Branch Dieback Research Articles

First Report of Neofusicoccum ribis Causing Cankers and Dieback Diseases on Apricot Trees in Canada and Worldwide.

Apricot trees (Prunus armeniaca L.) with cankers, gummosi and dieback symptoms were observed in a commercial orchard in Niagara-on-the-Lake, Ontario, Canada. In October 2018, up to 44.9% disease incidence (n = 318) was observed on 2-year-old 'Harostar™' trees grafted onto 'Haggith' rootstocks. Fungal colonies were consistently isolated and purified from small sections of wood collected from canker margins of symptomatic trunk and shoot tissue, as described by Ilyukhin et al. (2023). Purified mycelial isolates sharing similar morphological characteristics were categorized into five distinct morphotypes. One representative isolate from each morphotype was used to inoculate excised apricot shoots as described by Ilyukhin and Ellouze (2023). One morphotype displayed necrotic lesions on the shoots consistently yielded abundant white aerial mycelium that turned grey-brown on PDA after 7 days (Figure S1) and produced black pycnidia three weeks following incubtion at 22°C in the dark. Conidia were hyaline, one-celled, fusiform, with dimensions of 19.7 - 24.2 × 3.6 - 4.8 μm (average 22.1 × 4.3 μm, n = 30), the typical morphology of a Neofusicoccum sp. (Crous et al. 2006). Species identification was verified by extracting genomic DNA of the representative isolate M1-105, amplifying and sequencing the internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB2) gene regions with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b. Nucleotide sequences (GenBank Accession No. ITS: OK287034; EF1-α: OK346636; TUB2: OK346633) have 100%, 99.61% and 99.55% identity with Neofusicoccum ribis isolates from different hosts and countries (MT587514, DQ235142, OL455952, respectively). Randomized accelerated maximum likelihood analysis (Stamatakis et al. 2008), using ITS, EF1-α and TUB2 sequence data, clustered M1-105 with the highest bootstrap support values with the N. ribis ex-epitype CBS 115475 (Figure S2). A living culture of M1-105 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252247). Pathogenicity was verified using 5 potted healthy 1-year-old 'Haroblush™' apricot cultivar grafted onto 'Krymsk® 86' rootstocks. Trunks and shoots were inoculated in a shallow wound made by a scalpel with mycelial plugs from a 5-day-old culture of M1-105. Five control trees were inoculated with sterile plugs. Trees were put in an open-air area and watered as needed. Canker symptoms appeared 7 days after inoculation, and spread above and below the inoculation point. Fifteen days post-inoculation, the upper portion of inoculated shoots showed necrosis, gummosis and wilt (Fig. S1). Neofusicoccum ribis was re-isolated from all infected trees and species identity was confirmed by sequencing as described above. Controls remained symptom-free and no fungi were isolated from the wood. Therefore, Koch's postulates were completed. Neofusicoccum ribis was reported to cause branch dieback of olive trees in Spain (Romero et al. 2005) and pistachio in Italy (Corazza et al. 1986), stem blight and dieback of blueberry in Michigan (Heger et al. 2023) and Florida (Wright and Harmon 2010) and postharvest decay of apple fruit from cold storage in Pennsylvania (Jurick et al. 2013). To the best of our knowledge, this is the first report of N. ribis causing canker and shoot dieback of apricot trees in Canada and worldwide. This report reveals N. ribis as a potential threat, causing canker and dieback. Without proper management, it could lead to significant losses in apricot orchards and the stone fruit industry. This study paves the way for crucial research on N. ribis outbreaks and effective disease control.

