, respectively. Significant associations were observed between the frequency of high-magnitude pendelluft and IL-8, IL-18, and Caspase-1 changes (T0/T4 ratio). These results suggest that the frequency of high magnitude pendelluft may be a potential determinant of inflammatory response related to inspiratory efforts in ARDS patients transitioning to partial support ventilation. Future studies are needed to confirm these results.

Occurrence of leaf rust disease in Oxalis triangularis caused by Puccinia oxalidis in Valdivia, Chile.

False shamrock (Oxalis triangularis), native to South America, is an ornamental and popular plant bulb, commercialized for their attractive shape and color (purple triangular leaves) (Taha et al., 2013). In Chile, a rust was detected in O. triangularis plants growing from April to June in several gardens (n=10) in the city of Valdivia, estimating a disease incidence between 80 and 100%. The symptoms appeared as diffuse chlorotic spots from the upperside of leaves, where infected tissues eventually become completely necrotic, and yellow rust pustules were observed on the underside of leaves. Severe symptoms on infected leaves were consistently observed, showing necrosis on entire leaves. Symptomatic plants (n=50) were collected, and three representative isolates from different localities (OX1, OX2, and OX3) were used for morphological and genetic identification. Uredinia (n=20) were hypophyllous, erumpent, yellow, and irregularly distributed, with sizes from 340 to 850 µm in diameter. Urediniospores (n=150) were yellow, subglobose to globose, equinulate, with measures of 14.2 - 17.7 x 14.7 - 17.2 µm. Teliospores were absent. Based on morphological characters, this rust was identified as a Puccinia sp. These morphological characteristics coincided with those indicated by Safránková (2014), Abbasi et al. (2018), and Khouader et al. (2018). To classify this rust genetically, sequences analyses were performed using the ITS region of the rDNA (ITS4/ITS5) (White et al., 1990). The DNA was extracted using a commercial kit. The results indicated 99% similarity with two reference sequences of P. oxalidis (MH325473 and MH325474) available at GenBank (NCBI http://www.ncbi.nlm.nih.gov/BLAST/). The sequences obtained were deposited in GenBank (ON259085 to ON259087). Based on the maximum parsimony phylogenetic tree, the sequences of Chilean isolates were clustered with those of P. oxalidis references. Pathogenicity tests were conducted using three isolates (OX1 to OX3). Surface disinfection of leaves of O. triangularis (n=36 plants), were performed by spraying 1% NaOCl solution for 1 min. Subsequently, 2 mL of urediniospores suspensions of each isolate (OX1 to OX3) at a concentration of 106 urediniospores/mL, were sprayed with an atomizer on the underside of the leaves of all plants. Urediniospores were obtained following the methodology proposed by Ferrada et al. (2020). Control leaves were disinfested and inoculated with sterile distilled water. Plants of O. triangularis of 90-day-old were incubated in a humid chamber (24°C, 80% HR), with a photoperiod of 12 light /12 dark. At 11 days post-inoculation, all leaves inoculated developed chlorosis spots and pulverulent pustules (averaged 10.9 to 25.4 pustules per leaf), and then at 26 days post-inoculation, affected leaves showed necrotic tissues. The identity of these isolates was confirmed morphologically. The symptoms in the control leaves were negative. To our knowledge, this is the first report of multiple occurrences of the leaf rust disease on gardens of false shamrock caused by P. oxalidis in Valdivia, south of Chile. Previously, P. oxalidis has been reported to cause leaf rust disease in O. triangularis in the Czech Republic (Safránková, 2014) and O. debilis in Korea (Lee et al., 2018). The leaf rust disease could represent a threat to the ornamental gardens of O. triangularis in Valdivia. Currently, epidemiological studies of leaf rust disease are necessary to develop management strategies in gardens of O. triangularis.

Dothiorella sarmentorum Causing Branch Dieback of English Walnut in Maule Region, Chile.

