Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards

Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards Klaudia Zawadzka, Karolina Oszust, Michał Pylak, Agata Gryta, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8779190/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bull’s eye rot, caused by fungi of the genus Neofabraea (syn. Pezicula , Phlyctema, Gloeosporium ), is an important postharvest disease of apples worldwide. This study aimed to identify and characterize Neofabraea species associated with Bull’s Eye Rot in Polish orchards. Apples with disease symptoms were collected from 53 locations across five voivodships. The developed protocol for the selective isolation of fungal strains of the genus Neofabraea from symptomatic apples allowed the collection of 155 strains. This protocol involved apple tissue surface sterilization with 70% ethanol and incubation at 18°C, effectively reducing contamination by non-target fungi. Molecular identification using Sanger sequencing, followed by a Whole-Genome Sequencing approach, confirmed that the isolates belonged to Neofabraea alba . The isolates were assessed for chemical sensitivity using Biolog™ PM21D plates, revealing growth inhibition by several substrates, including sodium selenite, magnesium chloride, trifluoperazine, and nystatin, with substrate-dependent variability within functional groups. The fungicide-sensitivity test showed that three of ten commercial fungicides exhibited the highest inhibitory effects, while four fungicides showed no activity, indicating complete resistance. The Biolog™ ECO Plate analysis showed differences in the stress response of microbial communities inhabiting the BER symptomatic tissue of apples, depending on variety. Additionally, metataxonomic analyses revealed slight variety-dependent differences, with fungi belonging to Dermateceae predominating at infection sites. These findings improve understanding of Neofabraea sp. pathogens in Poland and support the development of sustainable disease management strategies aligned with the European Green Deal. Biological sciences/Microbiology Biological sciences/Plant sciences Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Fungi, as the most diverse group of organisms, significantly impact biodiversity and agriculture. Many of these species are fungal pathogens associated with plant diseases 1 , 2 . Effective control of fungal plant pathogens is crucial to minimizing the economic and environmental impact of crop diseases. It plays a key role in delivering high-quality products. One of the challenges fruit growers face in terms of quality and crop loss reduction is the occurrence of storage diseases 3 , 4 . One of the most commonly produced fruits, ranking third globally are apples (Statista, 2025). The largest producers are China, the United States, Turkey, and Poland (FAOSTAT, 2025). The occurrence of Bull’s Eye Rot (BER), caused by fungi of the Neofabraea (syn. Pezicula , Phlyctema, Gloeosporium ) genus, is one of the challenges encountered in apple cultivation 5 – 7 . Due to the latent nature of the disease, it is difficult to assess fruit quality at harvest, as its symptoms appear only when fruits are stored at low temperatures in cold stores 7 , 8 . Bull’s eye rot can be caused by various species belonging to the genus Neofabraea , which are widespread in many regions of the world 9 – 12 . Research has shown that the species most commonly found in Polish orchards is Neofabraea alba , followed by Neofabraea perennans , and occasionally Neofabraea kienholzii 13 . Although BER is one of the most common storage diseases, the diversity of pathogens causing it remains understudied 14 . Therefore, it is important to test multiple strains from different locations and apple varieties to understand their distribution and develop effective disease control practices 6 , 13 , 14 . However, existing research on these species is relatively old, and it's worth updating this knowledge with new findings. Furthermore, ongoing climate change is influencing the habitats of individual fungal species. This may lead to the spread of pathogens in previously non-endemic areas 15 , 16 . Furthermore, species identification using multiple markers increases the likelihood of confirming a specific species and of detecting the diversity present in a given area. Therefore, the first aim of our research was to determine whether only one species, Neofabraea sp., occurs in Polish apple orchards or whether species diversity exists. In addition to understanding the diversity of the pathogen attacking apple trees, it is also important to assess its chemical sensitivity within the fungicides used, which is of great importance in light of changing legislation in the area of ​​pathogen control. Fungicides used in plant protection are highly effective in controlling numerous pathogens and improving crop quality 17 , 18 . However, these chemicals have a long waiting period and can reduce the diversity of beneficial fungi 19 . With this in mind, and with the aim of ensuring sustainable agriculture, the European Commission introduced the European Green Deal and the Farm to Fork document, which is an important pillar of the deal 20 . Therefore, it is necessary to seek new potential substances to control and limit the development of the phytopathogen of the Neofabraea genus in fruit growing, which was the next aim of our research. Research on fruit during harvest or affected by the disease has so far focused only on checking for appropriate fruit color, developing methods to indicate the presence of a given disease 21 , and determining the degree of fruit ripeness at harvest 22 . Imported fruit entering the market has also been examined to prevent the spread of pathogens 23 . However, sections of affected apples have not yet been examined for their microbial communities. Therefore, the third aim of our study was to examine apples with BER symptoms for the presence of monocultures, to examine their metabolic profile, and to determine whether this profile is dependent on apple variety. Based on these objectives, we formulated the following hypotheses: (i) the fungi colonizing apples in Polish orchards belong to a single species, and (ii) infected apple fragments are colonized by fungal monocultures, and their metabolic profile depends on the apple variety from which they originated. 2. Materials and methods 2.1. Samples collection Apples exhibiting visible symptoms of Bull’s Eye Rot were collected from 53 orchards across five voivodeships in Poland. Data are presented in Table S1 . A map showing the locations of orchards, along with descriptions of the localities, is presented in Fig. 1 . 2.2. Obtaining Neofabraea spp. isolates To obtain Neofabraea spp. strains, infected apple fragments were cut out and placed on Petri dishes containing potato dextrose lab agar medium (PDA) supplemented with antibiotics, streptomycin, and chlortetracycline. The fruit surfaces were previously sterilized in 70% ethanol and rinsed with sterile distilled water. The Petri dishes were then incubated at 18°C ​​for 8 days. A lower culture temperature was used due to the nature of the disease (development occurs at low temperatures during fruit storage) and to limit the growth of other fungi 24 , 25 . Then, based on morphological features, Neofabraea -like isolates were selected and identified. 2.3. DNA extraction, amplification, and Sanger sequencing Sanger sequencing followed by PCR analysis using at least one of four different markers: ITS1, α-EF, D2LSU, and β-tubulin to confirm the affiliation of the obtained strains to the genus Neofabraea was performed. Firstly, DNA was extracted from isolates grown on the plate using the EURx GeneMATRIX Plant & Fungi DNA Purification Kit (EURx®, Gdansk, Poland). The DNA concentration was measured using a Nanodrop®, yielding a concentration of > 5 ng µl − 1 . The fragments of fungal DNA were amplified to identify the fungi. We used the following conditions: 95°C for 3 min for initial denaturation, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s (for β-tubulin, 45°C), and 72°C for 60 s, with a final step at 72°C for 5 min. 2.4. Whole Genome Sequencing by Illumina ® - genetic identification of isolates obtained from infected apples Whole-genome sequencing (WGS) analysis was performed using Illumina® MiSeq v3 (2x300) technology of four pathogenic Neofabraea spp. isolates (G255/23, G284/23, G333/22, G799/22). WGS libraries were prepared with the Illumina DNA Prep kit. Raw sequencing bcl data were basecalled with RTA (Illumina) and demultiplexed with bcl2fastq (Illumina), then quality assessment and quality trimming were performed on the obtained fastq files with fastQC 26 and Cutadapt 27 . After that, de novo sequence assembly was performed with SPAdes 28 , 29 Genome Assembler, and fasta 30 files containing contigs and scaffolds were obtained. QUAST 31 , 32 Icarus 33 and BUSCO 34 analysis and benchmark were performed to asses quality of assemblies. WGS analysis enabled species identification of isolates obtained from infected apples to determine which species dominate in apple orchards in Poland. Raw sequence reads were submitted to the Sequence Read Archive (SRA) database within BioProjects PRJNA1225134. 2.5. Chemical sensitivity An experiment was performed to assess the chemical sensitivity of pathogenic fungal isolates from the genus Neofabraea : G111/23 (accession number: PX417719), G24/23 (accession number: PX417698), and G788/22 (accession number: PX417812). Phenotypic analysis was performed using the Biolog™ plate system and the PM21D plate suitable for fungal cultures (Biolog™, Hayward, CA, USA). An appropriate amount of fungal spores, according to the manufacturer’s protocol, was mixed with the inoculum fluid to achieve a 62%T transmittance. The plate containing lyophilized substrates was inoculated with a 100 µl suspension of microorganisms. The plates were incubated at 18°C, and absorbance was measured at 750 nm at 24-hour intervals for 10 consecutive days. The wavelength of 750 nm was used to measure optical density, which correlates with changes in microbial biomass production 35 . Chemical sensitivity tests were performed on the plates, including anions, cations, membrane function compounds, chelators, cyclic compounds, organic compounds, antibiotics, and nitrogen compounds. The PM21D plate included 24 chemical compounds, grouped according to Panek et al. 36 . The analysis was carried out under the manufacturer's protocols 36 . 