Pathogenicity of Botryosphaeriaceae and Phytophthora species associated with Paulownia dieback, canker and root rot in Italy

In recent years, an unusual decline and mortality has been observed in Paulownia plantations throughout the Marche region (Central Italy). Given the economic importance of this emerging forest crop, a study was conducted to determine which pathogens are directly involved in this syndrome. Field surveys performed in two plantations revealed the widespread occurrence of severe disease symptoms such as leaf chlorosis, crown thinning, shoot and branch dieback, sunken cankers, epicormic shoots and root rot. Disease incidence was also assessed by aerial remote sensing (RS) technologies using drones. Symptomatic samples collected from both stem and root tissues yielded fungal and fungal-like colonies representing two distinct families: Botryosphaeriaceae and Peronosporaceae. Morphological and DNA sequence data revealed five distinct species, identified as Macrophomina phaseolina and Botryosphaeria dothidea (Botryosphaeriaceae), Phytophthora pseudocryptogea, P. citrophthora and P. erythroseptica (Peronosporaceae). Given that all species are reported here for the first time on Paulownia, Koch’s postulates were satisfied inoculating the three Phytophthora species and two Botryosphaeriaceae at the collar of the stem of potted 1-year-old rooted cuttings in June 2023. Thirty days after inoculation, all plants showed the same symptoms as those observed in the field.

Identification and Characterization of Fungal Pathogens Contributing to Citrus Twig Dieback in Kerman Province, Iran

Citrus decline with complex symptoms was recently observed in Kerman province, reducing the annual yield of citrus fruits. Agents that cause citrus decline include fungi, bacteria, viruses, nematodes, and abiotic factors. One of the most important symptoms of citrus decline is branch dieback. In this study, the fungal agent responsible for dieback was identified. To achieve this goal, sampling was conducted in citrus orchards with symptoms of twig dieback and twig canker in the summer of 2020. Samples were collected from different areas of the citrus growing region of Kerman province, including Bam, Rigan, Narmashir, Fahraj, Shahdad, and Orzueeh. Fungi were isolated from diseased branches on nutrient culture medium. The resulting fungi were identified using morphological characteristics. The following fungi were isolated from diseased plants: Alternaria spp., Nattrassia mangiferae, Ulocladium spp., Bipolaris spp., Fusarium spp., Phoma sp., Rhizoctonia sp., Paecilomyces spp., Chaetomium spp., Acremonium spp., Stemphylium sp., and Cladosporium spp. Notably, Alternaria spp. exhibited the highest frequency and widest distribution among them, making it the most prevalent fungus throughout all citrus-growing regions of Kerman province. N. mangiferae was the second most frequently isolated fungus. Pathogenicity tests were performed by artificially inoculating two-year-old lemon trees under controlled conditions. All fungal genera except Chaetomium sp. and Cladosporium spp. were virulent. N. mangiferae was the most virulent fungus and caused the most severe cankers six weeks after inoculation. It also had the greatest discoloration compared to other fungi in these tests

Exploring the Phylogenetic Diversity of Botryosphaeriaceae and Diaporthe Species Causing Dieback and Shoot Blight of Blueberry in Serbia.

Identifying the precise pathogens responsible for specific plant diseases is imperative for implementing targeted and efficient interventions and mitigating their spread. Dieback and shoot blight significantly diminish the lifespan and productivity of blueberries, yet the causative agents remain largely unidentified. To determine the identity and prevalence of the causal agents of branch dieback and shoot blight, we conducted multiyear and multisite sampling of diseased highbush blueberries (Vaccinium corymbosum cultivar Duke) in Serbia. Sixty-nine monosporic isolates were collected and characterized based on morphological, physiological features and multilocus phylogenetic analysis of internal transcribed spacer, β-tubulin, and translation elongation factor 1-α sequence data. Five species were identified as causal agents: Diaporthe eres (36 isolates), D. foeniculina (3 isolates), Neopestalotiopsis vaccinii (9 isolates), Neopestalotiopsis rosae (6 isolates), and Neofusicoccum parvum (15 isolates). The results of the pathogenicity tests performed with the 23 representative isolates confirmed the role of these species as primary pathogens in causing dieback and shoot blight of blueberry, with Neofusicoccum parvum being the most aggressive and D. eres the least. Our study underscores the diversity of genera and species of ascomycetes capable of causing blueberry dieback and shoot blight. Furthermore, our findings indicate that the agents responsible for the disease in Serbia differ from those identified in other regions worldwide.

