Sphaeropsis Sapinea Research Articles

The Diplodia Tip Blight Pathogen Sphaeropsis sapinea Is the Most Common Fungus in Scots Pines' Mycobiome, Irrespective of Health Status-A Case Study from Germany.

The opportunistic pathogen Sphaeropsis sapinea (≡Diplodia sapinea) is one of the most severe pathogens in Scots pine, causing the disease Diplodia tip blight on coniferous tree species. Disease symptoms become visible when trees are weakened by stress. Sphaeropsis sapinea has an endophytic mode in its lifecycle, making it difficult to detect before disease outbreaks. This study aims to record how S. sapinea accumulates in trees of different health status and, simultaneously, monitor seasonal and age-related fluctuations in the mycobiome. We compared the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018, and filamentous fungi were isolated. The mycobiome was analyzed by high-throughput sequencing (HTS) of the ITS2 region. A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Sphaeropsis sapinea was the most common endophyte isolated and the second most common in the HTS data. The fungus was highly abundant in symptomless (healthy) trees, presenting in its endophytic mode. Our results highlight the ability of S. sapinea to accumulate unnoticed as an endophyte in healthy trees before the disease breaks out, representing a sudden threat to Scots pines in the future, especially with increasing drought conditions experienced by pines.

Differences in the virulence of Sphaeropsis sapinea strains originating from Scots pine and non‐pine hosts

AbstractThe virulence of 15 endophytic and pathogenic Sphaeropsis sapinea strains was tested towards Scots pine (Pinus sylvestris). Two‐thirds of the strains had been isolated from Scots pines with varying health status: five isolates originated from healthy tissue (endophytic fungal stage) and five from diseased tissue (pathogenic stage). One‐third of the strains were isolated from symptomatic tissues of non‐pine hosts: black alder (Alnus glutinosa), European beech (Fagus sylvatica), European larch (Larix decidua), Norway spruce (Picea abies) and Douglas fir (Pseudotsuga menziesii). The S. sapinea strain isolated from black alder is the first proof that this fungus can form associations with alder species. On four‐year‐old P. sylvestris, one isolate per plant was inoculated on three side‐shoots of seven plants in a greenhouse (21 inoculations/strain). Differences in necrosis size caused by the isolates were measured 55 days after inoculation. The pathogenic S. sapinea isolates originating from diseased Scots pine and from non‐pine hosts were found to cause significantly longer necroses when compared to the endophytic isolates of S. sapinea from symptomless pines.

Biosynthesis of D-Mannitol by Diplodia pinea

Research with fungi in search of substances with potential biological activity is gaining more prominence since they are sustainable sources of new molecules. Diplodia pinea is a phytopathogenic fungus found in forest species, which has shown few reports of molecules with biological activity and may present promising biotechnological potential. Thus, the objective of this study was to detect substances produced by this fungus in a liquid medium, under stressful conditions. A metabolite was detected in the alcoholic fraction characterized by X-ray diffraction as D-mannitol. This is the first description of this monosaccharide produced by D. pinea.

Sphaeropsis sapinea and Associated Endophytes in Scots Pine: Interactions and Effect on the Host Under Variable Water Content

The ascomycete Sphaeropsis sapinea is the causal agent of the Diplodia Tip Blight disease on pines and other conifer species. This fungus has a symptomless endophytic life stage. Disease symptoms become visible when trees have been weakened by abiotic stress, usually related to warmer temperatures and drought. Currently, this disease is observed regularly in Scots pine (Pinus sylvestris) sites in parts of Europe, such as Germany, increasing dramatically in the last decade. Changes in climatic conditions will gradually increase the damage caused by this fungus, because it is favored by elevated temperature. Thus, host trees with reduced vitality due to climate change-related environmental stress are expected to be more susceptible to an outbreak of Diplodia Tip Blight disease. There is currently no established and effective method to control S. sapinea. This project aims to reveal the nature of the endophyte community of Scots pine. Utilizing the antagonistic core community of endophytes could serve as a novel tool for disease control. Results from this study provide a starting point for new solutions to improve forest health and counter S. sapinea disease outbreaks. We screened potential antagonistic endophytes against S. sapinea and infected Scots pine seedlings with the most common endophytes and S. sapinea alone and combination. The host was stressed by limiting access to water. The antagonism study revealed 13 possible fungi with the ability to inhibit the growth of S. sapinea in vitro, for example Sydowia polyspora. None of the tested co-infected fungi (Desmazierella acicola, Didymellaceae sp., Microsphaeropsis olivacea, Sydowia polyspora, and Truncatella conorum-piceae) showed strong necrosis development in vivo, even when host stress increased due to drought. However, the infection experiment demonstrated that drought conditions enhance the effect of the disease outbreak, triggering S. sapinea to cause more necrosis in the infected twigs.