English walnut (Juglans regia), cv. Chandler is the most cultivated tree nut in Chile, with 43,734 ha. In Maule Region, central Chile, English walnut plantings have expanded over an additional 7,000 ha in the last five years. During a routine orchard survey in 2019, branch and twig dieback symptoms were observed in two commercial orchards located in San Rafael (10 years old) and Longaví (12 years old) in the Maule Region, with an incidence of 45% to 65% of affected trees, respectively. Symptomatic branch samples (n = 15) were collected from the two commercial orchards and transported to the laboratory in a cooler and then surface sterilized in 96% ethanol for 3 s and briefly flamed. Cross-section of symptomatic branches revealed brown to dark-brown wedge-shaped wood cankers. Small (5 mm) pieces of wood from the edge of cankered tissues were placed on Potato Dextrose Agar (PDA, 2%) amended with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (PDAm) (Díaz and Latorre 2014) and incubated at 25°C for five days in the dark. Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Each fungal isolate was recovered from a single diseased branch (47%). Seven isolates (Dsar-1 to Dsar-7) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown pycnidia after 15 days at 25°C. Conidia were hyaline and aseptate, dark brown, 1-septate, with a brown wall, ovoid with a broadly rounded apex and truncated base, (17.5-) 19.5 ±1.2 (-22.0) x (7.6-) 8.9 ± 0.6 (-10.1) µm (n = 30). These isolates were tentatively identified morphologically as Dothiorella sp. (Phillips et al. 2005). Molecular identification was performed using ITS1/ITS4 and EF1-728F/EF1-986R primers (White et al. 1990; Dissanayake et al. 2015) of the internal transcribed spacer (ITS1-5.8S-ITS2) region and part of the translation elongation factor (EF1-) genes, respectively. A MegaBlast search in GenBank showed a 100% similarity to isolate CBS 115038, the ex-type of Dothiorella sarmentorum. The sequences were added to GenBank (OM161950 to OM161956 for ITS; OM177188 to OM177194 for EF1-). Pathogenicity of two isolates (Dsar-2 and Dsar-7) was tested in the orchard on freshly made pruning wounds on attached branches of 2-year-old-pruned English walnut trees cv. Chandler. A second pathogenicity test was done on freshly made pruning wounds in 1-year-old rooted cuttings (n=15) (40 cm of long) of English walnut cv. Chandler. Each pruning wound was inoculated with 40 µL conidial suspension (105 conidia/mL). Sterile distilled water was used as a control treatment. Both pathogenicity tests were repeated once. After seven months for attached branches and four months for rooted plants, necrotic streaks with a mean length of 81.3 and 44.5 mm were observed below the inoculated pruning wounds, respectively. No necrotic streaks were observed in any of the control wounds. Dothiorella sarmentorum was 100% reisolated from symptomatic tissues of inoculated branches and molecularly identified (EF1-), thus fulfilling Koch's postulates. Recently, D. sarmentorum has been reported causing English walnut dieback in Spain (López-Moral et al. 2020). To our knowledge, this is the first report of D. sarmentorum causing branch dieback of English walnut in Chile. Further studies are needed to know the impact and extent of canker and branch dieback of walnut in commercial orchards in the Maule Region, central Chile.

Severe Outbreak of Dry Core Rot in Apple Fruits cv. Fuji Caused by Kalmusia variispora During Pre-harvest in Maule Region, Chile.

