Acta agriculturae Slovenica, 120/3, 1–8, Ljubljana 2024 doi:10.14720/aas.2024.120.3.16128 Original research article / izvirni znanstveni članek Phaeoacremonium hungaricum, a species causing grapevine wood necroses in Iraq Raed Abduljabbar HALEEM 1, 2 Received September 13, 2023,; accepted June 30, 2024. Delo je prispelo 13. september 2023, sprejeto 30. junij 2024 1 University of Duhok, College of Agricultural Engineering Sciences, Plant Protection Department, Kurdistan; 2 corensponding author: region-Iraqraed.haleem@uod.ac Phaeoacremonium hungaricum, a species causing grapevine wood necroses in Iraq Abstract: This paper describes the symptoms caused by Phaeoacremonium hungaricum Essakhi, Mugnai, Surico & P.W. Crous on grapevine seedlings observed in pathogenicity tests. The tested isolate was obtained from a 15 to 20-year-old grapevine in the Duhok province, Kurdistan region, Iraq. It was identified based on morphological characters and phylogenetic inferences using ITS sequences. Disease symptoms included interveinal chlorosis progressing to necrosis, defoliation, wilt- ing, and shoot-tip dieback. Additionally, dark brown to black streaking became evident in artificially wounded shoots ap- proximately two months post-inoculation. This is the first re- port of Phaeoacremonium hungaricum causing young vine de- cline in Iraq. Key words: molecular identification, ITS, P. hungaricum, grapevine. Phaeoacremonium hungaricum, vrsta, ki povzroča nekroze lesa na žlahtni vinski trti v Iraku Izvleček: Članek opisuje simptome ki jih povzroča gliva Phaeoareminium hungaricum Essakhi, Mugnai, Surico & P.W. Crous na sejankah žlahtne vinske trte v testih patogenosti. Pre- iskušani sev je bil dobljen iz 15 do 20 let starih trt, v provinci- Duhok na območju Kurdistana v Iraku. Sev je bil določen na osnovi morfoloških znakov in filogenetskih razmerij z uporabo ITS zaporedij. Simptomi bolezni so obsegali medžilne kloroze, ki so prehajale v nekroze, odpadanje in venenje listov ter odmi- ranje vršičkov poganjkov. Dodatno so bile na umetno ranjenih poganjkih v dveh mesecih po inokulaciji vidne temnorjave do črne proge. To je prvo poročilo o pojavu te glive, ki povzroča propadanje mladih žlahtnih vinskih trt v Iraku. Ključne besede: molekularna identifikacija, ITS, P. hun- garicum, žlahtna vinska trta Acta agriculturae Slovenica, 120/3 – 20242 R. A. HALEEM et al. 1 INTRODUCTION Grapevine trunk diseases represent a significant economic challenge in all grape-growing regions, pro- foundly affecting grape production and long-term sus- tainability. Eutypa and Botryosphaeria diebacks, black foot, and Esca diseases are primary culprits, causing sub- stantial financial losses in the sector (Billones-Baaijens and Savocchia, 2019; Wicks and Davies, 1999; Whitelaw- Weckert et al., 2013). These issues are particularly promi- nent in older vineyards, typically over 10 years old, where diseases that affect the grapevine trunk are pervasive. However, even young grapevines in newly established vineyards exhibit signs of decline, a condition commonly referred to as Petri disease or young esca (Scheck et al., 1998). Phaeoacremonium species contribute significantly to this complex of diseases, with grape wood necrosis be- ing a primary symptom. This necrosis manifests as brown internal streaking, foliar discoloration, and desiccation (Haleem et al., 2013). The early detection of grapevine trunk diseases presents a growing challenge, given that symptoms typically manifest over years. Fungi primarily infiltrate through pruning wounds, as noted by Gramaje et al. (2018). There is a suspected pivotal role in prevent- ing pathogen infections affecting grapevine trunks by treating pruning wounds (Mondello et al., 2018). As the pathogen progresses, it breaks down the wood, ultimately leading to the vine's demise. The genus Phaeoacremoni- um was established by Crous et al. (1996), but identifying Phaeoacremonium species remains arduous. Traditional techniques such as isolation, culturing, and subsequent morphological trait analyses are employed for identifi- cation. Despite the existence of several morphological identifying keys (Moster et al., 2005; Crous et al., 1996; Dupont et al., 2000), distinguishing characteristics can be challenging, resulting in misidentifications. Further- more, Phaeoacremonium species are slow-growing, often requiring more than 20 days to develop on a microbio- logical media. This slow growth allows other