In vitro Safety Assessment of Extracts and Compounds From Plants as Sunscreen Ingredients.

This work investigated the safety of extracts obtained from plants growing in Colombia, which have previously shown UV-filter/antigenotoxic properties. The compounds in plant extracts obtained by the supercritical fluid (CO2) extraction method were identified using gas chromatography coupled to mass spectrometry (GC/MS) analysis. Cytotoxicity measured as cytotoxic concentration 50% (CC50) and genotoxicity of the plant extracts and some compounds were studied in human fibroblasts using the trypan blue exclusion assay and the Comet assay, respectively. The extracts from Pipper eriopodon and Salvia aratocensis species and the compound trans-ß-caryophyllene were clearly cytotoxic to human fibroblasts. Conversely, Achyrocline satureioides, Chromolaena pellia, and Lippia origanoides extracts were relatively less cytotoxic with CC50 values of 173, 184, and 89 µg/mL, respectively. The C. pellia and L. origanoides extracts produced some degree of DNA breaks at cytotoxic concentrations. The cytotoxicity of the studied compounds was as follows, with lower CC50 values representing the most cytotoxic compounds: resveratrol (91 µM) > pinocembrin (144 µM) > quercetin (222 µM) > titanium dioxide (704 µM). Quercetin was unique among the compounds assayed in being genotoxic to human fibroblasts. Our work indicates that phytochemicals can be cytotoxic and genotoxic, demonstrating the need to establish safe concentrations of these extracts for their potential use in cosmetics.

The effect of cigarette smoking on human seminal parameters, sperm chromatin structure and condensation.

Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11-20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking.

A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species.

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high-precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue-stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.

Ameliorating the in vivo antimalarial efficacy of artemether using nanostructured lipid carriers.

Cerebral malaria (CM) is a fatal neurological complication of Plasmodium falciparum infection that affects children (below five years old) in sub-Saharan Africa and adults in South-East Asia each year having the fatality rate of 10-25%. The survivors of CM also have high risk of long term neurological or cognitive deficits. The objective of the present investigation was to develop optimised nanostructured lipid carriers (NLCs) of artemether (ARM) for enhanced anti-malarial efficacy of ARM. NLCs of ARM were prepared by a combination of high speed homogenisation (HSH) and probe sonication techniques. Preliminary solubility studies for ARM showed highest solubility in trimyristin (solid lipid), capmul MCM NF (liquid lipid) and polysorbate 80 (surfactant). Trimyristin and capmul showed superior miscibility at a ratio of 70:30.The optimised NLC formulation has the particle size (PS) of: 48.59 ± 3.67 nm, zeta potential (ZP) of: -32 ± 1.63 mV and entrapment efficiency (EE) of: 91 ± 3.62%. In vitro cell line (human embryonic kidney fibroblast cell line (HEK 293 T)) cytotoxicity studies showed that prepared formulation was non-toxic. The results of in vivo studies in CM induced mice prevented the recrudescence of parasite after administration of NLCs of ARM. Additionally, NLCs of ARM showed better parasite clearance, higher survival (60%) in comparison to ARM solution (40%). Also it was observed that lesser entrapment of Evans blue stain (prepared in PBS as solution) in the NLCs of ARM treated brains of C57BL/6 mice than ARM solution treated mice. Hence NLCs of ARM may be a better alternative for improving therapeutic efficacy than ARM solution.

Genetic and genomic evidence of niche partitioning and adaptive radiation in mountain pine beetle fungal symbionts.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle's life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.

Intrahepatic cholangiocarcinoma.

Cholangiocarcinoma, also referred to as cholangiocellular carcinoma (particularly in Japan), develops along the biliary tract. The tumor may be intra- or extrahepatic and have different features with specific treatments based on the site of origin. Guidelines for diagnosis and management of cholangiorcarcinoma, such as those proposed by EASL (European Association for the Study of the Liver)1 and the Mayo Clinic2 classify the tumor into intrahepatic, perihilar, and distal cholangiocarcinoma. There are three main macroscopic patterns of growth of cholangiocarcinoma: mass-forming, periductal-infiltrating and intraductal. A combination of mass-forming and periductal infiltrating tumors have been shown to have a poor prognosis.3 Intrahepatic cholangiocarcinoma (ICC) comprises two microscopic subtypes: bile duct and cholangiolar.4 The bile duct subtype has tall columnar cells that form large glands, whereas cholangiolar tumors are composed of cuboidal and low columnar cells. Patients with cholangiolar tumors, referred to as cholangiolocellular carcinoma, reportedly have a better 5-year survival rate than those with the bile duct type.4.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients.

