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.

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.

Characterization and Pathogenicity of Botryosphaeriaceae Species Obtained from Avocado Trees with Branch Canker and Dieback and from Avocado Fruit with Stem End Rot in Chile.

Several species of the Botryosphaeriaceae family have been associated with branch canker, dieback, and stem end rot in avocado (Persea americana Mill.). In Chile, the incidence of diseases affecting the avocado tree increased from 2011 to 2016, which coincided with a severe drought that affected avocado production. Moreover, distant countries importing avocados from Chile also reported an increase of stem end rot of ripe avocados. Therefore, the aims of this study were to identify the pathogen species associated with branch canker, dieback, and stem end rot of avocado in Chile and to study their pathogenicity. This study was conducted between 2015 and 2016 in 'Hass' avocado orchards located in the main avocado-producing regions in Chile. A diverse collection of fungal species was recovered from both necrotic woody tissue and necrotic tissue on harvested ripe fruit. On the basis of morphology and phylogenetic analyses of the internal transcribed spacer region (ITS1-5.8S-ITS2) and the translation elongation factor 1-α (TEF1-α) gene, eight species in the Botryosphaeriaceae family were identified: Diplodia mutila, D. pseudoseriata, D. seriata, Dothiorella iberica, Lasiodiplodia theobromae, Neofusicoccum australe, N. nonquaesitum, and N. parvum. For each of these species, pathogenicity studies were conducted on 1-year-old healthy Hass avocado plants. All isolates produced brown gum exudate and caused necrosis in the vascular system 3 weeks after inoculation. N. nonquaesitum, N. parvum, and D. pseudoseriata were the most virulent species. Necrotic lesions and cavities with white mycelia near the peduncle union were observed on Hass avocado fruit inoculated postharvest. L. theobromae, N. australe, and N. parvum were significantly more virulent than the other tested species in the Botryosphaeriaceae family. This study identified and characterized the pathogenicity of Botryosphaeriaceae species in Chile, which will prove useful to future research on these pathogens directed at establishing effective control strategies in avocado.

Identification and Characterization of Alternaria Species Associated with Moldy Core of Apple in Chile.

Moldy core (MC) of apple is an important disease in Chile, with prevalence observed between 4 and 46% in Fuji, Oregon Spur Red Chief, and Scarlet apple in the 2014-15 and 2015-16 growing seasons. However, there is no information on the identity of the causal agents associated with MC in Chile. The analysis of 653 MC fruit revealed the presence of several genera of filamentous fungi. However, species of Alternaria (67.7%) were by far the most frequently fungi isolated. In total, 41 Alternaria isolates were characterized morphologically and molecularly using Alternaria major allergen Alt a1, calmodulin, and plasma membrane ATPase gene regions. Six small-spored Alternaria spp. were identified; namely, in order of importance, Alternaria tenuissima, A. arborescens, A. alternata, and A. dumosa in sect. Alternaria; A. frumenti in sect. Infectoriae; and A. kordkuyana in sect. Pseudoalternaria. MC symptoms were reproducible and consisted of a light gray to dark olive-green mycelium over the carpel and seed of immature and mature fruit, confirming that the isolates of these Alternaria spp. were pathogenic. These isolates caused brown necrotic lesions with concentric rings on wounded detached apple leaves. This study demonstrated that at least six Alternaria spp. are the cause of MC of apple in Chile. These Alternaria spp. were isolated alone, or with two or more species coexisting in the same fruit. This is the first report of A. tenuissima, A. arborescens, A. frumenti, A. dumosa, and A. kordkuyana associated with MC of apple in Chile and the first report of A. frumenti, A. kordkuyana, and A. dumosa causing MC of apple worldwide.

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.

Identification and Characterization of Botrytis Blossom Blight of Japanese Plums Caused by Botrytis cinerea and B. prunorum sp. nov. in Chile.