microor- ganisms to overgrow Phaeoacremonium, complicating identification and prolonging the process. Molecular tools have proven invaluable in identifying Phaeoacremo- nium species. For instance, Phaeoacremonium parasiti- cum Ajello, Georg & Wang was differentiated from Phae- oacremonium inflatipes W. Gams, Crous & M.J. Wingf. using the ITS region, certain Phaeoacremonium species associated with diseased grapevines were identified us- ing partial protein-encoding genes such as the β-tubulin gene (Dupont et al., 2002; Tegli et al., 2000). Species- specific primers based on the ITS region, actin, and β-tubulin genes from rDNA have facilitated the detec- tion and identification of Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai, and Phaeoacr- emonium chlamydosporum W. Gams, Crous, M.J. Wingf. & Mugnai from various locations worldwide (Dam and Fourie, 2005; Groenewald et al., 2000; Retief et al., 2005, Mostert et al. 2006, Saccà et al. 2018).The two important fungal diseases of grapevines belonging to Phaeoacremo- nium species are Petri disease of young vines and Esca disease of adult vines (Moster et al. 2006). By using the ITS regions 1 and 2, including the 5.8S rDNA (Dupont et al. 2000), and the β-tubulin gene (Groenewald et al. 2001, Mostert et al 2005), sixteen species of Phaeoacr- emonium have been identified on the grapevine. Further- more, Multiplex PCR tests based on the use of TUB and ACT primers, twenty-two Phaeoacermonium species as- sociated with grapevine showing esca diseases identified (Gramaje et al.,2009; Essakhi et al., 2008; Mostert et al, 2006). The Phaeoacremonium isolates studied here are derived from all over the Dohuk province in Iraq, mostly from Sendori, Badi, and Baribuhar, where local grape va- rieties are grown. We aimed at identifying Phaeoacremo- nium species isolated from grapevines showing disease symptoms and pruning wounds of trees not yet showing disease symptoms. 2 MATERIALS AND METHODS 2.1 FUNGAL ISOLATION During the summer season, fifteen-year-old Resh- mew local cultivar of Vitis vinifera L. branches (10 cm di- ameter) and trunks exhibiting symptoms such as brown streaking, necrosis, and brown-red wood were sampled from five distinct grapevine yards in the Duhok gover- norate of Iraq. To ensure proper surface disinfection, small pieces of tissue from the margin between necrotic and healthy tissue were immersed in 70  % ethanol for 30 seconds, followed by a 1-minute treatment in 1  % NaOCl solution, and then rinsed again in 70 % ethanol for an additional 30 seconds. Subsequently, the surface disinfected pieces were dried on filter paper and then plated on Potato Dextrose Agar (PDA) supplemented with 0.25 mg ml-1 chloramphenicol (Himedia Laborato- ries Pvt. Ltd., India). The growing hyphae from the plated tissue pieces were carefully transferred and subcultured onto fresh PDA plates. The plates were then incubated at 25 °C, following the method described by Van Niekerk et al. (2004). To stimulate sporulation, the isolates were cultured on autoclaved grapevine cane pieces embedded in 2 % water agar, and maintained at 25 °C under a 12/12 hour photoperiod, as outlined by Luque et al. (2005). This Acta agriculturae Slovenica, 120/3 – 2024 3 Phaeoacremonium hungaricum, a species causing grapevine wood necroses in Iraq process aimed to facilitate the growth and development of the isolates for further analyses and identification. 2.2 MORPHOLOGY IDENTIFICATION In this study, morphological characteristics such as conidia, conidiophore length, mycelial texture, and phialide shape (Type I, II, and III) were utilized to dif- ferentiate between various species of Phaeoacremonium isolated during a sampling that was carried out in five grapevines yards of Duhok governorate. The central point of the grape vineyard was selected in each loca- tion, four directions were determined from this point, and then 15–20 samples were taken randomly from each one to ensure identification, 40 conidia and phialides were measured for length and width for each isolate. All isolates were cultivated on potato dextrose agar (PDA) at 25  °C, either in the absence of light or under NUV + fluorescent illumination with a 12-hour photoperiod using Philips 36W bulbs. This cultivation process aimed to stimulate the sporulation of the isolates for further ex- amination. Identification of the isolated fungus was car- ried out based on morphological characters outlined by Crous et al. (1996, 2009) and Essakhi et al. (2008). 