Pathogenicity, Aggressiveness, and Temperature Response of Paramicrosphaeropsis eriobotryae Associated with Almond Twig Dieback and Wood Necrosis

Pathogenicity, Aggressiveness, and Temperature Response of Paramicrosphaeropsis eriobotryae Associated with Almond Twig Dieback and Wood Necrosis | 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 Short Report Pathogenicity, Aggressiveness, and Temperature Response of Paramicrosphaeropsis eriobotryae Associated with Almond Twig Dieback and Wood Necrosis Hamed Negahban, Zeinab Bolboli, Reza Mostowfizadeh-Ghalamfarsa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7105314/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Sep, 2025 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted 6 You are reading this latest preprint version Abstract In this study, Paramicrosphaeropsis eriobotryae is identified and characterized for the first time as a novel and aggressive pathogen responsible for twig dieback in almond orchards. Field surveys across major almond-growing regions of Iran, the world’s seventh-largest producer, revealed trees exhibiting vascular necrosis, wood discoloration, and progressive dieback symptoms. Fungal isolates from symptomatic tissues exhibited morphological characteristics consistent with those of a member of the Didymellaceae family, within Ascomycota . Phylogenetic analyses based on protein-coding loci β-tubulin ( tub2 ) and RNA polymerase II second-largest subunit ( rpb2 ) confirmed the identity of isolates as P. eriobotryae . Pathogenicity assays on detached shoots and one-year-old almond saplings fulfilled Koch’s postulates, inducing dark brown necrotic lesions and internal vascular discoloration that mirrored field symptoms. Notably, principal component analysis (PCA) confirmed significant geographic variation in aggressiveness, with southern isolates exhibiting markedly higher aggressiveness than western counterparts. Disease development was strongly temperature-dependent, peaking at 25–30°C and substantially suppressed at 15°C and 35°C. Importantly, previous research has demonstrated the capacity for latent infection in asymptomatic hosts such as loquat and olive, underscoring a considerable biosecurity risk to global almond production. This study establishes P. eriobotryae as an emerging threat characterized by temperature-sensitive pathogenicity and regional variations in aggressiveness, underscoring the need for integrated management strategies. Aggressiveness Ascomycota Almond dieback Didymellaceae Principal Component Analysis Phylogenetic analyses Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Almond ( Prunus dulcis Mill. D.A. Webb; family Rosaceae ), a Mediterranean crop of significant global economic and social value (Massantini and Frangipane 2022 ) originated in south-central Asia. Almond cultivation faces persistent threats from diseases that compromise yield and quality, including anthracnose, red leaf blotch, mosaic disease, viral bud failure, canker, and dieback (Martelli and Savino 1997 ; López-Moral et al. 2020 ; Miarnau et al. 2021 ; Negi and Handa 2023; Endes 2024 ). Over the past decade, canker and dieback syndromes have emerged as major concerns in nut crops, frequently linked to fungal pathogens (Luo et al. 2024 ). Almond decline, in particular, has been associated with diverse Ascomycete families, notably Botryosphaeriaceae , Ceratocystidaceae , Cytosporaceae , Diaporthaceae , Diatrypaceae , Nectriaceae , Pleurostomataceae , and Togniniaceae (Holland et al. 2019; Moral et al. 2019 ; Anton-Domínguez et al. 2023; Aloi et al. 2024 ; Endes 2024 ; Oren et al. 2025). Among these, Botryosphaeriaceae species, including Botryosphaeria dothidea (Moug.) Ces. & De Not., Diplodia corticola A.J.L. Phillips, A. Alves & J. Luque, D. mutila (Fr.) Fr., D. seriata De Not., Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Neofusicoccum arbuti (D.F. Farr & M. Elliott) Crous, Slippers & A.J.L. Phillips, Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum vitifusiforme (Van Niekerk & Crous) Crous, Slippers & A.J.L. Phillips, and Neoscytalidium dimidiatum (Penz.) Crous & Slippers, are frequently implicated in almond branch cankers and dieback (Holland et al. 2019; Anton-Domínguez et al. 2023; Aloi et al. 2024 ; Endes 2024 ). Our recent surveys of almond twig dieback in Iran yielded isolates of Paramicrosphaeropsis sp. closely related to P. eriobotryae Tavakolian, Mostowf. & Crous ( Didymellaceae ), a canker pathogen originally described on loquat in Fars Province (Tavakolian et al. 2023 ). Although a single isolate of P. eriobotryae was previously reported from an almond tree within an infected loquat orchard (Tavakolian et al. 2023 ), its role as a primary pathogen of almond remains unconfirmed. Critically, the prevalence of P. eriobotryae across almond-growing regions, its aggressiveness on almond hosts, and key environmental drivers (e.g., temperature) are unknown, hindering the development of effective disease management strategies. This study addresses these critical knowledge gaps through four objectives: (i) Morpho-molecular characterization of Paramicrosphaeropsis sp. isolates recovered from symptomatic almond twigs; (ii) Aggressiveness assessment of geographically diverse isolates (western and southern Iran) on detached almond shoots; (iii) Pathogenicity validation on one-year-old almond saplings under controlled conditions; (iv) Temperature response profiling to define optimal conditions for lesion development. By establishing Paramicrosphaeropsis dieback as an emerging threat to almond health, this work provides critical data for future disease mitigation efforts. 2. Materials and Methods 2.1. Sampling and fungal isolation During spring 2024, surveys were conducted in almond orchards exhibiting symptoms of twig necrosis and dieback in Fars (southern Iran) and Kermanshah Provinces (western Iran). Symptomatic one-year-old twigs were collected. The internal discoloration was assessed by examining transverse sections. All plant material was stored at 4°C until laboratory processing. For fungal isolation, small pieces (0.5 × 0.5 cm) were excised from the margins between symptomatic (discolored) and asymptomatic tissues of the sampled twigs. These pieces were rinsed in sterile distilled water and surface-disinfected by immersion in 1% sodium hypochlorite (prepared from 20% commercial bleach) for one minute. After disinfection, the samples were rinsed twice in sterile distilled water and surface-dried on sterile paper towels. The disinfected tissue segments were then plated onto potato dextrose agar (PDA; containing extract from 350 g/L boiled potato, 20 g/L dextrose, 16 g/L agar, and distilled water) amended with 100 mg/L tetracycline to inhibit bacterial growth. Petri dishes were incubated in the dark at room temperature for seven days. The resulting fungal isolates were purified using the single hyphal tip method on water agar (WA; 20 g/L agar, and distilled water). 2.2. Morphological and cultural characterization Pure cultures isolated from almond trees exhibiting twig dieback were morphologically characterized. Isolates were grown on PDA and incubated at 20°C in darkness for two weeks. Colony characteristics, including surface texture and pigmentation (obverse and reverse), were recorded. Microscopic features were assessed using light microscopy on lactic acid-mounted slides (Tavakolian et al. 2023 ). For each isolate selected, mature pycnidia and conidia were examined, with measurements taken of pycnidial dimensions (length and width), conidial size, and chlamydospore diameter (n = 30 per structure). To determine optimal growth temperature, selected isolates from infected trees were incubated on PDA at eight temperatures (5–40°C in 5°C increments). Radial growth rates were measured using four replicates per temperature. 2.3. Molecular identification 2.3.1. DNA extraction, PCR, and sequencing Genomic DNA was extracted from fungal mycelia using the DNG-PLUS extraction kit (CinnaGen, Tehran, Iran) following the manufacturer’s protocol (Negahban et al. 2024a ). DNA concentration and purity were assessed using an MD-1000 Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For selected Paramicropshaeropsis sp. isolates, partial sequences of the protein-coding loci β-tubulin ( tub2 ) and RNA polymerase II second-largest subunit ( rpb2 ) were amplified. The tub2 locus was amplified using primers Btub2Fd/Btub4Rd (Woudenberg et al. 2009 ), and rpb2 using primers fRPB2-5F/fRPB2-7cR (Liu et al. 1999 ). The thermal cycling program for tub2 amplification followed Tavakolian et al. ( 2023 ), while the rpb2 amplification program consisted of an initial denaturation step (95°C, 300 s), followed by 35 cycles of 94°C (45 s), 55.5°C (90 s), and 72°C (120 s), and a final extension (72°C, 600 s). Each 30 µL PCR mixture contained 1 µL of genomic DNA (~ 100 ng/µL), 1 µL of each primer (10 pM), 15 µL of 2× Taq DNA Polymerase Master Mix RED (Amplicon, Odense, Denmark), and 12 µL of PCR-grade water. Amplified PCR products were then sequenced using the same primer pairs via dye terminator cycle sequencing (Codon Genetic Group, Tehran, Iran). 2.3.2. Phylogenetic analyses Sequence identity was assessed using the BLASTn tool (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Multiple sequence alignments were generated using MAFFT v.7 (Katoh et al. 2019 ). Phylogenetic analyses based on individual and concatenated tub2 and rpb2 sequences were reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML) in TrEase (Mishra et al. 2023 ) under default settings. Trees were visualized and edited using MEGA v.11 (Tamura et al. 2021 ). 2.4. Pathogenicity assessment 2.4.1. Aggressiveness assay of isolates To evaluate the aggressiveness of Paramicrosphaeropsis sp. isolates obtained from almond trees in two geographically distinct regions of Iran (south and west), a detached shoot inoculation assay was performed to quantify symptom severity. Asymptomatic one-year-old almond shoots (diameter: 10 ± 1 mm) were collected, surface-disinfected by flaming with 98% ethanol, and sectioned into 20–30 cm segments. Each isolate was inoculated onto four replicate shoot segments following the protocol described by Bolboli et al. ( 2022 ). The inoculated shoots were then incubated at 25 ± 1°C under a 16-h light/8-h dark photoperiod in a plant growth chamber, arranged in a completely randomized design (CRD). After 8 days of incubation, bark tissue was aseptically removed from the inoculation sites. Lesion progression was quantified by measuring upward lesion progression (ULP), downward lesion progression (DLP), and lesion width (LW) using a digital caliper (Insize®, 0.01 mm precision). To fulfill Koch’s postulates, wood fragments from lesion margins were transferred to PDA amended with 100 mg/L tetracycline. The re-isolated fungi were identified based on cultural and morphological characteristics (Tavakolian et al. 2023 ). Statistical analyses were conducted to assess isolates aggressiveness based on lesion progression metrics (ULP, DLP, LW). All data were first checked for normality using the Shapiro-Wilk test (Shapiro and Wilk 1965 ) in IBM SPSS Statistics 25. A one-way analysis of variance (ANOVA) was performed using the General Linear Model (GLM) procedure in SAS (v.9.4, SAS Institute, Cary, NC, USA) to determine the effect of Paramicrosphaeropsis sp. isolates (fixed effect) on each pathogenicity trait. Where ANOVA indicated significant differences ( P ≤ 0.05), means were separated using Tukey's Honestly Significant Difference (HSD) test. Additionally, Principal Component Analysis (PCA) was conducted in R version 3.4.0 ( http://www.r-project.org/ ). to visualize relationships among the 12 Paramicrosphaeropsis sp. isolates and determine patterns of variance in aggressiveness traits. 