Chemical Signals of Vector Beetle Facilitate the Prevalence of a Native Fungus and the Invasive Pinewood Nematode.

In China, the invasive Bursaphelenchus xylophilus, the vector Monochamus alternatus beetle, and associated fungi exhibit a symbiotic relationship causing serious losses to pine forests. Although this complex system has been intensively investigated, the role of vector beetles on the development of associated fungi and their indirect contribution to the prevalence of pinewood nematode (PWN) is yet unknown. Here, three of the highly prevalent fungal species, viz., Sporothrix sp. 1, Ophiostoma ips, and Sporothrix sp. 2 were isolated from beetle chambers in diseased trees in Guangdong province, southeast China. Pairwise cultivation of isolated fungi demonstrated the dominance of Sporothrix sp. 1 over O. ips and Sporothrix sp. 2. On the other hand, two fatty acid ethyl esters (FAEE), ethyl palmitate (EP) and ethyl linoleate (EL), isolated from the body surface of the vector beetle enhanced the growth of Sporothrix sp. 1. When PWN were cultured on Sporothrix sp. 1, the fecundity and the body length were increased significantly as compared with when cultured on O. ips and Sporothrix sp. 1. Our results suggest that the vector beetles promote Sporothrix sp. 1 to occupy more niches by rapid growth and spread, which in turn better support PWN population, hence facilitate PWN pathogenicity in the invasive regions.

Differences in defence responses of Pinus contorta and Pinus banksiana to the mountain pine beetle fungal associate Grosmannia clavigera are affected by water deficit.

We tested the hypotheses that responses to the mountain pine beetle fungal associate Grosmannia clavigera will differ between the evolutionarily co-evolved host lodgepole pine (Pinus contorta var. latifolia) and the naïve host jack pine (Pinus banksiana) and that these responses will be influenced by water availability. G. clavigera inoculation resulted in more rapid stem lesion development in lodgepole than in jack pine; water deficit delayed lesion development in both species. Decreased hydraulic conductivity was observed in inoculated lodgepole pine seedlings, likely because of tracheid occlusion by fungal hyphae and/or metabolite accumulation. Drought but not inoculation significantly impacted bark abscisic acid levels. Jasmonic and salicylic acid were implicated in local and systemic responses of both species to G. clavigera, with salicylic acid appearing to play a greater role in jack pine response to G. clavigera than lodgepole pine. Water deficit increased constitutive levels and/or attenuated induced responses to G. clavigera for several monoterpenes in lodgepole but not jack pine. Instead, inoculation of well-watered but not water deficit jack pine resulted in a greater number of xylem resin ducts. These findings reveal mechanisms underlying differences in G. clavigera-induced responses between lodgepole and jack pine hosts, and how water availability modulates these responses.

Quantitation of Mucin by Densitometry of an Alcian Blue-Stained Membrane.

It is a challenging task to quantify mucin using conventional protein quantification methods due to the large number of glycans attached to the peptide, which make up approximately 50-90% of its molecular weight. To address this issue, we propose a simple quantification method that involves spotting mucins onto a membrane and staining them with Alcian blue.

Validation of an automated iron stain process for use with bone marrow aspirate smear slides.