First report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

Seasonal Susceptibility of Sweet Cherry Pruning Wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

Fungal canker pathogens commonly infect trees at pruning wounds leading to branch dieback and loss of productivity in sweet cherry orchards. However, the seasonal susceptibility of sweet cherry pruning wounds to Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata is not well understood. This study compared the susceptibility of sweet cherry pruning wounds made during the dormant season (January) and the postharvest season (late May to June) to infection by main canker pathogens in California. Field trials were conducted in three cherry orchards and trees were pruned at the different periods over 2 years. Fresh pruning wounds were inoculated with spores of each pathogen, and pathogen recovery was assessed through microbiological isolations at 3 to 4 months after inoculations. Pruning wounds made in late May and June resulted in significantly higher infection by Cal. pulchella compared to pruning wounds made in January. Pruning wounds made during both seasons were generally equally susceptible to Cyt. sorbicola and E. lata infections. However, there was one orchard where dormant pruning wounds were more susceptible to infection by E. lata and there was one particularly cold winter where Cyt. sorbicola did not infect pruning wounds. Overall, our findings suggest that Cal. pulchella infections of cherry pruning wounds are more likely to occur during periods of warm temperatures such as late spring and early summer. However, infections by Cyt. sorbicola and E. lata can occur year-round if inoculum is present and if winter temperatures are not abnormally low for California. Finally, our results suggest that the emergence of Cal. pulchella as a major canker pathogen of sweet cherry in California may be the result of a shift from dormant to after-harvest pruning of sweet cherry trees.

New Melastomataceae hosts of Chrysoporthe species in Brazil

AbstractThe angiosperm Melastomataceae family is one of the most abundant plant families worldwide and in the Brazilian cerrado, with significant environmental importance in regenerating degraded areas, especially those previously occupied by pastures. Recently, Chrysoporthe Gryzenhout & M. J. Wingf. species were reported in Brazil, causing canker, branch dieback, and mortality in native Melastomataceae. This leads to the demand for further investigation and understanding of these pathosystems. During field surveys, typical signs and symptoms associated with Chrysoporthe infection were found in Rhynchanthera grandiflora (Aubl.) D C. and Miconia theaezans (Bonpl.) Cogn. in southern Minas Gerais. Through phylogenetic analysis of the BT1 and BT2 fragments of the β‐tubulin gene and morphological characterization of the isolates obtained, it was possible to identify C. doradensis Gryzenh. & M. J. Wingf. occurring in R. grandiflora and C. puriensis M. E. S. Oliv., T. P. F. Soar. & M. A. Ferr. occurring in R. grandiflora and M. theaezans. Furthermore, pathogenicity assays confirmed the pathogenicity of both species to their hosts.

Molecular Identification and Pathogenicity of Diaporthe eres and D. hongkongensis (Diaporthales, Ascomycota) Associated with Cherry Trunk Diseases in China.

This study aimed to identify fungal species associated with trunk diseases of sweet cherries (Prunus avium) in several commercial cherry orchards in Beijing, Guizhou and Shandong provinces, China. In total, eighteen fungal strains that fitted well into the species concept of Diaporthe were isolated. Based on both morphological and multi-locus phylogenetic analyses of internal transcribed spacer region (ITS), beta-tubulin (tub-2), calmodulin (Cal) and translation elongation factor 1-α (tef1-α) sequencing data, fourteen isolates were identified as Diaporthe eres, while four isolates were classified as D. hongkongensis. Here, we report D. hongkongensis causing sweet cherry branch dieback disease and, further, we confirmed the host association of D. eres with sweet cherries in China. A pathogenicity assay revealed the ability of both D. eres and D. hongkongensis to cause shoot necrosis and stem lesions on Prunus avium cv. 'Brooks' (mean lesion lengths of 1.86 cm and 1.56 cm, respectively). The optimal temperature for the growth of both Diaporthe species was tested. The optimal growth temperature for D. hongkongensis was 30 °C, and the 25-28 °C temperatures were the most favorable for the growth of D. eres strains. This research advances the understanding of fungal trunk diseases in fruit crops, particularly gummosis and branch dieback disease in Chinese cherry orchards, and will aid growers in making decisions about cultural practices and disease management.