2.4.2. Pathogenicity test on almond saplings To confirm pathogenicity, the most aggressive isolate (previously identified on detached shoots) was inoculated onto one-year-old almond saplings. A sterile 5-mm cork borer was used to create uniform wounds on the main stem of each sapling. Each wound was inoculated with a 5 × 5 × 2 mm mycelial plug excised from the actively growing margin of a two-week-old culture grown on PDA. The inoculation site was immediately sealed with Parafilm ® (Bemis Packaging, USA). Control saplings received sterile PDA plugs prepared identically (Gonzalez-Domínguez et al. 2016 ). The experiment employed a completely randomized design with four replicate saplings per treatment. Inoculated plants were maintained in a greenhouse (20–35°C; natural light cycle) and irrigated as needed to sustain soil moisture at field capacity. After a two-month incubation period, external and internal symptoms (ULP, DLP, and LW) were assessed. Koch’s postulates were fulfilled by re-isolating the fungus from the margin of symptomatic wood tissue onto tetracycline-amended (100 mg/L) PDA, followed by morphological identification consistent with the original isolate. 2.4.3. Effect of temperature on aggressiveness of P. eriobotryae To assess the impact of temperature on the aggressiveness of P. eriobotryae , detached almond shoots were inoculated with the isolate previously identified as the most aggressive in detached shoot assays (Section 2.4.1 ). The inoculation protocol followed Bolboli et al. ( 2022 ). Inoculated and control shoots were placed in growth chambers under five constant temperature regimes: 15, 20, 25, 30, and 35°C (± 0.5°C) with a 16-hour light/8-hour dark photoperiod. The experiment was set up in a completely randomized design with four replicate shoots per temperature treatment, including controls. Ten days after inoculation, the bark was carefully removed from each shoot, and pathogenicity was evaluated by measuring ULP, DLP, and LW. Measurements were taken using digital calipers (precision ± 0.01 mm). Statistical analysis was conducted using SAS v.9.4 (SAS Institute, Cary, NC, USA). Data normality for ULP, DLP, and LW was confirmed using the Shapiro-Wilk test (Shapiro and Wilk 1965 ) within the UNIVARIATE procedure. The impact of temperature on each pathogenicity trait was analyzed separately through ANOVA performed with the GLM procedure. Significant differences indicated by ANOVA ( P ≤ 0.05) were further analyzed using Tukey’s Honestly Significant Difference (HSD) test to separate means (Negahban et al. 2024b ). Results are presented as mean ± standard error (SE). 3. Results 3.1. Disease symptoms and fungal isolations Field surveys in almond orchards across two Iranian provinces revealed trees exhibiting canker and dieback symptoms. Affected twigs and branches showed external necrosis and dieback, leading to progressive decline and death. Transverse sections displayed internal vascular discoloration (brown to dark brown) and wood necrosis (Fig. 1 ). Fungal isolates from the families Didymellaceae and Pleosporaceae were recovered from symptomatic tissues using culturing techniques and identified based on morphology. 3.2. Morphological characteristics Twelve of 24 isolates obtained from twig dieback were morphologically identified as Didymellaceae . On PDA at 20°C (after 14 days), colonies exhibited a cottony texture, white to pale pinkish obverse, and pinkish to olivaceous brown reverse (Fig. 2 ). Mean radial growth was 3.35 ± 0.3 mm/day at 20°C, declining to 0.18 ± 0.02 mm/day at 40°C. Pycnidia (produced after ~ 1 month at room temperature) were pale to dark brown, globose to subglobose, solitary or confluent, semi-immersed, and averaged 246.41 ± 80.51 µm in radius (range: 102.45–488.48 µm; n = 100). Conidia were solitary, greenish-brown, variably shaped (globose to subcylindrical), and measured 7.99 ± 1.53 × 4.82 ± 0.71 µm (range: 4.24–11.48 × 3.54–6.55 µm; n = 60). Chlamydospores occurred in chains, were globose to ellipsoid, smooth, thick-walled, and measured 12.31 ± 2.61 × 8.41 ± 1.39 µm (range: 6.53–16.67 × 6.37–11.24 µm; n = 30). Hyphae averaged 3.85 ± 0.68 µm in width (range: 2.62–5.29 µm; n = 50) (Fig. 2 ). 3.3. Phylogenetic analyses Preliminary BLASTn analysis of the tub2 and rpb2 nucleotide sequences revealed that three isolates, BA03 and BA05 (Fars Province) and KA09 (Kermanshah Province), shared 100% similarity and coverage with P. eriobotryae isolate CBS 149854 (Tavakolian et al. 2023 ) (Supplementary Table 1). Based on phylogenetic analysis of individual and concatenated tub2 and rpb2 sequences, the isolates clustered within a monophyletic clade comprising P. eriobotryae isolates. High posterior probability and bootstrap values (Fig. 3 ) robustly support their identification as P. eriobotryae. 3.3. Pathogenicity assessment 3.3.1 Assessing the aggressiveness of the isolates from almond twig dieback disease Detached shoot inoculations confirmed the pathogenicity of all 12 P. eriobotryae isolates inducing dark brown necrotic lesions on almond shoots and fulfilling Koch’s postulates. ANOVA revealed highly significant differences in lesion progression metrics (ULP, DLP, LW) among isolates ( P < 0.0002; Supplementary Table 2), with BA05 exhibiting the greatest aggressiveness (ULP: 4.03 ± 0.15 mm; DLP: 4.53 ± 0.55 mm; LW: 2.90 ± 0.10 mm; Tukey’s HSD, P ≤ 0.05; Fig. 4 ), while KA01, KA04, and KA08 were less aggressive. Principal Component Analysis of lesion traits segregated isolates into three distinct groups (Fig. 5 ), explaining 95.9% of the total variance (PC1: 82.6%, PC2: 13.3%). Group A contained the aggressive isolate, BA05 (from southern Iran), Group B included moderately aggressive isolates BA02-BA04 (from southern Iran), and Group C comprised less aggressive isolates–KA01, KA02, KA04, KA05, KA07, KA08, KA09 (from western Iran) and BA01 (from southern Iran) (Fig. 5 ). 3.3.2. Pathogenicity assessment on almond sapling Pathogenicity assays on one-year-old almond saplings confirmed that P. eriobotryae induced disease symptoms consistent with almond twig dieback. By two months post-inoculation, all inoculated saplings exhibited distinct external symptoms, including dark brown necrotic lesions radiating from the inoculation point and longitudinal bark cracking (Fig. 6 a & b). Internal symptom assessment following bark removal revealed extensive brown-to-black vascular discoloration extending longitudinally through the wood (Fig. 6 c–e). Quantification of the lesions demonstrated significant progression: upward lesion length (ULP: 28.75 ± 4.79 mm), downward lesion length (DLP: 21.25 ± 0.96 mm), and lesion width (LW: 11.00 ± 1.08 mm). In contrast, control saplings inoculated with sterile PDA plugs showed no disease development, exhibiting only minor wound callusing at the inoculation site (Fig. 6 f & g). Paramicrosphaeropsis eriobotryae was consistently re-isolated from the margin of necrotic tissues onto PDA and morphologically identified, thereby fulfilling Koch's postulates. 3.3.3. Effect of temperature on symptom progression Temperature had a significant impact on the progression of necrotic lesions caused by P. eriobotryae on detached almond shoots (Fig. 7 ). One-way ANOVA showed a significant influence of temperature on all pathogenicity traits, ultimate ULP, DLP, and LW ( P < 0.005) (Supplementary Table 3). Post-hoc analysis (Tukey’s HSD) demonstrated maximum symptom development at 25°C and 30°C. At 10 days post-inoculation, ULP reached 6.25 ± 1.26 mm and 8.78 ± 1.31 mm, DLP measured 5.62 ± 1.25 mm and 5.25 ± 0.65 mm, and LW was 5.00 ± 1.41 mm and 5.50 ± 0.71 mm at 25°C and 30°C, respectively (Fig. 8 ). Conversely, symptom expression was significantly reduced at 15°C and 35°C (Fig. 8 ). 4. Discussion This study established P. eriobotryae as a causal agent of almond twig dieback. We provide the first validated evidence of its isolation from symptomatic almond trees in Fars and Kermanshah Provinces, Iran, alongside comprehensive pathogenicity confirmation. This finding is particularly significant given Iran’s position as the world’s seventh-largest almond producer, with an annual harvest of 102,414.06 tons from 43,342 hectares (FAO, 2023), highlighting P. eriobotryae as a newly identified threat to this commercially vital crop. While a previous study reported isolating a single P. eriobotryae isolate from almond wood tissue without a pathogenicity assessment in live trees (Tavakolian et al. 2023 ), our work definitively demonstrates pathogenicity through a multifaceted approach. This approach included rigorous pathogenicity testing on almond saplings, assessment of aggressiveness among multiple isolates using several key traits, and evaluation of temperature effects on infection using detached shoots. In this survey, P. eriobotryae isolates were recovered from symptomatic almond twigs and one-year-old branches exhibiting vascular internal wood discoloration and necrosis. Morphological and molecular analyses confirmed significant similarity between these almond-derived isolates and the P. eriobotryae type strain (CBS 148866; (Tavakolian et al. 2023 )), initially identified as a canker pathogen of loquat ( Eriobotrya japonica ) in Fars Province, southern Iran. While members of the Botryosphaeriaceae are often implicated as primary causal agents of almond canker and dieback, our findings highlight that these diseases involve a broader community of Ascomycete fungi. To date, at least 84 species associated with almond canker and dieback symptoms, representing families such as Ceratocystidaceae , Cytosporaceae , Diaporthaceae , Diatrypaceae , Nectriaceae , Pleurostomataceae , and Togniniaceae (Holland et al. 2019; Moral et al. 2019 ; Anton-Domínguez et al. 2023; Aloi et al. 2024 ; Endes 2024 ; Oren et al. 2025), which may primarily act as secondary pathogens or endophytic colonizers. Further research is needed to understand the complex interactions between P. eriobotryae and these diverse fungal associates, as well as the key biotic and abiotic factors influencing almond canker and dieback development. Furthermore, the isolation of P. eriobotryae from asymptomatic olive trees by Tavakolian et al. (Tavakolian et al. 2023 ) demonstrates its capacity for latent infection within natural host tissues. This latency poses an insidious threat to agricultural biosecurity. Such pathogens persist cryptically within hosts, enabling undetected colonization and facilitating widespread dissemination, often via asymptomatic nursery stock (Havenga et al. 2019 ; Luo et al. 2024 ). Indeed, latent infection by canker-causing fungi is a well-established phenomenon