The assessment of bone marrow iron stores is typically performed on an aspirate smear slide that has been manually stained by a technologist using a commercially available kit. This approach can contribute to inconsistent results and limit the broad use of iron staining in bone marrow specimens, particularly when laboratories have low staffing and/or high specimen volumes. Here, we describe the adaptation and validation of the Ventana Benchmark automated stainer and iron stain kit for routine clinical use of staining iron in bone marrow aspirate smear slides. We assessed accuracy and precision of the Ventana automated iron staining protocol compared to the Perls Prussian blue manual iron staining index method. Hematopathologists assigned Gale scores and enumerated the percentages of erythroid sideroblasts on paired patient bone marrow aspirate smear slides stained by the automated method and the manual iron staining method. We found a similar level of performance of the Ventana automated iron stain relative to the index manual method (as assessed by Pearson correlation and Bland-Altman analyses). In addition, there was low imprecision between replicates performed via the automated iron stain protocol. We also report superior qualitative findings of the automated method in ease of localization of iron storage, visualization of sideroblasts, and counterstain consistency. Automated iron staining of bone marrow aspirate smear slides performed similarly to the manual method and may allow for accurate routine evaluation of bone marrow iron stores as part of bone marrow analysis.

Ophiostoma haidaense, sp. nov., a new member of the O. piceae species complex associated with yellow-cedar, Callitropsis nootkatensis.

Ophiostoma haidanensis is described as a new species of the Ophiostoma piceae complex isolated from yellow-cedar (Callitropsis nootkatensis (D. Don) Oerst. ex D.P. Little) sapwood in the Haida Gwaii island archipelago and the North Coast of British Columbia, Canada. The fungus is characterized by the production of a typical sporothrix-like asexual morph but is distinguished morphologically from other members of the O. piceae species complex by its large, multiseptate primary conidia. Phylogenetic analysis of DNA sequences from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the ß-tubulin (BTUB) and translation elongation factor 1-α (TEF1) genes supports the inclusion of O. haidensis as a distinct member within the O. piceae complex. To our knowledge, this is the first report of a blue stain fungus infecting yellow-cedar, an ecologically, culturally, and economically important conifer naturally distributed along the coastal forests of the Pacific Northwest in North America.

Cellulose Nano Crystals (CNC) as Additive for a Bio-Based Waterborne Acrylic Wood Coating: Decay, Artificial Weathering, Physical and Chemical Tests.

Wood coatings prolong the service life of wood-based products, but they are usually of synthetic origin. The aim of the present article is to reduce the fossil-based compounds in a commercial waterborne acrylic coating by CNC addition and to test its performance. The coatings were applied on European beech and Norway spruce wood in order to test durability against Gloeophyllum trabeum (brown wood rot) and Trametes versicolor (white wood rot). Artificial weathering and blue stain, contact angle, physical tests (adhesion, impact and scratch test), chemical (FTIR) and morpho-anatomical analysis (SEM) were carried out. CNC addition increased viscosity, limiting the spreading of the coating into wood pores as visible after SEM observation, which reduced coating adhesion on the substrate. CNC improved fungal resistance as seen by a reduced mass loss and FTIR spectroscopy thanks to crosslinks formation, which reduced water sorption as well. Color change was not significant, and, on the other hand, glossiness was reduced but resulted as more homogeneous than control. CNC addition gave good results also in blue stain protection. CNC improved scratch resistance, but no visible change to impact was registered. CNC has promising results in coatings depending on wood and fungal species and presence of further commercial additives (biocides).

Utility of Perl's Prussian Blue Stain in Exfoliated Buccal Cells of Thalassemia and Sickle Cell Anemia Patients and Their Correlation With Serum Ferritin Levels.

Introduction Iron is essential for all living beings. Excess iron, on the other hand, is dangerous because it causes the creation of free radicals. As a result, iron absorption is carefully managed to maintain a balance between absorption and iron loss in the body. Due to the lack of particular excretory channels for iron in humans, iron excess in the tissues is common. It can be caused by a number of factors, including increased iron absorption, as seen in hemochromatosis, or frequent parenteral iron treatment, as seen in thalassemia and sickle cell anemia patients (a transfusional overload). Aim The study aims to demonstrate Perl's Prussian blue stain to identify iron overload at a preliminary stage and correlate with serum ferritin levels in patients with thalassemia and sickle cell anemia who frequently receive blood transfusions. Materials and methods The present study comprised 62 confirmed cases of thalassemia and sickle cell anemia patients undergoing repeated blood transfusions of a minimum of 15/more, along with 62 clinically healthy individuals between December 2016 and November 2018. The patients with thalassemia and sickle cell anemia were confirmed by hemoglobin electrophoresis (Bio-Rad D-10, Bio-Rad Laboratories, Inc, California, United States). The buccal smears were obtained from these patients along with the controls, and these slides were stained by Perl's Prussian blue stain and were examined under a light microscope. Results Sixty-two cases and 62 controls were considered in the current investigation. Forty-seven of the 62 people had thalassemia, and 15 had sickle cell anemia. Thirty-nine out of the 47 patients with thalassemia and six of the 15 individuals with sickle cell anemia had positive results for Perl's Prussian blue stain. All patients had elevated blood ferritin levels, with varying ranges associated with positive results for Perl's Prussian blue stain. Conclusion The objective of this study was to demonstrate the utility of oral exfoliative cytology in thalassemia and sickle cell anemia patients who often receive blood transfusions as a screening and diagnostic tool. The exfoliative cytology methods' acceptability and simplicity, along with their correlation with serum ferritin levels and Perl's Prussian blue reaction, make this noninvasive procedure an excellent screening and diagnostic tool for all patients who receive repeated blood transfusions.