Apple (Malus × domestica) is an important fruit crop in Chile, with a cultivated area of 32,313 ha concentrated (63%) in Maule Region (35°25' S). Unusual core rot on 'Fuji' apples was observed at harvest in a commercial orchard in Curicó, Maule Region, with an incidence ranged between 22 to 35% in 2018 and 2019. Previously, in 2017, an incidence of 30% was estimated on 'Fuji' fruits destined to the Asian market. Internal decay symptoms consisted of dry, corky light to dark-brown tissue, within the seed locules initially. In moderate to severe cases, the necrotic lesion progresses deeper into the mesoderm. External symptoms were quite subtle, and typically, the disease goes unnoticed. However, infected fruit ripen earlier. Small pieces (2-3 mm) from the internal lesion margin of symptomatic apples (n = 50) were placed on Potato Dextrose Agar (PDA) (2%) and incubated at 20°C for 10 days. Pure cultures (n = 41) were obtained and transferred to Malt Extract Agar (MEA) (2%). Colonies on MEA produced an even to slight undulating buff margin with white woolly aerial mycelium, and immerse ochreous in the center, changing gray to olivaceous aerial mycelium with age. On the underside, colonies were umber and buff in the center and margin, respectively. After 10 days, numerous densely aggregate dark-brown mature pycnidia were observed. Aseptate conidia were subglobose to cylindrical, straight, and sometimes curved with rounded at both ends, that was initially hyaline to pale olive, thin, smooth wall with mean dimensions of (2.9-) 3.4 (-4.4) x (1.5-) 1.8 (2.2) µm (n=50). Based on morphology, the fungus was identified as Kalmusia variispora (Verkley et al. 2014). The internal transcribed space (ITS), portion of ß-tubulin (TUB), and large subunits of the nuclear ribosomal RNA (LSU) loci were used for molecular identification, using primers ITS4/ITS5, Bt2a/Bt2b, and LR0R/LR5 (Ariyawansa et al., 2014). BLAST searches indicated 100% identity with K. variispora (ex-type CBS 121517). The maximum parsimony phylogenetic analysis placed Chilean isolates in the K. variispora clade. Sequences were deposited in GenBank (OL711706 to OL711709, OL739499 to OL739502 and OL711710 to OL711713 for ITS, TUB and LSU, respectively). Pathogenicity tests were conducted using four K. variispora isolates. 'Fuji' apples (n = 20) were surface disinfested (75% ethanol, 30 s) and then wounded and inoculated with conidial suspension (50 L of 106 conidia/mL) deposited in the middle and into the core region using a sterile fine-tipped micropipette. Additionally, 20 one-year dormant rooted cuttings 'Fuji' and 'Cripps Pink' were pruned and immediately inoculated on the pruning wound. An equal number of apples and rooted cuttings treated with sterile water were used as controls. The experiments were repeated once. All inoculated fruits developed lateral lesions (22 to 37 mm) and dry core rot (18 to 36 mm) symptoms identical to those described in the original outbreak, after 20 days at 20°C in a commercial packing box. The inoculated cuttings produced canker lesions of 10 to 21 mm in length, and dieback symptoms were observed after 3 months. No symptoms were observed on the negative controls. Koch's postulates were fulfilled by 100% reisolating K. variispora. Previously, Alternaria spp. have been reported as the primary pathogen associated with moldy core and dry core rot of apples worldwide (McLeod et al., 2014) and in Chile (Elfar et al., 2018). However, Kalmusia spp. have been associated with dry core rot in apples (McLeod et al., 2014) and have been isolated from canker symptoms on apples in Chile (Díaz et al. 2021). To our knowledge, this is the first report of a severe outbreak of K. variispora causing dry core rot in apples in Chile and worldwide.

Characterization and Pathogenicity of Diplodia, Lasiodiplodia, and Neofusicoccum Species Causing Botryosphaeria Canker and Dieback of Apple Trees in Central Chile.

In recent years, the number of apple trees affected by Botryosphaeria cankers and dieback has considerably increased in central Chile. This study aimed to identify the species of Botryosphaeriaceae associated with canker and dieback symptoms, estimate disease incidence and distributions, and study their pathogenicity and virulence on apple and other fruit crops. A field survey of 34 commercial orchards of apple (7 to 30 years old) was conducted in 16 localities, obtaining 270 symptomatic branch and trunk samples in 2017 and 2018 growing seasons. The incidence of Botryosphaeria canker and dieback ranged between 5 and 40%, and a total of 255 isolates of Botryosphaeriaceae spp. were obtained from 238 cankers. Morphological identification along with phylogenetic studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the translation elongation factor 1-α (tef1-α), and part of the ß-tubulin (tub2) genes allowed us to identify Diplodia mutila (n = 49 isolates), D. seriata (n = 136 isolates), Lasiodiplodia theobromae (n = 16 isolates), and Neofusicoccum arbuti (n = 54 isolates). L. theobromae was isolated mainly from apple dieback from northern localities. All pathogens tested were pathogenic, causing canker and dieback symptoms on lignified twigs of apple, pear, walnut, and green grapevine shoots in the field. Isolates of N. arbuti were the most virulent, reproducing more severe cankers on the lignified tissues inoculated. This study reports, for the first time, D. mutila and L. theobromae associated with Botryosphaeria canker and dieback in Chile, and it is the first description of N. arbuti causing apple dieback worldwide.

