The Mycobiota Diversity Associated with Mistletoe (Viscum album L.) in Poland

Abstract

The spread of mistletoe, which mainly affects Scots pine in Europe’s managed forests, has become a serious ecological and economic problem, exacerbated by climate change and drought stress. Research into mistletoe-associated mycobiota provides crucial insights into the biology, ecology and distribution of these fungi and forms the basis for sustainable plant protection and effective management strategies. The aim of this study was to identify the fungal species associated with mistletoe (Viscum album L.) at 75 sites across Poland, with a focus on the northeastern regions of the country. The field work included a systematic and targeted search using the transect method, followed by laboratory analysis, during which fungal samples were prepared and examined under both light and scanning electron microscopes. The genetic analysis was carried out using ITS sequencing to confirm the presence of pathogen-specific fungi in the majority of V. album samples examined. The results showed four fungal species associated with the mycobiota of mistletoe: Sphaeropsis visci, Plenodomus visci, Cylindrodendrum hubeiense and Septoria krystynae. It is noteworthy that two of these species (P. visci and C. hubeiense) have not yet been detected in Poland. Based on these findings, the distribution areas of the identified fungal species were mapped accordingly.

1. Introduction

Mistletoe (Viscum album L.), a perennial, evergreen semi-parasite spread by birds, mainly colonizes branches of woody plants. It attaches itself to the xylem of the host via haustoria and extracts water, minerals and carbohydrates, which leads to stunting of the host, defoliation and increased susceptibility to pathogens [1,2,3,4,5,6].

The spread of mistletoe, especially in managed pine forests, is a major forestry problem in Central Europe, with Scots pine (Pinus sylvestris) being severely affected in Poland, taking up over 60% of forest areas [7]. An inventory from 2019 revealed an infestation of 167,000 hectares due to drought stress caused by climate change, which has favoured the spread of mistletoe from southern to northern Poland [5,8]. Recent reports from ICP Forests identify V. album as a major biotic factor damaging Scots and Austrian pines across Europe [9]. Regions such as Switzerland, Germany and Hungary report similar problems, with mistletoe infestations in Brandenburg’s pine forests increasing from 1% in 2009 to 11% in 2015, further threatening forest health through increased susceptibility to pests such as the pine bark beetle (Ips acuminatus) [7,10].

The term mycobiota includes all fungi that occur in a specific area or are associated with a specific organism [11,12]. Our analysis shows that the mycobiota of V. album are still largely unexplored, as previous studies focused only on selected fungal taxa and not on the entire assemblage. The identified fungi on mistletoe include both parasitic and saprotrophic species, and a total of 12 taxa are known [2,13,14,15,16].

This study investigates fungal communities associated with V. album and specifically analyzes phytomycoses, host trees and coexisting saprotrophic and parasitic fungi with the aim of mapping the fungal diversity of mistletoe and its host-fungal relationships. Research methods include the collection of symptomatic plant parts, microscopic analysis, taxonomic identification and the genetic qPCR method [13].

1.1. Biological and Morphological Characteristics of Mistletoe

The life expectancy of V. album is typically between 10 and 20 years, but in rare cases this hemiparasite can persist for 30 years or more [2,13]. Once established, mistletoe infestation often lasts the entire lifespan of the host tree, often accelerating host decline and contributing to its eventual death [17].

Nutrients are absorbed by V. album via a system of haustoria, which are lateral branches of the suction roots that penetrate the cambium and xylem of the host tree [18]. In times of water scarcity, the host plants close their stomata to inhibit gas exchange and prevent photosynthesis. However, the mistletoe continues to transpire and deprives the host plant of water and nutrients, which leads to further stress for the host plants during dry periods [5,6,18,19].

Due to its dispersal mechanisms, V. album can parasitize the same host over several generations. The continuous deposition of berries often leads to repeated infections by the hemiparasite [17]. Mistletoe infects trees over long distances through the spread of diaspores by birds via defecation or regurgitation and over short distances through spontaneous fruit drop from the host tree [20,21]. The sticky substance in the berries, known as viscin (a viscous plant mucilage), makes it easier for the seeds to adhere to branches and trunks. Once the seed adheres, it germinates and forms haustoria, which penetrate the host’s xylem to obtain water and nutrients.

In the course of its development, mistletoe initially takes on a fan-shaped form, which later transforms into a spherical shape with a diameter of up to 150 cm [2,3]. The leaves are smooth, leathery, evergreen, with entire margins, stalkless, broad-lanceolate and green or yellow–green in colour, opposite or occasionally arranged in whorls of three, four or five [2]. Leaf lengths range from 1.3 to 10.7 cm, widths from 0.3 to 4.3 cm. Leaf size and shape vary considerably, even for V. album on the same host (Figure 1A) [1,2,3]. The stems are olive green, dichotomously branched, with annual growth forming a new branch (Figure 1B) [2].

