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Genetic diversity and virulence variability of Diplodia mutila isolates recovered from grapevines in New Zealand were in‐ vestigated. The universally primed PCR (UP‐PCR) and vegeta‐ tive compatibility group (VCG) methods were used to investi‐ gate the genetic diversity. Pathogenicity tests with ‘Sauvignon Blanc’ detached shoots and potted vines were used to de‐ termine the virulence diversity. UP‐PCR analysis determined eight genetic groups of D. mutila with 70% of the population within one group. Phylogenetic analysis also determined that New Zealand isolates were more closely related to Australi‐ an isolates than Californian isolates. Vegetative compatibility grouping analysis placed the isolates into three VCG groups with 57% of isolates belonging to all three VCGs. Vegetative compatibility reactions were observed among isolates, but this was not correlated with the genetic clustering. Viru‐ lence assays proved that all isolates tested were pathogenic on grapevine stems. Differences in necrotic lesions lengths caused by D. mutila isolates were identified, indicating differ‐ ent virulence levels among isolates, however, no relationship was found between the genetic groups and the virulence. The results of the study indicated movement of D. mutila isolates between nurseries, vineyards, and other sources in New Zea‐ land. This information will inform control strategies to lim‐ it the further spread of this pathogen into vineyards in the same region or new regions. [ABSTRACT FROM AUTHOR]

In der vorliegenden Arbeit wurde der Erstnachweis des Auftretens von Diplodia mutila an Ligustrum vulgare erbracht. Im Rahmen eines Infektionsversuches wurde die Pathogenität eines von Liguster stammenden Isolates von D. mutila bei Ligustrum vulgare ‘Atrovirens‘, Prunus avium und Thuja occidentalis ‘Smaragd‘ bestätigt, nicht jedoch bei den Gehölzarten Crataegus monogyna und Forsythia × intermedia. Im Rahmen eines Wachstumsversuches wurde für das Isolat von Liguster 25°C als optimale Temperatur des Myzelwachstums ermittelt. DOI: 10.5073/JfK.2014.04.03, https://doi.org/10.5073/JfK.2014.04.03

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina ( New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards ( Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. ( I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease-free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co-inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature. [ABSTRACT FROM AUTHOR]

This study encompassed 137 cultures of Diplodia mutila isolated from necrotic tissue of 3- to 20-year-old trees of Fraxinus excelsior with decline symptoms, originating from three geographic regions of Poland differing by climatic conditions. Fungus identification was conducted on the basis of morphological traits of cultures and confirmed by analysis of ribosomal DNA sequences. The structure and genetic variation of D. mutila populations was determined with the use of RAMS markers. Neighbor Joining analysis of ITS sequences of the examined isolates showed the greatest similarity to sequences of D. mutila isolated from F. excelsior and deposited in GenBank. Intra-population variation expressed by Dice coefficient and Shannon index was high and increased along with the shifting of a population's location from mild climate (northern Poland) to more severe climate, characteristic for upland and mountainous areas (southern Poland). The smallest genetic distance was shown by pairs of populations close to one another, inside individual regions. AMOVA showed great share (91%) of genetic variation inside a population, whereas variation was very small between populations (6.4%) and regions (2.6%). Principal component analysis showed grouping of fungus populations in accordance with their geographic location. [ABSTRACT FROM AUTHOR]

1. Climate change predictions in the Amazon have largely focused on carbon-water relations, while the impacts of increased air temperature and reduced precipitation on host-pathogen relationships have not been extensively explored. These relationships are known to affect recruitment of many Amazonian plant species. 2. Host-pathogen relationships are well suited to a dynamical analysis of the effects of climate change due to the direct linkages between pathogen behaviour and abiotic factors such as temperature and rainfall. 3. Seedlings of the palm Iriartea deltoidea experience significant mortality due to infection by the fungus Diplodia mutila. This host-pathogen interaction was examined by combining a semi-analytical model with field data illustrating the temperature sensitivity of D. mutila reproductive rates and I. deltoidea seedling mortality in response to infection. 4. The data-model combination shows that projected climatic shifts in rainfall and temperature for the Amazon region will tend to reduce recruitment by altering pathogen activity and reducing palm fecundity. The magnitude of the reduction is sensitive to the details of the epidemiology of the D. mutila-I. deltoidea host-pathogen system, and ranges from 10% to 56% under plausible scenarios. 5. Although considerable uncertainty remains, the proposed model provides a blueprint for research on one aspect of ecosystem change in future climate models. 6. Synthesis. The study illustrates the potential for ecosystem responses to climate change, which can be investigated through tractable models simple enough to assimilate into climate modelling frameworks. Particular environmental sensitivities in fungal dynamics are identified. The implications of combined plant physiological stress and enhanced pathogenic activity under future climate scenarios are highlighted as critical issues for projecting forest response. [ABSTRACT FROM AUTHOR]