Duration of the susceptibility of pruning wounds of different ages to infections by Phaeomoniella chlamydospora on grapevine cv. Cabernet Sauvignon in Central Chile.

Grapevine trunk diseases (GTDs) are one of the most important phytosanitary problems that affect grapevines (Vitis vinifera) worldwide. In Chile, Phaeomoniella chlamydospora is the major fungal trunk pathogen associated with GTDs. In the vineyards, the natural infections by P. chlamydospora are associated with air-borne conidia dispersed onto fresh pruning wounds from pycnidia. These pruning wounds are considered an important entrance for fungal trunk pathogens such as P. chlamydospora in the host in the field. However, the duration of the susceptibility of grapevine annual pruning wounds to P. chlamydospora is still unknown in Chile. Therefore, this study aimed to evaluate the period of susceptibility of pruning wounds of different ages to artificial infection of P. chlamydospora on grapevine cv. Cabernet Sauvignon, Central Chile. Artificial inoculations of a conidial suspension (105 conidia/mL) of P. chlamydospora were used to determine the susceptibility of pruning wounds of different ages, from 1, 15, 30, and 45 days after pruning. The experiments were conducted on lignified cuttings in a greenhouse, and on vine spurs in two vineyards (Buin and Nancagua, Central Chile) during two consecutive seasons. The results indicated that the pruning wounds of grapevine cv. Cabernet Sauvignon were very susceptible to infections by P. chlamydospora, with a percentage of pruning wounds infected from 97 to 71% for cuttings, and 96% to 60% for spurs, during the first 15 days after pruning. However, the susceptibility of pruning wounds of different ages in cuttings and spurs of grapevine, generally decreased as the time from pruning to inoculation increased. Moreover, the pruning wounds the pruning wounds remained susceptible to artificial inoculation by P. chlamydospora for up 45 days after pruning with percent of wounds infected from 8.0 to 12.2, and 8.3 to 18.8% on cuttings and spurs of grapevine, respectively. Finally, this study constitutes study constitutes the first research focalized on the susceptibility of pruning wounds of various ages of grapevine cv. Cabernet Sauvignon to artificial inoculations by P. chlamydospora in Central Chile.

Clinical characteristics and in-hospital mortality of patients with COVID-19 in Chile: A prospective cohort study.

AIMS OF THIS STUDY: To describe the Latin American population affected by COVID-19, and to determine relevant risk factors for in-hospital mortality. METHODS: We prospectively registered relevant clinical, laboratory, and radiological data of adult patients with COVID-19, admitted within the first 100 days of the pandemic from a single teaching hospital in Santiago, Chile. The primary outcome was in-hospital mortality. Secondary outcomes included the need for respiratory support and pharmacological treatment, among others. We combined the chronic disease burden and the severity of illness at admission with predefined clinically relevant risk factors. Cox regression models were used to identify risk factors for in-hospital mortality. RESULTS: We enrolled 395 adult patients, their median age was 61 years; 62.8% of patients were male and 40.1% had a Modified Charlson Comorbidity Index (MCCI) ≥5. Their median Sequential Organ Failure Assessment (SOFA) score was 3; 34.9% used a high-flow nasal cannula and 17.5% required invasive mechanical ventilation. The in-hospital mortality rate was 14.7%. In the multivariate analysis, were significant risk factors for in-hospital mortality: MCCI ≥5 (HR 4.39, P < .001), PaO2 /FiO2 ratio ≤200 (HR 1.92, P = .037), and advanced chronic respiratory disease (HR 3.24, P = .001); pre-specified combinations of these risk factors in four categories was associated with the outcome in a graded manner. CONCLUSIONS AND CLINICAL IMPLICATIONS: The relationship between multiple prognostic factors has been scarcely reported in Latin American patients with COVID-19. By combining different clinically relevant risk factors, we can identify COVID-19 patients with high-, medium- and low-risk of in-hospital mortality.

Severe outbreak of Fusarium wilt on common beans (Phaseolus vulgaris) caused by Fusarium oxysporum in the Maule Region, central Chile.