The flowers of the common mistletoe are yellow–green, unisexual, insect-pollinated and rarely wind-pollinated. They form buds that are unable to self-pollinate and are located at the intersections of the branches [4,20,22]. The flowers are small, 2–3 mm in size, with the male flowers typically slightly larger than the female flowers [2]. Both sexes produce nectar, with the female flowers producing larger quantities [2,22]. The flowering period is from February to April [1,2]. The berries are spherical or ovoid, white and have a diameter of 6 to 10 mm. They ripen from fall to early winter (Figure 1C). The berries of V. album consist of a translucent exocarp, a viscid filled mesocarp and a thin layer of endocarp attached to the seed (Figure 1D). The seeds contain one to four embryos [1].

The spread of V. album is mainly favoured by numerous bird species, especially from the family of thrushes (Turdidae) and waxwings (Bombycillidae) [10,23]. These birds can consume up to 100 berries per day to meet their energy needs [24]. Understanding the morphology, biology and ecology of V. album is crucial for developing effective management strategies to reduce its impact on host trees and forest ecosystems.

1.2. Subspecies of V. album in Poland

In Poland, three subspecies of the common mistletoe—V. album L. subsp. album, V. album subsp. austriacum, and V. album subsp. abietis—can be identified, although the distinction by morphology alone is difficult and is mainly based on the characteristics of the berries rather than the leaves [4,13,22,25]. Each subspecies is associated with specific host preferences [8,19].

V. album subsp. album is the most widespread species, parasitizing 452 species, especially in Salix, Populus, Acer and Malus (Figure 2A), and accounting for 78.4% of mistletoe cases in Poland [1,24,25]. Some species, Fagus and certain oaks, show resistance to the infection [1].

V. album subsp. austriacum primarily infests conifers, especially Scots pine, and accounts for 15.6% of the areas infested by mistletoe in Poland (Figure 2B) [5,8,13]. Its spread, which is probably due to water scarcity, gives rise to serious forestry concerns in central and southern Poland [5].

The fir mistletoe, V. album subsp. abietis, is rare in Poland but is increasingly damaging European silver fir, causing tree mortality, wood degradation and lower yields [18,24,26].

Similarly, an increase in finds of the fir mistletoe, V. album subsp. abietis, has been observed throughout Europe. In combination with pest infestations, the increased occurrence of the fir mistletoe often leads to the death of European silver fir (Abies alba) stands. This subspecies is responsible for considerable losses in fir forests but has never caused the complete destruction of entire stands [24,26]. V. album subsp. abietis is the least widespread subspecies in Poland, accounting for about 6% of all recorded occurrences of V. album, which is related to its exclusive occurrence on fir trees (Abies spp.) [8,10,27]. Of all the subspecies discussed, V. album subsp. abietis has proven to be the most damaging to host trees and commercial forests, as it contributes significantly to tree mortality, wood destruction and reduced timber yield [18]. This subspecies can be distinguished from the scattered common mistletoe (V. album subsp. austriacum) by the shape and length of its leaves, while the typical common mistletoe (V. album subsp. album) is characterised by a smaller number of embryos in its fruits and straight-edged berries [13].

1.3. Characterisation of Fungal Associations with the Common Mistletoe (V. album)

Fungi associated with mistletoe (V. album L.) are either parasitic or saprotrophic and affect the health and ecology of mistletoe. Parasitic fungi can have a negative impact on mistletoe by causing disease, while saprotrophic fungi contribute to the decomposition of dead mistletoe tissue [13,28,29].

To date, thirteen parasitic and saprotrophic fungal species have been identified on V. album, including several important pathogens and common saprotrophs (

Table 1. List of fungal species detected on V. album with corresponding sources.

Fungal Species	Citation
Alternaria alternata (Fr.) Keissl.	[2,16]
Aureobasidium harposporum (Bres. & Sacc.) Herm.-Nijh.	[16]
Botryosphaeria dothidea (Moug.) Ces. & De Not.	[2,14,16]
Chordomyces albus Giraldo, Deanna A. Sutton & Guarro	[16]
Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.	[2,15]
Cylindrodendrum hubeiense (Zhuang, Nong & Luo) L. Lombard & Crous	[16]
Fusarium sarcochroum (Desm.) Sacc.	[15]
Plenodomus visci (Moesz) Gruyter, Aveskamp & Verkley	[16,30]
Phyllosticta visci (Sacc.) Allesch.	[16,31]
Rhabdospora visci (Bres.) Died.	[16,30]
Septoria krystynae Ruszk.-Mich.	[32]
Sphaeropsis demersa (Bonord.) Sacc.	[33]
Sphaeropsis visci (Alb. & Schwein.) Sacc.	[2,14,33]

).