Primary laryngeal cryptococcosis mimicking laryngeal malignancy: a case report.

Primary laryngeal cryptococcosis is an extremely rare infection and presents with non-specific symptoms such as hoarseness or sore throat, resulting in delayed diagnosis. Here, we report the patient of a 56-year-old female patient with primary laryngeal cryptococcosis, who was being treated with oral and inhaled steroids for rheumatoid arthritis and bronchial asthma. The patient suffered from prolonged hoarseness and sore throat, and endoscopic biopsy was performed several times under local anesthesia, demonstrating only inflammatory cell infiltration. Considering the possibility of laryngeal malignancy, a third biopsy was performed by endoscopic laryngomicrosurgery under general anesthesia. Intraoperative frozen section revealed non-neoplastic laryngeal mucosa with erosion and severe inflammatory cell infiltration. However, we could not confirm the definite diagnosis of the lesion in the intraoperative consultation. Postoperative histopathological examination revealed a small number of yeast-type fungi and a definitive diagnosis was established by special stains including Alcian blue stain. Finally, the patient was diagnosed as primary laryngeal cryptococcosis. Daily oral administration of fluconazole (400 mg/day) was performed for 6 months according to the treatment protocol for pulmonary cryptococcosis. The symptoms gradually improved, and endoscopy revealed no recurrence 6 months post-treatment. Clinicians should consider the possibility of laryngeal cryptococcosis when severe inflammation is found in the larynx and discuss the disease history and pathological results with pathologists more closely.

Fungal Communities of the Pine Wilt Disease Complex: Studying the Interaction of Ophiostomatales With Bursaphelenchus xylophilus.

Considered one of the most devastating plant-parasitic nematodes worldwide, Bursaphelenchus xylophilus (commonly known as pinewood nematode, PWN) is the causal agent of the pine wilt disease in the Eurasian coniferous forests. This migratory parasitic nematode is carried by an insect vector (Monochamus spp.) into the host tree (Pinus species), where it can feed on parenchymal cells and reproduce massively, resulting in the tree wilting. In declining trees, PWN populations are strongly dependent on fungal communities colonizing the host (predominantly ophiostomatoid fungi known to cause sapwood blue-staining, the blue-stain fungi), which not only influence their development and life cycle but also the number of individuals carried by the insect vector into a new host. Our main aim is to understand if PWN-associated mycobiota plays a key role in the development of PWD, in interaction with the PWN and the insect vector, and to what extent it can be targeted to disrupt the disease cycle. For this purpose, we characterized the fungal communities of Pinus pinaster trees infected and non-infected with PWN in three collection sites in Continental Portugal with different PWD temporal incidences. Our results showed that non-infected P. pinaster mycoflora is more diverse (in terms of abundance and fungal richness) than PWN-infected pine trees in the most recent PWD foci, as opposed to the fungal communities of long-term PWD history sites. Then, due to their ecological importance for PWN survival, representatives of the main ophiostomatoid fungi isolated (Ophiostoma, Leptographium, and Graphilbum) were characterized for their adaptative response to temperature, competition in-between taxa, and as food source for PWN. Under the conditions studied, Leptographium isolates showed promising results for PWN control. They could outcompete the other species, especially O. ips, and significantly reduce the development of PWN populations when compared to Botrytis cinerea (routinely used for PWN lab culturing), suggesting this to be a natural antagonist not only for the other blue-stain species but also for the PWN.