In Chile, the planted area of European hazelnut ( Corylus avellana L.) reaches around 30,000 hectares, mainly concentrated in the central and southern area of the country where climate and soil provide a natural environment well suited to growing this species. Only a few diseases affect this nut tree in Chile. During the spring seasons in 2018 and 2020, European hazelnut plants (6 to 20% of incidence) exhibited wood necrosis and vascular discoloration of branches, with reduced growth, cankers and wilt branches, in orchards located in San Clemente and Curicó, Maule Region, Bulnes and El Carmen, Ñuble Region, Chile (36°45'-36°54' S; 71°03'-72°26' W). Symptomatic tissues were surface disinfected using a ~1% commercial sodium hypochlorite solution. Disinfected tissues were cut longitudinally, placed onto potato dextrose agar (PDA, Difco) plates, and incubated at 25 °C in the dark for 48 hours. Fungal hyphal tips were taken and placed on PDA medium. A fungal species was consistently isolated from these lignified tissues. The mycelium was initially translucent (turning white in appearance), while the mature mycelium was aerial, varying in color from pale to dark gray (Munsell color code: colony edge mycelium 6Y-6 4 / 5G and colony center mycelium B6-PB 7 / 5PB). The production of pycnidia and conidia was induced using pine needles in water agar medium and incubated in the dark for 10 days. Hyaline unicellular conidia of 25 ± 1.1 µm (range 23.9 to 26.1 µm) long and 11 ± 0.5 µm (Range 10.5 to 11.5 µm) wide (n = 50) were obtained from black pycnidia. Based on the cultural and morphological characteristics observed, the pathogen was identified as a possible species of the family Botryosphaeriaceae (20 isolates). Molecular techniques were used to identify the species of pathogen, and three isolates (F154, F199, and F167) were analyzed by using Multilocus sequence typing to confirm the identity of the pathogen. Genes ITS (internal transcribed spacer region), tef-1 (translation elongation factor 1-alpha) and β-tub (β-tubulin) were amplified using endpoint PCR, with primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone & Kohn, 1999) and Bt2a/Bt2b (Glass & Donaldson, 1995), respectively. The segments were sequenced using the same primers, deposited in Gen Bank, and the accession numbers for each isolate were OM993582, OM993583, ON003481 for ITS, ON054936, ON054938, ON054937 for tef1 and ON054939, ON054941, ON054940 for β-tub , respectively. A phylogenetic tree was constructed using the maximum likelihood statistical method with the Tamura-Nei model based on a concatenated dataset of ITS region, tef1 and β-tubulin gene using Mega-X, and the three Chilean isolates (F154, F199, and F167) formed a single clade with the reference isolates of Diplodia mutila (Fr.) Mont. BLAST algorithm analyses indicated 100% identity to D. mutila for ITS (accession NR_144906), for tef-1 (accession MK573559), and for β-tubulin (accession MG952719). The pathogenicity of the three isolates was validated through Koch's postulates. For this purpose, a trial was established in 6-year-old European hazelnut plants cv. Tonda Di Giffoni. Ten healthy branches were individually inoculated using actively growing mycelial discs from each isolate, while a disc of PDA without fungus was used as a control. Holes of 5-mm diameter were inoculated, making sure the mycelium was in contact with the wood. Finally, the wounds were sealed with plastic film to prevent external contamination and improve humidity conditions. After 120 days, each branch was cut longitudinal-sectioned to verify the presence of wood necrosis which arose between 3.0 to 16.2 mm of length around the point of inoculation. No necrosis was observed in the control. To confirm pathogenicity, infected tissues were cut into small pieces with sterile knives and scalpels, and surface disinfected with a 1% sodium hypochlorite solution for 1 min. The disinfected tissues were placed on PDA medium and incubated at 25°C in the dark until fungal growth was observed. Hyphal tips were taken from the mycelia developed from the pieces of wood, and placed on PDA medium in order to obtain pure isolates. The pathogenicity of the D. mutila isolates F154 and F199 was observed in 100% of the inoculated branches, while isolate F167 showed symptoms in 85% of the branches. The reisolated strains showed similar mycelial growth and microscopic fungal structures to those observed in the isolates used for inoculation. This is the first report of D. mutila affecting European hazelnut in Chile. This fungus has been recently reported affecting hazelnut in Oregon, USA (Wiman et al., 2019), causing similar symptoms to those observed in our study. In addition, D. mutila has been reported infecting walnut in Chile (Diaz et al. 2018) and native forest trees, specifically Araucaria araucana in Chile (Besoain et al., 2017). The presence of D. mutila in commercial hazelnut orchards in Chile highlights the need for epidemiological studies in order to understand the characteristics and impact of this pathogen and, based on this, develop adequate phytosanitary programs for its control.