Some of these fungi, such as Botryosphaeria dothidea and Colletotrichum gloeosporioides, are known pathogens that can exacerbate host plant decline, while others contribute to nutrient cycling through decomposition. Understanding the diversity of fungi associated with V. album is critical to understanding its ecology and developing management strategies for mistletoe-infested trees.

It is not uncommon for a single fungal species to act as a hyperparasite on V. album and switch to a saprotrophic lifestyle after the plant dies. Hyperparasitic fungi that colonise mistletoe are responsible for tissue damage, necrosis and decay, which can lead to reduced host growth, defoliation and, in rare cases, the death of V. album [2,19,34].

2. Materials and Methods

2.1. Geographical Distribution of the Sampling Locations

The main study area was located in the Warmian–Masurian and Podlaskie voivodeships in northeastern Poland. Some additional sites in the Mazovian and Łódź voivodeships were included due to the distribution area of the scattered common mistletoe (V. album subsp. austriacum). Plant fragments of symptomatic mistletoe showing signs of fungal infection were collected from a total of 75 sites in 17 districts in four voivodeships. The amount of material collected varied from region to region: 55 samples from Podlaskie Voivodeship, 16 from Warmian–Masurian Voivodeship, 3 from Lodzkie and 1 from Mazovian. The distribution of sample collection points and the specific identified fungal taxa on the plots are shown in Figure 3.

The collection areas included natural habitats such as forest districts (FDs), urban parks, areas within the Natura 2000 network, ecological sites and buffer zones of national parks, as well as human-altered environments such as urban areas, roadsides, cemeteries and suburban woodlands.

2.2. Sampling Techniques

The fieldwork included both systematic and targeted searches for V. album. We conducted the fieldwork using the transect method, where transects of 100 m in length were established at each site to systematically search for mistletoe-infected trees. In addition, targeted searches were carried out in areas with high V. album infestations. We assessed mistletoe health on infected trees and sampled mistletoe plants that showed visible symptoms of fungal infection, such as leaf necrosis, stem cankers, deformation of plant parts or discolouration (Figure 4). The samples were collected from the stems, leaves, fruits or flowers of V. album. Each site was recorded with an assigned number, collection date, host tree species, environmental context and precise coordinates determined via GPS or compartment numbers from the Forest Data Bank (for forested areas).

2.3. Methodology for Sample Preparation and Fungal Identification

Taxonomic identifications were made using fungal identification keys, including:

• Illustrated Genera of Ascomycetes Volume 1 [30];
• The Genera of Hyphomycetes [35];
• A Revision of the Species Described in Phyllosticta [31];
• British Stem- and Leaf-Fungi (Coelomycetes) Volume 1: Sphaeropsidales [33].

Due to widespread occurrence of the investigated fungi, microscopic work and available keys for their identification were sufficient to analyse the abovementioned fungi. The necessary measurements of the fungal fruiting bodies were made and more than a hundred photographs were taken (Figure 5), which are included in the Supplementary Materials. This approach saved us the tedious phase of isolating and purifying fungal cultures to obtain pure cultures, which are superfluous for species identification. However, we are planning to carry out genetic analyses for the direct identification of the abovementioned fungi in plant material in the future. Obtaining pure cultures of mistletoe pathogens will be necessary in the future to test the suitability of these fungi for the production of biopreparations to reduce mistletoe populations.

Fungal occurrence data were analysed to evaluate host associations. All necessary permits for sampling were obtained from the local environmental authorities.

2.4. Molecular Identification of Fungal Pathogens

ITS sequencing was conducted to confirm the presence of fungal pathogens associated with V. album. The analysis aimed to validate pathogen identification based on macro- and microscopic morphological characteristics by examining DNA extracts from both healthy and infected V. album plants.

Three leaves from healthy V. album plants and three leaves from V. album plants infected with specific fungal pathogens were randomly selected for ITS sequencing. Genomic DNA was isolated using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Each sample was extracted in 50 $\mu$L of dilution buffer and subsequently stored at −20 °C until further analysis.

3. Results

3.1. Identified Fungal Species on V. album and Their Morphological Characterisation

Mycological and morphological analyses were performed on 75 samples of V. album collected during fieldwork, including both living and dead plants, from different regions of northeastern Poland to assess the diversity of fungal species associated with V. album. These samples showed symptoms of fungal infection on different plant parts such as leaves, stems, branches and fruits (Figure 1). Additionally, we documented uninfected V. album plants, classifying subspecies and morphological structures (Figure 2) and recorded damage and diseases caused by fungal phytopathogens (Figure 4). The analysis confirmed the presence of four fungal species on V. album, which were identified as either parasites or saprotrophs (Figure 6). These species are listed below.