Blossom blight is a destructive disease of plums (Prunus salicina) when humid and temperate weather conditions occur in Chile. Disease incidence ranging from 4 to 53% has been observed. Symptoms include light brown petal necrosis, starting as light brown mottles or V-shaped necrosis at the margins of the petals, progressing to the stamen and pistils. In this study, the etiology of blossom blight of plums was determined. High- and low-sporulating isolates of Botrytis were obtained consistently from blighted blossoms and apparently healthy flowers of plums. Based on colony morphology, conidial production and molecular phylogenetic analysis, these high- and low-sporulating isolates were identified as B. cinerea and B. prunorum sp. nov., respectively. Phylogenetic analysis of the genes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) grouped B. prunorum isolates in a single cluster, distantly from B. cinerea and other Botrytis species. The phylogenetic analysis of necrosis and ethylene-inducing protein (NEP1 and NEP2) genes corroborated these results. Analysis of the internal transcribed spacer region and large-subunit (26S) ribosomal DNA and detection of Boty and Flipper transposable elements, were not useful to differentiate between these Botrytis species. Both species were pathogenic on plum flowers and the fruit of plums, apples, and kiwifruits. However, B. prunorum was less virulent than B. cinerea. These pathogens were re-isolated from inoculated and diseased tissues; thus, Koch's postulates were fulfilled, confirming its role in blossom blight of plums. B. cinerea was predominant, suggesting that B. prunorum may play a secondary role in the epidemiology of blossom blight in plums in Chile. This study clearly demonstrated that the etiology of blossom blight of plums is caused by B. cinerea and B. prunorum, which constitute a species complex living in sympatry on plums and possibly on other stone fruit trees.

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.

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.

Characterization of the Bull's Eye Rot of Apple in Chile.

Apple fruit in Chile are primarily produced for export to Asia, Europe, and the United States, which typically requires 15 to 40 days of maritime transportation. Therefore, Chilean apple production must fulfill the sanitization requirements imposed by the receiving countries. Under these circumstances, it was important to clarify the etiology of bull's eye rot that can severely affect 'Cripps Pink' apple and other late-harvest cultivars in Chile. Based on morphological characteristics and the partial sequence analysis of the internal transcribed spacer sequences and ß-tubulin genes, Neofabraea alba was identified as the causal agent of the bull's eye rot of Chilean apple. These results were further corroborated using species-specific primers. The incidence of bull's eye rot varied considerably; for instance, in 2009, 0.0 to 58.7% in 38 Cripps Pink orchards surveyed in the relatively arid and humid apple-growing areas of Chile, respectively. There was no evidence for the presence of N. malicorticis or N. perennans, which are commonly identified as causal agents of bull's eye rot in other apple-producing countries. Altogether, these data suggest that N. alba might represent the predominant and possibly the only cause of bull's-eye rot of Chilean apple.

Ochratoxigenic Aspergillus species on grapes from Chilean vineyards and Aspergillus threshold levels on grapes.

This study reports the incidence of ochratoxigenic strains of Aspergillus on Chilean grapes (Vitis vinifera) and wineries, and production of OTA levels in wines with grapes having different levels of contamination with OTA-producing Aspergillus carbonarius was studied. A. carbonarius, A. niger, A. niveus, A. paradoxus, A. versicolor, A. wentii, and A. westerdijkiae were identified on apparently healthy clusters of red and white grape cultivars. However, A. carbonarius and A. niger were the most frequently identified species, more abundant on red than white grape cultivars. Aspergillus spp. populations increased between veraison and harvest, but the isolation frequencies were relatively low over the entire growing season. At the winery, A. carbonarius, A. niger and A. westerdijkiae were occasionally found in the air, exclusively during winemaking. OTA-producing strains were only found among isolates of A. carbonarius, A. niger, A. wenti, and A. westerdijkiae, producing 2 to 17 microg/L of OTA in liquid medium; however, A. westerdijkiae produced the highest OTA concentration in vitro. Red wines elaborated with 0.5% of grapes infected with an OTA-producing strain of A. carbonarius (Aspuc-SB36) exceeded the 2 microg/L of OTA tolerance established for wines by the European Community. Therefore, a threshold below 0.5% infected berries is proposed for red wines. ELISA tests proved to be useful for detecting OTA in broth culture as in wine samples.