Common bean (Phaseolus vulgaris L.) is an important grain legume cultivated worldwide as food for humans and livestock (Schwartz et al., 2005). Common beans in central Chile reach up to 3,893 ha from which 1,069 ha are located in the Maule region. Common bean is produced by small farmers who have limited access to fertilization, technical irrigation, and crop protection. In spring 2018, bean plants initially showed a slight yellowing and premature senescence 50 days after sowing (das) until showing wilting symptoms (70 -100 das) in Curepto fields (35 05'S; 72 01'W), Maule region. The basal part of affected plants displayed internal reddish-brown discoloration of the vascular tissues. Based on the plant external symptoms, we estimated an incidence between 15% and 45% in bean fields. Nine symptomatic plants were collected, and surface washed with sterile water and disinfested with 75% ethanol (v/v). Then small fragments (5-mm) from damage vascular tissue from each plant were cut and placed on Petri dishes containing PDA acidified with 0.5 ml/l of 92% lactic acid (APDA, 2%). The isolations were incubated for seven days at 25°C. Nine Fusarium-like isolates from single-spore on APDA (2%) became pale vinaceous, floccose with abundant aerial mycelium and dark vinaceous reverse colony, with a growing rate of 10.8 to 11.6 mm/d at 25°C (Lombard et al., 2019). Phialides were short, singular growing laterally on the mycelium. Macroconidia were hyaline, fusiform with basal foot cells shaped to pointed and apical cells tapered, 2-5 septate, and 28.6 to 47.6 (av. 38.1) µm long x 2.2 to 3.6 (av. 3.1) µm wide. Microconidia were hyaline, oval to ellipsoid, one-celled, and 4.5 to 10.9 (av. 6.1) µm long and 2.2 to 3.3 (av. 2.7) µm wide (n=50 spore). For molecular identification, three isolates (Curi-3.1, Be-8.1, and Be-11.3) were sequenced using PCR amplification of the partial sequences of beta-tubulin (BT) and translation elongation factor 1-α gene (TEF) (Lombard et al., 2019). NCBI BLAST analysis showed 99 to 100% similarity with sequences (TEF; BT) of strain CPC 25822 of Fusarium oxysporum. The maximum-likelihood phylogenetic analysis placed the Chilean isolates in the F. oxysporum complex clade. Chilean sequences were deposited into GenBank under accession numbers MW419125, MW419126, MW419127 (TEF) and MW419128, MW419129, MW419130 (BT). Pathogenicity tests (isolates Curi-3.1, Be-8.1, and Be-11.3) were conducted under greenhouse (15-28°C, 85%RH) on healthy bean plants (n=30) cv. Blanco Español INIA cultivated in pots (sand/peat moss/soil) at the University of Talca. Plants that are 30 days-old were inoculated using 200 µl of conidial suspension (106 conidia/ml) on wounded roots (crown). Control plants (n=10) were similarly inoculated with sterile distilled water. After 45 days, all inoculated plants with F. oxysporum isolates developed necrotic lesions on vascular tissue, and chlorosis, and wilting while control plants remained healthy. This experiment was conducted twice. The pathogen was reisolated (100%) from diseased plants and molecularly identified as F. oxysporum. To our knowledge, this is the report of a severe outbreak of F. oxysporum causing Fusarium yellows in P. vulgaris in the Maule region, Chile. Previously, F. oxysporum has been reported affecting tomato (Sepúlveda-Chavera et al., 2014) and blueberry in Chile (Moya-Elizondo et al., 2019).

Phytohormones Trigger Drought Tolerance in Crop Plants: Outlook and Future Perspectives.

In the past and present, human activities have been involved in triggering global warming, causing drought stresses that affect animals and plants. Plants are more defenseless against drought stress; and therefore, plant development and productive output are decreased. To decrease the effect of drought stress on plants, it is crucial to establish a plant feedback mechanism of resistance to drought. The drought reflex mechanisms include the physical stature physiology and biochemical, cellular, and molecular-based processes. Briefly, improving the root system, leaf structure, osmotic-balance, comparative water contents and stomatal adjustment are considered as most prominent features against drought resistance in crop plants. In addition, the signal transduction pathway and reactive clearance of oxygen are crucial mechanisms for coping with drought stress via calcium and phytohormones such as abscisic acid, salicylic acid, jasmonic acid, auxin, gibberellin, ethylene, brassinosteroids and peptide molecules. Furthermore, microorganisms, such as fungal and bacterial organisms, play a vital role in increasing resistance against drought stress in plants. The number of characteristic loci, transgenic methods and the application of exogenous substances [nitric oxide, (C28H48O6) 24-epibrassinolide, proline, and glycine betaine] are also equally important for enhancing the drought resistance of plants. In a nutshell, the current review will mainly focus on the role of phytohormones and related mechanisms involved in drought tolerance in various crop plants.