in perennial fruit and nut crops, extensively documented across diverse pathosystems (Havenga et al. 2019 ; Luo et al. 2019 , 2021 ; Avenot et al. 2022; Lopez-Moral et al. 2023; Luo et al. 2024 ; Negahban et al. 2024a ; Bolboli et al. 2025 ; Ghaedi et al. 2025 ). The scale is significant, surveys have revealed high proportions of nursery trees harbor latent infections, exemplified by Californian almond nurseries, where Lasiodiplodia spp. and Neofusicoccum spp. predominate (Luo et al. 2024 ). This underscores the latent phase as a primary pathway for regional and global pathogen spread. Consequently, understanding latent infection dynamics is critical for devising effective management strategies. Given the inherent challenge of detecting cryptic colonization, advanced molecular diagnostics, particularly PCR-based assays, have become indispensable tools for early and accurate pathogen identification, enabling proactive intervention (Luo et al. 2019 , 2021 ; Negahban et al. 2024a ; Bolboli et al. 2025 ; Ghaedi et al. 2025 ). Pathogen aggressiveness was rigorously quantified using a detached almond shoot assay, a validated method for rapid and reliable assessment of canker-causing fungi (Garkava-Gustavsson et al. 2014; Nouri et al. 2019 ; Bolboli et al. 2022 ). Isolates of P. eriobotryae from almond twig dieback showed significant variation in lesion development and wood necrosis severity. Principal components analysis (PCA), incorporating three key pathogenicity traits, separated the isolates into three distinct groups. Notably, upward (ULP) and downward (DLP) lesion progression emerged as the primary indicators of aggressiveness, explaining 82.6% of the variance on PC1. This approach also revealed a significant geographic pattern in this grouping: isolates from Fars Province (southern Iran) clustered together, while those from Kermanshah Province (western Iran) were mainly grouped separately. Particularly, isolate BA05 (from Fars Province) showed significantly higher aggressiveness compared to all others, and isolates from Fars Province were more aggressive than those from Kermanshah Province, suggesting that regional climatic differences influence pathogen populations (Quesada et al. 2019 ). Subsequent pathogenicity trials on one-year-old almond saplings in controlled greenhouse conditions confirmed the pathogenicity. Within two months post-inoculation, saplings displayed necrosis, internal wood discoloration, and dieback, validating the ecological relevance of our aggressiveness metrics. Our data establish a thermal optimum for P. eriobotryae pathogenicity, with peak disease severity (lesion development) occurring between 25°C and 30°C. This contrasts sharply with significantly suppressed symptom expression at 15°C and 35°C. Such pronounced temperature dependence strongly implicates P. eriobotryae as a significant threat within temperate to warm-temperate almond-growing regions. Integrating these findings with the likely latency of P. eriobotryae in asymptomatic olive trees (Tavakolian et al. 2023 ) support a hypothesis that abiotic stressors, particularly temperature extremes and drought, serve as key triggers converting latent P. eriobotryae infections into aggressive disease. This hypothesis aligns with existing literature that elucidates the impact of temperature on the development of various canker-causing pathogens (Lopez-Moral et al. 2022a; Luo et al. 2022 ; Avenot et al. 2023 ; Negahban et al. 2024b ). Notably, the temperature-dependent patterns of lesion growth observed in almond shoots vary among different canker pathogens, with B. dothidea and Lasiodiplodia citricola exhibiting increased aggressiveness at higher temperatures (30°C), while Cytospora leucostoma (Pers.) Sacc. and Neofusicoccum mediterraneum display optimal symptom development at 25°C and 20°C, respectively (Luo et al. 2022 ). This study establishes P. eriobotryae as a novel, aggressive causal agent of twig dieback in commercially critical almond orchards across Iran, a leading global producer. Our survey confirmed its significant pathogenic potential, revealed a pronounced geographic difference in isolate aggressiveness between Fars and Kermanshah Provinces, and defined a critical thermal optimum (25–30°C) for disease development. Priority research must now focus on mapping the epidemiological reservoir by assessing P. eriobotryae prevalence in non-almond hosts within and bordering orchard ecosystems and surrounding landscapes. Implementing non-destructive molecular diagnostics (e.g., qPCR, LAMP) for proactive surveillance in nurseries, orchards, and potential alternative hosts is essential. Additionally, screening commercial almond cultivars to evaluate their susceptibility remains a high priority. Addressing these critical gaps is fundamental for developing integrated management strategies to safeguard existing orchards and secure sustainable new almond plantings against this emerging pathogen. Declarations CRediT authorship contribution statement Hamed Negahban : Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Zeinab Bolboli : Writing – review & editing, Writing – original draft, Validation, Methodology, Funding acquisition. Reza Mostowfizadeh-Ghalamfarsa : Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Funding This work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4038664. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Plant Pathol. 66: 77–78. https://doi.org/10.1111/ppa.12556 Havenga M, Gatsi GM, Halleen F, Spies CF, van der Merwe R, Mostert L (2019) Canker and wood rot pathogens present in young apple trees and propagation material in the Western Cape of South Africa. Plant Dis. 103(12): 3129–3141. https://doi.org/10.1094/PDIS-04-19-0867-RE Holland LA, Trouillas FP, Nouri MT, Lawrence DP, Crespo M, Doll DA, ..., Fichtner EJ (2021) Fungal pathogens associated with canker diseases of almond in California. Plant Dis. 105(2): 346–360. https://doi.org/10.1094/PDIS-10-19-2128-RE Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 20: 1160–1166. https://doi.org/10.1093/bib/bbx108 Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Mol. Biol. Evol. 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092 López-Moral A, Agustí-Brisach C, Lovera M, Arquero O, Trapero A (2020) Almond anthracnose: current knowledge and future perspectives. Plants 9(8): 945. https://doi.org/10.3390/plants9080945 López-Moral A, Lovera M, Antón-Domínguez BI, Gámiz AM, Michailides TJ, Arquero O, ..., Agustí-Brisach C (2022) Effects of cultivar susceptibility, branch age, and temperature on infection by Botryosphaeriaceae and Diaporthe fungi on English walnut ( Juglans regia ). Plant Dis. 106(11): 2920–2926. https://doi.org/10.1094/PDIS-09-21-2042-RE López-Moral A, Lovera M, Antón-Domínguez BI, Michailides TJ, Arquero O, Trapero A, Agustí-Brisach C (2023) Effects of cultivar susceptibility, fruit maturity, and natural wounds on the infection of English walnut ( Juglans regia L.) fruits by Botryosphaeriaceae and Diaporthe fungi. J. Plant Pathol. 105(4): 1391–1401. https://doi.org/10.1007/s42161-023-01492-0 Luo Y, Ma R, Barrera E, Gusella G, Michailides TJ (2022) Effects of temperature on development of canker-causing pathogens in almond and prune. Plant Dis. 106(9): 2424–2432. https://doi.org/10.1094/PDIS-01-22-0048-RE Luo Y, Niederholzer F, Camiletti BX, Michailides TJ (2024) Survey on latent infection of canker-causing pathogens in budwood and young trees from almond and prune nurseries in California. Plant Dis. 108(3): 550–557. https://doi.org/10.1094/PDIS-07-23-1449-SR Luo Y, Niederholzer FJ, Lightle DM, Felts D, Lake J, Michailides TJ (2021) Limited evidence for accumulation of latent infections of canker-causing pathogens in shoots of stone fruit and nut crops in California. Phytopathology 111(11): 1963–1971. https://doi.org/10.1094/PHYTO-01-21-0009-R Luo Y, Niederholzer FJ, Lightle DM, Felts DG, Michailides TJ (2019) Understanding the process of latent infection of canker-causing pathogens in stone fruit and nut crops in California. Plant Dis. 103(9): 2374–2384. https://doi.org/10.1094/PDIS-11-18-1963-RE Martelli GP, Savino V (1997) Infectious diseases of almond with special reference to the mediterranean area 1. EPPO Bull. 27(4): 525–534. https://doi.org/10.1111/j.1365-2338.1997.tb00679.x Massantini R, Frangipane MT (2022) Progress in almond quality and sensory assessment: an overview. Agriculture 12(5): 710. https://doi.org/10.3390/agriculture12050710 Miarnau X, Zazurca L, Torguet L, Zúñiga E, Batlle I, Alegre S, Luque J (2021) Cultivar susceptibility and environmental parameters affecting symptom expression of red leaf blotch of almond in Spain. Plant Dis. 105(4): 940–947. https://doi.org/10.1094/PDIS-04-20-0869-RE Mishra B, Ploch S, Weiland C, Thines M (2023) The TrEase web service: inferring phylogenetic trees with ease. Mycol. Prog. 22(12): 84. https://doi.org/10.1007/s11557-023-01931-3 Moral J, Morgan D, Trapero A, Michailides TJ (2019) Ecology and epidemiology of diseases of nut crops and olives caused by Botryosphaeriaceae fungi in California and Spain. Plant Dis. 103: 1809–1827. https://doi.org/10.1094/PDIS-03-19-0622-FE Negahban H, Bolboli Z, Mostowfizadeh-Ghalamfarsa R (2024a) Development of PCR-based assays for the detection of the evident and latent infection with Stilbocrea banihashemiana , the causal agent of fruit tree cankers. Crop Prot. 181: 106677. https://doi.org/10.1016/j.cropro.2024.106677 Negahban H, Mostowfizadeh-Ghalamfarsa R, Bolboli Z, Salami M, Jafari M (2024b) Potential host range of Stilbocrea banihashemiana and susceptibility of economically important trees to this emergent fungal canker-causing pathogen. J. Plant Dis. Prot. : 1–12. https://doi.org/10.1007/s41348-024-00930-0 Negi G, Handa A (n.d.) Serological and Molecular Detection of Prunus necrotic ringspot virus infecting Almond (Prunus dulcis (Mill.) DA Webb) causing bud failure disease in North Western Himalyan region. Nouri MT, Lawrence DP, Holland LA, Doll DA, Kallsen CE, Culumber CM, Trouillas FP (2019) Identification and pathogenicity of fungal species associated with canker diseases of pistachio in California. Plant Dis. 103(9): 2397–2411. https://doi.org/10.1094/PDIS-10-18-1717-RE Ören E, Bayraktar H (2025) Identifying fungi responsible for trunk and scaffold diseases in almonds in Türkiye. Physiol. Mol. Plant Pathol. 102729. https://doi.org/10.1016/j.pmpp.2025.102729 Quesada T, Lucas S, Smith K, Smith J (2019) Response to temperature and virulence assessment of Fusarium circinatum isolates in the context of climate change. Forests 10(1): 40. https://doi.org/10.3390/f10010040 Shapiro SS, Wilk MB (1965) An analysis of variance test for normality (complete samples). Biometrika 52(3-4): 591–611. https://doi.org/10.1093/biomet/52.3-4.591 Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38(7): 3022–3027. https://doi.org/10.1093/molbev/msab120 Tavakolian B, Mostowfizadeh-Ghalamfarsa R, Crous PW (2023) Paramicrosphaeropsis eriobotryae sp. nov., a new agent of loquat canker in Iran. Plant Pathol. 