A new disease of kumquat (Fortunella margarita) caused by Colletotrichum karsti: twig and branch dieback

Citrus fruit crops are important in many countries. Anthracnose, post bloom fruit drop, fruit stem-end rot, twig and branch dieback and gummosis, caused by Colletotrichum spp., are diseases that seriously threaten citrus production. Surveys of kumquat (Fortunella margarita) orchards were conducted in Eastern Sicily, Southern Italy, during 2022-23. Fungi isolated from twig and branch dieback of F. margarita were identified as Colletotrichum karsti through multi-locus (gapdh, tub2 and act) phylogeny. Pathogenicity and aggressiveness on detached apple fruit and kumquat plants were confirmed for a selection of representative isolates, although with different levels of disease incidence observed. This is the most comprehensive study on identification of C. karsti as the causal agent of twig and branch dieback of kumquat.

A Survey on Ambrosia Beetle (Coleoptera: Curculionidae) Problems in Ornamental and Pecan Industries in Georgia

Abstract The ambrosia beetles, mainly Xylosandrus crassiusculus (Motschulsky) (Coleoptera: Curculionidae: Scolytinae), are serious pests of young trees in the ornamental industry and pecan orchards as infested trees develop branch dieback with occasional tree mortality. Surveys were conducted in the ornamental industry in 2020 and 2021 and the pecan industry in 2020 because clientele perspective on the extent of damage, phenology, monitoring, management, and loss related to the ambrosia beetle was unclear. Of 35 and 40 ornamental industry respondents, 68% and 82% indicated problems with this pest in 2020 and 2021, respectively. Of 66 pecan industry respondents, 52% reported beetle problems in 2020. About 85% of ornamental and 58% of pecan respondents indicated that 1–10 trees were attacked by ambrosia beetles and 1–30 or more trees were culled annually. The beetle problem persists throughout the growing season but appears greater during the spring than in other periods. About 73% of respondents indicated that current monitoring tools helped them with management decisions; however, a proportion did not use recommended monitoring tools but instead relied on visual signs to determine attacks on trees. In the 2020 surveys, only 37% of ornamental respondents and 43% of the pecan respondents used insecticide sprays, whereas in the 2021 survey, 71% of the ornamental clientele (mostly nurseries) sprayed pyrethroid insecticides for ambrosia beetle management. In 2020 surveys, about 48% and 56% of ornamental and pecan respondents, respectively, spent <$500 USD for ambrosia beetle management.

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The etiology of shisham (Dalbergia sissoo) die-back has been a subject of considerable scientific debate for hundred years. No single pathogen species is responsible for die-back outbreaks, making shisham die-back challenging to study. Symptoms of the disease include yellowing, the water-soaked appearance of leaves, thinning of leaves and crown, branch die-back (single or whole), bark splitting, gummosis, and lesion development. Symptomatic shisham were sampled from 117 sites across Pakistan. One hundred and ninety-two fungal isolations were made from 1170 shisham samples and were identified as Ceratocystis sp. and Fusarium sp. based on cultural morphology. Koch's postulates were completed using 23 isolates of Ceratocystis sp. and four isolates of Fusarium sp. on three shisham age groups a) 6-12-month-old nursery seedlings growing under greenhouse conditions, b) 1-3-year-old plants growing under natural field conditions, and c) 10-15-year-old tree growing under natural field conditions. Inoculation with Ceratocystis isolates resulted in typical shisham die-back symptoms. Inoculation with Fusarium sp. did not produce any symptoms alone or in combination with Ceratocystis sp. Four fungicides were evaluated for efficacy against Ceratocystis sp. using in vitro tests and field trials. In in vitro test, propiconazole resulted in the highest level of inhibition (88.88%) at day seven, followed by difenoconazole (84.75%), hexaconazole (81.11%), while benzimidazole (77.73%) was least effective against the tested fungus. The efficacy of these fungicides has declined throughout greenhouse trials conducted in this study. Additionally, these fungicides could not prevent tree loss in trees inoculated with Ceratocystis sp. in the field trials. Thus, shisham die-back continues to be of concern in shisham-growing areas in Indo- Pakistan. However, chemical management and continuing research on the epidemiology of this disease may provide a solution

Evaluation of Spore Acquisition, Spore Production, and Host Survival Time for Tea Shot-Hole Borer, Euwallacea perbrevis, Adults after Exposure to Four Commercial Products Containing Beauveria bassiana.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6-8 days) compared to beetles dipped in the other fungal products (10-11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

First report of Diplodia mutila causing canker and branch dieback of walnut trees in Xinjiang, China.