Systemic reprogramming of phytohormone profiles and metabolic traits by virulent Diplodia infection in its pine (Pinus sylvestris L.) host.

The widespread ascomycetous fungus Diplodia pinea is a latent, necrotrophic pathogen in Pinus species causing severe damages and world-wide economic losses. However, the interactions between pine hosts and virulent D. pinea are largely not understood. In the present study, systemic defence responses were investigated in non-inoculated, asymptomatic needles and roots of D. pinea infected saplings of two P. sylvestris provenances under controlled greenhouse conditions. Here, we show that D. pinea infection induced a multitude of systemic responses of the phytohormone profiles and metabolic traits. Shared systemic responses of both pine provenances in needles and roots included increased abscisic acid and jasmonic acid levels. Exclusively in the roots of both provenances, enhanced salicylic acid and reduced indole-3-acetic acid levels, structural biomass, and elevated activities of anti-oxidative enzymes were observed. Despite these similarities, the two pine provenances investigated different significantly in the systemic responses of both, phytohormone profiles and metabolic traits in needles and roots. However, the different systemic responses did not prevent subsequent destruction of non-inoculated needles, but rather prevented damage to the roots. Our results provide a detailed view on systemic defence mechanisms of pine hosts that are of particular significance for the selection of provenances with improved defence capacity.

Synergistic Ability of Chitosan and Trichoderma harzianum to Control the Growth and Discolouration of Common Sapstain Fungi of Pinus radiata

An environmentally compatible method for controlling sapstain fungi in wood was evaluated, using a combination of chitosan and an albino strain of Trichoderma harzianum, a biological control agent (BCA). The growth and penetration into the wood of the sapstain fungi Ophiostoma piceae, Leptographium procerum, and Sphaeropsis sapinea were assessed in radiata pine wafers treated with chitosan and BCA, both alone and in combination. Several mycological and microscopic techniques were used, including a gfp (green fluorescent protein) transformed strain of O. piceae for assessing the depth of penetration in the wood samples. The synergy between the chitosan and BCA was evident, and for two tested fungi, only the combination of chitosan and BCA afforded protection. The synnemata (recognized by erect conidiogenous cells bearing conidia) was observed on the surface of the wafers inoculated with L. procerum and O. piceae, but the hyphae were unable to penetrate and melanise. The results suggest that the limited ability of chitosan to penetrate deeply into the wood was compensated by the fast growth of T. harzianum in the inner wood.

Endosphere Bacterial Diversity within Mixed Forest of Pinus densiflora Infected by Different Incidences of Sphaeropsis sapinea

Endosphere Bacterial Diversity within Mixed Forest of Pinus densiflora Infected by Different Incidences of Sphaeropsis sapinea

Blue‐stain fungi isolated from freshly felled Scots pine logs in Poland, including Leptographium sosnaicola sp. nov

AbstractWounds on felled tree stems provide entrance to inner wood for many fungi, including blue‐staining fungi, which cause permanent discoloration of wood. The aim of work presented here was to explore the diversity of blue‐stain fungi and assess the influence of the felling method and storage time/sampling period on their abundance and species diversity. Scots pines (Pinus sylvestris) grown in Poland were cut in April and July 2016, and fungal isolations from 20 stained logs that were obtained by mechanized harvesting and chainsaw harvesting, were carried out 3, 6, 9 and 12 weeks after felling. In total, eight fungal taxa were identified, including seven known and one novel species. Among the blue‐staining fungi detected, seven taxa belonged to Ophiostomatales, and one was identified as Sphaeropsis sapinea. The most frequently isolated fungi were S. sapinea, Ophiostoma minus and Ophiostoma ips. Additionally, Graphilbum acuminatum, Leptographium sosnaicola sp. nov., O. floccosum, O. piliferum and Sporothrix eucastaneae were found. More fungal isolates were obtained from summer‐harvested logs than spring‐harvested logs and from chainsaw‐harvested logs than mechanically harvested logs.