2.6. Fungicide sensitivity In this experiment, a range of ten commercially available fungicides, F1-F10, were prepared in solution at specific concentrations to be applied as in agricultural doses. The fungicides were based on the following active substances: F1 with boscalid 252 g kg − 1 and pyraclostrobin 128 g kg − 1 , F2 with ditianon 700 g kg − 1 , F3 with trifloxystrobin 500 g kg − 1 , F4 with captan 800 g kg − 1 , F5 with cyprodynil 750 g kg − 1 , F6 with sulfur 800 g kg − 1 , F7 with copper oxychloride 500 g kg − 1 , F8 with fluopyram and tebuconazole 200 g kg − 1 , F9 with fludioxonil 500 g kg − 1 and F10 with cyprodinil 375 g kg − 1 and fludioxonil 250 g kg − 1 . The final concentrations of the fungicide solutions were as follows: F1 at 0.16%, F2 at 0.25%, F3 at 0.04%, F4 at 0.4%, F5 at 0.35%, F6 at 1.25%, F7 at 0.3%, F8 at 0.15%, F9 at 0.15%, and F10 at 0.125%. The fungicide sensitivity test was performed using a well test Petri Plate with PDA medium + antibiotics. The following strains were used in the experiment: G344/22 (accession number: PX417793), G361/22 (accession number: PX417803), G9/23 (accession number: PX417688), G17/23 (accession number: PX417694), G267/23 (accession number: PX417758), G271/23 (accession number: PX417760), G278/23 (accession number: PX417763), G297/23 (accession number: PX417774), G299/23 (accession number: PX417775). Fungal isolates were sown on a PDA medium. The 100 µl of the material was pipetted onto the plates and smoothed into the medium with a spatula. Transmittance (%T) was determined at 70% for all isolates tested. Then, 50 µl of fungicide at the appropriate concentration was added to the center. The plates were incubated at 18°C. The growth inhibition of Neofabraea spp. was measured after 7 days of incubation. 2.7. Microbial communities inhabiting apples with Bull’s Eye Rot symptoms The communities of microorganisms inhabiting areas with BER symptoms were characterized by apple variety. Apple surfaces were sterilized with 96% ethanol, and the skin was removed to minimize the risk of contamination with other fungal strains. A section of the apple was then collected, showing necrotic lesions of similar diameter in all varieties, indicating a similar stage of pathogen development. Metabolic and metataxonomic profiling was performed using Biolog™ and Next-Generation Sequencing (NGS), respectively. For analysis, infected apples of similar size were used. 2.7.1. Biolog ™ ECO Plates The metabolic profiling of microbial communities followed Biolog™ ECO plate reads (at both 590 nm and 750 nm wavelengths), as described in detail by Pylak et al. 37 and according to previous research 38 , 39 . The results were presented as diversity indices such as Richness (R) and Shannon (H) (followed by data obtained at 590 nm and 750 nm wavelengths), average well color development (AWCD) (based on 590 nm), average well density development (AWDD) (based on 750 nm), substrate stress index (SST), calculated as the ratio of 590 nm/750 nm values. The lower the SST value, the higher the biomass production at lower substrate utilization, which corresponds to better growing conditions for the microbial community in given substrates 40 . 2.7.2. NGS by Illumina® - apple fungal communities metataxonomic profiling The metataxonomic analyses were conducted using Next-Generation Sequencing (NGS) technology, specifically Illumina® SBS (sequencing by synthesis) (San Diego, USA), with the ITS1 fungal phylogenetic marker, as summarized by Siegieda et al. 41 . Information on the apples used for analysis is provided in Table 1 . Firstly, the environmental DNA from six infected apples was extracted using the GeneMATRIX Environmental DNA & RNA Purification Kit (Eurx®, Gdańsk, Poland). The DNA concentration was measured using a Nanodrop®, yielding a concentration of > 5 ng µl − 1 . The ITS1 fragment was considered using the methods employed to obtain high taxonomic resolution 42 . To determine which fungi were present in each apple sample, the ITS1 fragment of fungal DNA was amplified using primers suggested by Bellmain et al. 43 . The KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cape Town, South Africa) was used for the amplification. We used the following conditions: 95°C for 3 min for initial denaturation, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s, with a final step at 72°C for 5 min. The obtained amplicons were then purified using magnetic beads CleanNGS (CleanNA, Netherlands, Gouda). Each sample was indexed with a unique Illumina® sequencing adapter (Illumina Nextera set a, b, and c) using the following program: 95°C for 3 min and 10 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s with a final step of 72°C for 5 min. Samples were subsequently purified using magnetic beads and quantified using the Quantus fluorometer with QuantiFluor® ONE reagents (Promega, Madison, WI, USA). Library size was analyzed using D1000 DNA ScreenTape on TapeStation 4150 (Agilent Technologies Inc., Santa Clara, USA), and samples were diluted to the same concentration with PCR-grade water and sequenced on the Illumina® MiSeq platform. The data were processed and analyzed using QIIME2 2023.9 44 . In QIIME2, ITSxpress 45 was used for primer trimming, and DADA2 (Divisive Amplicon Denoising Algorithm v.2 ) 46 was used for denoising, chimera removal (method: consensus and minimal fold parent overabundance parameter set to 12), merging, and amplicon sequence variant (ASV) calling 47 . Taxonomy was assigned using the Scikit-learn (sklearn) classifier 48 trained on a UNITE 8.3 database (ver. dynamic, 98.5% similarity level) 49 , 50 . Quantitative positive controls were applied for the target microorganism groups (the MOCK community) 51 . Raw sequence reads were submitted to the Sequence Read Archive (SRA) database within BioProjects SUB12531419. Table 1 The metataxonomic profile of microbial communities from sites with symptoms of Bull’s Eye Rot. Number of ASVs indicating a match to the Dermateceae family according to the FUNGuild database. ASV ID Jonagored Golden Delicious Beni Shogun Red Prince Jonagold Gala Taxonomy FUNGuild ASV45 99.47 99.38 99.40 99.41 0.00 0.00 k__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae ASV12 0.00 0.00 0.00 0.00 100.00 2.63 k__Fungi;p__Ascomycota;c__Sordariomycetes;o__Glomerellales;f__Glomerellaceae ASV29 0.53 0.58 0.56 0.56 0.00 0.00 k__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae ASV69 0.00 0.01 0.00 0.00 0.00 97.37 not identified ASV34 0.00 0.04 0.04 0.04 0.00 0.00 k__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae Localization Siedliska Pierwsze Góry Markuszowskie Siedliska Pierwsze Gutanów Wilków Wilków 2.8. Statistical analysis and visualization Both data normality and homogeneity of variance were initially checked to determine the significance of the difference in Statistica 13.1 software, and adequate tests were applied. The Kruskal-Wallis test and the Student’s t-test were used to evaluate differences in chemical sensitivity of isolates Neofabraea spp. for each group of substrates in 192 h of incubation. The ANOVA with Tukey HSD post hoc test (following Welch’s t-test) was used to analyze differences in sensitivity for fungicides. The CA analysis, including the grouping of treatments (apple variety) and features (response to substrates in Biolog™ ECO plates), was performed on the standardized absorbance average values obtained at 72 h of incubation, as these were the most representative. A dendrogram representing the similarities in carbon utilization patterns from substrates in Biolog™ ECO plates was set on scaled axis bond distances (Ward’s method, within Euclidean distance), with boundaries marked according to Sneath’s criteria (restrictive, 33% or less; less restrictive, 66%) 52,53 . 3. Results 3.1. Obtained isolates All 155 isolated strains are listed in Table S1 , along with their accession numbers. The obtained sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI). Sanger sequencing of at least one marker confirmed that the strains studied belonged to the genus Neofabraea . The WGS analysis of selected strains showed that they belonged to the species Neofabraea alba ( Pezicula vagabunda ), so it can be assumed that this one species dominates in apple orchards in Poland. They were given the following accession numbers: Neofabraea sp. G799/22: JBMPNT000000000, Neofabraea sp. G233/23: JBMPNU000000000, Neofabraea sp. G284/23: JBMPNV000000000, Neofabraea sp. G255/23: JBMPNW000000000. 3.2. Characteristics of selected Neofabraea strains 3.2.1. Chemical sensitivity of pathogenic isolates The sensitivity of the tested microorganisms to several chemical compounds within individual groups was evaluated, and the results are presented for the highest concentration. Figure 2 shows the results for changes in the biomass of the Neofabraea spp. isolates within 192 h with 24 -hour intervals. Strains were found to be sensitive to certain substrates, e.g., sodium selenite, dodecyltrimethyl ammonium bromide, magnesium chloride, copper (II) sulfate, nystatin, 1-hydroxypyridine-2-thione, trifluoperazine, D-cyclocloserine, or thiourea. Their biomass increased on the first day of incubation with a given substrate, then decreased or remained stable over the following days after exposure. This indicates that they are not suitable for effective biomass growth of the pathogen. It is worth emphasizing, however, that the tested isolates also did not demonstrate sensitivity to certain substrates, e.g., sodium dichromate, guanidine hydrochloride, cetylpyridinium chloride, EDTA, or L-aspartic acid b-hydroxamate. Despite their presence, the strains increased their biomass over the following days of incubation. Figure 3 presents the chemical sensitivity results for individual substrates after 192 h of incubation. A similar biomass increase is observed for substrates belonging to the cyclic compounds group (Fig. 3 b) and antibiotics (Fig. 3 f). However, it is worth noting that in some cases, substrates belonging to the same group may cause different biomass increase efficiencies, as seen for anions (Fig. 3 a), nitrogen compounds (Fig. 3 c), organic compounds (Fig. 3 d), chelators (Fig. 3 e), and membrane compounds (Fig. 3 h). However, statistical differences were shown only for the groups of anions (Fig. 3 a) and chelators (Fig. 3 e). 3.2.2. Fungicide sensitivity The study’s findings highlight the varying efficacy of different fungicides in inhibiting fungal growth. Figure 4 presents the inhibition of nine Neofabraea spp. isolates: G344/22, G361/22, G9/23, G17/23, G267/23, G271/23, G278/23, G297/23, G299/23. In this case, the greatest effect was achieved with the captan-based fungicide F4, which inhibited the growth of all tested isolates, with an average inhibition of 20 mm. A similar effect was achieved with the fungicide F1, containing boscalid and pyraclostrobin, with an average inhibition of 15.7 mm. A high average inhibition of 15.6 mm was observed with the fungicide F8, containing fluopyram and tebuconazole, although it inhibited the growth of five of the tested isolates. It was also shown that some of the tested fungicides did not inhibit the growth of the pathogen isolates, such as F2 (ditianon), F6 (sulfur), F7 (copper in the form of copper oxychloride), and F9 (fludioxonil). Overall, the results indicate that F1 (boscalid + pyraclostrobin) and F4 (captan) are the most effective fungicides against Neofabraea spp. F8 (fluopyram + tebuconazole) is efficient against most isolates tested. Some fungicides, such as F2 (ditianon), F6 (sulfur), F7 (copper in the form of copper oxychloride), and F9 (fludioxonil), show no significant effect on any of the isolates, suggesting a lack of efficacy against the pathogen. It is worth mentioning that, in the case of growth-inhibiting fungicides, statistical differences were found among individual isolates; isolate G299/23 showed no differences for both growth-inhibiting and no-effect fungicides. 