72(7): 1247–1259. https://doi.org/10.1111/ppa.13746 Woudenberg JHC, Aveskamp MM, De Gruyter J, Spiers AG, Crous PW (2009) Multiple Didymella teleomorphs are linked to the Phoma clematidina morphotype. Persoonia 22(1): 56–62. https://doi.org/10.3767/003158509X427808 Table Table 1 List of Paramicrosphaeropsis eriobotryae isolates recovered from twig dieback symptoms in infected almond trees of Fars and Kermanshah Provinces, Iran. Isolates code location Date latitude longitude BA01 Bajgah, Fars Province 21-Apr-2024 29.725902 52.588418 BA02 Bajgah, Fars Province 21-Apr-2024 29.725902 52.588418 BA03 Bajgah, Fars Province 21-Apr-2024 29.725902 52.588418 BA04 Bajgah, Fars Province 21-Apr-2024 29.725902 52.588418 BA05 Bajgah, Fars Province 21-Apr-2024 29.725902 52.588418 KA01 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA02 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA04 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA05 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA07 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA08 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 KA09 Kermanshah, Kermanshah Province 12-Apr-2024 34.390619 47.156130 Supplementary Files Graphicalabstract.tif SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 15 Sep, 2025 Read the published version in Journal of Plant Diseases and Protection → Version 1 posted Editorial decision: Minor revisions 10 Aug, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers invited by journal 25 Jul, 2025 Editor invited by journal 16 Jul, 2025 Editor assigned by journal 14 Jul, 2025 First submitted to journal 11 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7105314","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":491036889,"identity":"dc187b75-5739-4f2c-bd40-27a3bdc45a9e","order_by":0,"name":"Hamed Negahban","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hamed","middleName":"","lastName":"Negahban","suffix":""},{"id":491036890,"identity":"556668dc-919c-4c90-b216-34bc44363f8b","order_by":1,"name":"Zeinab Bolboli","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Bolboli","suffix":""},{"id":491036891,"identity":"252a7f45-39d9-4f19-b226-6e17f907e36e","order_by":2,"name":"Reza Mostowfizadeh-Ghalamfarsa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3RMYvCMBjG8XeqywtdI/b8DC8EdPSrNAidinOHo8Slo938NA4JgZtyuAou16WTQ0WQG0SMiwhie+fkkP+W4UfyEACf730bYggI6naOu4ACjn35XwKk8I8vGi+NOTQZRXz9rU32mQvZMz9QrZ6TaJskTFnC0WYWa/sVCIkJQVw/J2yQjkAXJ0eQtAxQSEjdFtVKeKMLQl5aR85MyHDXSYhdCUFKel6QkKzzFrfFui1scyWLmBesJtVOpqbJMpqEpeV7ecw/ynBaVb8t5LEA4O5PfT6fz/dSF8w+UM7PfI5NAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5572-918X","institution":"Shiraz University","correspondingAuthor":true,"prefix":"","firstName":"Reza","middleName":"","lastName":"Mostowfizadeh-Ghalamfarsa","suffix":""}],"badges":[],"createdAt":"2025-07-12 03:01:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7105314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7105314/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s41348-025-01156-4","type":"published","date":"2025-09-15T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87750599,"identity":"7421f74e-19ec-4926-8a3b-717a26f0b110","added_by":"auto","created_at":"2025-07-28 14:59:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4012327,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms of \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e infection on almond trees in Fars and Kermanshah provinces, Iran\u003cem\u003e. \u003c/em\u003eTwig and branch dieback disease (a–e); Internal wood discoloration in one-year-old branches (f–j).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/bea1983ebb48e56b2b7bace5.png"},{"id":87749760,"identity":"f5ad8c83-966a-4970-86bf-68a538b292b3","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5321138,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characteristics of \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e (isolate BA05) obtained from infected almond twigs. Colony appearance (front and reverse) on PDA incubated at 20 °C after 12 days (a, b); Pycnidia developing on PDA (c–e); Conidia (f); Chlamydospores (g). Scale bars: d, e = 200 µm; f, g = 20 µm.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/cdcf6861d82d9926b9e1f701.png"},{"id":87749754,"identity":"8701c5c9-2c48-4e9f-beb7-1ee730dc9999","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1893566,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e isolates associated with twig dieback of almond trees in Iran. Bayesian inference trees based on \u003cem\u003etub2\u003c/em\u003e (β-tubulin)\u003cstrong\u003e \u003c/strong\u003e(a), and \u003cem\u003erpb2\u003c/em\u003e (RNA polymerase II subunit 2) (b) gene loci, and combined \u003cem\u003etub2\u003c/em\u003e + \u003cem\u003erpb2\u003c/em\u003e datasets (c) shows the phylogenetic placement of isolates from Fars (BA03, BA05) and Kermanshah (KA09) Provinces within \u003cem\u003eDidymellaceae\u003c/em\u003e. Bayesian posterior probabilities (BI-PP, left) and Maximum Likelihood bootstrap support values (ML-BS, right) are indicated at nodes. Branches with BI–PP= 1/ML–BS= 100 are considered fully supported. Trees were rooted using \u003cem\u003eNothophoma brennandiae\u003c/em\u003e and \u003cem\u003eNothophoma quercina\u003c/em\u003e. Isolates from this study are highlighted in bold.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/969357553202766dbbbad2d2.png"},{"id":87749757,"identity":"0bfeffb7-5d7c-4f07-a117-f80b82f360e0","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":985353,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of three pathogenicity characteristics in inoculated detached shoots of almond trees with twelve \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e isolates, eight days post-inoculation. The figure displays the distribution of upward (a) and downward (b) lesion progression, as well as lesion width (c). Values followed by different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e ≤ 0.05. Error bars represent the standard deviation of replicates (n = 3).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/c8b84d7c892f5dd78a40002f.png"},{"id":87749755,"identity":"c790e738-53a9-4d28-93de-0ef410ddb21f","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":927734,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of the aggressiveness assessment dataset of \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e isolates on almond detached shoots. The PCA biplot, which includes the first (PC1) and second (PC2) principal components based on three pathogenicity traits clustered the isolates into three groups: group A, aggressive isolate (5: BA05); group B, moderately aggressive isolates (2: BA02; 3: BA03; 4: BA04); and group C, \u0026nbsp;less aggressive isolates (1: BA01; 6: KA01; 7: KA02; 8: KA04; 9: KA05; 10: KA07; 11: KA08; 12: KA09). The percentage of explained variance for each of the three dimensions (principal components) is shown in (a). The bar chart displays the contribution percentage of each variable (pathogenicity traits), including upward lesion progression (ULP), downward lesion progression (DLP), and lesion width (LW) in the first principal component (b).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/d456a3cae93b8015d7c07102.png"},{"id":87749769,"identity":"dd07c08c-45d2-4e6a-8b36-5084d5a6b0d8","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3246965,"visible":true,"origin":"","legend":"\u003cp\u003eAlmond saplings stems inoculated with \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e (isolate BA05), two months after inoculation. Wood necrosis and dieback observed in inoculated stems (a \u0026amp; b); Internal discolored wood tissue (c–e); Negative controls inoculated with sterile PDA disks (f \u0026amp; g). Arrows indicate inoculation sites; dotted lines show symptom progression.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/a56b8a206b8ddd155fb8f895.png"},{"id":87749777,"identity":"d319dafe-e3bd-478f-951b-e11c323f0542","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2804640,"visible":true,"origin":"","legend":"\u003cp\u003eLesion development caused by \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e (isolate BA05) on detached almond shoots at various temperatures, observed 10 days after inoculation. Wood discoloration associated with infection is shown at 15 °C (a), 20 °C (b), 25 °C (c), 30 °C (d), and 35 °C (e), compared to the negative control (f). Arrows indicate inoculation points; dotted lines delineate symptom progression.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/3b8be6709b598ebf6e4e5c7f.png"},{"id":87750609,"identity":"4ef92319-7f1e-4fbb-af57-2e6640d51fdc","added_by":"auto","created_at":"2025-07-28 14:59:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":885533,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenicity of \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e (isolate BA05) on detached almond shoots at different temperatures, evaluated 10 days after inoculation. The figure shows upward lesion progression (a), downward lesion progression (b), and lesion width (c). Varations with statistically significant differences (\u003cem\u003eP\u003c/em\u003e ≤ 0.05) are denoted by different letters. Error bars represent standard deviation (n = 4).\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/9b10508df890f5bae38afbfa.png"},{"id":91889798,"identity":"a1738fc2-a8a4-467c-86e8-b016f0f3da1c","added_by":"auto","created_at":"2025-09-22 16:02:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20033519,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/894c35da-4557-4f0b-a7f3-b1f60e672d6d.pdf"},{"id":87750600,"identity":"0c513c5e-672d-4a8b-83dc-681179a5c66e","added_by":"auto","created_at":"2025-07-28 14:59:46","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1748776,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/8c5fbd4941a097ea44a510f5.tif"},{"id":87749752,"identity":"3b3cee56-7f55-4351-9b26-5b00f2a7b028","added_by":"auto","created_at":"2025-07-28 14:51:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23582,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7105314/v1/2c9e185fe1807c3144c24ede.docx"}],"financialInterests":"","formattedTitle":"Pathogenicity, Aggressiveness, and Temperature Response of Paramicrosphaeropsis eriobotryae Associated with Almond Twig Dieback and Wood Necrosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlmond (\u003cem\u003ePrunus dulcis\u003c/em\u003e Mill. D.A. Webb; family \u003cem\u003eRosaceae\u003c/em\u003e), a Mediterranean crop of significant global economic and social value (Massantini and Frangipane \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) originated in south-central Asia. Almond cultivation faces persistent threats from diseases that compromise yield and quality, including anthracnose, red leaf blotch, mosaic disease, viral bud failure, canker, and dieback (Martelli and Savino \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; L\u0026oacute;pez-Moral et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Miarnau et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Negi and Handa 2023; Endes \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOver the past decade, canker and dieback syndromes have emerged as major concerns in nut crops, frequently linked to fungal pathogens (Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Almond