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Sphaeropsis sapinea Diplodia sapinea Pinus sylvestris S. sapinea S. sapinea S. sapinea S. sapinea

Sphaeropsis sapinea and fungal endophyte diversity in twigs of Scots pine (Pinus sylvestris) in Germany

Sphaeropsis sapinea is the causal fungal agent of Diplodia tip blight disease of Scots pine (Pinus sylvestris) and other coniferous trees of relevance to forestry in Germany. In this study, the distribution and occurrence of S. sapinea and accompanying endophytic fungi in twigs of healthy and diseased Scots pine was investigated on a spatial and temporal scale. Sampling of 26,000 twig segments from trees in 105 temperate coniferous forest stands in Germany resulted in isolation of 33,000 endophytic fungi consisting of 103 species identified based on morphological and ITS-DNA sequence analyses. Approximately 98% of the sample was represented by fungi in the Ascomycota, with only two species (Peniophora pini and Coprinellus sp.) belonging to the Basidiomycota. Four species were detected in a frequency greater than 10% (Sphaeropsis sapinea, Sydowia polyspora, Microsphaeropsis olivacea, and Truncatella conorum-piceae) from the collective sample. A typical inhabitant of Scots pine twigs Desmazierella acicola was isolated and additionally typical hardwood colonizers like Biscogniauxia spp. were detected. S. sapinea, an endophytic plant pathogen with saprobic capabilities, was isolated from more than 80% of the studied pine trees, but the majority of trees sampled showed no symptoms of Diplodia tip blight. No invasive, pathogenic quarantine fungi for Germany were isolated from healthy or diseased Scots pines. Advantages and disadvantages of isolation-based endophyte studies over studies using direct DNA-isolation are discussed. Knowledge of the fungal endophyte communities in twigs of Scots pine allowed for identification S. sapinea and other potential pathogens of pines and other forest trees that may possibly contribute to increased disease under repeated periods of drought and heat stress in the future.

Antifungal Activity of β-Pinene-Based Hydronopyl Quaternary Ammonium Salts Against Phytopathogenic Fungi

β-Pinene can be used as a cheap source to synthesize a large number of high value-added derivatives. In this study, a series of β-pinene derivatives was prepared, and the antifungal activities of the compounds were assessed against phytopathogenic fungi. Eight N-alkyl hydronopyl diethyl ammonium halide salts were synthesized by the reaction of hydronopyl diethyl ammonium halide with 8 halogenated alkanes. The structures of the synthesized products were characterized by Fourier-transform infrared spectroscopy and nuclear magnetic resonance spectroscopy and mass spectrometry. The antifungal activities of these derivatives were tested against 11 plant pathogens, and the preliminary structure-activity relationship is discussed. Some derivatives exhibited moderate to significant antifungal activity due to the fusion of the hydronopyl, a long-chain alkyl, bromine, and iodine anionic groups. In contrast to the structure-activity relationship of compounds 2a, 2b, and 2c, iodine ions in 2f, 2g, and 2f had a significant effect on enhancing the antifungal activity against Colletotrichum gloeosporioides, S clerotinia sclerotiorum, Phytophthora capsici, Phomopsis, Sphaeropsis sapinea, Glomerella cingulata, and Fusicoccum aesculi. A higher molecular weight could increase the antifungal activity against Fusarium proliferatum, Alternaria kikuchiana, Sclerotinia sclerotiorum, P. capsici, Phomopsis, and S. sapinea. Compounds 2d and 2e exhibited broad-spectrum antifungal activity against the tested strains. These derivatives are expected to be used as precursor molecules for novel pesticide development in further research.

Pine engraver beetle Ips acuminatus as a potential vector of Sphaeropsis sapinea

The pine engraver beetle Ips acuminatus Gyll. is a potential vector of the Sphaeropsis tip blight pathogen according to Leach’s postulates. The specimens of I. acuminatus were associated with numerous fungi species, namely Sphaeropsis sapinea (Fr.) Dyko &amp; B. Sutton and ophiostomatoid species. The association between opportunistic pathogen S. sapinea and I. acuminatus has been confirmed for 62.9 % of all branches (44 % of needle samples and 82 % of wood samples). The presence of S. sapinea in the galleries and on the surface of the beetle indicates that I. acuminatus may transport the pathogen and later introduce it into healthy trees. The bark beetle can transfer pathogenic fungus during maturation feeding on the shoots of healthy pine crowns and into the branches during making galleries.