Characterization of Botrytis cinerea and B. prunorum From Healthy Floral Structures and Decayed 'Hayward' Kiwifruit During Post-Harvest Storage.

Gray mold is the primary postharvest disease of 'Hayward' kiwifruit (Actinidia deliciosa) in Chile, with a prevalence of 33.1% in 2016 and 7.1% in 2017. Gray mold develops during postharvest storage, which is characterized by a soft, light to brown watery decay that is caused by Botrytis cinerea and B. prunorum. However, there is no information on the role of B. prunorum during the development and storage of kiwifruit in Chile. For this purpose, asymptomatic flowers and receptacles were collected throughout fruit development and harvest from five orchards over two seasons in the Central Valley of Chile. Additionally, diseased kiwifruits were selected after storage for 100 days at 0°C and 2 days at 20°C. Colonies of Botrytis sp. with high and low conidial production were consistently obtained from apparently healthy petals, sepals, receptacles, and styles and diseased kiwifruit. Morphological and phylogenetic analysis of three partial gene sequences encoding glyceraldehyde-3-phosphate dehydrogenase, heat shock protein 60, and DNA-dependent RNA polymerase subunit II were able to identify and separate B. cinerea and B. prunorum species. Consistently, B. cinerea was predominantly isolated from all floral parts and fruit in apparently healthy tissue and diseased kiwifruit. During full bloom, the highest colonization by B. cinerea and B. prunorum was obtained from petals, followed by sepals. In storage, both Botrytis species were isolated from the diseased fruit (n = 644), of which 6.8% (n = 44) were identified as B. prunorum. All Botrytis isolates grew from 0°C to 30°C in vitro and were pathogenic on kiwifruit leaves and fruit. Notably, B. cinerea isolates were always more virulent than B. prunorum isolates. This study confirms the presence of B. cinerea and B. prunorum colonizing apparently healthy flowers and floral parts in fruit and causing gray mold during kiwifruit storage in Chile. Therefore, B. prunorum plays a secondary role in the epidemiology of gray mold developing in kiwifruit during cold storage.

Identification and Pathogenicity of Diplodia, Neofusicoccum, Cadophora, and Diaporthe Species Associated with Cordon Dieback in Kiwifruit cultivar Hayward in Central Chile.

Dieback symptoms associated with fungal trunk pathogens cause significant economic losses for farmers of kiwifruit and other woody fruit trees worldwide. This study represents the first attempt to identify and characterize the fungal trunk pathogens associated with cordon dieback disease of kiwifruit in central Chile. Field surveys were conducted throughout the main kiwifruit-growing regions in central Chile to determine the incidence and characterize the fungal trunk pathogens associated with cordon dieback of kiwifruit cultivar Hayward through morphological, molecular, and pathogenicity studies. A total of 250 cordon samples were collected and isolations were performed on 2% acidified potato dextrose agar (APDA) plus antibiotics and Igepal. The incidence of kiwifruit cordon dieback ranged between 5% and 85% in all surveyed areas in central Chile. A total of 246 isolates were isolated and identified using culture and morphological features belonging to three fungal taxa: Diaporthaceae spp. (Diaporthe ambigua and D. australafricana; n = 133 isolates); Botryosphaeriaceae spp. (Diplodia seriata and Neofusicoccum parvum; n = 89 isolates); and Ploettnerulaceae spp. (Cadophora luteo-olivacea and C. malorum; n = 24 isolates). These were identified using phylogenetics studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the ß-tubulin gene (tub2), and part of the translation elongation factor 1-α gene (tef1-α). Isolates of N. parvum and D. seriata were the most virulent, causing internal brown lesions and dieback symptoms in attached green shoots, attached lignified canes, and young inoculated kiwifruits. This report is the first to describe D. seriata and C. luteo-olivacea associated with kiwifruit cordon dieback in Chile. It presents the first description of N. parvum causing kiwifruit dieback worldwide.