3.3. Microbial communities of apple parts with Bull’s Eye Rot-like symptoms 3.3.1. Metabolic profile – Biolog ™ ECO Plates Differences in the responses of communities from infected parts of apples to the presence of substrates in the ECO plate were assessed. Variation in the response of the communities was noted depending on the apple variety (Fig. 5 ). The highest absorbance values ​​for both wavelengths, 590 nm and 750 nm, and all studied indicators were observed for the Honeycrisp variety. This indicates that the microbial communities inhabiting these apples demonstrated both high respiratory activity and biomass growth. However, the lowest values ​​were observed for the microorganisms inhabiting the Rubinstar variety. It is worth noting that these differences are statistically significant. Clustering analyzes were also performed and presented in the form of a heat map and dendogram (Fig. 6 ). Cluster analysis grouping objects allows to compare substrate consumption and biomass growth depending on the community of microorganisms inhabiting a given apple variety in the presence of different substrates (Fig. 6 b). There was a predominance of respiratory processes over biomass production in the communities in the 'Rubinstar' and 'Golden delicious' varieties, which indicates the occurrence of a situation of metabolic stress for the communities in these two varieties. The stress response was observed primarily upon exposure to the following substrates: D-Galactonic Acid Lactone, Glycyl-L-Glutamic Acid, D-Glucosaminic Acid, and D-Galacuronic Acid. Additionally, considering the functional diversity of microbial communities using the restrictive Sneath criterion (33%), the microorganisms inhabiting the individual apple varieties shown in the dendrogram (Fig. 6 a) are visibly distinct from one another. However, given the results for the less restrictive Sneath criterion (66%), it is clear that the Golden Delicious apple variety shows less similarity in respiratory activity and biomass growth than the other varieties. 3.3.2. Metataxonomic composition Metataxonomic analysis revealed the presence of fungi from the genus Neofabraea sp. and associated microbiota, Bull’s Eye Rot symptoms. Based on ASVs data, the FUNGuild, a community‑annotated database, showed that four apples were most heavily infected (> 98%) with strains from the Dermateceae family, which includes Neofabraea sp. The Warcup database/library showed that four of the apples examined were colonized by Neofabraea alba strains (> 99%). One apple was dominated by Colletotrichum clavatum . Additionally, the analysis also revealed the presence of other fungi, such as Kalmanozyma fusiformata and Leptosphaeria rubefaciens , which constitute the natural apple microbiome. 4. Discussion 4.1. Characteristics of the obtained Neofabraea sp. isolates Pathogenic fungi cause diseases that lead to post-harvest losses of fresh fruit, which can amount to up to 30% of the yield 54 . Apples are one of the most commonly produced fruits, and Poland is a leading exporter (Statista, 2025). Therefore, it is crucial to develop effective methods to control fungal pathogens without disrupting microbiological diversity and incorporating the latest European Union legislation on sustainable agriculture 55 , 56 . The main disease threatening apple trees is Bull’s Eye Rot, a storage disease caused by fungi of the genus Neofabraea 57 . Therefore, to confirm the first aim of our study, we examined the diversity of Neofabraea sp. strains occurring in Polish orchards, based on studies of various apple varieties from locations across five voivodeships. The development of an effective method for isolating strains from apples and then culturing them at the appropriate temperature allowed us to recover 155 Neofabraea sp. strains from orchards in Poland. Sanger sequencing using four different markers and NGS metataxonomic analysis confirmed that the isolates belonged to the single species Neofabraea alba ( Pezicula vagabunda ). These results are important for developing biocontrol methods against pathogens. It is possible that, despite belonging to the same genus, individual species may differ in fungicide sensitivity or plant pathogenicity 58 , 59 . The next stages of the research allowed us to examine the chemical sensitivity or resistance of the pathogen isolates we obtained to commonly used fungicides. The use of chemical plant protection products significantly impacts the control of harmful fungi in agriculture, increasing yields and improving their quality 60 . However, they can negatively impact the biodiversity of microorganisms in the environment 19 . Additionally, studies on the development of fungal pathogen resistance to fungicides were also conducted, demonstrating that mutations in genes encoding fungicide resistance can occur, posing a threat to effective crop protection 61 . The sensitivity of Neofabraea spp. strains to fungicide use were also assessed. Studies conducted in the Pacific Northwest (PNW) region of the United States demonstrated the effectiveness of the three fungicides tested against Neofabraea perennans and Neofabraea kienholzii . However, the authors emphasize that despite positive results, fungicide doses should be used with caution due to the potential for fungicide resistance 62 . Weber and Palm 63 tested the effectiveness of thiophanate-methyl against Neofabraea perennans , demonstrating that strains widespread throughout the Lower Elbe region (Germany) responded with moderate to high resistance to the applied plant protection product. It is worth emphasizing that these studies covered geographically diverse regions and Neofabraea species different from those identified in Poland. Therefore, when developing effective methods for controlling the pathogen, it is crucial to understand the sensitivity of strains occurring in a given area 37 . Our results on the inhibition of Neofabraea alba growth by commonly used fungicides show that, despite their antifungal properties, only 2 of the 10 tested fungicides inhibited the growth of all isolates. The average zone of inhibition for all fungi by fungicides F1 and F4 was 15.7 mm and 20 mm, respectively. Additionally, F8 inhibited the growth of half of the tested isolates within 15.6 mm. However, these differences were not statistically significant. In light of these results, it is reasonable to conclude that, despite the use of commercially available fungicides to combat fungal pathogens, it is important to develop more targeted methods that will also be safer for the environment. Chemical sensitivity is another important characteristic in developing effective methods to control pathogens. The response of microorganisms to specific chemical substrates can indicate which active substances are potentially suitable for inhibiting pathogen growth and for developing formulations for their control 36 , 64 . The Neofabraea spp. isolates we tested showed sensitivity to some of the substrates found in the PM screening plate test. Sodium selenite and trifluoperazine are compounds whose antifungal activity has been confirmed in studies. Sodium selenite proved effective in the formation of sclerotia of the fungus Sclerotinia sclerotiorum , while other researchers have demonstrated its reduction of damage caused by wilt caused by Fusarium oxysporum 65 , 66 . Buchan et al. 67 demonstrated an inhibitory effect on the growth of C. albicans after in vitro application of trifluoperazine. This confirms our results that in the presence of certain chemicals, Neofabraea spp. showed growth only in the initial hours of incubation or was inhibited with subsequent days of exposure to a given substrate. Comparison of chemical sensitivity to substances within the same group on the last day of incubation demonstrates that selecting appropriate compounds should not be guided by general group characteristics, but rather by testing specific substances. This is evident in the differences between sodium dichromate and sodium selenite, both anions, or L-aspartic acid b-hydroxamate and trifluoperazine, both organic compounds. However, these differences are not statistically significant. 4.2. Microbial communities of the BER-infected parts of apples Most studies focus on fungal pathogens, but there is no examination of the microbial communities that inhabit fruit infection sites. We demonstrated differences between the communities depending on the infected apple variety. A heatmap of substrate-stress results showed a stress response in communities inhabiting the Golden Delicious apple variety across six tested substrates, whereas the Rubinstar variety showed a similar response to three substrates. The communities inhabiting the remaining varieties most likely share a similar composition of microorganisms performing specific functions in the environment 68 . The results of dendrogram clustering confirm differences in the functioning of Golden Delicious apple communities compared to the other five varieties (less restrictive Sneath criterion, 66%). After conducting NGS analysis and based on dominant ASVs, it was confirmed that in apples affected by Bull’s Eye Rot symptoms, mainly isolates belonging to the Dermateaceae family, to which Neofabraea sp. belongs, occur (> 99%). The presence of isolates from the Glomerellaceae family was also detected, which includes Colletotrichum sp., an apple pathogen causing similar symptoms, which may contribute to the misdiagnosis of the disease during harvesting 69 – 71 . This may also indicate secondary infection with this pathogen. The conducted research and the obtained results allow us to accept both hypotheses: that the species inhabiting apples in Polish orchards belong to one species - Neofabraea alba , and the fragments of infected apples are mainly inhabited by a fungal monoculture, the metabolic profile of which depends on the apple variety. 