2.3 DNA EXTRACTION AND POLYMERASE CHAIN REACTION Pure culture of fungal isolate was done by hyphal tip method by cutting out the tip of a single hyphae grow- ing from a single spore colony. Fungal isolates from the single spore were sub-cultured in potato dextrose broth and then incubated at 25 °C for six days to extract ge- nomic DNA. Under aseptic conditions, mycelia were purified and then frozen at –20 °C. The manufacturer's instructions were followed when extracting DNA using a Jena (Jena Bioscience, Germany) kit for extracting yeast DNA. PCR was done with primer set ITS1/ITS4 (White, Species Isolate ID GenBank accession number Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai CBS 631.94 AF266647.1 Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai strain 30 DQ404355.1 Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai STE-U 3080 AF197996.1 Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai PVFi 157 AF266654.1 Phaeoacremonium hungaricum Essakhi, Mugnai, Surico & Crous PHD45 MW355028.1 Phaeoacremonium hungaricum Essakhi, Mugnai, Surico & Crous JAMA5 OK649973.1 Phaeoacremonium hungaricum Essakhi, Mugnai, Surico & Crous IHBF 2229 MF326630.1 Phaeoacremonium prunicola L. Mostert, Damm & Crous STE-U5967 NR_135938.1 Phaeoacremonium viticola J. Dupont CBS 101738 MH862747.1 Phaeoacremonium viticola J. Dupont LCP 93 3886 AF118137.1 Phaeoacremonium alvesii L. Mostert, Summerb. & Crous KMU10268 LC508975.1 Phaeoacremonium sp. W. Gams, Crous & M.J. Wingf MRHf10 MK120896.1 Phaeoacremonium italicum A. Carlucci & M.L. Raimondo, ColPat-676 MT022463.1 Phaeoacremonium parasiticum (Ajello, Georg & C.J.K. Wang) W. Gams, Crous & M.J. Wingf. UZ577 17 MF363156.1 Phaeoacremonium parasiticum (Ajello, Georg & C.J.K. Wang) W. Gams, Crous & M.J. Wingf. KMU 9954 LC203479.1 Phaeoacremonium scolyti L. Mostert, Summerb. & Crous voucher DUCC404 KC013299.1 Phaeoacremonium hispanicum Gramaje, Armengol & L. Mostert Y549-09-3b JF275865.1 Phaeoacremonium inflatipes W. Gams, Crous & M.J. Wingf CBS 391.71 MH860178.1 Phaeoacremonium sphinctrophorum L. Mostert, Summerb. & Crous 51561 KT989682.1 Ulocladium atrum Preuss, Linnaea ATCC 18040 AF229486.1 Table 1: Species name, Isolate number and NCBI GenBank accession numbers included in this study (Phaeoacremonium hungari- cum Isolate PHD 45). Acta agriculturae Slovenica, 120/3 – 20244 R. A. HALEEM et al. et al. 1990), Ex-Taq PCR Master Mix (Jena Bioscience, Germany), 1 µl of each forward and reverse primer (20 pmol), 6 µl of template DNA (30-100 ng µl-1), and 12 µl RNase-free water to reach a final volume of 40 µl. Reac- tions took place on an Applied Biosystems ABI Geneamp 9700 PCR-thermal cycler. PCR was carried out using a denaturation step of 95 °C for 4 min; 10 cycles with 94 °C for 30 s, then 60 °C for 45 s, 72 °C for 1:30 s: 25 cycles with 94 °C for 30 s, 55 °C for 45 s, 72 °C of 1:30 s; and finally, an extension step of 72°C for 10 min. A 1 % agarose gel was used to visualize PCR products with EvaGreen ® Fluorescent Gel Stain.+ 2.4 SEQUENCING AND MOLECULAR PHYLOG- ENY Sequence data was edited with BioEdit sequence alignment editor (BioEditv7) before depositing the se- quence in GenBank. The NCBI's BLAST method was employed to assess the degree of similarity between the generated sequence to others stored in GenBank (Table 1). Selected sequences were aligned using ClustalW. The evolutionary history of Phaeoacremonium spp. was in- ferred with the maximum likelihood method and imple- mentation of the Tamura-Nei model (Tamura and Nei, 1993) by using MEGA11 Software (Tamura et al., 2021). The tree that has the highest log likelihood is displayed (-2569.41). Bootstrap support values were derived from 1000 replicates and were printed below branches.ITS se- quences from 20 taxa were compared. Branches with less than 50 % bootstrap support collapsed. The final dataset included 859 positions in total. 