Biocontrol Potential of Grapevine Endophytic and Rhizospheric Fungi Against Trunk Pathogens.

Grapevine Trunk Diseases (GTDs) are a major challenge to the grape industry worldwide. GTDs are responsible for considerable loss of quality, production, and vineyard longevity. Seventy-five percent of Chilean vineyards are estimated to be affected by GTDs. GTDs are complex diseases caused by several fungi species, including members of the Botryosphaeriaceae family and Phaeomoniella chlamydospora, considered some of the most important causal agents for these diseases in Chile. In this study, we isolated 169 endophytic and 209 rhizospheric fungi from grapevines grown under organic and conventional farming in Chile. Multiple isolates of Chaetomium sp., Cladosporium sp., Clonostachys rosea, Epicoccum nigrum, Purpureocillium lilacinum, and Trichoderma sp. were evaluated for their potential of biocontrol activity against Diplodia seriata, Neofusicoccum parvum, and Pa. chlamydospora. Tests of antagonism were carried out using two dual-culture-plate methods with multiple media types, including agar containing grapevine wood extract to simulate in planta nutrient conditions. Significant pathogen growth inhibition was observed by all isolates tested. Clonostachys rosea showed 98.2% inhibition of all pathogens in the presence of grapevine wood extract. We observed 100% pathogen growth inhibition when autoclaved lignified grapevine shoots were pre-inoculated with either C. rosea strains or Trichoderma sp. Overall, these results show that C. rosea strains isolated from grapevines are promising biocontrol agents against GTDs.

Identification and Characterization of Diaporthe ambigua, D. australafricana, D. novem, and D. rudis Causing a Postharvest Fruit Rot in Kiwifruit.

Diaporthe spp. are important plant pathogens causing wood cankers, blight, dieback, and fruit rot in a wide range of hosts. During surveys conducted during the 2013 and 2014 seasons, a postharvest rot in Hayward kiwifruit (Actinidia deliciosa) was observed in Chile. In order to identify the species of Diaporthe associated with this fruit rot, symptomatic fruit were collected from seven kiwifruit packinghouses located between San Francisco de Mostazal and Curicó (central Chile). Twenty-four isolates of Diaporthe spp. were identified from infected fruit based on morphological and cultural characters and analyses of nucleotides sequences of three loci, including the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequences of the ß-tubulin, and translation elongation factor 1-α genes. The Diaporthe spp. identified were Diaporthe ambigua, D. australafricana, D. novem, and D. rudis. Multilocus phylogenetic analysis revealed that Chilean isolates were grouped in separate clades with their correspondent ex-types species. All species of Diaporthe were pathogenic on wounded kiwifruit after 30 days at 0°C under normal and controlled-atmosphere (2% O2 and 5% CO2) storage and they were sensitive to benomyl, pyraclostrobin, and tebuconazole fungicides. D. ambigua isolates were the most virulent based on the lesion length measured in inoculated Hayward and Jintao kiwifruit. These findings confirm D. ambigua, D. australafricana, D. novem, and D. rudis as the causal agents of kiwifruit rot during cold storage in Chile. The specie D. actinidiae, a common of Diaporthe sp. found associated with kiwifruit rot, was not identified in the present study.

Infection Caused by Phaeomoniella chlamydospora Associated with Esca-like Symptoms in Grapevine in Chile.

Trunk diseases such as esca have been recognized as an economically important problem of grapevine worldwide. A study was conducted to characterize the distribution of Phaeomoniella chlamydospora in Chile. A field survey of young and mature grapevines from 67 vineyards located along a 1,315-km north-south axis demonstrated that P. chlamydospora was present in 94.9% of the grapevine samples showing the black-wood streaking symptom (BWS) but not the characteristic foliar symptoms of esca. Phylogenetic analysis of the ribosomal DNA internal transcribed spacer (ITS) combined with ß-tubulin (BT) genes grouped Chilean isolates together with reference isolates from South Africa and the United States, whereas Spanish isolates were clustered separately. Chilean isolates differed by only 2 to 3 bp for BT and ITS, respectively. Conidia germinated at 5 to 35°C, with an optimal temperature of 25 to 30°C. Isolates were pathogenic, and Koch's postulates were fulfilled in separate sets of inoculations of axenic plantlets, cuttings, 2-year-old plants, spurs, and shoots of V. vinifera. This study showed that P. chlamydospora was associated consistently with BWS and no other apparent symptom in young and mature grapevines, including nursery plants, in Chile. Inoculum was absent from the soil, grapevine pruning debris, sap samples, and herbaceous weeds, which is in contrast to past studies. At this time, Vitis spp. are the only known hosts of P. chlamydospora in Chile.