decline, in particular, has been associated with diverse Ascomycete families, notably \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e, \u003cem\u003eCeratocystidaceae\u003c/em\u003e, \u003cem\u003eCytosporaceae\u003c/em\u003e, \u003cem\u003eDiaporthaceae\u003c/em\u003e, \u003cem\u003eDiatrypaceae\u003c/em\u003e, \u003cem\u003eNectriaceae\u003c/em\u003e, \u003cem\u003ePleurostomataceae\u003c/em\u003e, and \u003cem\u003eTogniniaceae\u003c/em\u003e (Holland et al. 2019; Moral et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Anton-Dom\u0026iacute;nguez et al. 2023; Aloi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Endes \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Oren et al. 2025). Among these, \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e species, including \u003cem\u003eBotryosphaeria dothidea\u003c/em\u003e (Moug.) Ces. \u0026amp; De Not., \u003cem\u003eDiplodia corticola\u003c/em\u003e A.J.L. Phillips, A. Alves \u0026amp; J. Luque, \u003cem\u003eD. mutila\u003c/em\u003e (Fr.) Fr., \u003cem\u003eD. seriata\u003c/em\u003e De Not., \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e (Pat.) Griffon \u0026amp; Maubl., \u003cem\u003eNeofusicoccum arbuti\u003c/em\u003e (D.F. Farr \u0026amp; M. Elliott) Crous, Slippers \u0026amp; A.J.L. Phillips, \u003cem\u003eNeofusicoccum mediterraneum\u003c/em\u003e Crous, M.J. Wingf. \u0026amp; A.J.L. Phillips, \u003cem\u003eNeofusicoccum parvum\u003c/em\u003e (Pennycook \u0026amp; Samuels) Crous, Slippers \u0026amp; A.J.L. Phillips, \u003cem\u003eNeofusicoccum vitifusiforme\u003c/em\u003e (Van Niekerk \u0026amp; Crous) Crous, Slippers \u0026amp; A.J.L. Phillips, and \u003cem\u003eNeoscytalidium dimidiatum\u003c/em\u003e (Penz.) Crous \u0026amp; Slippers, are frequently implicated in almond branch cankers and dieback (Holland et al. 2019; Anton-Dom\u0026iacute;nguez et al. 2023; Aloi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Endes \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur recent surveys of almond twig dieback in Iran yielded isolates of \u003cem\u003eParamicrosphaeropsis\u003c/em\u003e sp. closely related to \u003cem\u003eP. eriobotryae\u003c/em\u003e Tavakolian, Mostowf. \u0026amp; Crous (\u003cem\u003eDidymellaceae\u003c/em\u003e), a canker pathogen originally described on loquat in Fars Province (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although a single isolate of \u003cem\u003eP. eriobotryae\u003c/em\u003e was previously reported from an almond tree within an infected loquat orchard (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), its role as a primary pathogen of almond remains unconfirmed. Critically, the prevalence of \u003cem\u003eP. eriobotryae\u003c/em\u003e across almond-growing regions, its aggressiveness on almond hosts, and key environmental drivers (e.g., temperature) are unknown, hindering the development of effective disease management strategies. This study addresses these critical knowledge gaps through four objectives: (i) Morpho-molecular characterization of \u003cem\u003eParamicrosphaeropsis\u003c/em\u003e sp. isolates recovered from symptomatic almond twigs; (ii) Aggressiveness assessment of geographically diverse isolates (western and southern Iran) on detached almond shoots; (iii) Pathogenicity validation on one-year-old almond saplings under controlled conditions; (iv) Temperature response profiling to define optimal conditions for lesion development. By establishing Paramicrosphaeropsis dieback as an emerging threat to almond health, this work provides critical data for future disease mitigation efforts.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sampling and fungal isolation\u003c/h2\u003e\u003cp\u003eDuring spring 2024, surveys were conducted in almond orchards exhibiting symptoms of twig necrosis and dieback in Fars (southern Iran) and Kermanshah Provinces (western Iran). Symptomatic one-year-old twigs were collected. The internal discoloration was assessed by examining transverse sections. All plant material was stored at 4\u0026deg;C until laboratory processing. For fungal isolation, small pieces (0.5 \u0026times; 0.5 cm) were excised from the margins between symptomatic (discolored) and asymptomatic tissues of the sampled twigs. These pieces were rinsed in sterile distilled water and surface-disinfected by immersion in 1% sodium hypochlorite (prepared from 20% commercial bleach) for one minute. After disinfection, the samples were rinsed twice in sterile distilled water and surface-dried on sterile paper towels. The disinfected tissue segments were then plated onto potato dextrose agar (PDA; containing extract from 350 g/L boiled potato, 20 g/L dextrose, 16 g/L agar, and distilled water) amended with 100 mg/L tetracycline to inhibit bacterial growth. Petri dishes were incubated in the dark at room temperature for seven days. The resulting fungal isolates were purified using the single hyphal tip method on water agar (WA; 20 g/L agar, and distilled water).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Morphological and cultural characterization\u003c/h2\u003e\u003cp\u003ePure cultures isolated from almond trees exhibiting twig dieback were morphologically characterized. Isolates were grown on PDA and incubated at 20\u0026deg;C in darkness for two weeks. Colony characteristics, including surface texture and pigmentation (obverse and reverse), were recorded. Microscopic features were assessed using light microscopy on lactic acid-mounted slides (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For each isolate selected, mature pycnidia and conidia were examined, with measurements taken of pycnidial dimensions (length and width), conidial size, and chlamydospore diameter (n\u0026thinsp;=\u0026thinsp;30 per structure). To determine optimal growth temperature, selected isolates from infected trees were incubated on PDA at eight temperatures (5\u0026ndash;40\u0026deg;C in 5\u0026deg;C increments). Radial growth rates were measured using four replicates per temperature.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Molecular identification\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. DNA extraction, PCR, and sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from fungal mycelia using the DNG-PLUS extraction kit (CinnaGen, Tehran, Iran) following the manufacturer\u0026rsquo;s protocol (Negahban et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). DNA concentration and purity were assessed using an MD-1000 Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For selected \u003cem\u003eParamicropshaeropsis\u003c/em\u003e sp. isolates, partial sequences of the protein-coding loci β-tubulin (\u003cem\u003etub2\u003c/em\u003e) and RNA polymerase II second-largest subunit (\u003cem\u003erpb2\u003c/em\u003e) were amplified. The \u003cem\u003etub2\u003c/em\u003e locus was amplified using primers Btub2Fd/Btub4Rd (Woudenberg et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and \u003cem\u003erpb2\u003c/em\u003e using primers fRPB2-5F/fRPB2-7cR (Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The thermal cycling program for \u003cem\u003etub2\u003c/em\u003e amplification followed Tavakolian et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), while the \u003cem\u003erpb2\u003c/em\u003e amplification program consisted of an initial denaturation step (95\u0026deg;C, 300 s), followed by 35 cycles of 94\u0026deg;C (45 s), 55.5\u0026deg;C (90 s), and 72\u0026deg;C (120 s), and a final extension (72\u0026deg;C, 600 s). Each 30 \u0026micro;L PCR mixture contained 1 \u0026micro;L of genomic DNA (~\u0026thinsp;100 ng/\u0026micro;L), 1 \u0026micro;L of each primer (10 pM), 15 \u0026micro;L of 2\u0026times; Taq DNA Polymerase Master Mix RED (Amplicon, Odense, Denmark), and 12 \u0026micro;L of PCR-grade water. Amplified PCR products were then sequenced using the same primer pairs via dye terminator cycle sequencing (Codon Genetic Group, Tehran, Iran).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Phylogenetic analyses\u003c/h2\u003e\u003cp\u003eSequence identity was assessed using the BLASTn tool (NCBI; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignments were generated using MAFFT v.7 (Katoh et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Phylogenetic analyses based on individual and concatenated \u003cem\u003etub2\u003c/em\u003e and \u003cem\u003erpb2\u003c/em\u003e sequences were reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML) in TrEase (Mishra et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) under default settings. Trees were visualized and edited using MEGA v.11 (Tamura et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Pathogenicity assessment\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Aggressiveness assay of isolates\u003c/h2\u003e\u003cp\u003eTo evaluate the aggressiveness of \u003cem\u003eParamicrosphaeropsis\u003c/em\u003e sp. isolates obtained from almond trees in two geographically distinct regions of Iran (south and west), a detached shoot inoculation assay was performed to quantify symptom severity. Asymptomatic one-year-old almond shoots (diameter: 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm) were collected, surface-disinfected by flaming with 98% ethanol, and sectioned into 20\u0026ndash;30 cm segments. Each isolate was inoculated onto four replicate shoot segments following the protocol described by Bolboli et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The inoculated shoots were then incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a 16-h light/8-h dark photoperiod in a plant growth chamber, arranged in a completely randomized design (CRD). After 8 days of incubation, bark tissue was aseptically removed from the inoculation sites. Lesion progression was quantified by measuring upward lesion progression (ULP), downward lesion progression (DLP), and lesion width (LW) using a digital caliper (Insize\u0026reg;, 0.01 mm precision). To fulfill Koch\u0026rsquo;s postulates, wood fragments from lesion margins were transferred to PDA amended with 100 mg/L tetracycline. The re-isolated fungi were identified based on cultural and morphological characteristics (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStatistical analyses were conducted to assess isolates aggressiveness based on lesion progression metrics (ULP, DLP, LW). All data were first checked for normality using the Shapiro-Wilk test (Shapiro and Wilk \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1965\u003c/span\u003e) in IBM SPSS Statistics 25. A one-way analysis of variance (ANOVA) was performed using the General Linear Model (GLM) procedure in SAS (v.9.4, SAS Institute, Cary, NC, USA) to determine the effect of \u003cem\u003eParamicrosphaeropsis\u003c/em\u003e sp. isolates (fixed effect) on each pathogenicity trait. Where ANOVA indicated significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), means were separated using Tukey's Honestly Significant Difference (HSD) test. Additionally, Principal Component Analysis (PCA) was conducted in R version 3.4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org/\u003c/span\u003e\u003cspan address=\"http://www.r-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). to