Walnut (Juglans regia L.) has become an important economic fruit tree in China. In April 2022, branch dieback was observed in 15-year-old walnut trees (cv Wen 185) in a commercial orchard in Aksu, Xinjiang, China (40°21'55''N, 80°1'48''E), with an incidence of 2% (4 out of 200 trees) of affected trees. The symptoms observed, included depressed and shrunken cankers, twigs and branches dieback. Cross-sections of diseased branches revealed dark-brown wedge-shaped lesions. To isolate the potential causal pathogen, four specimens were isolated from diseased branches, and small pieces taken from the edge of canker samples (0.5 × 0.5 cm), were disinfected by immersion in 75% ethanol for 30 s and 2% NaClO solution for 3 min, and rinsed three times with sterile water. The disinfected wood samples were then placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for 2-3 days. Then, we applied the mycelial tip purification method and repeated this purification process until a single colony was formed. Four pure isolates (3-1-51A, W-2-54, K-1-43A, K-1-43B) developed white to white-gray fast-growing colonies with abundant aerial mycelium after seven days at 25°C on PDA and gradually became dark olive green over subsequent growth stages. Conidia production was then induced on 2% w/v water agar containing sterilized pine needles under near-U/V light (Alves et al., 2004). The conidia were initially hyaline, thick-walled, oblong to ovoid with one septum and a size range of 19.47-24.16 × 9.78-13.51 µm (n = 40). Based on morphological characteristics these isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al., 2004).To confirm the pathogen identified, the representative isolate 3-1-51A was amplified and sequenced using specific primer pairs (ITS1/ITS4, EF1-986/EF1-728E, BT2a/BT2b) to the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB) (White et al. 1990; Carbone and Kohn 1999; O' Donnell and Cigelnik 1997), respectively. The sequences showed 100% similarity to two D. mutila strains CBS230.30 and CBS112553. Maximum likelihood analysis was performed based on a concatenated dataset (ITS + TEF1-α + TUB) gene using MEGA 11. 0 and isolate 3-1-51A formed a single clade with the reference ex-type of D. mutila. The isolate 3-1-51A was deposited into GenBank as OP006733, OP373140, and OP373139 for ITS, TEF1-α, and TUB, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate 3-1-51A on one-year-old healthy walnut branches cv. Wen 185 of walnut trees (n=5). Five twigs of healthy walnut branches were cleaned, submerged in 1% NaClO for 15 minutes and then dried. Then, a sterile hole punch (5mm in diameter) was used to create a wound in the middle of each walnut branch, and placing mycelial plugs(3 days old; 5 mm in diameter) and sealed with parafilm. An equal number of twigs inoculated with sterile agar plugs served as controls. On the 7th day after inoculation, dark brown coloration was developed on the branches with symptoms of shrinkage dryness, and dieback. D. mutila isolate was re-isolated only from the inoculated branches. In negative control twigs, lesions and re-isolated were absent, thus fulfilling Koch's postulates. D. mutila has been previously reported causing canker and branch dieback in walnut trees in Chile (Díaz et al. 2018) and California (Chen et al. 2014). Previously, D. seriata (Zhang et al. 2017), Botryosphaeria dothidea (Guo et al. 2016), Lasiodiplodia pseudotheobromae (Guo et al. 2016), Dothiorella gregaria (Liu et al. 1986) and Neofusicoccum parvum (Yu et al. 2015) have been identified on walnut trees in China. To our knowledge, this is the first report of canker and branch dieback caused by D. mutila in walnut trees in Xinjiang, China. Further studies are now required to better understand the etiology of canker and branch dieback on walnut trees from different areas in China.