5. Conclusions Many fungi present in the environment are plant pathogens. The use of chemical control agents significantly limits the growth and activity of pathogens, thus improving crop quality. Apples, one of the most commonly produced fruits in the world, are susceptible to storage diseases, among others. One of these is Bull’s Eye Rot (BER), caused by fungi of the genus Neofabraea . Despite the frequent occurrence of the BER, the pathogens responsible for it are not well-studied. Therefore, we aimed to investigate fungal pathogens of the genus Neofabraea occurring in Poland. To increase the diversity of the isolates tested, apples with symptoms of the BER were collected from 53 locations across five voivodships. Refining the method for isolating strains from infected apples – rubbing the skin with 70% alcohol and incubating the strains at a low temperature (18°C) – reduced the likelihood of culturing other associated fungi and obtaining isolates belonging to the Neofabraea species. Identification of the strains after Sanger sequencing using four primer pairs and WGS analysis confirmed that all isolates belong to the species Neofabraea alba . In subsequent stages of the study, the obtained strains were characterized for chemical and fungicide sensitivity. Biolog™ analysis using PM21D plates enabled the identification of which of the available substrates inhibited the growth of the tested isolates. It was confirmed that the isolates were sensitive to several agents, including sodium selenite, magnesium chloride, trifluoperazine, and nystatin. In their presence, growth of the tested isolates was observed only on the first day of incubation, after which it was inhibited or decreased with each passing day for 192 hours. The sensitivity of the isolates to the substrates of the individual groups was also examined during the last 192 hours of incubation. It was demonstrated that within a single group, Neofabraea sp. growth inhibition can vary across substrates. Fungicide susceptibility was tested for nine isolates in the presence of 10 commercially available fungicides. Fungicides F4, F1, and F8 showed the highest mean growth inhibition of all isolates tested, at 20, 15.7, and 15.6 mm, respectively. It was also noted that four of the tested fungicides did not inhibit the growth of any of the nine isolates, indicating that they were completely resistant to those fungicides. For the first time, the microorganism communities inhabiting areas with symptoms of Bull’s Eye Rot in apples were also analyzed. The heatmap results indicated that the microorganisms inhabiting the Golden Delicious variety, and to a lesser extent Rubinstar, exhibited the greatest stress response to the substrates present on the plate. This is confirmed by the Ward dendrogram, which shows that the Golden Delicious variety communities are less similar in their metabolic profiles compared to the other varieties. Next-generation sequencing enabled us to analyze the metataxonomic profile of six apples from different varieties and originating from four locations. Based on the individual databases, we found that most OTUs indicate that the microorganisms inhabiting the apple infection sites belong to the family Dermateceaea, which includes Neofabraea sp. The analyses conducted allowed us to identify and characterize fungal pathogens belonging to the Neofabrea genus, occurring in Polish orchards. Characterizing these pathogens allows us to develop appropriate methods and preparations that will help combat them while also aligning with the European Green Deal, ensuring sustainable agriculture and safety. Examining the microbial communities from infected areas may be important in the context of future research, which may allow for a better understanding of the pathogens' mechanisms of action depending on the fruit variety, which may be effective in combating them. Declarations Funding This paper was financed by the National Centre for Research and Development within the framework of the project LIDER XII (acronym: APPAT(f)REE), contract number LIDER/7/0054/L-12/20/NCBR/2021. CRediT authorship contribution statement Klaudia Zawadzka: Conceptualization, Methodology, Investigation, Formal analysis, Software, Visualization, Writing – original draft, Writing – review & editing. Karolina Oszust: Funding acquisition, Project administration, Conceptualization, Methodology, Investigation, Visualization, Validation, Formal analysis, Resources, Writing – review & editing, Supervision. Michał Pylak: Methodology, Investigation, Formal analysis, Validation, Software, Visualization, Writing – review & editing. Agata Gryta: Methodology, Investigation, Formal analysis, Validation, Writing – review & editing. Jacek Panek: Methodology, Software, Investigation, Validation, Writing – review & editing. Tomasz Lipa: Writing – review & editing. Artur Zdunek: Resources, Writing – review & editing. Magdalena Frąc: Conceptualization, Methodology, Supervision, Resources, Writing – review & editing. Declaration of competing interests The authors declare that they have no conflict of interest. Data availability The datasets generated and analysed during the current study are available in the NCBI BioProject database under accession number PRJNA1225134, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1225134, for the genetic identification of the obtained isolates, and under accession number PRJNA923268, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA923268, for the apple fungal community metataxonomic profiling. The raw dataset for Biolog TM PM21 and ECO, as well as fungicide sensitivity, is provided in the “Supplementary raw data” file available with this online publication. References Fisher, M. C. et al. Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture. Am Soc. Microbiol 13 , (2020). 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error bars indicate a standard deviation.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/d424a27f463916322131ef9a.png"},{"id":103212293,"identity":"fa49ecbb-2440-411b-893e-8ba3f3117a36","added_by":"auto","created_at":"2026-02-23 08:52:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":410197,"visible":true,"origin":"","legend":"\u003cp\u003eThe differences in chemical sensitivity/resistance of \u003cem\u003eNeofabraea\u003c/em\u003e sp. isolates to substrates depending on the group after 192 h of incubation: (a) anions, (b) cyclic compounds, (c) nitrogen compounds, (d) organic compounds, (e) chelators, (f) antibiotics, (g) cations, (h) membrane compounds; error bars indicate a standard deviation and different letters above the bars indicate significant differences (according to the Kruskal-Wallis test and the Student’s t-test, p \u0026lt; 0.05, n=3).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/7e219defa47cf2ab54de8b35.png"},{"id":103212294,"identity":"bda2be5c-3a99-4b21-a297-a6254487b9f2","added_by":"auto","created_at":"2026-02-23 08:52:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221092,"visible":true,"origin":"","legend":"\u003cp\u003eThe fungicide resistance of \u003cem\u003eNeofabraea\u003c/em\u003e sp. isolates. Error bars indicate a standard deviation, and different letters above the bars indicate significant differences (according to the ANOVA Tukey HSD test, p \u0026lt; 0.05, following Welch’s t-test, n=3).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/c74555522ce3baa708a52c2a.png"},{"id":104808140,"identity":"6d5d4ef7-632c-410d-b112-9b9199f408be","added_by":"auto","created_at":"2026-03-17 12:18:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":288625,"visible":true,"origin":"","legend":"\u003cp\u003eThe biodiversity indices for two wavelengths of microbial communities inhabiting infected apple sites. (a) Richness, (b) Average well color/density development, (c) Shannon's Diversity Index. Error bars indicate a standard deviation, and different letters above the bars indicate significant differences (according to the ANOVA Tukey HSD test, p \u0026lt; 0.05, following Welch’s t-test, n=3).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/31aae445fadc22f9043eec63.png"},{"id":103212298,"identity":"7bfa5ba9-d593-4f90-b549-cf1f1068dfb3","added_by":"auto","created_at":"2026-02-23 08:52:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":296144,"visible":true,"origin":"","legend":"\u003cp\u003eThe metabolic profiles of microbial communities inhabiting the infected apples according to apple variety, following Biolog\u003csup\u003eTM\u003c/sup\u003e ECO plates analyses. Dendrogram (a) and heat map (b) based on substrate stress index (SST). Statistical analyses included Ward's Agglomerative Hierarchical Clustering Method within Sneath`s criteria (strict, 33%, and less restrictive, 66%).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/da7e9cf2369b0d3af5c77ec0.png"},{"id":106725727,"identity":"aa9e7a20-9c33-4fad-a4f3-d946b5397a70","added_by":"auto","created_at":"2026-04-12 18:33:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5033571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/4ba6a955-a2de-40d5-a79b-94c697ba8cd6.pdf"},{"id":103505591,"identity":"4fab3172-0ddb-4a39-8bf7-fceff93cfdec","added_by":"auto","created_at":"2026-02-26 13:32:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28023,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. The factsheet with the numbers of strains identified as \u003cem\u003eNeofabraea\u003c/em\u003e spp., the collection site, province, apple variety from which the isolate was obtained, and the assigned accession number from the GenBank database.\u003c/p\u003e","description":"","filename":"Tab.S1..xlsx.docx","url":"https://assets-eu.researchsquare.com/files/rs-8779190/v1/f082d37398687e0e424387e3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFungi, as the most diverse group of organisms, significantly impact biodiversity and agriculture. Many of these species are fungal pathogens associated with plant diseases \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Effective control of fungal plant pathogens is crucial to minimizing the economic and environmental impact of crop diseases. It plays a key role in delivering high-quality products. One of the challenges fruit growers face in terms of quality and crop loss reduction is the occurrence of storage diseases \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of the most commonly produced fruits, ranking third globally are apples (Statista, 2025). The largest producers are China, the United States, Turkey, and Poland (FAOSTAT, 2025). The occurrence of Bull\u0026rsquo;s Eye Rot (BER), caused by fungi of the \u003cem\u003eNeofabraea\u003c/em\u003e (syn. \u003cem\u003ePezicula\u003c/em\u003e, \u003cem\u003ePhlyctema, Gloeosporium\u003c/em\u003e) genus, is one of the challenges encountered in apple cultivation \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Due to the latent nature of the disease, it is difficult to assess fruit quality at harvest, as its symptoms appear only when fruits are stored at low temperatures in cold stores \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Bull\u0026rsquo;s eye rot can be caused by various species belonging to the genus \u003cem\u003eNeofabraea\u003c/em\u003e, which are widespread in many regions of the world \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Research has shown that the species most commonly found in Polish orchards is \u003cem\u003eNeofabraea alba\u003c/em\u003e, followed by \u003cem\u003eNeofabraea perennans\u003c/em\u003e, and occasionally \u003cem\u003eNeofabraea kienholzii\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Although BER is one of the most common storage diseases, the diversity of pathogens causing it remains understudied \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, it is important to test multiple strains from different locations and apple varieties to understand their distribution and develop effective disease control practices \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, existing research on these species is relatively old, and it's worth updating this knowledge with new findings. Furthermore, ongoing climate change is influencing the habitats of individual fungal species. This may lead to the spread of pathogens in previously non-endemic areas \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, species identification using multiple markers increases the likelihood of confirming a specific species and of detecting the diversity present in a given area. Therefore, the first aim of our research was to determine whether only one species, \u003cem\u003eNeofabraea\u003c/em\u003e sp., occurs in Polish apple orchards or whether species diversity exists.