Effects of prone positioning on lung protection in patients with acute respiratory distress syndrome.

RATIONALE: Positive end-expiratory pressure (PEEP) and prone positioning may induce lung recruitment and affect alveolar dynamics in acute respiratory distress syndrome (ARDS). Whether there is interdependence between the effects of PEEP and prone positioning on these variables is unknown. OBJECTIVES: To determine the effects of high PEEP and prone positioning on lung recruitment, cyclic recruitment/derecruitment, and tidal hyperinflation and how these effects are influenced by lung recruitability. METHODS: Mechanically ventilated patients (Vt 6 ml/kg ideal body weight) underwent whole-lung computed tomography (CT) during breath-holding sessions at airway pressures of 5, 15, and 45 cm H2O and Cine-CTs on a fixed thoracic transverse slice at PEEP 5 and 15 cm H2O. CT images were repeated in supine and prone positioning. A recruitment maneuver at 45 cm H2O was performed before each PEEP change. Lung recruitability was defined as the difference in percentage of nonaerated tissue between 5 and 45 cm H2O. Cyclic recruitment/de-recruitment and tidal hyperinflation were determined as tidal changes in percentage of nonaerated and hyperinflated tissue, respectively. MEASUREMENTS AND MAIN RESULTS: Twenty-four patients with ARDS were included. Increasing PEEP from 5 to 15 cm H2O decreased nonaerated tissue (501 ± 201 to 322 ± 132 grams; P < 0.001) and increased tidal-hyperinflation (0.41 ± 0.26 to 0.57 ± 0.30%; P = 0.004) in supine. Prone positioning further decreased nonaerated tissue (322 ± 132 to 290 ± 141 grams; P = 0.028) and reduced tidal hyperinflation observed at PEEP 15 in supine patients (0.57 ± 0.30 to 0.41 ± 0.22%). Cyclic recruitment/de-recruitment only decreased when high PEEP and prone positioning were applied together (4.1 ± 1.9 to 2.9 ± 0.9%; P = 0.003), particularly in patients with high lung recruitability. CONCLUSIONS: Prone positioning enhances lung recruitment and decreases alveolar instability and hyperinflation observed at high PEEP in patients with ARDS.

Characterization of Diaporthe australafricana and Diaporthe spp. Associated with Stem Canker of Blueberry in Chile.

Stem canker and dieback are important factors that limit the longevity and reduce the yield of blueberry (Vaccinium spp.) in Chile. In this study, species of Diaporthe associated with blueberry were isolated and identified. The internal transcribed spacer (ITS) regions of ribosomal DNA of 30 isolates and the translation elongation factor 1-α (EF1-α) of 14 isolates were sequenced, analyzed, and compared with their morphological and pathological characteristics. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Diaporthe ambigua, D. australafricana, D. neotheicola, D. passiflorae, and Diaporthe sp. 1. However, morphology alone was insufficient to identify these species. The combined analysis of ITS and EF1-α gene sequences grouped the Chilean blueberry isolates in the same five groups obtained in the ITS analysis. Pathogenicity tests conducted with attached and detached blueberry shoots (<1 year old) and stems (1 to 2 years old) confirmed that isolates of these Diaporthe spp. were pathogenic. The symptoms were reproducible and consisted of necrotic reddish-brown cankers on blueberry shoots and stems. These isolates were capable of infecting blueberry fruit, causing a soft decay, suggesting that they were tissue nonspecific and were also pathogenic on shoots of apple, grapevine, and pear. D. australafricana was the most frequently isolated species and D. ambigua, D. australafricana, and D. passiflorae were highly virulent in shoots, stems, and fruit of blueberry. This study showed that at least four species of Diaporthe are primary pathogens, capable of causing stem canker symptoms on blueberry, and this is the first report of D. ambigua, D. neotheicola, and D. passiflorae attacking this host.