2.5 PATHOGENICITY TEST Pathogenicity tests were done at the College of Ag- ricultural Engineering Sciences at Duhok University in Iraq by using rooting cuttings of Reshmew and Taefi cultivars of Vitis vinifera L., planted in 15 kg pots with autoclaved sandy loam: peat moss (3:1) soil. Trunks were inoculated above the fourth shoot internode with Phaeoacremonium sp., isolate PHD 45. Grapevine shoots were wounded to a depth of 8 mm using a sterilized and sharp blade. Plugs of an agar culture (4 mm in diame- ter) were taken from the edge of a two-week-old PDA culture and inserted into the wound with the mycelium facing inward. Inoculated wounds were sealed with para- film. After 48 hours, the parafilm was taken off. Negative control plants were equally treated but inoculated with a plug from a sterile PDA plate. Pieces of tissue from the lesions’ margins were placed on PDA plates at the end of the experiment to check for the presence of the pathogen (Philips, 1998), After four months, the length of the can- ker on the inoculated shoot was measured. A Complete Randomized Design (CRD) was applied by using 3 in- oculated trees and 3 negative control plants per cultivar. 3 RESULTS AND DISCUSSION 3.1 MORPHOLOGY IDENTIFICATION Phaeoacremonuim hungaricum Essakhi, Mugnai, Surico & P.W. Crous (Isolate PHD 45) was isolated from grapevine wood tissues exhibiting internal wood discol- oration in vineyards aged between 15–20 years in the Duhok province. On potato dextrose agar (PDA), the col- onies displayed a flat, felt-like texture with entire edges. After 16 days of culture, the colony’s color was whitish- grey. The mycelium hyphae were observed to be branched and septate, appearing singly or bundled, sometimes ex- ceeding 14 in number. They exhibited a width ranging from 1–3.5 µm, with a smooth texture varying from sub- hyaline to medium brown, occasionally presenting ver- ruculose characteristics. Conidiophores typically bore a single terminal phialide, ranging from subhyaline to pale brown, becoming paler towards the tip, and displaying a smooth to verruculose surface. They measured 27 to 30 millimeters in length and 2.5 to 3.5 millimeters in width. These conidiophores were unbranched, short, erect, and simple in structure. Phialides, measuring approximately 1 µm in length and 1.6 µm in width, were predominantly subhyaline and located laterally. They exhibited a smooth to verruculose texture and were monophialidic. Type I phialides, the most prevalent, displayed an elongated am- pulliform shape, often with a constricted or attenuated base, while others exhibited a cylindrical form, measur- ing 7–14 × 2.5–3 µm. Type II phialides were navicular or subulate, sometimes appearing subcylindrical. The hya- line conidia were typically subcylindrical or cylindrical, occasionally allantoid, with dimensions of 3–5 × 1.5–2 µm. Accordingly, the isolate was identified as Phaeoacr- emonuim hungaricum. (Figure 1). 3.2 TAXONOMY Taxon name: Phaeoacremonium hungaricum Es- sakhi, Mugnai, Surico & Crous, Persoonia, 21, 127 (2008) [MB#506948] Basionym: Phaeoacremonium hungaricum Essakhi, Mugnai, Surico & Crous, Persoonia, 21, 127 (2008) [MB#506948] Material examined: Iraq, Kurdistan region, Dohuk Acta agriculturae Slovenica, 120/3 – 2024 5 Phaeoacremonium hungaricum, a species causing grapevine wood necroses in Iraq province, College of Agricultural Engineering sciences, Plant Protection Department, from grapevine trunk, cv. Rashmew, collected and isolated by R. Haleem (Isolate No. PHD 45; GenBank MW355028). 3.3 SEQUENCING AND PHYLOGENIC ANALYSIS Blast searches based on obtained ITS sequence identified isolate PH45 as Phaeoacremonium hungaricum by the unique universal primer pairs ITS1/ITS4 region ITS sequences of the isolate from Iraq and CBS 123036 (ex holotype strain of Phaeoacremonium hungaricum, NR_135953, were identical. ITS rDNA blast analysis backed up the morphological analysis. Maximum likeli- hood analysis (ML) was used to generate a phylogenetic tree for Phaeoacremonium hungaricum that was divided into eleven clades using 18 Phaeoacremonium reference strains, by using Ulocladium atrum Preuss, Linnaea as outgroup (Fig. 2). The isolate of this study (No. PH45) clade together with Phaeoacremoinum hungaricum JAMA 5 strain with 96 % ML value. 3.4 PATHOGENICITY TEST The inoculation of Phaeoacremonium hungaricum on shoots (Fig. 3) resulted in the characteristic symp- toms of vascular fungal infection in plants, evidenced by brownish to black vascular discolorations. Evaluation of canker lengths on shoots revealed that Phaeoacremo- nium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai induced significant canker formation on ‘Taefi’ shoots after four months of inoculation, with an average length of 12.67 mm. Within 20 days of inoculation, symptoms of P. hungaricum were observed on ‘Selemani’ leaves as chlorotic spots between veins and along margins. These initial spots later progressed to necroses. These