\u003c/p\u003e \u003cp\u003eIn addition to understanding the diversity of the pathogen attacking apple trees, it is also important to assess its chemical sensitivity within the fungicides used, which is of great importance in light of changing legislation in the area of ​​pathogen control. Fungicides used in plant protection are highly effective in controlling numerous pathogens and improving crop quality \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, these chemicals have a long waiting period and can reduce the diversity of beneficial fungi \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. With this in mind, and with the aim of ensuring sustainable agriculture, the European Commission introduced the European Green Deal and the Farm to Fork document, which is an important pillar of the deal \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, it is necessary to seek new potential substances to control and limit the development of the phytopathogen of the \u003cem\u003eNeofabraea\u003c/em\u003e genus in fruit growing, which was the next aim of our research.\u003c/p\u003e \u003cp\u003eResearch on fruit during harvest or affected by the disease has so far focused only on checking for appropriate fruit color, developing methods to indicate the presence of a given disease \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and determining the degree of fruit ripeness at harvest \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Imported fruit entering the market has also been examined to prevent the spread of pathogens \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, sections of affected apples have not yet been examined for their microbial communities. Therefore, the third aim of our study was to examine apples with BER symptoms for the presence of monocultures, to examine their metabolic profile, and to determine whether this profile is dependent on apple variety. Based on these objectives, we formulated the following hypotheses: (i) the fungi colonizing apples in Polish orchards belong to a single species, and (ii) infected apple fragments are colonized by fungal monocultures, and their metabolic profile depends on the apple variety from which they originated.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Samples collection\u003c/h2\u003e \u003cp\u003eApples exhibiting visible symptoms of Bull\u0026rsquo;s Eye Rot were collected from 53 orchards across five voivodeships in Poland. Data are presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. A map showing the locations of orchards, along with descriptions of the localities, is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Obtaining \u003cem\u003eNeofabraea\u003c/em\u003e spp. isolates\u003c/h2\u003e \u003cp\u003eTo obtain \u003cem\u003eNeofabraea\u003c/em\u003e spp. strains, infected apple fragments were cut out and placed on Petri dishes containing potato dextrose lab agar medium (PDA) supplemented with antibiotics, streptomycin, and chlortetracycline. The fruit surfaces were previously sterilized in 70% ethanol and rinsed with sterile distilled water. The Petri dishes were then incubated at 18\u0026deg;C ​​for 8 days. A lower culture temperature was used due to the nature of the disease (development occurs at low temperatures during fruit storage) and to limit the growth of other fungi \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Then, based on morphological features, \u003cem\u003eNeofabraea\u003c/em\u003e-like isolates were selected and identified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. DNA extraction, amplification, and Sanger sequencing\u003c/h2\u003e \u003cp\u003eSanger sequencing followed by PCR analysis using at least one of four different markers: ITS1, α-EF, D2LSU, and β-tubulin to confirm the affiliation of the obtained strains to the genus \u003cem\u003eNeofabraea\u003c/em\u003e was performed. Firstly, DNA was extracted from isolates grown on the plate using the EURx GeneMATRIX Plant \u0026amp; Fungi DNA Purification Kit (EURx\u0026reg;, Gdansk, Poland). The DNA concentration was measured using a Nanodrop\u0026reg;, yielding a concentration of \u0026gt;\u0026thinsp;5 ng \u0026micro;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The fragments of fungal DNA were amplified to identify the fungi. We used the following conditions: 95\u0026deg;C for 3 min for initial denaturation, followed by 35 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s (for β-tubulin, 45\u0026deg;C), and 72\u0026deg;C for 60 s, with a final step at 72\u0026deg;C for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cb\u003eWhole Genome Sequencing by Illumina\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026reg;\u003c/b\u003e\u003c/sup\u003e - \u003cb\u003egenetic identification of isolates obtained from infected apples\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWhole-genome sequencing (WGS) analysis was performed using Illumina\u0026reg; MiSeq v3 (2x300) technology of four pathogenic \u003cem\u003eNeofabraea\u003c/em\u003e spp. isolates (G255/23, G284/23, G333/22, G799/22). WGS libraries were prepared with the Illumina DNA Prep kit. Raw sequencing bcl data were basecalled with RTA (Illumina) and demultiplexed with bcl2fastq (Illumina), then quality assessment and quality trimming were performed on the obtained fastq files with fastQC \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and Cutadapt \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. After that, de novo sequence assembly was performed with SPAdes \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Genome Assembler, and fasta \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e files containing contigs and scaffolds were obtained. QUAST \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Icarus \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and BUSCO \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e analysis and benchmark were performed to asses quality of assemblies. WGS analysis enabled species identification of isolates obtained from infected apples to determine which species dominate in apple orchards in Poland. Raw sequence reads were submitted to the Sequence Read Archive (SRA) database within BioProjects PRJNA1225134.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Chemical sensitivity\u003c/h2\u003e \u003cp\u003eAn experiment was performed to assess the chemical sensitivity of pathogenic fungal isolates from the genus \u003cem\u003eNeofabraea\u003c/em\u003e: G111/23 (accession number: PX417719), G24/23 (accession number: PX417698), and G788/22 (accession number: PX417812). Phenotypic analysis was performed using the Biolog\u0026trade; plate system and the PM21D plate suitable for fungal cultures (Biolog\u0026trade;, Hayward, CA, USA). An appropriate amount of fungal spores, according to the manufacturer\u0026rsquo;s protocol, was mixed with the inoculum fluid to achieve a 62%T transmittance. The plate containing lyophilized substrates was inoculated with a 100 \u0026micro;l suspension of microorganisms. The plates were incubated at 18\u0026deg;C, and absorbance was measured at 750 nm at 24-hour intervals for 10 consecutive days. The wavelength of 750 nm was used to measure optical density, which correlates with changes in microbial biomass production \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChemical sensitivity tests were performed on the plates, including anions, cations, membrane function compounds, chelators, cyclic compounds, organic compounds, antibiotics, and nitrogen compounds. The PM21D plate included 24 chemical compounds, grouped according to Panek et al. \u003csup\u003e36\u003c/sup\u003e. The analysis was carried out under the manufacturer's protocols \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Fungicide sensitivity\u003c/h2\u003e \u003cp\u003eIn this experiment, a range of ten commercially available fungicides, F1-F10, were prepared in solution at specific concentrations to be applied as in agricultural doses. The fungicides were based on the following active substances: F1 with boscalid 252 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and pyraclostrobin 128 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F2 with ditianon 700 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F3 with trifloxystrobin 500 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F4 with captan 800 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F5 with cyprodynil 750 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F6 with sulfur 800 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F7 with copper oxychloride 500 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F8 with fluopyram and tebuconazole 200 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, F9 with fludioxonil 500 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and F10 with cyprodinil 375 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and fludioxonil 250 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The final concentrations of the fungicide solutions were as follows: F1 at 0.16%, F2 at 0.25%, F3 at 0.04%, F4 at 0.4%, F5 at 0.35%, F6 at 1.25%, F7 at 0.3%, F8 at 0.15%, F9 at 0.15%, and F10 at 0.125%.\u003c/p\u003e \u003cp\u003eThe fungicide sensitivity test was performed using a well test Petri Plate with PDA medium\u0026thinsp;+\u0026thinsp;antibiotics. The following strains were used in the experiment: G344/22 (accession number: PX417793), G361/22 (accession number: PX417803), G9/23 (accession number: PX417688), G17/23 (accession number: PX417694), G267/23 (accession number: PX417758), G271/23 (accession number: PX417760), G278/23 (accession number: PX417763), G297/23 (accession number: PX417774), G299/23 (accession number: PX417775). Fungal isolates were sown on a PDA medium. The 100 \u0026micro;l of the material was pipetted onto the plates and smoothed into the medium with a spatula. Transmittance (%T) was determined at 70% for all isolates tested. Then, 50 \u0026micro;l of fungicide at the appropriate concentration was added to the center. The plates were incubated at 18\u0026deg;C. The growth inhibition of \u003cem\u003eNeofabraea\u003c/em\u003e spp. was measured after 7 days of incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Microbial communities inhabiting apples with Bull\u0026rsquo;s Eye Rot symptoms\u003c/h2\u003e \u003cp\u003eThe communities of microorganisms inhabiting areas with BER symptoms were characterized by apple variety. Apple surfaces were sterilized with 96% ethanol, and the skin was removed to minimize the risk of contamination with other fungal strains. A section of the apple was then collected, showing necrotic lesions of similar diameter in all varieties, indicating a similar stage of pathogen development. Metabolic and metataxonomic profiling was performed using Biolog\u0026trade; and Next-Generation Sequencing (NGS), respectively. For analysis, infected apples of similar size were used.