• Sphaeropsis visci (Alb. & Schwein.) Sacc.;
• Plenodomus visci (Moesz) Gruyter, Aveskamp & Verkley;
• Cylindrodendrum hubeiense (W.Y. Zhuang, Nong & J. Luo) L. Lombard & Crous;
• Septoria krystynae Ruszk.-Mich.

3.1.1. Sphaeropsis visci

This species occurs as a saprotroph or parasite on the leaves, twigs, flowers and fruits of V. album. It produces numerous black pycnidia (asexual, spore-bearing structures) that are 300–400 $\mu$m in size and cause yellowing and death of the infected parts. The spores are ovoid to ovoid-elongated and measure 40–54 $\mu$m × 18–26 $\mu$m. This species has already been described several times in the scientific literature as occurring in Poland [16,36]. We present examples of microscopic images of S. visci in Figure 4A and Figure 5A.

3.1.2. Plenodomus visci

This species occurs as a saprotroph or parasite on the leaves and twigs of V. album and forms numerous tufts of black pycnidia with diameters of 200 to 250 $\mu$m. These pycnidial bundles cause localised dark orange or brown necroses on V. album. The spores are ovoid-elongated and measure 4–5 $\mu$m × 1.5–2.5 $\mu$m. To our knowledge, this taxon has not yet been recorded in Poland [16,33]. We present examples of macro and microscopic images of P. visci in Figure 4B and Figure 5B.

3.1.3. Cylindrodendrum hubeiense

This fungal taxon was identified on the leaves of V. album. Its development is characterised by the appearance of a white mycelial mat, which leads to the death of the infected leaf parts. The spores are cylindrical to cylindrical-elliptical and measure 8–18 $\mu$m × 1.8–2.7 $\mu$m. The spores can contain up to three septa (partitions) or be completely aseptic. This species has not yet been recorded in Poland [16,37]. We present an example of a microscopic image of C. hubeiense in Figure 5C.

3.1.4. Septoria krystynae

This fungus species forms subcircular, yellowish-white leaf spots with a slightly concave surface and a reddish-brown, blistered edge. The conidiomata are primarily epiphyllous, i.e., they are immersed in the leaf tissue and are often located under the stomatal cells. They are single to aggregated, pycnidial structures with a diameter of 150–180 $\mu$m, brown in colour and with a central ostiole. The conidiomatal wall consists of three to five layers of medium brown textura angularis. The conidiogenous cells line the inner cavity and are holoblastic, hyaline, smooth-walled and amphullate, with a size of 10–12 $\mu$m × 3–3.5 $\mu$m. The conidia are solitary, straight or irregularly curved, 1–3-septate, hyaline, smooth, guttulate, subcylindrical to narrowly ellipsoidal and measure 14–20 $\mu$m × 2–2.5 $\mu$m. This species, newly described in 2024, has only three confirmed occurrences in the scientific literature. We present an example of a microscopic image of S. krystynae in Figure 5D.

3.2. Molecular Identification

ITS sequencing was conducted to confirm the presence of pathogens by analyzing DNA extracts from V. album, which were morphologically identified as infected with specific fungal pathogens. All ITS sequences of fungal isolates have been deposited in GenBank, with the corresponding accession numbers provided in

Table 2. Morphological identification of isolates, their closest genetic matches, and corresponding GenBank accession numbers.

Morphological Identification	Isolate	GenBank Acc. No.	Closest Match
			GenBank Acc. No.	Identity (%)
Sphaeropsis visci	1	PQ661328	OP699910.1	99.16
Sphaeropsis visci	2	PQ661329	OP699910.1	99.33
Sphaeropsis visci	3	PQ661330	OP699910.1	99.15
Plenodomus visci	1	PQ661331	MT223832.1	99.12
Plenodomus visci	2	PQ661332	MT223832.1	98.58
Plenodomus visci	3	PQ661333	MT223832.1	98.94
Cylindrodendrum hubeiense	1	PQ661334	MZ078580.1	99.30
Cylindrodendrum hubeiense	2	PQ661335	MZ078580.1	99.13
Cylindrodendrum hubeiense	3	PQ661336	MZ078580.1	99.29
Septoria krystynae	1	PQ661337	OP295001.1	99.16
Septoria krystynae	2	PQ661338	OP295001.1	98.08
Septoria krystynae	3	PQ661339	OP295001.1	98.95

, which also includes the morphological identification, isolate numbers, closest matches, and identity percentages relative to the closest match for each fungal species. The analysis includes three sequencing replicates for each of the following fungal species: Sphaeropsis visci, Plenodomus visci, Cylindrodendrum hubeiense and Septoria krystynae.

The results of ITS sequencing for the four fungal species confirmed the morphological identification performed in the preliminary analysis. This agreement between molecular and morphological methods confirms the accuracy of the initial morphological assessment as a reliable approach for pathogen identification.