observa- tions align with previous reports by Mugnai et al. (1999), Sands et al. (1997), Harrington et al. (2000), Edwards et al. (2001), and Feliciano et al. (2004), who documented similar signs and symptoms associating Phaeoacremo- nium infections. Through the morphological and cultural charac- teristics, coupled with DNA sequence data analysis, the Figure 1: Phaeoacremonuim hungaricum. A, B = Colony growth on PDA. C, D = Lateral monophialides. E = Hyphae sometimes forming coils. F = Hyaline subcylindrical conidia Scale bar C, D = 15µm, E,F = 10µm.. Figure 2. Phylogenetic maximum likelihood analysis based on ITS sequences. The tree identifies PHD 45 isolate from Iraq as Phaeoacremonium hungaricum. Acta agriculturae Slovenica, 120/3 – 20246 R. A. HALEEM et al. presence of Phaeoacremonium hungaricum in the sam- pled region was recorded in five grapevine orchards of the Duhok governorate. Key morphological features such as conidia and phialide size and shape, along with the structure of conidiophores, proved instrumental in iden- tifying one of the obtained isolates as P. hungaricum. Par- ticularly, distinguishing characteristics included short, typically unbranched conidiophores arising from aerial or sunken hyphae, erect and simple in structure, often with a single terminal phialide, and displaying a smooth to verruculose surface. Despite the importance of mor- phological and cultural characteristics, challenges per- sist in species determination solely based on these traits. The overlapping nature of morphological features among species complicates accurate identification. Hence, DNA sequence data analysis remains crucial for comprehen- sive and reliable species description. Mostert et al. (2006) developed a polyphasic identification key incorporating multiple aspects of Phaeoacremonium species, including DNA sequence data, and morphological, and cultural characteristics. This tool combines various data sources to differentiate between Phaeoacremonium species effec- tively. Due to the diverse range of species found on grape- vines, including some that are infectious to humans, ac- curate identification poses a significant challenge. While morphological identification has limitations, molecular- based approaches offer valuable tools for detecting and identifying these species effectively. It is essential to gen- erate sequences of protein-encoding genes especially when describing new species. Especially the variable in- trons from the actin and beta-tubulin gene could be used for the generating of species-specific primer pairs and as species-specific molecular detection tools. By leveraging these genes, researchers can develop robust molecular as- says capable of accurately identifying Phaeoacremonium species, aiding in research and disease management ef- forts. PCR offers significant advantages in the detection of Phaeoacremonium, notably rapidity and sensitivity, both crucial factors in managing this pathogen effective- ly. However, the time required for identification is often prolonged when using enriched growth media, as Phaeo- acremonium species exhibit slow growth on such media. Consequently, Phaeocremonium may often be obscured or intermingled with other pathogens or saprophytic or- ganisms, potentially leading to ambiguous detection re- sults. Comparative studies between traditional and PCR methods for Phaeocremonium detection have consist- ently demonstrated the superior sensitivity of PCR. The vascular discoloration observed at inoculation sites dur- ing this investigation likely stems from the oxidation and transfer of various breakdown products of plant cells, re- sulting from fungal enzymes attacking the plant. Indeed, P. hungaricum, like other phytopathogenic fungi, pro- duces numerous enzymes that degrade macromolecules within plant tissues, including polysaccharides. Valtaud et al. (2009) corroborated this observation, noting xyla- nase and glucosidase activity in the culture medium of this fungus. Accurate identification of the causal organ- ism, as facilitated by PCR-based strategies, holds promise for enhancing Iraq's capacity to mitigate grapevine trunk diseases effectively. In summary, the PCR-based approach outlined in this study enables sensitive, rapid, and reliable identifica- tion of Phaeoacremonium species associated with grape- vine decline. 4 REFERENCES Agrios, G. N. (2005). Plant Pathology (5th ed.). 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