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Biolog\u003csup\u003e\u0026trade;\u003c/sup\u003e ECO Plates\u003c/h2\u003e \u003cp\u003eThe metabolic profiling of microbial communities followed Biolog\u0026trade; ECO plate reads (at both 590 nm and 750 nm wavelengths), as described in detail by Pylak et al. \u003csup\u003e37\u003c/sup\u003e and according to previous research \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The results were presented as diversity indices such as Richness (R) and Shannon (H) (followed by data obtained at 590 nm and 750 nm wavelengths), average well color development (AWCD) (based on 590 nm), average well density development (AWDD) (based on 750 nm), substrate stress index (SST), calculated as the ratio of 590 nm/750 nm values. The lower the SST value, the higher the biomass production at lower substrate utilization, which corresponds to better growing conditions for the microbial community in given substrates \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. NGS by Illumina\u0026reg; - apple fungal communities metataxonomic profiling\u003c/h2\u003e \u003cp\u003eThe metataxonomic analyses were conducted using Next-Generation Sequencing (NGS) technology, specifically Illumina\u0026reg; SBS (sequencing by synthesis) (San Diego, USA), with the ITS1 fungal phylogenetic marker, as summarized by Siegieda et al. \u003csup\u003e41\u003c/sup\u003e. Information on the apples used for analysis is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Firstly, the environmental DNA from six infected apples was extracted using the GeneMATRIX Environmental DNA \u0026amp; RNA Purification Kit (Eurx\u0026reg;, Gdańsk, Poland). The DNA concentration was measured using a Nanodrop\u0026reg;, yielding a concentration of \u0026gt;\u0026thinsp;5 ng \u0026micro;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The ITS1 fragment was considered using the methods employed to obtain high taxonomic resolution \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. To determine which fungi were present in each apple sample, the ITS1 fragment of fungal DNA was amplified using primers suggested by Bellmain et al. \u003csup\u003e43\u003c/sup\u003e. The KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cape Town, South Africa) was used for the amplification. We used the following conditions: 95\u0026deg;C for 3 min for initial denaturation, followed by 35 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 60 s, with a final step at 72\u0026deg;C for 5 min. The obtained amplicons were then purified using magnetic beads CleanNGS (CleanNA, Netherlands, Gouda). Each sample was indexed with a unique Illumina\u0026reg; sequencing adapter (Illumina Nextera set a, b, and c) using the following program: 95\u0026deg;C for 3 min and 10 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 60 s with a final step of 72\u0026deg;C for 5 min. Samples were subsequently purified using magnetic beads and quantified using the Quantus fluorometer with QuantiFluor\u0026reg; ONE reagents (Promega, Madison, WI, USA). Library size was analyzed using D1000 DNA ScreenTape on TapeStation 4150 (Agilent Technologies Inc., Santa Clara, USA), and samples were diluted to the same concentration with PCR-grade water and sequenced on the Illumina\u0026reg; MiSeq platform. The data were processed and analyzed using QIIME2 2023.9 \u003csup\u003e44\u003c/sup\u003e. In QIIME2, ITSxpress \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e was used for primer trimming, and DADA2 (Divisive Amplicon Denoising Algorithm \u003cem\u003ev.2\u003c/em\u003e) \u003csup\u003e46\u003c/sup\u003e was used for denoising, chimera removal (method: consensus and minimal fold parent overabundance parameter set to 12), merging, and amplicon sequence variant (ASV) calling \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Taxonomy was assigned using the Scikit-learn (sklearn) classifier \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e trained on a UNITE 8.3 database (ver. dynamic, 98.5% similarity level) \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Quantitative positive controls were applied for the target microorganism groups (the MOCK community) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Raw sequence reads were submitted to the Sequence Read Archive (SRA) database within BioProjects SUB12531419.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe metataxonomic profile of microbial communities from sites with symptoms of Bull\u0026rsquo;s Eye Rot. Number of ASVs indicating a match to the Dermateceae family according to the FUNGuild database.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJonagored\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGolden Delicious\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeni Shogun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRed Prince\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJonagold\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGala\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTaxonomy FUNGuild\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek__Fungi;p__Ascomycota;c__Sordariomycetes;o__Glomerellales;f__Glomerellaceae\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e97.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enot identified\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASV34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek__Fungi;p__Ascomycota;c__Leotiomycetes;o__Helotiales;f__Dermateaceae\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocalization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSiedliska Pierwsze\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\u0026oacute;ry Markuszowskie\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSiedliska Pierwsze\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGutan\u0026oacute;w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWilk\u0026oacute;w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWilk\u0026oacute;w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis and visualization\u003c/h2\u003e \u003cp\u003eBoth data normality and homogeneity of variance were initially checked to determine the significance of the difference in \u003cem\u003eStatistica 13.1\u003c/em\u003e software, and adequate tests were applied. The Kruskal-Wallis test and the Student\u0026rsquo;s t-test were used to evaluate differences in chemical sensitivity of isolates \u003cem\u003eNeofabraea\u003c/em\u003e spp. for each group of substrates in 192 h of incubation. The ANOVA with Tukey HSD post hoc test (following Welch\u0026rsquo;s t-test) was used to analyze differences in sensitivity for fungicides. The CA analysis, including the grouping of treatments (apple variety) and features (response to substrates in Biolog\u0026trade; ECO plates), was performed on the standardized absorbance average values obtained at 72 h of incubation, as these were the most representative. A dendrogram representing the similarities in carbon utilization patterns from substrates in Biolog\u0026trade; ECO plates was set on scaled axis bond distances (Ward\u0026rsquo;s method, within Euclidean distance), with boundaries marked according to Sneath\u0026rsquo;s criteria (restrictive, 33% or less; less restrictive, 66%) \u003csup\u003e52,53\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Obtained isolates\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll 155 isolated strains are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, along with their accession numbers. The obtained sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI). Sanger sequencing of at least one marker confirmed that the strains studied belonged to the genus \u003cem\u003eNeofabraea\u003c/em\u003e. The WGS analysis of selected strains showed that they belonged to the species \u003cem\u003eNeofabraea alba\u003c/em\u003e (\u003cem\u003ePezicula vagabunda\u003c/em\u003e), so it can be assumed that this one species dominates in apple orchards in Poland. They were given the following accession numbers: \u003cem\u003eNeofabraea\u003c/em\u003e sp. G799/22: JBMPNT000000000, \u003cem\u003eNeofabraea\u003c/em\u003e sp. G233/23: JBMPNU000000000, \u003cem\u003eNeofabraea\u003c/em\u003e sp. G284/23: JBMPNV000000000, \u003cem\u003eNeofabraea\u003c/em\u003e sp. G255/23: JBMPNW000000000.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characteristics of selected \u003cem\u003eNeofabraea\u003c/em\u003e strains\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Chemical sensitivity of pathogenic isolates\u003c/h2\u003e \u003cp\u003eThe sensitivity of the tested microorganisms to several chemical compounds within individual groups was evaluated, and the results are presented for the highest concentration.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results for changes in the biomass of the \u003cem\u003eNeofabraea\u003c/em\u003e spp. isolates within 192 h with 24 -hour intervals. Strains were found to be sensitive to certain substrates, e.g., sodium selenite, dodecyltrimethyl ammonium bromide, magnesium chloride, copper (II) sulfate, nystatin, 1-hydroxypyridine-2-thione, trifluoperazine, D-cyclocloserine, or thiourea. Their biomass increased on the first day of incubation with a given substrate, then decreased or remained stable over the following days after exposure. This indicates that they are not suitable for effective biomass growth of the pathogen. It is worth emphasizing, however, that the tested isolates also did not demonstrate sensitivity to certain substrates, e.g., sodium dichromate, guanidine hydrochloride, cetylpyridinium chloride, EDTA, or L-aspartic acid b-hydroxamate. Despite their presence, the strains increased their biomass over the following days of incubation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the chemical sensitivity results for individual substrates after 192 h of incubation. A similar biomass increase is observed for substrates belonging to the cyclic compounds group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). However, it is worth noting that in some cases, substrates belonging to the same group may cause different biomass increase efficiencies, as seen for anions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), nitrogen compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), organic compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), chelators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), and membrane compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). However, statistical differences were shown only for the groups of anions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and chelators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Fungicide sensitivity\u003c/h2\u003e \u003cp\u003eThe study\u0026rsquo;s findings highlight the varying efficacy of different fungicides in inhibiting fungal growth. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the inhibition of nine \u003cem\u003eNeofabraea\u003c/em\u003e spp. isolates: G344/22, G361/22, G9/23, G17/23, G267/23, G271/23, G278/23, G297/23, G299/23. In this case, the greatest effect was achieved with the captan-based fungicide F4, which inhibited the growth of all tested isolates, with an average inhibition of 20 mm. A similar effect was achieved with the fungicide F1, containing boscalid and pyraclostrobin, with an average inhibition of 15.7 mm. A high average inhibition of 15.6 mm was observed with the fungicide F8, containing fluopyram and tebuconazole, although it inhibited the growth of five of the tested isolates. It was also shown that some of the tested fungicides did not inhibit the growth of the pathogen isolates, such as F2 (ditianon), F6 (sulfur), F7 (copper in the form of copper oxychloride), and F9 (fludioxonil).