Systemic Tree Injection of Propizol (14.3% wt./wt. propiconazole ME) in Austrian Pine (Pinus nigra) for Control of Diplodia Tip Blight (Diplodia pinea)

"Diplodia tip blight (Diplodia pinea) affects mature pines, including Austrian (Pinus nigra) and Ponderosa (P. ponderosa) pines. Infections spread from needle fascicles to branch and, if unchecked, to the entire tree. Efficacy studies of fungicide injections in conifers are limited. Minute vascular tissues and resin exudate, a response to drilling, present impediments to injection. The efficacy of Propizol (14.3% propiconazole) for control of Diplodia tip blight in Austrian pines was evaluated. We evaluated (1) time of year, (2) injection spacing, and (3) fungicide dilution with respect to injection efficiency. Late fall injections expedited uptake, which is consistent with the reduced monoterpene emission rates in autumn and winter reported by Kim et al. 2005 and Lim et al. 2008. The time required for the dose to be administered was recorded for close and wide spacing of injection sites. Close spacing had the greatest impact on reducing the application time, irrespective of time of year. Low volume injections required less time to apply compared to high volume. Regardless of the application method, we observed a significant decrease in disease incidence in Propizol-treated trees. Injections applied in late fall resulted in a mean reduction in infections of new candles in the next growing season. Injections in the following spring, however, did not result in improvement in candle condition until a year later. We believe that these differences are based on whether the fungicide was applied prior to or after infection. Based on these findings, we recommend Propizol prior to infection for optimal results.

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

AbstractSphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.

Response of Scots pine (Pinus sylvestris) seedlings subjected to artificial infection with the fungus Sphaeropsis sapinea

The ascomycete Sphaeropsis sapinea (syn. Diplodia pinea), the causal agent of Diplodia blight of pine, is also known to be an endophyte with a latent pathogenic potential on pine trees. Due to climate change effects, especially warming and more prolonged or frequent drought events, the hosts are weakened and S. sapinea could become more aggressive in the future. Normally, pines which are 25 years old or older are more susceptible to the disease as compared with younger trees. However, if younger trees are growing on poor sites or are located close to affected trees, they may also become infected. Here, we analyzed the interaction of young pine seedlings (4 years old) with the fungus in vitro, in order to examine the changes in gene activity for example of genes encoding metabolites and therewith contributing to an induced resistance against the fungus. During different stages of infection (control without infection and 14 days after infection), pine needles of four samples per stage (eight samples all together) were collected and analyzed by mRNA sequencing. The comparison between the two stages showed that, nearly independent of the genotype of the plant, 5691 genes were differentially expressed. In the severe stage of infection, elevated transcript levels of genes involved in lignin- and phytoalexin-biosynthesis and of pathogenesis-related genes were found. Furthermore, some of these genes were also validated in a natural population of Pinus sylvestris by reverse transcriptase (RT)-PCR in order to analyze induced defense responses.

Characterization of a novel victorivirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is an important pathogenic fungus that causes serious diseases in fruits and trunks of many wood plant species worldwide. In this study, 28 B. dothidea strains isolated from pear trunk samples showing stem wart or canker symptoms were used to detect double-stranded RNA (dsRNA) viruses. The dsRNA bands with the size of ~ 1.0 to ~ 6.0 kbp were examined from ten strains. Here, we reported a novel dsRNA mycovirus, tentatively named as Botryosphaeria dothidea victorivirus 2 (BdVV2), isolated from the B. dothidea strain MSD53. BdVV2 contained spherical virions that were ~ 38nm in diameter consisting of a single linear dsRNA genome of 5,090bp in length. The BdVV2 genome contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), which shared significant amino acid identities of 68% and 60% with the corresponding proteins of Sphaeropsis sapinea RNA viruses 1 (SsRV1). Phylogenetic analyses based on the aa sequences of CP and RdRp both showed that BdVV2 was phylogenetically related to the members of the genus Victorivirus in the family Totiviridae. BdVV2 is thus a novel victorivirus isolated from the phytopathogenic fungus B. dothidea.