Characterization of Cladosporium Rot in Grapevines, a Problem of Growing Importance in Chile.

Cladosporium rot (Cladosporium spp.) of grapevine (Vitis vinifera) is a common disease in Chile, particularly in Cabernet Sauvignon and other red wine grape cultivars. It is favored by delayed harvest to obtain the phenolic maturity necessary for high-quality red wine. This study expands on previous investigations of the specific causal agents, the histopathological host:pathogen relationship, and the population dynamics of Cladosporium spp. during the seasonal development of grape clusters. Over 100 isolates were obtained and identified as C. cladosporioides and C. herbarum, confirming previous results. The identity of a subset of isolates was confirmed by molecular analysis. Isolates of both C. cladosporioides and C. herbarum from grapevines were pathogenic on inoculated table grapes and wine grapes. These pathogens were reisolated, fulfilling Koch's postulates. Berry injuries and total soluble solids content above 15% were necessary for Cladosporium spp. to infect wine grapes. The mycelia of C. cladosporioides and C. herbarum grew between 0 and 30°C, but no growth was obtained at 35°C in vitro. The histological studies showed that Cladosporium spp. superficially colonize mature V. vinifera berries, invading the epidermis but scarcely penetrating the hypodermis. The Cladosporium populations obtained on apparently healthy berries of V. vinifera cvs. Cabernet Sauvignon and Chardonnay were significantly larger (P = 0.05) than the populations obtained under similar conditions on berries of V. champini cv. Ramsey and hybrids Kober 5BB and Couderc 1613. Considering the importance of Cladosporium rot in Chile compared with other grape production areas, the development of control strategies is needed to prevent high disease severity, which affects both yield and wine quality.

Canker and Twig Dieback of Blueberry Caused by Pestalotiopsis spp. and a Truncatella sp. in Chile.

Blueberry (Vaccinium spp.) has great economic importance in Chile, which currently has about 8,500 ha being cultivated. Recently, the presence of canker and dieback symptoms has been observed along the productive blueberry zone of Chile. Species of Pestalotiopsis and Truncatella were consistently isolated from diseased samples in 22 different locations. Therefore, the objective of this study was to identify and characterize the species of Pestalotiopsis and Truncatella associated with canker and twig dieback symptoms on blueberry. Forty-nine isolates were obtained on acidified potato dextrose agar in 2006 and 2007. These isolates were identified as Pestalotiopsis clavispora, P. neglecta, and Truncatella (=Pestalotia) angustata on the basis of colony characteristics and conidial morphology. This identification was verified by internal transcribed spacer analysis of DNA. Isolates of P. clavispora, P. neglecta, and T. angustata were pathogenic on apple, kiwifruit, and blueberry fruit. Similarly, isolates of P. clavispora were pathogenic on detached blueberry twigs of cv. O'Neal. Additionally, three selected isolates of P. clavispora induced light-brown canker lesions, surrounded by a reddish halo, and shoot dieback after twig inoculations on 2-year-old twigs of blueberry cvs. O'Neal, Bluecrop, Brightwell, Brigitta, Duke, Elliot, and Misty. Among blueberry cultivars, Brightwell and O'Neal were the most susceptible and Bluecrop and Misty the least susceptible, while Elliot, Brigitta, and Duke were moderately susceptible to P. clavispora. These pathogens were isolated consistently from inoculated plants, confirming Koch's postulates. P. clavispora was highly sensitive to fludioxonil and pyraclostrobin with a median effective concentration of 0.06 to 0.08 and 0.04 to 0.8 µg/ml, respectively. Therefore, the results of this study indicate that P. clavispora, P. neglecta, and T. angustata are primary pathogens that can cause canker lesions and dieback symptoms on blueberry not previously described in Chile. However, these results do not exclude that other species of these genera or other plant-pathogenic fungi (e.g., Botryosphaeria, Pestalotia, and Phomopsis spp.) may eventually be involved in this syndrome of blueberry.