First Report of Neofusicoccum australe Causing Branch Dieback of English Walnut cv. Chandler in Central Chile.

The English walnut (Juglans regia L.) is the second most important fruit crop of importance in Chile, with 43,700 hectares mainly in the Central Valley (www.odepa.cl, 2022). For several seasons symptoms of a branch dieback have been observed in walnut orchards with 3 to 50% of trees incidence levels. During the 2020 winter season (July) a total of 150 symptomatic spurs of 15 trees were sampled from an 8-year-old walnut cv. Chandler orchard located in Buin (33°42' S, 70° 42' W). The collected spurs showed external and internal brown necroses, starting from the tip with well-defined margins. The symptomatic tissue was cut in to small pieces (5 x 4 x 2 mm), surface disinfected by dipping in a 10% solution made from a commercial bleach solution (4,9% NaOHCl), rinsed twice in sterile water and plated on APDA (PDA Difco laboratories acidified with lactic acid (2,5 ml of 25% (vol/vol) per liter of medium). After five days at 20 °C in darkness, fast-growing, white-grey turning to black colonies were obtained, tentatively classified as a member of the Botryosphaeraceae family and two single-spore isolates (SS1, SS2) were selected for identification. Colony mycelia were first white and turned to light grey, dark grey or black, with tufts of mouse gray aerial mycelia. The pycnidia and conidia production was induced by inoculating autoclaved pine needles placed on APDA an incubation for 25 to 30 days at 20 °C in darkness. Black pycnidia solitary and globose were obtained producing hyaline, aseptate, fusiform to obovoid conidia with truncated ends with dimensions of (22.6-) 19.1 ± 1.4 (-13.3) x (6.7-) 5.5 ± 0.5 (-3.7) µm and 3.5 length/width ratio (n=100). Both isolates were identified using dichotomous keys confirming the description of Crous et al, 2006 as Neofusicoccum australe. The identification was molecularly confirmed by amplifying the nuclear ribosomal gene 5,8S (ITS1-5.8S-ITS2) using the ITS1/ITS4 primers, a partial region of β-tubulin gene (Bt2a/Bt2b), and the translation elongation factor 1-α gene (TEF1) with TEF1-728F/TEF1-986R primers. The BLASTn search revealed 100% of identity for ITS and TEF according to sequences of N. australe reference strains MT587467.1 and MK759852.1, respectively; and over 99% for β-tubulin compared to N. australe strain KX464929.1. The DNA sequences were submitted to the GenBank (ITS, OP142414, OP142416; BT, OP209981, OP209978; and TEF OP209979, OP209980) for SS1 and SS2 isolates, respectively, and deposited in the fungal collection of CChRGM - INIA, Chillán, Chile (RGM 3409 and 3410). Pathogenicity of both isolates was tested in 8-year-old asymtomatic English walnut cv. Chandler in the field during 2020 spring season, by cutting transversally 15 twigs of different tress and inoculating with a 5 day-old PDA plug. An equal number of wounded twigs were inoculated with a sterile PDA plug and served as control. After six months, all inoculated twigs developed the same necrotic lesions observed in field of 2.0 to 10.1 cm (SS1) and 1.9 to 10.8 cm (SS2) in length while control twigs showed only a scar without any dieback tissues. The inoculated pathogens of N. australe were recovered from the diseased tissues, thus fulfilling Koch's postulates. A similar dieback of walnut was reported in Chile, which caused Diplodia mutila (Díaz et al, 2018), and N. parvum (Luna et al, 2022) while N. australe has been reported in other hosts (Auger et al, 2013, Besoain et al, 2013). To the best of our knowledge, this is the first report of N. australe associated with walnut branch dieback in Chile.

Fungal Pathogens Associated with Almond Decline Syndrome, an Emerging Disease Complex in Intensive Almond Crops in Southern Spain.