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the results indicate that F1 (boscalid\u0026thinsp;+\u0026thinsp;pyraclostrobin) and F4 (captan) are the most effective fungicides against \u003cem\u003eNeofabraea\u003c/em\u003e spp. F8 (fluopyram\u0026thinsp;+\u0026thinsp;tebuconazole) is efficient against most isolates tested. Some fungicides, such as F2 (ditianon), F6 (sulfur), F7 (copper in the form of copper oxychloride), and F9 (fludioxonil), show no significant effect on any of the isolates, suggesting a lack of efficacy against the pathogen. It is worth mentioning that, in the case of growth-inhibiting fungicides, statistical differences were found among individual isolates; isolate G299/23 showed no differences for both growth-inhibiting and no-effect fungicides.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Microbial communities of apple parts with Bull\u0026rsquo;s Eye Rot-like symptoms\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Metabolic profile \u0026ndash; Biolog\u003csup\u003e\u0026trade;\u003c/sup\u003e ECO Plates\u003c/h2\u003e \u003cp\u003eDifferences in the responses of communities from infected parts of apples to the presence of substrates in the ECO plate were assessed. Variation in the response of the communities was noted depending on the apple variety (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The highest absorbance values ​​for both wavelengths, 590 nm and 750 nm, and all studied indicators were observed for the Honeycrisp variety. This indicates that the microbial communities inhabiting these apples demonstrated both high respiratory activity and biomass growth. However, the lowest values ​​were observed for the microorganisms inhabiting the Rubinstar variety. It is worth noting that these differences are statistically significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClustering analyzes were also performed and presented in the form of a heat map and dendogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Cluster analysis grouping objects allows to compare substrate consumption and biomass growth depending on the community of microorganisms inhabiting a given apple variety in the presence of different substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). There was a predominance of respiratory processes over biomass production in the communities in the 'Rubinstar' and 'Golden delicious' varieties, which indicates the occurrence of a situation of metabolic stress for the communities in these two varieties. The stress response was observed primarily upon exposure to the following substrates: D-Galactonic Acid Lactone, Glycyl-L-Glutamic Acid, D-Glucosaminic Acid, and D-Galacuronic Acid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, considering the functional diversity of microbial communities using the restrictive Sneath criterion (33%), the microorganisms inhabiting the individual apple varieties shown in the dendrogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) are visibly distinct from one another. However, given the results for the less restrictive Sneath criterion (66%), it is clear that the Golden Delicious apple variety shows less similarity in respiratory activity and biomass growth than the other varieties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Metataxonomic composition\u003c/h2\u003e \u003cp\u003eMetataxonomic analysis revealed the presence of fungi from the genus \u003cem\u003eNeofabraea\u003c/em\u003e sp. and associated microbiota, Bull\u0026rsquo;s Eye Rot symptoms. Based on ASVs data, the FUNGuild, a community‑annotated database, showed that four apples were most heavily infected (\u0026gt;\u0026thinsp;98%) with strains from the Dermateceae family, which includes \u003cem\u003eNeofabraea\u003c/em\u003e sp. The Warcup database/library showed that four of the apples examined were colonized by \u003cem\u003eNeofabraea alba\u003c/em\u003e strains (\u0026gt;\u0026thinsp;99%). One apple was dominated by \u003cem\u003eColletotrichum clavatum\u003c/em\u003e. Additionally, the analysis also revealed the presence of other fungi, such as \u003cem\u003eKalmanozyma fusiformata\u003c/em\u003e and \u003cem\u003eLeptosphaeria rubefaciens\u003c/em\u003e, which constitute the natural apple microbiome.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Characteristics of the obtained \u003cem\u003eNeofabraea\u003c/em\u003e sp. isolates\u003c/h2\u003e \u003cp\u003ePathogenic fungi cause diseases that lead to post-harvest losses of fresh fruit, which can amount to up to 30% of the yield \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Apples are one of the most commonly produced fruits, and Poland is a leading exporter (Statista, 2025). Therefore, it is crucial to develop effective methods to control fungal pathogens without disrupting microbiological diversity and incorporating the latest European Union legislation on sustainable agriculture \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The main disease threatening apple trees is Bull\u0026rsquo;s Eye Rot, a storage disease caused by fungi of the genus \u003cem\u003eNeofabraea\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Therefore, to confirm the first aim of our study, we examined the diversity of \u003cem\u003eNeofabraea\u003c/em\u003e sp. strains occurring in Polish orchards, based on studies of various apple varieties from locations across five voivodeships. The development of an effective method for isolating strains from apples and then culturing them at the appropriate temperature allowed us to recover 155 \u003cem\u003eNeofabraea\u003c/em\u003e sp. strains from orchards in Poland. Sanger sequencing using four different markers and NGS metataxonomic analysis confirmed that the isolates belonged to the single species \u003cem\u003eNeofabraea alba\u003c/em\u003e (\u003cem\u003ePezicula vagabunda\u003c/em\u003e). These results are important for developing biocontrol methods against pathogens. It is possible that, despite belonging to the same genus, individual species may differ in fungicide sensitivity or plant pathogenicity \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe next stages of the research allowed us to examine the chemical sensitivity or resistance of the pathogen isolates we obtained to commonly used fungicides. The use of chemical plant protection products significantly impacts the control of harmful fungi in agriculture, increasing yields and improving their quality \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. However, they can negatively impact the biodiversity of microorganisms in the environment \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, studies on the development of fungal pathogen resistance to fungicides were also conducted, demonstrating that mutations in genes encoding fungicide resistance can occur, posing a threat to effective crop protection \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The sensitivity of \u003cem\u003eNeofabraea\u003c/em\u003e spp. strains to fungicide use were also assessed. Studies conducted in the Pacific Northwest (PNW) region of the United States demonstrated the effectiveness of the three fungicides tested against \u003cem\u003eNeofabraea perennans\u003c/em\u003e and \u003cem\u003eNeofabraea kienholzii\u003c/em\u003e. However, the authors emphasize that despite positive results, fungicide doses should be used with caution due to the potential for fungicide resistance\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Weber and Palm\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e tested the effectiveness of thiophanate-methyl against \u003cem\u003eNeofabraea perennans\u003c/em\u003e, demonstrating that strains widespread throughout the Lower Elbe region (Germany) responded with moderate to high resistance to the applied plant protection product. It is worth emphasizing that these studies covered geographically diverse regions and \u003cem\u003eNeofabraea\u003c/em\u003e species different from those identified in Poland. Therefore, when developing effective methods for controlling the pathogen, it is crucial to understand the sensitivity of strains occurring in a given area \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our results on the inhibition of \u003cem\u003eNeofabraea alba\u003c/em\u003e growth by commonly used fungicides show that, despite their antifungal properties, only 2 of the 10 tested fungicides inhibited the growth of all isolates. The average zone of inhibition for all fungi by fungicides F1 and F4 was 15.7 mm and 20 mm, respectively. Additionally, F8 inhibited the growth of half of the tested isolates within 15.6 mm. However, these differences were not statistically significant. In light of these results, it is reasonable to conclude that, despite the use of commercially available fungicides to combat fungal pathogens, it is important to develop more targeted methods that will also be safer for the environment.\u003c/p\u003e \u003cp\u003eChemical sensitivity is another important characteristic in developing effective methods to control pathogens. The response of microorganisms to specific chemical substrates can indicate which active substances are potentially suitable for inhibiting pathogen growth and for developing formulations for their control \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eNeofabraea\u003c/em\u003e spp. isolates we tested showed sensitivity to some of the substrates found in the PM screening plate test. Sodium selenite and trifluoperazine are compounds whose antifungal activity has been confirmed in studies. Sodium selenite proved effective in the formation of sclerotia of the fungus \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, while other researchers have demonstrated its reduction of damage caused by wilt caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Buchan et al. \u003csup\u003e67\u003c/sup\u003e demonstrated an inhibitory effect on the growth of \u003cem\u003eC. albicans\u003c/em\u003e after \u003cem\u003ein vitro\u003c/em\u003e application of trifluoperazine. This confirms our results that in the presence of certain chemicals, \u003cem\u003eNeofabraea\u003c/em\u003e spp. showed growth only in the initial hours of incubation or was inhibited with subsequent days of exposure to a given substrate. Comparison of chemical sensitivity to substances within the same group on the last day of incubation demonstrates that selecting appropriate compounds should not be guided by general group characteristics, but rather by testing specific substances. This is evident in the differences between sodium dichromate and sodium selenite, both anions, or L-aspartic acid b-hydroxamate and trifluoperazine, both organic compounds. However, these differences are not statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Microbial communities of the BER-infected parts of apples\u003c/h2\u003e \u003cp\u003eMost studies focus on fungal pathogens, but there is no examination of the microbial communities that inhabit fruit infection sites. We demonstrated differences between the communities depending on the infected apple variety. A heatmap of substrate-stress results showed a stress response in communities inhabiting the Golden Delicious apple variety across six tested substrates, whereas the Rubinstar variety showed a similar response to three substrates. The communities inhabiting the remaining varieties most likely share a similar composition of microorganisms performing specific functions in the environment \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. The results of dendrogram clustering confirm differences in the functioning of Golden Delicious apple communities compared to the other five varieties (less restrictive Sneath criterion, 66%).