A comparative characteristic of fungal communities associated with Ips acuminatus in different regions of Ukraine

Pine bark beetles are typically associated with complexes of fungi that could reveal different functional interaction. Thus, previously nonaggressive bark beetle Ips acuminatus is considering now to be among the most serious pests of pine forest in Ukraine and other European countries and vectored fungal community is very important to assess total harm of this bark beetle. The aim of this study was to reveal the vectored fungal community associated with the pine engraver beetle, I. acuminatus with special emphasis on pathogenic fungi for further evaluation of harm bark-beetle - fungi association for Ukrainian forest.&#x0D; In total, 288 adult beetles were collected from Scots pine trees at six different sites through Ukraine. DNA sequencing as fungal culturing from all beetles resulted in 1681 isolates and amplicons representing 42 fungal taxa. NCBI BLAST search revealed that the overall fungal community was composed of 94 species, of which 80.85% were Ascomycota, followed by Basidiomycota and unidentified fungal group, which accounted for 10.6% and 8.5 % of the total sequences, respectively. Among these, the most commonly detected fungi for pooling dataset were Sphaeropsis sapinea (23.6%), Cladosporium pini-ponderosae (19.44%), Ophiostoma ips (19.1%), Ophiostoma canum (19.1%) and Cladobotryum mycophilum (18.06%). In the pooled dataset of isolates and amplicons for each site, Shannon diversity indices ranged between 1.9 and 2.9 while Simpson diversity index varied between 0.69 and 0.89 indicating rich species diversity.&#x0D; In total twelve ophiostomatoid species were detected. All ophiostomatoid fungi were showing varying degrees of virulence and O. minus was the most aggressive fungus in previous studies. It is concluded that I. acuminatus vectors a species-rich fungal community including pathogens such as ophiostomatoid fungi, Sphaeropsis sapinea, different needle pathogens and wood decay fungi that seems to be very important for the assessment of threat of I. acuminatus to the pine forest in Ukraine.

Antagonistic activity of Diplodia pinea against phytopathogenic fungi.

The ability of Diplodia pinea to inhibit Armillaria sp., Bjerkandera adusta, Botrytis cinerea, and Rhizoctonia sp. mycelium growth was analyzed using the double-culture method. Wild-type fungal strains were incubated in a biochemical oxygen demand incubator using potato agar dextrose medium at 24 ± 2°C for 35days in darkness. D. pinea significantly inhibited the growth of all fungi species tested (30.75 to 98.37% inhibition) and showed moderate antagonistic activity (antagonistic index, 14.5). Chemical analysis of D. pinea culture broth extracts revealed steroids, triterpenes, and phenolic compounds. Alkaloids were qualitatively detected in the mycelium crude extract. The presence of these compounds may be related to the antagonistic activity observed. The inhibition ability of D. pinea is due to competition with the tested fungi for substrate and space.

Current Problems and Tasks of Forest Protection in Poland

Abstract Current problems of forest protection concern the declining health of forest stands due to climate change and the resulting extreme weather events such as heat waves, droughts, hurricane winds, heavy rainfalls and floods. Repeated impacts of these factors increase susceptibility of forest stands to pest insects and fungal pathogens. Norway spruce (Picea abies [L.] H. Karst) is sensitive to high air temperatures and water shortage. Long lasting droughts during the last two decades, have been one of the reasons behind Norway spruce dieback due to severe outbreak of European spruce bark beetle Ips typographus (L.) in the mountainous regions of southern Poland. In the Scots pine (Pinus sylvestris L.) stands, water balance disorders have enhanced the colonization of weekend trees by steelblue jewel beetle Phaenops cyanea (F.) and engraver beetle Ips acuminatus (Gyll.), as well as contributed to the spread of fungal diseases caused by Gremmeniella abietina (Lagerb.) Cenangium ferruginosum Fr. and Sphaeropsis sapinea Fr. fungi. Water related stress leads to weakening of oak stands, which are attacked by Agrilus spp. beetles and pathogens from the genus Phytophthora. It is possible that long lasting droughts initiated the spread of infectious ash disease caused by Hymenoscyphus fraxineus (= Chalara fraxinea), which resulted in the epidemic of ash dieback throughout Europe. Until recently, the use of plant protection products was the most common method of forest protection against pest insects and pathogens. Poland’s accession to the European Union has affected the marketing and use of plant protection products in the country. The implementation of the EU legislation (Directive 91/414/EEC, Directive 2009/128/ EC and Regulation No 1107/2009) has resulted in decreased assortment of pesticides registered for the protection of forests. High costs and long registration process considerably limited the interest of producers in placing the plant protection products on the market. Systematic decrease in the number of plant protection products possible to register for use in forestry, as well as the principles of integrated plant protection established in the EU in 2014 call for seeking plant protection methods based on the natural enemies of pests, such as pathogenic microorganisms, parasites and predators. Therefore, contemporary forest protection requires the advancement of integrated methods for pest insect and disease control through developing methods of forecasting forest dangers, the use of natural enemies and agro-technical methods for regulation of pests, as well as the development of decision support systems as a tool facilitating introduction of integrated forest protection principles. Such support systems help to establish optimal terms for the implementation of protection measures, so as to increase their efficiency while limiting the use of chemical pesticides to an absolute minimum.