In 2016, an almond (Prunus dulcis) decline syndrome (ADS) emerged in intensive almond plantations in the Andalusia region (southern Spain), showing branch dieback, gummosis, and general tree decline. The aim of this work was to elucidate the etiology of this disease complex. For this purpose, surveys were conducted across the Andalusia region, and a wide collection of fungi was recovered from wood samples showing gum and internal discoloration. Representative isolates were selected and identified by sequencing ITS, TEF1, TUB, ACT, LSU, and/or RPB2 genes. The following fungal species were identified to be associated with the disease: Botryosphaeria dothidea, Diplodia corticola, Di. seriata, Dothiorella iberica, Lasiodiplodia viticola, Macrophomina phaseolina, Neofusicoccum mediterraneum, N. parvum, N. vitifusiforme, Diaporthe neotheicola, Dia. rhusicola, Dia. ambigua, Eutypa lata, E. tetragona, Eutypella citricola, Eu. microtheca, Fusarium oxysporum s.l., Pleurostoma richardsiae, Phaeoacremonium iranianum, Pm. krajdenii, Pm. parasiticum, and Cytospora sp. All isolates were tested for pathogenicity by inoculating detached or attached almond shoots. Di. corticola and N. parvum were the most aggressive species, showing the largest lesions and most gummosis in attached shoots. The results suggest that the species belonging to Botryosphaeriaceae play a key role in disease development, while the remaining identified species may act as secondary pathogens or endophytes. However, further research to determine the interaction between all these fungal species and other biotic and abiotic factors in the ADS progress is needed.

Field Resistance of American Sycamore ‘Davis’ to Canker Pathogens

Abstract American sycamores (Platanus occidentalisL.) are found in many ecosystems and planted in urban landscapes worldwide. The trees are highly susceptible to anthracnose and canker pathogens, causing leaf blight and branch dieback. On The Ohio State University campus in Columbus, Ohio, an American sycamore was observed to thrive among many symptomatic sycamores. The healthy tree, subsequently protected as cultivar ‘Davis,’ was vegetatively propagated and tested for field resistance to natural infection of canker pathogens compared to the wildtype. Incidence and severity of leaf necrosis, incidence of dieback, and tree death were evaluated for 2 consecutive seasons. The incidence of leaf necrosis was disconnected from the incidence of dieback and tree mortality, as little to no leaves were produced on the wildtype trees. By the end of the second season, 7 out of 12 wildtype trees were dead, while all 12 ‘Davis’ trees were alive. Several canker pathogens were recovered from both ‘Davis’ and the wildtype, includingApiognomonia plataniandDiaporthe eres. The latter had not been previously reported on American sycamore. Pathogenicity tests confirmed thatD. eresis indeed pathogenic on sycamores and also that ‘Davis’ is significantly more resistant than wildtype to canker development and should be preferred over the wildtype in the urban landscape.

First Report of Olive Branch Dieback in Croatia Caused by Cytospora pruinosa Défago.

Olive (Olea europaea L.) is a very important crop grown in the Mediterranean part of Croatia. Olive branch and fruit dieback symptoms were observed in two olive orchards in Istria, Croatia. The samples from symptomatic trees were collected and brought to the laboratory for analysis. Based on their morphological characterization, isolated fungi were identified as Cytospora sp. Two representative isolates (one per orchard) were taken for molecular analysis, and based on DNA sequence data of the ITS and TUB gene regions, and phylogenetic analysis of the sequences, the isolates were identified as Cytospora pruinosa Défago. To determine pathogenicity, pathogenicity tests were conducted on detached olive branches and two-year-old olive trees in the greenhouse. This is the first report of C. pruinosa causing olive branch and fruit dieback in Croatia.

Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding.