Gastrointestinal Mast Cell Tumor in an African Dormouse (Graphiurus sp.).

Mast cell tumors (MCTs) are well-known neoplasms derived from either mucosal or connective tissue mast cells. While well studied in several domestic species, MCTs are rarely documented in rodents. A three-year-old, male African dormouse (Graphiurus sp.) presented with a history of vomiting and anorexia for 3 months. Sonography revealed thickened gastric mucosa and hyperperistalsis. The patient died after receiving symptomatic treatment for 2 months. At necropsy, locally extensive, pale, thickened mucosal foci obscuring the first half of the stomach lumen was noted. Histological examination revealed moderately polymorphic, round, oval to spindle cells with amphophilic cytoplasmic granules infiltrating the mucosa to tunica muscularis, with moderate numbers of eosinophils. The mucosa was severely ulcerated with the proliferation of granulation tissue. The granules in most tumor cells exhibited metachromasia with the toluidine blue stain. Neoplastic cells revealed positive membranous immunoreactivity to KIT. Herein, we report the first case report of MCT in dormouse but also the first gastrointestinal MCT in a rodent species.

Efficacy of Csaba stain to demonstrate mature and immature mast cells in oral inflammatory lesions: An in-vitro study.

Background: Mast cells (MCs) are immune cells of the myeloid lineage and are present in connective tissues throughout the body. Mastocytosis regulates many physiologic processes and affects the pathogenesis of allergic conditions, anaphylactic reactions, autoimmune disorders and leukemias. Toluidine blue is commonly employed as a special stain for MCs that uniformly imparts blue color to both immature and mature cells. Csaba stain that distinguishes mature from immature MCs has not been widely explored. However, its use in differentiating mature and immature cells has not been reported in the literature. The identification of mature and immature MCs may provide a major clue for the diagnosis of any unrecognized systemic pathologies. Objectives: To evaluate the staining efficacy of Csaba stain, toluidine blue and Leishman's stains in the identification of MCsTo compare the effectiveness of Csaba stain, toluidine blue and Leishman's stains to identify MCs in inflammatory pathologic lesions. Materials and Methods: Fifty sections of inflammatory lesions were subjected to Csaba stain, toluidine blue and Leishman's stains each for studying MCs. The staining efficacy of the three stains was compared for parameters such as homogeneity, specificity, staining intensity, granular staining property and differentiation between mature and immature cells. Results: The Csaba, toluidine blue and Leishman's stains showed statistically insignificant P = 0.50, 0.95, 0.29, 0.48 for homogeneity, specificity, staining intensity and granular staining property respectively, but Csaba stain showed statistically significant P = 0.0001* in differentiating mature from immature cells. Conclusion: Csaba stain is more effective in differentiating mature from immature MCs compared to the other special stains.

Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.

Ophiostomatales Associated with Mediterranean Pine Engraver, Orthotomicus erosus (Coleoptera, Curculionidae) in Dalmatia, Croatia.

Mediterranean pine engraver, Orthotomicus erosus was never considered as a significant pest in Croatia and did not appear in high population densities until 2017, when it reached outbreak level in Aleppo pine stands. The beetle was first detected in Marjan Forest Park, Split, and was soon recorded in other parts of the Dalmatian coast. Soon after the outbreak occurred, we observed that all of the attacked trees exhibit severe blue staining in the sapwood which indicated fungal infection caused by the Ophiostomatales group of fungi. This raised the need to investigate their relationship with O. erosus and the pine decline, and the main aim of this study was to isolate and identify them. Isolates were obtained from adult O. erosus beetles, their galleries, and blue-stained sapwood, and identified according to the morphological characteristics and DNA sequencing. A total of six Ophiostomatales (Ophiostoma ips, O. piceae, Graphilbum cf. rectangulosporium, O. floccosum, Sporothrix pseudoabietina and Ceratocystiopsis cf. minuta) were identified in the study. This is the first record of Ophiostomatales as organisms associated with the pest O. erosus and pine species in Croatia.