\u003c/p\u003e \u003cp\u003eAfter conducting NGS analysis and based on dominant ASVs, it was confirmed that in apples affected by Bull\u0026rsquo;s Eye Rot symptoms, mainly isolates belonging to the Dermateaceae family, to which \u003cem\u003eNeofabraea\u003c/em\u003e sp. belongs, occur (\u0026gt;\u0026thinsp;99%). The presence of isolates from the Glomerellaceae family was also detected, which includes \u003cem\u003eColletotrichum\u003c/em\u003e sp., an apple pathogen causing similar symptoms, which may contribute to the misdiagnosis of the disease during harvesting \u003csup\u003e\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This may also indicate secondary infection with this pathogen.\u003c/p\u003e \u003cp\u003eThe conducted research and the obtained results allow us to accept both hypotheses: that the species inhabiting apples in Polish orchards belong to one species - \u003cem\u003eNeofabraea alba\u003c/em\u003e, and the fragments of infected apples are mainly inhabited by a fungal monoculture, the metabolic profile of which depends on the apple variety.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eMany fungi present in the environment are plant pathogens. The use of chemical control agents significantly limits the growth and activity of pathogens, thus improving crop quality. Apples, one of the most commonly produced fruits in the world, are susceptible to storage diseases, among others. One of these is Bull\u0026rsquo;s Eye Rot (BER), caused by fungi of the genus \u003cem\u003eNeofabraea\u003c/em\u003e. Despite the frequent occurrence of the BER, the pathogens responsible for it are not well-studied. Therefore, we aimed to investigate fungal pathogens of the genus \u003cem\u003eNeofabraea\u003c/em\u003e occurring in Poland.\u003c/p\u003e \u003cp\u003eTo increase the diversity of the isolates tested, apples with symptoms of the BER were collected from 53 locations across five voivodships. Refining the method for isolating strains from infected apples \u0026ndash; rubbing the skin with 70% alcohol and incubating the strains at a low temperature (18\u0026deg;C) \u0026ndash; reduced the likelihood of culturing other associated fungi and obtaining isolates belonging to the \u003cem\u003eNeofabraea\u003c/em\u003e species. Identification of the strains after Sanger sequencing using four primer pairs and WGS analysis confirmed that all isolates belong to the species \u003cem\u003eNeofabraea alba\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn subsequent stages of the study, the obtained strains were characterized for chemical and fungicide sensitivity. Biolog\u0026trade; analysis using PM21D plates enabled the identification of which of the available substrates inhibited the growth of the tested isolates. It was confirmed that the isolates were sensitive to several agents, including sodium selenite, magnesium chloride, trifluoperazine, and nystatin. In their presence, growth of the tested isolates was observed only on the first day of incubation, after which it was inhibited or decreased with each passing day for 192 hours. The sensitivity of the isolates to the substrates of the individual groups was also examined during the last 192 hours of incubation. It was demonstrated that within a single group, \u003cem\u003eNeofabraea\u003c/em\u003e sp. growth inhibition can vary across substrates.\u003c/p\u003e \u003cp\u003eFungicide susceptibility was tested for nine isolates in the presence of 10 commercially available fungicides. Fungicides F4, F1, and F8 showed the highest mean growth inhibition of all isolates tested, at 20, 15.7, and 15.6 mm, respectively. It was also noted that four of the tested fungicides did not inhibit the growth of any of the nine isolates, indicating that they were completely resistant to those fungicides.\u003c/p\u003e \u003cp\u003eFor the first time, the microorganism communities inhabiting areas with symptoms of Bull\u0026rsquo;s Eye Rot in apples were also analyzed. The heatmap results indicated that the microorganisms inhabiting the Golden Delicious variety, and to a lesser extent Rubinstar, exhibited the greatest stress response to the substrates present on the plate. This is confirmed by the Ward dendrogram, which shows that the Golden Delicious variety communities are less similar in their metabolic profiles compared to the other varieties.\u003c/p\u003e \u003cp\u003eNext-generation sequencing enabled us to analyze the metataxonomic profile of six apples from different varieties and originating from four locations. Based on the individual databases, we found that most OTUs indicate that the microorganisms inhabiting the apple infection sites belong to the family Dermateceaea, which includes \u003cem\u003eNeofabraea\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003eThe analyses conducted allowed us to identify and characterize fungal pathogens belonging to the \u003cem\u003eNeofabrea\u003c/em\u003e genus, occurring in Polish orchards. Characterizing these pathogens allows us to develop appropriate methods and preparations that will help combat them while also aligning with the European Green Deal, ensuring sustainable agriculture and safety. Examining the microbial communities from infected areas may be important in the context of future research, which may allow for a better understanding of the pathogens' mechanisms of action depending on the fruit variety, which may be effective in combating them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis paper was financed by the National Centre for Research and Development within the framework of the project LIDER XII (acronym: APPAT(f)REE), contract number LIDER/7/0054/L-12/20/NCBR/2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKlaudia Zawadzka:\u003c/strong\u003e Conceptualization, Methodology, Investigation, Formal analysis, Software, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eKarolina Oszust: \u003c/strong\u003eFunding acquisition, Project administration, Conceptualization, Methodology, Investigation, Visualization, Validation, Formal analysis, Resources, Writing \u0026ndash; review \u0026amp; editing, Supervision. \u003cstrong\u003eMichał Pylak:\u003c/strong\u003e Methodology, Investigation, Formal analysis, Validation, Software, Visualization, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eAgata Gryta:\u003c/strong\u003e Methodology, Investigation, Formal analysis, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJacek Panek:\u003c/strong\u003e Methodology, Software, Investigation, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eTomasz Lipa: \u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eArtur Zdunek:\u003c/strong\u003e Resources, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMagdalena Frąc:\u003c/strong\u003e Conceptualization, Methodology, Supervision, Resources, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the NCBI BioProject database under accession number PRJNA1225134, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1225134, for the genetic identification of the obtained isolates, and under accession number PRJNA923268, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA923268, for the apple fungal community metataxonomic profiling. The raw dataset for Biolog\u003csup\u003eTM\u003c/sup\u003e PM21 and ECO, as well as fungicide sensitivity, is provided in the \u0026ldquo;Supplementary raw data\u0026rdquo; file available with this online publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFisher, M. C. et al. Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture. \u003cem\u003eAm Soc. Microbiol\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAguilar-Marcelino, L. et al. Chapter 26 - Using molecular techniques applied to beneficial microorganisms as biotechnological tools for controlling agricultural plant pathogens and pest. in \u003cem\u003eMolecular Aspects of Plant Beneficial Microbes in Agriculture\u003c/em\u003e (eds. Sharma, V., Salwan, R. \u0026amp; Al-Ani, L. K. T. B. T.-M. A. of P. B. 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Fungi\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 660 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8779190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8779190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBull\u0026rsquo;s eye rot, caused by fungi of the genus \u003cem\u003eNeofabraea\u003c/em\u003e (syn. \u003cem\u003ePezicula\u003c/em\u003e, \u003cem\u003ePhlyctema, Gloeosporium\u003c/em\u003e), is an important postharvest disease of apples worldwide. This study aimed to identify and characterize \u003cem\u003eNeofabraea\u003c/em\u003e species associated with Bull\u0026rsquo;s Eye Rot in Polish orchards. Apples with disease symptoms were collected from 53 locations across five voivodships. The developed protocol for the selective isolation of fungal strains of the genus \u003cem\u003eNeofabraea\u003c/em\u003e from symptomatic apples allowed the collection of 155 strains. This protocol involved apple tissue surface sterilization with 70% ethanol and incubation at 18\u0026deg;C, effectively reducing contamination by non-target fungi. Molecular identification using Sanger sequencing, followed by a Whole-Genome Sequencing approach, confirmed that the isolates belonged to \u003cem\u003eNeofabraea alba\u003c/em\u003e. The isolates were assessed for chemical sensitivity using Biolog\u0026trade; PM21D plates, revealing growth inhibition by several substrates, including sodium selenite, magnesium chloride, trifluoperazine, and nystatin, with substrate-dependent variability within functional groups. The fungicide-sensitivity test showed that three of ten commercial fungicides exhibited the highest inhibitory effects, while four fungicides showed no activity, indicating complete resistance. The Biolog\u0026trade; ECO Plate analysis showed differences in the stress response of microbial communities inhabiting the BER symptomatic tissue of apples, depending on variety. Additionally, metataxonomic analyses revealed slight variety-dependent differences, with fungi belonging to Dermateceae predominating at infection sites. These findings improve understanding of \u003cem\u003eNeofabraea\u003c/em\u003e sp. pathogens in Poland and support the development of sustainable disease management strategies aligned with the European Green Deal.\u003c/p\u003e","manuscriptTitle":"Neofabraea alba, an agent of apple Bull’s Eye Rot – multi-approach insights from Polish orchards","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 08:52:14","doi":"10.21203/rs.3.rs-8779190/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78b0f45c-a474-4aa9-b133-70aea551dd97","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63306704,"name":"Biological sciences/Microbiology"},{"id":63306706,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-10T13:11:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 08:52:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8779190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8779190","identity":"rs-8779190","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}