Walnut dieback can be caused by several fungal pathogenic species, which are associated with symptoms ranging from branch dieback to fruit necrosis and blight, challenging the one pathogen-one disease concept. Therefore, an accurate and extensive description of the walnut fungal pathobiome is crucial. To this end, DNA metabarcoding represents a powerful approach provided that bioinformatic pipelines are evaluated to avoid misinterpretation. In this context, this study aimed to determine (i) the performance of five primer pairs targeting the ITS region in amplifying genera of interest and estimating their relative abundance based on mock communities and (ii) the degree of taxonomic resolution using phylogenetic trees. Furthermore, our pipelines were also applied to DNA sequences from symptomatic walnut husks and twigs. Overall, our results showed that the ITS2 region was a better barcode than ITS1 and ITS, resulting in significantly higher sensitivity and/or similarity of composition values. The ITS3/ITS4_KYO1 primer set allowed to cover a wider range of fungal diversity, compared to the other primer sets also targeting the ITS2 region, namely, GTAA and GTAAm. Adding an extraction step to the ITS2 sequence influenced both positively and negatively the taxonomic resolution at the genus and species level, depending on the primer pair considered. Taken together, these results suggested that Kyo set without ITS2 extraction was the best pipeline to assess the broadest fungal diversity, with a more accurate taxonomic assignment, in walnut organs with dieback symptoms.

Pest categorisation of Neoscytalidium dimidiatum.

The EFSA Plant Health Panelperformed a pest categorisation of Neoscytalidium dimidiatum, a clearly defined plant pathogenic fungus of the family Botryosphaeriaceae. The pathogen affects a wide range of woody perennial crops and ornamental plants causing symptoms such as leaf spot, shoot blight, branch dieback, canker, pre- and post-harvest fruit rot, gummosis and root rot. The pathogen is present in Africa, Asia, North and South America, and Oceania. It has also been reported from Greece, Cyprus and Italy, with a restricted distribution. Nevertheless, there is a key uncertainty on the geographical distribution of N. dimidiatum worldwide and in the EU, because in the past, when molecular tools were not available, the two synanamorphs of the pathogen (Fusicoccum-like and Scytalidium-like) might have been misidentified based only on morphology and pathogenicity tests. N. dimidiatum is not included in Commission Implementing Regulation (EU) 2019/2072. Because of the wide host range of the pathogen, this pest categorisation focuses on those hosts for which there is robust evidence that the pathogen was formally identified by a combination of morphology, pathogenicity and multilocus sequence analysis. Plants for planting, fresh fruits and bark and wood of host plants as well as soil and other plant growing media are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors occurring in parts of the EU are favourable for the further establishment of the pathogen. In the areas of its present distribution, including Italy, the pathogen has a direct impact on cultivated hosts. Phytosanitary measures are available to prevent the further introduction and spread of the pathogen into the EU. N. dimidiatum satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

Seasonal dynamics of inoculum of Botryosphaeriaceae in macadamia orchards in Australia

AbstractMacadamia (Macadamia integrifolia and M. tetraphylla and their hybrids) is a tree nut crop affected by Botryosphaeria branch dieback. The disease results in branch and tree death in commercial orchards, so reducing the productivity of the Australian macadamia industry. There is a dearth of knowledge of the disease cycle in macadamia, which confounds disease management. This study investigated the dynamics of pathogen inoculum associated with Botryosphaeria branch dieback and the influence of environmental factors (temperature, rainfall and relative humidity) on the seasonal spore release within the orchards. Diseased and asymptomatic plant parts from macadamia trees were collected each season for 3 years in commercial orchards. Fungal structures (pycnidia and pseudothecia) mainly were (71%) observed on symptomatic wood samples. A comparison of the number of spores released from the fungal structures between summer and autumn revealed high concentrations of Lasiodiplodia pseudotheobromae from diseased wood (4.8 × 104 conidia/cm2) and Neofusicoccum luteum from blighted leaves (2.1 × 103 conidia/cm2). The peak airborne conidia dispersal period, quantified using microscopy and quantitative PCR assays, was consistently detected after rainfall and strongly associated with relative humidity over 70% and temperatures between 20 and 27°C during summer and autumn. Botryosphaeriaceae isolates obtained from the pycnidia on the macadamia trees caused severe branch dieback symptoms when inoculated on detached macadamia twigs, which suggests both airborne and rain‐splashed spores from fungal structures on the trees contribute to the disease in macadamia orchards.