Apple (Malus pumila Mill.) is an important fruit crop in Xinjiang, China. In September 2021, apple tree canker was observed in a 21-year-old commercial apple orchard cv. Fuji in Xinjiang (38°17'51.43"N, 77°9'50.81"E) , northwest of China. Of the 200 plants surveyed, 25% were symptomatic. The diseased trees showed branch dieback and cankers. The cankers observed on the wood were sunken, shriveled, and discolored. After the bark was peeled off, the diseased wood was dark brown, and the necrosis was obvious on the cross-section of the diseased branch. To identify the causal agent, five symptomatic trees were collected and analyzed in the laboratory. Apple wood samples (0.5×0.5 cm) were surface-disinfected with 1% v/v sodium hypochlorite and 75% v/v ethanol, rinsed with sterile distilled water, transferred onto potato dextrose agar (PDA), and incubated in the dark at 25 °C for 5 days. Conidia were induced on sterilized pine needles covered with 2% w/v water agar under near-UV light. The colonies of five isolates were white to gray with sparse aerial mycelium that gradually became dark olive green in the later stage. Conidia were initially hyaline but becoming brown at maturity, 1-septate, oval, rounded at both ends, and with dimensions of 24.9-32.1 × 15.1-21.5 µm (n =50) and the aspect ratio of 1.6. Based on the cultural and morphological features of Phillips (2002), the isolates were identified initially as Diplodia mutila (Fr. : Fr.) Mont. To confirm species identification, genomic DNA was extracted from the representative isolate SC-8A. The primer ITS1/ITS4, EF1-728F/EF1-986R and BT2a/BT2b were used to amplify the rDNA sequences of, respectively, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α) gene, and a portion of beta-tubulin (tub2) gene. The nucleotide sequences indicated ≥99% identity to D. mutila (CBS 112553) for three DNA regions. Consensus sequences were deposited in GenBank. as accession numbers OM618108, OM676657 and OM676658 for ITS, EF1-α and tub2, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate SC-8A on one year old branches of cv. Fuji (n=5). Wounds were created in the middle of the branches using a sterilized hole punch (5mm diameter) and were immediately inoculated with mycelial plugs of the same diameter. For the control treatment, sterile agar plugs were used (n=5) in the branches. The inoculated and control branches were wrapped with sterile parafilm. On the 10th day after inoculation, canker lesions appeared on the inoculated branches, but no lesions were observed in the negative control. D. mutila was re-isolated from 100% of the inoculated shoots and was not re-isolated from any of the negative controls, the Koch's postulates were met. Previously, D. mutila has been reported in Canada (Úrbez-Torres et al., 2016), Argentina (Lódolo et al., 2022) and Chile (Díaz et al., 2022) causing Botryosphaeria canker and dieback in apples. To our knowledge, this is the first report of D. mutila causing Botryosphaeria canker and dieback in apple trees in China.

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 β-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.

Serpula himantiodes was found to be a causal agent of butt rot in Douglas fir and Japanese larch in northern Germany. The decay started predominantly from tap roots and main side roots and extended few meters into the stem. In final stages of rot the basal heartwood was completely destroyed and the residual width of healthy sapwood was reduced. Laboratory tests revealed a tolerance by the fungus to extremely acid substrates. [ABSTRACT FROM AUTHOR]

Two new phytotoxic tetrasubstituted 1,4-naphthoquinones, named diploquinones A and B, were isolated together with vanillic acid from Diplodia mutila (DAR78993), a grapevine pathogen involved in Botryosphaeria dieback in Australia. Diploquinones A and B were characterized as 6,7-dihydroxy-2-methoxy-5-methylnaphthalene-1,4-dione and 3,5,7-trihydroxy-2-methoxynaphthalene-1,4-dione using spectroscopic methods (essentially 1D and 2D 1 H and 13 C NMR and HR ESIMS). The already known vanillic acid was isolated for the first time as fungal phytotoxin and as metabolite of D. mutila. The three compounds were assayed on detached grapevine leaves ( Vitis vinifera cv. Shiraz) at concentrations of 10 -3 M and 2.5 × 10 -3 M. Vanillic acid showed the highest phytotoxic effect on grapevine leaves irrespective of the tested concentration, while diploquinones A and B showed varying degrees of toxicity.