First report of Diplodia sapinea on Cedrus libani in Turkey

Cedar of Lebanon (Cedrus libani) is an ecologically and economically important conifer with a natural distribution in southern Turkey. Currently, no serious diseases affect C. libani in its native range, although it is susceptible to pathogens such as Heterobasidion annosum and Diplodia pinea in inoculation experiments (Lehtijärvi et al., 1). Recent reports from the Balkans and Northern Europe, however, suggest that C. libani is susceptible to foliage and shoot diseases such as Diplodia pinea, Dothistroma septosporum and Sirococcus tsugae (Mullet et al., 2; Zlatkovic et al., 2016). During disease surveys in July 2017 in the Atatürk Arboretum, İstanbul (41°10'30.53“N, 28°59'5.22”E), dieback was observed on branches of a young C. libani (Fig. 1). Abundant black pycnidia were observed on dead needles under a binocular microscope (Fig. 2). Examination under a compound microscope revealed the presence of non- or one-septate, light to dark brown, oblong-ellipsoid conidiospores, characteristic of the genus Diplodia (Figs. 3-4). Isolations were made by plating spore masses onto 2% malt extract agar in 90 mm diameter Petri dishes under aseptic conditions and incubating at room temperature for ten days in the dark. Blackish-dark grey colonies with grey aerial mycelia were subcultured on 2% malt extract agar covered with cellophane membrane and incubated as described above. DNA was extracted from mycelia using an E.Z.N.A. fungal DNA extraction kit (Omega Bio-tek, USA) and amplified by PCR using species-specific primers for both D. pinea and D. scrobiculata (Smith & Stanosz, 4). The D. pinea primers amplified a product of 700 bp. The ITS region was also amplified and sequenced (White et al., 5), and the sequence of a representative isolate was deposited in GenBank (Accession No. MH368280). Comparison of the ITS sequences obtained from C. libani showed 100% nucleotide identity with those of reference strains of D. sapinea (e.g. NR_152452.1). Inoculations were made on two-year-old C. libani plants to determine the pathogenicity of the fungus, with five replicate plants each for stem and shoot inoculations. A circular 4-mm wound was made on stems approximately 10 cm above the root collar, by removing the bark tissues with a surface-sterilised cork borer. For shoot inoculations, a small wound of c. 3 mm was made on the shoots using a scalpel to remove the bark, at c. 5 cm from the tip of the terminal shoot. Agar plugs (4 and 2 mm diameter for stem and shoots, respectively) taken from the margins of an actively growing D. sapinea colony on potato dextrose agar (PDA), were placed mycelium-side onto the wounds. All treatments were wrapped in plastic paraffin film. Controls (three replicates for both stem and shoot inoculations) were wounded, as described above, and treated with sterile PDA plugs. All plants were incubated at 24°C for eight weeks in a climate chamber. Following incubation, dieback symptoms were observed on all inoculated plants; foliage and stems were dead above the inoculation point (Fig. 5). Lesion extension towards the root collar was 24.2 ± 2.0 mm for stem and 26.0 ± 1.16 mm for the shoot inoculations. The pathogen was successfully re-isolated from symptomatic stem and shoot tissues, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Diplodia sapinea causing dieback on C. libani in Turkey. Financial support from the Süleyman Demirel University (Project No.4984-GÜP-17) is gratefully acknowledged.