visualize relationships among the 12 \u003cem\u003eParamicrosphaeropsis\u003c/em\u003e sp. isolates and determine patterns of variance in aggressiveness traits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Pathogenicity test on almond saplings\u003c/h2\u003e\u003cp\u003eTo confirm pathogenicity, the most aggressive isolate (previously identified on detached shoots) was inoculated onto one-year-old almond saplings. A sterile 5-mm cork borer was used to create uniform wounds on the main stem of each sapling. Each wound was inoculated with a 5 \u0026times; 5 \u0026times; 2 mm mycelial plug excised from the actively growing margin of a two-week-old culture grown on PDA. The inoculation site was immediately sealed with Parafilm\u003csup\u003e\u0026reg;\u003c/sup\u003e (Bemis Packaging, USA). Control saplings received sterile PDA plugs prepared identically (Gonzalez-Dom\u0026iacute;nguez et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The experiment employed a completely randomized design with four replicate saplings per treatment. Inoculated plants were maintained in a greenhouse (20\u0026ndash;35\u0026deg;C; natural light cycle) and irrigated as needed to sustain soil moisture at field capacity. After a two-month incubation period, external and internal symptoms (ULP, DLP, and LW) were assessed. Koch\u0026rsquo;s postulates were fulfilled by re-isolating the fungus from the margin of symptomatic wood tissue onto tetracycline-amended (100 mg/L) PDA, followed by morphological identification consistent with the original isolate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Effect of temperature on aggressiveness of \u003cem\u003eP. eriobotryae\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo assess the impact of temperature on the aggressiveness of \u003cem\u003eP. eriobotryae\u003c/em\u003e, detached almond shoots were inoculated with the isolate previously identified as the most aggressive in detached shoot assays (Section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.4.1\u003c/span\u003e). The inoculation protocol followed Bolboli et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Inoculated and control shoots were placed in growth chambers under five constant temperature regimes: 15, 20, 25, 30, and 35\u0026deg;C (\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) with a 16-hour light/8-hour dark photoperiod. The experiment was set up in a completely randomized design with four replicate shoots per temperature treatment, including controls. Ten days after inoculation, the bark was carefully removed from each shoot, and pathogenicity was evaluated by measuring ULP, DLP, and LW. Measurements were taken using digital calipers (precision\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mm). Statistical analysis was conducted using SAS v.9.4 (SAS Institute, Cary, NC, USA). Data normality for ULP, DLP, and LW was confirmed using the Shapiro-Wilk test (Shapiro and Wilk \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1965\u003c/span\u003e) within the UNIVARIATE procedure. The impact of temperature on each pathogenicity trait was analyzed separately through ANOVA performed with the GLM procedure. Significant differences indicated by ANOVA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) were further analyzed using Tukey\u0026rsquo;s Honestly Significant Difference (HSD) test to separate means (Negahban et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Disease symptoms and fungal isolations\u003c/h2\u003e\u003cp\u003eField surveys in almond orchards across two Iranian provinces revealed trees exhibiting canker and dieback symptoms. Affected twigs and branches showed external necrosis and dieback, leading to progressive decline and death. Transverse sections displayed internal vascular discoloration (brown to dark brown) and wood necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fungal isolates from the families \u003cem\u003eDidymellaceae\u003c/em\u003e and \u003cem\u003ePleosporaceae\u003c/em\u003e were recovered from symptomatic tissues using culturing techniques and identified based on morphology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Morphological characteristics\u003c/h2\u003e\u003cp\u003eTwelve of 24 isolates obtained from twig dieback were morphologically identified as \u003cem\u003eDidymellaceae\u003c/em\u003e. On PDA at 20\u0026deg;C (after 14 days), colonies exhibited a cottony texture, white to pale pinkish obverse, and pinkish to olivaceous brown reverse (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Mean radial growth was 3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm/day at 20\u0026deg;C, declining to 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mm/day at 40\u0026deg;C. Pycnidia (produced after ~\u0026thinsp;1 month at room temperature) were pale to dark brown, globose to subglobose, solitary or confluent, semi-immersed, and averaged 246.41\u0026thinsp;\u0026plusmn;\u0026thinsp;80.51 \u0026micro;m in radius (range: 102.45\u0026ndash;488.48 \u0026micro;m; n\u0026thinsp;=\u0026thinsp;100). Conidia were solitary, greenish-brown, variably shaped (globose to subcylindrical), and measured 7.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 \u0026times; 4.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 \u0026micro;m (range: 4.24\u0026ndash;11.48 \u0026times; 3.54\u0026ndash;6.55 \u0026micro;m; n\u0026thinsp;=\u0026thinsp;60). Chlamydospores occurred in chains, were globose to ellipsoid, smooth, thick-walled, and measured 12.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61 \u0026times; 8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 \u0026micro;m (range: 6.53\u0026ndash;16.67 \u0026times; 6.37\u0026ndash;11.24 \u0026micro;m; n\u0026thinsp;=\u0026thinsp;30). Hyphae averaged 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 \u0026micro;m in width (range: 2.62\u0026ndash;5.29 \u0026micro;m; n\u0026thinsp;=\u0026thinsp;50) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Phylogenetic analyses\u003c/h2\u003e\u003cp\u003ePreliminary BLASTn analysis of the \u003cem\u003etub2\u003c/em\u003e and \u003cem\u003erpb2\u003c/em\u003e nucleotide sequences revealed that three isolates, BA03 and BA05 (Fars Province) and KA09 (Kermanshah Province), shared 100% similarity and coverage with \u003cem\u003eP. eriobotryae\u003c/em\u003e isolate CBS 149854 (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Supplementary Table\u0026nbsp;1). Based on phylogenetic analysis of individual and concatenated \u003cem\u003etub2\u003c/em\u003e and \u003cem\u003erpb2\u003c/em\u003e sequences, the isolates clustered within a monophyletic clade comprising \u003cem\u003eP. eriobotryae\u003c/em\u003e isolates. High posterior probability and bootstrap values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) robustly support their identification as \u003cem\u003eP. eriobotryae.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Pathogenicity assessment\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Assessing the aggressiveness of the isolates from almond twig dieback disease\u003c/h2\u003e\u003cp\u003eDetached shoot inoculations confirmed the pathogenicity of all 12 \u003cem\u003eP. eriobotryae\u003c/em\u003e isolates inducing dark brown necrotic lesions on almond shoots and fulfilling Koch\u0026rsquo;s postulates. ANOVA revealed highly significant differences in lesion progression metrics (ULP, DLP, LW) among isolates (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0002; Supplementary Table\u0026nbsp;2), with BA05 exhibiting the greatest aggressiveness (ULP: 4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mm; DLP: 4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 mm; LW: 2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mm; Tukey\u0026rsquo;s HSD, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while KA01, KA04, and KA08 were less aggressive. Principal Component Analysis of lesion traits segregated isolates into three distinct groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), explaining 95.9% of the total variance (PC1: 82.6%, PC2: 13.3%). Group A contained the aggressive isolate, BA05 (from southern Iran), Group B included moderately aggressive isolates BA02-BA04 (from southern Iran), and Group C comprised less aggressive isolates\u0026ndash;KA01, KA02, KA04, KA05, KA07, KA08, KA09 (from western Iran) and BA01 (from southern Iran) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Pathogenicity assessment on almond sapling\u003c/h2\u003e\u003cp\u003ePathogenicity assays on one-year-old almond saplings confirmed that \u003cem\u003eP. eriobotryae\u003c/em\u003e induced disease symptoms consistent with almond twig dieback. By two months post-inoculation, all inoculated saplings exhibited distinct external symptoms, including dark brown necrotic lesions radiating from the inoculation point and longitudinal bark cracking (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea \u0026amp; b). Internal symptom assessment following bark removal revealed extensive brown-to-black vascular discoloration extending longitudinally through the wood (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;e). Quantification of the lesions demonstrated significant progression: upward lesion length (ULP: 28.75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79 mm), downward lesion length (DLP: 21.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 mm), and lesion width (LW: 11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 mm). In contrast, control saplings inoculated with sterile PDA plugs showed no disease development, exhibiting only minor wound callusing at the inoculation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef \u0026amp; g). \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e was consistently re-isolated from the margin of necrotic tissues onto PDA and morphologically identified, thereby fulfilling Koch's postulates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e3.3.3. Effect of temperature on symptom progression\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTemperature had a significant impact on the progression of necrotic lesions caused by \u003cem\u003eP. eriobotryae\u003c/em\u003e on detached almond shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). One-way ANOVA showed a significant influence of temperature on all pathogenicity traits, ultimate ULP, DLP, and LW (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Supplementary Table\u0026nbsp;3). Post-hoc analysis (Tukey\u0026rsquo;s HSD) demonstrated maximum symptom development at 25\u0026deg;C and 30\u0026deg;C. At 10 days post-inoculation, ULP reached 6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 mm and 8.