3.3. Geographical Distribution of Fungi on V. album

The

Table A1. Summary of the information on the collection sites of V. album samples and the fungal taxa identified on them.

Sample Number	Collection Date	Collection Region	Collection Site	Host Tree Species	Identified Fungus Species
VA1	4 July 2019	Białowieża Primeval Forest	Compartment 385B	Betula pendula	S. visci, P. visci
VA2	3 May 2019	Forest Unit Złoty Potok, Mrągowo FD	Compartment 264	Tilia cordata	S. visci, P. visci
VA3	5 May 2020	Forest Unit Kromnice, Mrągowo FD	Compartment 01	Betula pendula	S. visci
VA4	23 July 2020	City of Hajnówka	52°${44}^{\prime }$27.6″ N 23°${35}^{\prime }$01.6″ E	Betula pendula	S. visci
VA5	24 July 2020	Białowieża Primeval Forest	Compartment 365C	Betula pendula	S. visci, P. visci
VA6	23 July 2020	City of Hajnówka	52°${44}^{\prime }$25.6″ N 23°${34}^{\prime }$52.5″ E	Betula pendula	S. visci, P. visci
VA7	24 July 2020	Białowieża Primeval Forest	Compartment 445A	Betula pendula	S. visci
VA8	6 May 2020	City of Ruciane Nida	53°${39}^{\prime }$23.9″ N 21°${32}^{\prime }$49.9″ E	Betula pendula	S. visci
VA9	24 July 2020	Dunajek, Świętajno County	54°${01}^{\prime }$53.6″ N 22°${17}^{\prime }$58.1″ E	Acer platanoides	S. visci
VA10	24 July 2020	Białobrzegi, near the Netta River	53°${48}^{\prime }$19.4″ N 22°${58}^{\prime }$04.3″ E	Salix alba	S. visci
VA11	6 May 2020	City of Ruciane Nida	53°${39}^{\prime }$00.8″ N 21°${33}^{\prime }$43.3″ E	Betula pendula	S. visci
VA12	29 June 2020	City of Choroszcz	53°${09}^{\prime }$05.7″ N 22°${59}^{\prime }$02.3″ E	Tilia cordata	S. visci
VA13	27 June 2019	Forest Unit Borowo, Mrągowo FD	Compartment 120	Betula pendula	P. visci
VA14	24 July 2020	City of Suwałki	54°${06}^{\prime }$15.5″ N 22°${53}^{\prime }$31.2″ E	Tilia cordata	S. visci
VA15	24 July 2020	Sulimy, Giżycko County	54°${02}^{\prime }$15.9″ N 21°${49}^{\prime }$29.8″ E	Tilia cordata	S. visci
VA16	17 June 2020	City of Bielsk Podlaski	52°${46}^{\prime }$29.2″ N 23°${11}^{\prime }$41.3″ E	Betula pendula	S. visci
VA17	3 August 2020	Kuraszewo, Czyże County	52°${49}^{\prime }$04.3" N 23°${28}^{\prime }$40.9" E	Populus nigra	Unidentified Species
VA18	23 July 2020	Białowieża Primeval Forest	Compartment 355C	Betula pendula	S. visci
VA19	24 July 2020	Filipów, near the Rospuda River	54°${10}^{\prime }$46.0″ N 22°${37}^{\prime }$46.5″ E	Salix alba	S. visci
VA20	24 July 2020	City of Suwałki	54°${06}^{\prime }$44.2″ N 22°${55}^{\prime }$43.7″ E	Tilia cordata	S. visci
VA21	20 July 2020	Dubiny, Hajnówka County	52°${46}^{\prime }$14.4″ N 23°${35}^{\prime }$11.2″ E	Betula pendula	S. visci
VA22	11 May 2020	Sorkwity, Mrągowo County	53°${50}^{\prime }$41.5″ N 21°${08}^{\prime }$33.9″ E	Betula pendula	S. visci, C. hubeiense
VA23	5 May 2020	Karwie, Mrągowo County	53°${49}^{\prime }$57.5″ N 21°${16}^{\prime }$02.9″ E	Betula pendula	S. visci