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 mm, DLP measured 5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 mm and 5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 mm, and LW was 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 mm and 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 mm at 25\u0026deg;C and 30\u0026deg;C, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Conversely, symptom expression was significantly reduced at 15\u0026deg;C and 35\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study established \u003cem\u003eP. eriobotryae\u003c/em\u003e as a causal agent of almond twig dieback. We provide the first validated evidence of its isolation from symptomatic almond trees in Fars and Kermanshah Provinces, Iran, alongside comprehensive pathogenicity confirmation. This finding is particularly significant given Iran\u0026rsquo;s position as the world\u0026rsquo;s seventh-largest almond producer, with an annual harvest of 102,414.06 tons from 43,342 hectares (FAO, 2023), highlighting \u003cem\u003eP. eriobotryae\u003c/em\u003e as a newly identified threat to this commercially vital crop. While a previous study reported isolating a single \u003cem\u003eP. eriobotryae\u003c/em\u003e isolate from almond wood tissue without a pathogenicity assessment in live trees (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), our work definitively demonstrates pathogenicity through a multifaceted approach. This approach included rigorous pathogenicity testing on almond saplings, assessment of aggressiveness among multiple isolates using several key traits, and evaluation of temperature effects on infection using detached shoots.\u003c/p\u003e\u003cp\u003eIn this survey, \u003cem\u003eP. eriobotryae\u003c/em\u003e isolates were recovered from symptomatic almond twigs and one-year-old branches exhibiting vascular internal wood discoloration and necrosis. Morphological and molecular analyses confirmed significant similarity between these almond-derived isolates and the \u003cem\u003eP. eriobotryae\u003c/em\u003e type strain (CBS 148866; (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)), initially identified as a canker pathogen of loquat (\u003cem\u003eEriobotrya japonica\u003c/em\u003e) in Fars Province, southern Iran. While members of the \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e are often implicated as primary causal agents of almond canker and dieback, our findings highlight that these diseases involve a broader community of Ascomycete fungi. To date, at least 84 species associated with almond canker and dieback symptoms, representing families such as \u003cem\u003eCeratocystidaceae\u003c/em\u003e, \u003cem\u003eCytosporaceae\u003c/em\u003e, \u003cem\u003eDiaporthaceae\u003c/em\u003e, \u003cem\u003eDiatrypaceae\u003c/em\u003e, \u003cem\u003eNectriaceae\u003c/em\u003e, \u003cem\u003ePleurostomataceae\u003c/em\u003e, and \u003cem\u003eTogniniaceae\u003c/em\u003e (Holland et al. 2019; Moral et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Anton-Dom\u0026iacute;nguez et al. 2023; Aloi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Endes \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Oren et al. 2025), which may primarily act as secondary pathogens or endophytic colonizers. Further research is needed to understand the complex interactions between \u003cem\u003eP. eriobotryae\u003c/em\u003e and these diverse fungal associates, as well as the key biotic and abiotic factors influencing almond canker and dieback development.\u003c/p\u003e\u003cp\u003eFurthermore, the isolation of \u003cem\u003eP. eriobotryae\u003c/em\u003e from asymptomatic olive trees by Tavakolian et al. (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrates its capacity for latent infection within natural host tissues. This latency poses an insidious threat to agricultural biosecurity. Such pathogens persist cryptically within hosts, enabling undetected colonization and facilitating widespread dissemination, often via asymptomatic nursery stock (Havenga et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Indeed, latent infection by canker-causing fungi is a well-established phenomenon in perennial fruit and nut crops, extensively documented across diverse pathosystems (Havenga et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Avenot et al. 2022; Lopez-Moral et al. 2023; Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Negahban et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Bolboli et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ghaedi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The scale is significant, surveys have revealed high proportions of nursery trees harbor latent infections, exemplified by Californian almond nurseries, where \u003cem\u003eLasiodiplodia\u003c/em\u003e spp. and \u003cem\u003eNeofusicoccum\u003c/em\u003e spp. predominate (Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This underscores the latent phase as a primary pathway for regional and global pathogen spread. Consequently, understanding latent infection dynamics is critical for devising effective management strategies. Given the inherent challenge of detecting cryptic colonization, advanced molecular diagnostics, particularly PCR-based assays, have become indispensable tools for early and accurate pathogen identification, enabling proactive intervention (Luo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Negahban et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Bolboli et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Ghaedi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePathogen aggressiveness was rigorously quantified using a detached almond shoot assay, a validated method for rapid and reliable assessment of canker-causing fungi (Garkava-Gustavsson et al. 2014; Nouri et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bolboli et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Isolates of \u003cem\u003eP. eriobotryae\u003c/em\u003e from almond twig dieback showed significant variation in lesion development and wood necrosis severity. Principal components analysis (PCA), incorporating three key pathogenicity traits, separated the isolates into three distinct groups. Notably, upward (ULP) and downward (DLP) lesion progression emerged as the primary indicators of aggressiveness, explaining 82.6% of the variance on PC1. This approach also revealed a significant geographic pattern in this grouping: isolates from Fars Province (southern Iran) clustered together, while those from Kermanshah Province (western Iran) were mainly grouped separately. Particularly, isolate BA05 (from Fars Province) showed significantly higher aggressiveness compared to all others, and isolates from Fars Province were more aggressive than those from Kermanshah Province, suggesting that regional climatic differences influence pathogen populations (Quesada et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Subsequent pathogenicity trials on one-year-old almond saplings in controlled greenhouse conditions confirmed the pathogenicity. Within two months post-inoculation, saplings displayed necrosis, internal wood discoloration, and dieback, validating the ecological relevance of our aggressiveness metrics.\u003c/p\u003e\u003cp\u003eOur data establish a thermal optimum for \u003cem\u003eP. eriobotryae\u003c/em\u003e pathogenicity, with peak disease severity (lesion development) occurring between 25\u0026deg;C and 30\u0026deg;C. This contrasts sharply with significantly suppressed symptom expression at 15\u0026deg;C and 35\u0026deg;C. Such pronounced temperature dependence strongly implicates \u003cem\u003eP. eriobotryae\u003c/em\u003e as a significant threat within temperate to warm-temperate almond-growing regions. Integrating these findings with the likely latency of \u003cem\u003eP. eriobotryae\u003c/em\u003e in asymptomatic olive trees (Tavakolian et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) support a hypothesis that abiotic stressors, particularly temperature extremes and drought, serve as key triggers converting latent \u003cem\u003eP. eriobotryae\u003c/em\u003e infections into aggressive disease. This hypothesis aligns with existing literature that elucidates the impact of temperature on the development of various canker-causing pathogens (Lopez-Moral et al. 2022a; Luo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Avenot et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Negahban et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Notably, the temperature-dependent patterns of lesion growth observed in almond shoots vary among different canker pathogens, with \u003cem\u003eB. dothidea\u003c/em\u003e and \u003cem\u003eLasiodiplodia citricola\u003c/em\u003e exhibiting increased aggressiveness at higher temperatures (30\u0026deg;C), while \u003cem\u003eCytospora leucostoma\u003c/em\u003e (Pers.) Sacc. and \u003cem\u003eNeofusicoccum mediterraneum\u003c/em\u003e display optimal symptom development at 25\u0026deg;C and 20\u0026deg;C, respectively (Luo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study establishes \u003cem\u003eP. eriobotryae\u003c/em\u003e as a novel, aggressive causal agent of twig dieback in commercially critical almond orchards across Iran, a leading global producer. Our survey confirmed its significant pathogenic potential, revealed a pronounced geographic difference in isolate aggressiveness between Fars and Kermanshah Provinces, and defined a critical thermal optimum (25\u0026ndash;30\u0026deg;C) for disease development. Priority research must now focus on mapping the epidemiological reservoir by assessing \u003cem\u003eP. eriobotryae\u003c/em\u003e prevalence in non-almond hosts within and bordering orchard ecosystems and surrounding landscapes. Implementing non-destructive molecular diagnostics (e.g., qPCR, LAMP) for proactive surveillance in nurseries, orchards, and potential alternative hosts is essential. Additionally, screening commercial almond cultivars to evaluate their susceptibility remains a high priority. Addressing these critical gaps is fundamental for developing integrated management strategies to safeguard existing orchards and secure sustainable new almond plantings against this emerging pathogen.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHamed Negahban\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eZeinab Bolboli\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Validation, Methodology, Funding acquisition. \u003cstrong\u003eReza Mostowfizadeh-Ghalamfarsa\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4038664.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are in supplementary tables or available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAloi F, Luque-Cruz C, Agust\u0026iacute;-Brisach C, Spadaro D, Guarnaccia V (2024) First report of almond decline syndrome caused by \u003cem\u003eNeofusicoccum parvum\u003c/em\u003e in Italy. 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Plant Dis. 106(11): 2920\u0026ndash;2926. https://doi.org/10.1094/PDIS-09-21-2042-RE\u003c/li\u003e\n \u003cli\u003eL\u0026oacute;pez-Moral A, Lovera M, Ant\u0026oacute;n-Dom\u0026iacute;nguez BI, Michailides TJ, Arquero O, Trapero A, Agust\u0026iacute;-Brisach C (2023) Effects of cultivar susceptibility, fruit maturity, and natural wounds on the infection of English walnut (\u003cem\u003eJuglans\u003c/em\u003e \u003cem\u003eregia\u003c/em\u003e L.) fruits by \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e and Diaporthe fungi. J. Plant Pathol. 