VA24	17 August 2020	City of Giżycko	54°${02}^{\prime }$37.1″ N 21°${45}^{\prime }$31.2″ E	Populus alba	S. visci
VA25	20 July 2020	City of Hajnówka	52°${44}^{\prime }$27.3″ N 23°${33}^{\prime }$53.3″ E	Malus domestica	S. visci, C. hubeiense
VA26	20 July 2020	City of Hajnówka	52°${44}^{\prime }$40.5″ N 23°${34}^{\prime }$52.5″ E	Salix alba	S. visci
VA27	21 August 2021	City of Mrągowo	53°${52}^{\prime }$04.8″ N 21°${17}^{\prime }$06.9″ E	Sorbus aucuparia	S. visci
VA28	28 August 2021	City of Mińsk Mazowiecki	52°${10}^{\prime }$44.8″ N 21°${36}^{\prime }$05.0″ E	Tilia cordata	S. visci
VA29	21 August 2021	City of Mrągowo	53°${52}^{\prime }$02.7″ N 21°${16}^{\prime }$58.6″ E	Tilia cordata	S. visci
VA30	14 July 2021	Choroszcz County	53°${09}^{\prime }$21.8″ N 23°${00}^{\prime }$54.4″ E	Salix alba	S. visci
VA31	14 July 2021	Rutki County	53°${04}^{\prime }$52.9″ N 22°${18}^{\prime }$37.8″ E	Populus alba	S. visci
VA32	14 July 2021	Rutki County	53°${04}^{\prime }$56.2″ N 22°${19}^{\prime }$31.6″ E	Populus alba	S. visci
VA33	14 July 2021	Gać Village, Łomża County	53°${05}^{\prime }$03.3″ N 22°${13}^{\prime }$31.5″ E	Populus nigra	S. visci
VA34	14 July 2021	Gać Village, Łomża County	53°${05}^{\prime }$01.8″ N 22°${13}^{\prime }$21.1″ E	Populus nigra	S. visci
VA35	15 July 2021	Białowieża Primeval Forest	Compartment 355A	Carpinus betulus	P. visci
VA36	15 July 2021	Białowieża Primeval Forest	Compartment 329E	Tilia cordata	S. visci
VA37	15 July 2021	Białowieża Primeval Forest	Compartment 355B	Tilia cordata	S. visci
VA38	15 July 2021	Białowieża Primeval Forest	Compartment 329E	Carpinus betulus	S. visci
VA39	15 July 2021	Białowieża Primeval Forest	Compartment 301A	Tilia cordata	S. visci, C. hubeiense
VA40	15 March 2021	Kolno County	53°${25}^{\prime }$49.5″ N 21°${51}^{\prime }$14.2″ E	Betula pubescens	Unidentified Species
VA41	15 March 2021	Kolno County	53°${25}^{\prime }$58.5″ N 21°${51}^{\prime }$15.7″ E	Betula pubescens	S. visci, P. visci
VA42	5 May 2020	Karwie, Mrągowo County	53°${49}^{\prime }$51.4″ N 21°${16}^{\prime }$00.9″ E	Populus alba	S. visci
VA43	15 March 2021	Kolno County	53°${26}^{\prime }$04.9″ N 21°${50}^{\prime }$54.9″ E	Betula pubescens	S. visci, C. hubeiense
VA44	30 June 2019	Sorkwity, Mrągowo County	53°${50}^{\prime }$34.2″ N 21°${09}^{\prime }$03.5″ E	Populus nigra	P. visci
VA45	30 June 2019	Sorkwity, Mrągowo County	53°${50}^{\prime }$40.4″ N 21°${08}^{\prime }$32.1″ E	Populus nigra	S. visci
VA46	5 March 2021	Białowieża Primeval Forest	Compartment 419D	Carpinus betulus	S. visci, P. visci
VA47	5 March 2021	Białowieża Primeval Forest	Compartment 445A	Ulmus minor	S. visci, C. hubeiense
VA48	5 March 2021	Białowieża Primeval Forest	Compartment 445A	Carpinus betulus	S. visci
VA49	14 June 2021	Bielsk Podlaski County	52°${42}^{\prime }$16.2″ N 23°${07}^{\prime }$55.3″ E	Betula pendula	P. visci
VA50	14 June 2021	Bielsk Podlaski County	52°${44}^{\prime }$27.3″ N 23°${09}^{\prime }$56.6″ E	Populus nigra	S. visci
VA51	14 June 2021	Bielsk Podlaski County	52°${44}^{\prime }$44.8″ N 23°${10}^{\prime }$06.9″ E	Betula pendula	P. visci
VA52	14 June 2021	Boćki County	52°${39}^{\prime }$20.1″ N 23°${03}^{\prime }$22.9″ E	Salix alba	P. visci
VA53	14 June 2021	Boćki County	52°${38}^{\prime }$12.5″ N 23°${01}^{\prime }$26.5″ E	Populus alba	S. visci
VA54	14 June 2021	Boćki County	52°${37}^{\prime }$12.8″ N 22°${59}^{\prime }$59.4″ E	Robinia pseudoacacia	S. visci
VA55	19 June 2021	Dziadkowice County	52°${35}^{\prime }$19.0″ N 22°${56}^{\prime }$36.4″ E	Betula pendula	S. visci
VA56	19 June 2021	Dziadkowice Town	52°${33}^{\prime }$42.6″ N 22°${54}^{\prime }$40.3″ E	Fraxinus pennsylvanica	C. hubeiense
VA57	19 June 2021	Siemiatycze County	52°${28}^{\prime }$31.3″ N 22°${52}^{\prime }$35.2″ E	Betula pendula	S. visci
VA58	20 June 2021	Czartajew Town	52°${27}^{\prime }$28.9″ N 22°${51}^{\prime }$31.9″ E	Sorbus aucuparia	S. visci
VA59	20 June 2021	Siemiatycze Town	52°${23}^{\prime }$39.4″ N 22°${52}^{\prime }$11.8″ E	Populus alba	S. visci
VA60	20 June 2021	Siemiatycze County	52°${23}^{\prime }$20.3″ N 22°${52}^{\prime }$19.9″ E	Populus alba	Unidentified Species
VA61	23 June 2021	Bielsk Podlaski County	52°${44}^{\prime }$23.3″ N 23°${12}^{\prime }$33.5″ E	Populus nigra	S. visci
VA62	23 June 2021	Bielsk Podlaski County	52°${44}^{\prime }$00.7″ N 23°${12}^{\prime }$44.9″ E	Tilia cordata	S. visci
VA63	1 July 2021	Kleszczele County	52°${36}^{\prime }$01.6″ N 23°${19}^{\prime }$38.4″ E	Betula pendula	S. visci, P. visci
VA64	1 July 2021	Kleszczele County	52°${35}^{\prime }$03.2″ N 23°${19}^{\prime }$46.1″ E	Populus alba	S. visci
VA65	11 August 2021	City of Białystok	53°${07}^{\prime }$04.4″ N 23°${09}^{\prime }$47.8″ E	Fraxinus pennsylvanica	S. visci, C. hubeiense
VA66	11 August 2021	City of Białystok	53°${07}^{\prime }$00.2″ N 23°${08}^{\prime }$22.3″ E	Populus alba	S. visci, C. hubeiense
VA67	11 August 2021	City of Białystok	53°${06}^{\prime }$52.9″ N 23°${07}^{\prime }$59.4″ E	Acer saccharinum	S. visci
VA68	11 August 2021	City of Białystok	53°${07}^{\prime }$25.0″ N 23°${09}^{\prime }$47.7″ E	Acer saccharinum	S. visci
VA69	2 July 2021	Tomaszów Mazowiecki County	51°${31}^{\prime }$39.1″ N 19°${57}^{\prime }$34.4″ E	Pinus sylvestris	P. visci
VA70	2 July 2021	Tomaszów Mazowiecki County	51°${31}^{\prime }$50.7″ N 19°${59}^{\prime }$21.9″ E	Pinus sylvestris	S. visci
VA71	2 July 2021	Tomaszów Mazowiecki County	51°${30}^{\prime }$55.4″ N  20°00’01.8″ E	Pinus sylvestris	P. visci
VA72	20 September 2021	City of Ełk	53°${49}^{\prime }$42.3″ N 22°${21}^{\prime }$39.3″ E	Betula pendula	S. visci
VA73	6 August 2022	City of Mońki	53°${24}^{\prime }$15.2″ N 22°${47}^{\prime }$51.1″ E	Betula pendula	S. visci
VA74	20 May 2019	Forest Unit Topiło, Białowieża FD	Compartment 452C	Betula pendula	S. krystynae
VA75	3 June 2019	Białowieża Forest	Compartment 476A	Tilia cordata	S. krystynae