105(4): 1391\u0026ndash;1401. https://doi.org/10.1007/s42161-023-01492-0\u003c/li\u003e\n \u003cli\u003eLuo Y, Ma R, Barrera E, Gusella G, Michailides TJ (2022) Effects of temperature on development of canker-causing pathogens in almond and prune. Plant Dis. 106(9): 2424\u0026ndash;2432. https://doi.org/10.1094/PDIS-01-22-0048-RE\u003c/li\u003e\n \u003cli\u003eLuo Y, Niederholzer F, Camiletti BX, Michailides TJ (2024) Survey on latent infection of canker-causing pathogens in budwood and young trees from almond and prune nurseries in California. Plant Dis. 108(3): 550\u0026ndash;557. https://doi.org/10.1094/PDIS-07-23-1449-SR\u003c/li\u003e\n \u003cli\u003eLuo Y, Niederholzer FJ, Lightle DM, Felts D, Lake J, Michailides TJ (2021) Limited evidence for accumulation of latent infections of canker-causing pathogens in shoots of stone fruit and nut crops in California. Phytopathology 111(11): 1963\u0026ndash;1971. https://doi.org/10.1094/PHYTO-01-21-0009-R\u003c/li\u003e\n \u003cli\u003eLuo Y, Niederholzer FJ, Lightle DM, Felts DG, Michailides TJ (2019) Understanding the process of latent infection of canker-causing pathogens in stone fruit and nut crops in California. Plant Dis. 103(9): 2374\u0026ndash;2384. https://doi.org/10.1094/PDIS-11-18-1963-RE\u003c/li\u003e\n \u003cli\u003eMartelli GP, Savino V (1997) Infectious diseases of almond with special reference to the mediterranean area 1. EPPO Bull. 27(4): 525\u0026ndash;534. https://doi.org/10.1111/j.1365-2338.1997.tb00679.x\u003c/li\u003e\n \u003cli\u003eMassantini R, Frangipane MT (2022) Progress in almond quality and sensory assessment: an overview. Agriculture 12(5): 710. https://doi.org/10.3390/agriculture12050710\u003c/li\u003e\n \u003cli\u003eMiarnau X, Zazurca L, Torguet L, Z\u0026uacute;\u0026ntilde;iga E, Batlle I, Alegre S, Luque J (2021) Cultivar susceptibility and environmental parameters affecting symptom expression of red leaf blotch of almond in Spain. Plant Dis. 105(4): 940\u0026ndash;947. https://doi.org/10.1094/PDIS-04-20-0869-RE\u003c/li\u003e\n \u003cli\u003eMishra B, Ploch S, Weiland C, Thines M (2023) The TrEase web service: inferring phylogenetic trees with ease. Mycol. Prog. 22(12): 84. https://doi.org/10.1007/s11557-023-01931-3\u003c/li\u003e\n \u003cli\u003eMoral J, Morgan D, Trapero A, Michailides TJ (2019) Ecology and epidemiology of diseases of nut crops and olives caused by \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e fungi in California and Spain. Plant Dis. 103: 1809\u0026ndash;1827. https://doi.org/10.1094/PDIS-03-19-0622-FE\u003c/li\u003e\n \u003cli\u003eNegahban H, Bolboli Z, Mostowfizadeh-Ghalamfarsa R (2024a) Development of PCR-based assays for the detection of the evident and latent infection with \u003cem\u003eStilbocrea banihashemiana\u003c/em\u003e, the causal agent of fruit tree cankers. Crop Prot. 181: 106677. https://doi.org/10.1016/j.cropro.2024.106677\u003c/li\u003e\n \u003cli\u003eNegahban H, Mostowfizadeh-Ghalamfarsa R, Bolboli Z, Salami M, Jafari M (2024b) Potential host range of \u003cem\u003eStilbocrea\u003c/em\u003e \u003cem\u003ebanihashemiana\u003c/em\u003e and susceptibility of economically important trees to this emergent fungal canker-causing pathogen. J. Plant Dis. Prot. : 1\u0026ndash;12. https://doi.org/10.1007/s41348-024-00930-0\u003c/li\u003e\n \u003cli\u003eNegi G, Handa A (n.d.) Serological and Molecular Detection of Prunus necrotic ringspot virus infecting Almond (Prunus dulcis (Mill.) DA Webb) causing bud failure disease in North Western Himalyan region.\u003c/li\u003e\n \u003cli\u003eNouri MT, Lawrence DP, Holland LA, Doll DA, Kallsen CE, Culumber CM, Trouillas FP (2019) Identification and pathogenicity of fungal species associated with canker diseases of pistachio in California. Plant Dis. 103(9): 2397\u0026ndash;2411. https://doi.org/10.1094/PDIS-10-18-1717-RE\u003c/li\u003e\n \u003cli\u003e\u0026Ouml;ren E, Bayraktar H (2025) Identifying fungi responsible for trunk and scaffold diseases in almonds in T\u0026uuml;rkiye. Physiol. Mol. Plant Pathol. 102729. https://doi.org/10.1016/j.pmpp.2025.102729\u003c/li\u003e\n \u003cli\u003eQuesada T, Lucas S, Smith K, Smith J (2019) Response to temperature and virulence assessment of \u003cem\u003eFusarium\u003c/em\u003e \u003cem\u003ecircinatum\u003c/em\u003e isolates in the context of climate change. Forests 10(1): 40. https://doi.org/10.3390/f10010040\u003c/li\u003e\n \u003cli\u003eShapiro SS, Wilk MB (1965) An analysis of variance test for normality (complete samples). Biometrika 52(3-4): 591\u0026ndash;611. https://doi.org/10.1093/biomet/52.3-4.591\u003c/li\u003e\n \u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38(7): 3022\u0026ndash;3027. https://doi.org/10.1093/molbev/msab120\u003c/li\u003e\n \u003cli\u003eTavakolian B, Mostowfizadeh-Ghalamfarsa R, Crous PW (2023) \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e sp. nov., a new agent of loquat canker in Iran. Plant Pathol. 72(7): 1247\u0026ndash;1259. https://doi.org/10.1111/ppa.13746\u003c/li\u003e\n \u003cli\u003eWoudenberg JHC, Aveskamp MM, De Gruyter J, Spiers AG, Crous PW (2009) Multiple \u003cem\u003eDidymella\u003c/em\u003e teleomorphs are linked to the \u003cem\u003ePhoma clematidina\u003c/em\u003e morphotype. Persoonia 22(1): 56\u0026ndash;62. https://doi.org/10.3767/003158509X427808\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e List of \u003cem\u003eParamicrosphaeropsis eriobotryae\u0026nbsp;\u003c/em\u003eisolates recovered from twig dieback symptoms in infected almond trees of Fars and Kermanshah Provinces, Iran.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"582\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolates code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elatitude\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003elongitude\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eBA01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eBajgah, Fars Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e21-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.725902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e52.588418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eBA02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eBajgah, Fars Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e21-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.725902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e52.588418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eBA03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eBajgah, Fars Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e21-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.725902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e52.588418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eBA04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eBajgah, Fars Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e21-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.725902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e52.588418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eBA05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eBajgah, Fars Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e21-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.725902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e52.588418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eKA09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eKermanshah, Kermanshah Province\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e12-Apr-2024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e34.390619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e47.156130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aggressiveness, Ascomycota, Almond dieback, Didymellaceae, Principal Component Analysis, Phylogenetic analyses","lastPublishedDoi":"10.21203/rs.3.rs-7105314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7105314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, \u003cem\u003eParamicrosphaeropsis eriobotryae\u003c/em\u003e is identified and characterized for the first time as a novel and aggressive pathogen responsible for twig dieback in almond orchards. Field surveys across major almond-growing regions of Iran, the world\u0026rsquo;s seventh-largest producer, revealed trees exhibiting vascular necrosis, wood discoloration, and progressive dieback symptoms. Fungal isolates from symptomatic tissues exhibited morphological characteristics consistent with those of a member of the \u003cem\u003eDidymellaceae\u003c/em\u003e family, within \u003cem\u003eAscomycota\u003c/em\u003e. Phylogenetic analyses based on protein-coding loci β-tubulin (\u003cem\u003etub2\u003c/em\u003e) and RNA polymerase II second-largest subunit (\u003cem\u003erpb2\u003c/em\u003e) confirmed the identity of isolates as \u003cem\u003eP. eriobotryae\u003c/em\u003e. Pathogenicity assays on detached shoots and one-year-old almond saplings fulfilled Koch\u0026rsquo;s postulates, inducing dark brown necrotic lesions and internal vascular discoloration that mirrored field symptoms. Notably, principal component analysis (PCA) confirmed significant geographic variation in aggressiveness, with southern isolates exhibiting markedly higher aggressiveness than western counterparts. Disease development was strongly temperature-dependent, peaking at 25\u0026ndash;30\u0026deg;C and substantially suppressed at 15\u0026deg;C and 35\u0026deg;C. Importantly, previous research has demonstrated the capacity for latent infection in asymptomatic hosts such as loquat and olive, underscoring a considerable biosecurity risk to global almond production. This study establishes \u003cem\u003eP. eriobotryae\u003c/em\u003e as an emerging threat characterized by temperature-sensitive pathogenicity and regional variations in aggressiveness, underscoring the need for integrated management strategies.\u003c/p\u003e","manuscriptTitle":"Pathogenicity, Aggressiveness, and Temperature Response of Paramicrosphaeropsis eriobotryae Associated with Almond Twig Dieback and Wood Necrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 14:51:41","doi":"10.21203/rs.3.rs-7105314/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-08-10T16:21:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-26T00:32:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-25T19:53:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2025-07-16T07:46:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T01:50:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2025-07-11T22:59:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c70e57a2-6240-4301-8d20-2772920618d7","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T15:59:30+00:00","versionOfRecord":{"articleIdentity":"rs-7105314","link":"https://doi.org/10.1007/s41348-025-01156-4","journal":{"identity":"journal-of-plant-diseases-and-protection","isVorOnly":false,"title":"Journal of Plant Diseases and Protection"},"publishedOn":"2025-09-15 15:57:24","publishedOnDateReadable":"September 15th, 2025"},"versionCreatedAt":"2025-07-28 14:51:41","video":"","vorDoi":"10.1007/s41348-025-01156-4","vorDoiUrl":"https://doi.org/10.1007/s41348-025-01156-4","workflowStages":[]},"version":"v1","identity":"rs-7105314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7105314","identity":"rs-7105314","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}