in Appendix A summarises the data for each sample, including the assigned collection number, the date of acquisition and the region where the samples were collected. The collection location is indicated by geographic coordinates or compartment numbers (in the case of forest areas where satellite coverage did not allow accurate use of Google Maps). The table also includes the host tree species and the fungal species identified on the sample.

The distribution of locations where individual fungal taxa were collected and identified is shown on contour maps of Poland, divided into counties (Figure 3). The species Sphaeropsis visci was collected at 60 locations, while the taxon Plenodomus visci was found at 14 locations. Cylindrodendrum hubeiense occurred at eight sites. The fungus Septoria krystynae was detected at two sites, both located in the Białowieża Forest (Figure 7).

3.4. Mistletoe Host Tree Associations of Fungal Taxa

Analysis of the results included the identification of host tree species for Viscum album on which a specific fungal taxon was observed. Sphaeropsis visci had the broadest host range and occurred on V. album, parasitizing a total of 15 different tree taxa. Plenodomus visci and Cylindrodendrum hubeiense were associated with V. album growing on seven different tree species. Additionally, Septoria krystynae was observed on V. album parasitizing two different tree species.

4. Discussion

In the last 40 years, mistletoe, especially V. album subsp. austriacum, has become a serious threat in managed pine forests, especially under climatic conditions with hot summers and mild winters that weaken drought-stricken stands [5]. Pine mistletoe has greater virulence and a higher mortality rate among hosts than common mistletoe, which threatens forest ecosystems and increases susceptibility to pests such as the pine bark beetle, which accelerates tree mortality [7,18,24].

In Poland, the control of V. album is mainly based on sanitary pruning and removal of heavily infested trees, which are costly and time-consuming methods in large forests [20,38]. Research on mistletoe-associated fungi is limited, and only 12 fungal taxa have been identified worldwide and only two (Sphaeropsis visci and Phyllosticta visci) have been documented in Poland [16,19]. Current mechanical control methods for mistletoe are often inefficient and expensive [18,20], highlighting the need for alternative strategies. The use of parasitic ascomycetes offers a promising biological control option that could help to reduce mistletoe populations in the context of integrated pest management (IPM). Our research on pathogens affecting different parts of V. album could make an important contribution to the development of an effective biopreparation to limit the spread of mistletoe V. album subsp. austriacum in forest ecosystems. These findings could support a coordinated approach to more efficient mistletoe management.

Only a few studies have investigated the associations of fungi with V. album subsp. austriacum compared to their host (Pinus sylvestris) and have shown that parasitic fungi could potentially control mistletoe populations. However, the effectiveness of fungi in reducing mistletoe on a global scale is still largely unexplored [39].

In our study, the fungi present on the mistletoe leaves were first identified using classical methods, including macroscopic and microscopic examination. These identifications were then confirmed by ITS sequencing. All fungal species examined in this study are plant pathogens common in Polish pine forests, with most appearing to be exclusively parasitic on V. album based on current research; these include S. visci, P. visci and S. krystnae. Other discovered species such as C. hubeiense show low pathogenicity towards living plants, including mistletoe, and typically develop on dead organic material.

The presence of these pathogens leads to necrosis of the mistletoe tissue, which reduces the reproductive potential of the mistletoe and in some cases leads to the death of the host plant. The most common species, Sphaeropsis visci, was found in 83.56% of infested sites and affected all parts of the mistletoe plant. The frequency of S. visci was highest on Betula, Populus and Tilia, suggesting that the type of host tree influences the fungal infection rate, possibly due to the chemical composition of the mistletoe, which varies depending on the host tree [40].

Two fungal species, Plenodomus visci and Cylindrodendrum hubeiense, which had not been previously reported in Poland, were identified in this study.

Sphaeropsis visci was the most widespread and infested all parts of the mistletoe plant. Its prevalence on Betula (32.8%), Populus poplar (21.3%) and Tilia (18%) suggests that the host species plays an important role in susceptibility to fungal infection, which may be influenced by the chemical composition of the host [40]. Plenodomus visci, which occurs mainly on birch (53.3%), shows a similar host preference, suggesting that mistletoes parasitising on birch are particularly susceptible to infection by ascomycetous fungi from the class Dothideomycetes.

In contrast, Cylindrodendrum hubeiense exhibited no significant host specificity, as it was identified on mistletoe parasitizing birch. This observation highlights the vulnerability of birch-associated mistletoe to fungal infections.

Most parasitic ascomycetes reproduce asexually, which complicates the identification of their anamorphic (asexual) and teleomorphic (sexual) stages, each of which can affect the host differently [41,42].

5. Conclusions

The conducted research significantly advances the understanding of national mycobiota and fungi associated with Viscum album (V. album). Analysis of 75 V. album samples with fungal infection symptoms from different regions revealed four parasitic or saprotrophic species, illustrating the diversity of mistletoe-associated fungi.

Among these, Plenodomus visci and Cylindrodendrum hubeiense were documented for the first time in Poland, extending the known distribution of these fungi on mistletoe.

The distribution area of Sphaeropsis visci has been considerably extended with 61 newly identified localities, which represents a significant increase compared to the few previously recorded occurrences worldwide. Two new localities were also documented for the recently described Septoria krystynae, providing further insights into its distribution and ecology. Sphaeropsis visci was the most frequently observed fungal species on mistletoe, while other species such as Septoria krystynae were found much less frequently, indicating the dominance of S. visci as a mistletoe-associated fungus.

A possible correlation exists between certain fungal taxa and the host tree species of V. album. In particular, Sphaeropsis visci and Plenodomus visci were predominantly associated with mistletoe on birch (Betula spp.). These results suggest that the host tree species may influence the composition of the fungal community on mistletoe.

In addition, the species identified in this study, in particular Sphaeropsis visci, may provide opportunities for future biocontrol applications aimed at containing the spread of V. album in sensitive ecosystems such as pine stands. This research thus lays the foundation for future studies focusing on biosecurity measures and the sustainable management of semi-parasitic mistletoe populations.