Mycobiota Associated with the Vascular Wilt of Poplar

In 2017, a 560-ha area of hybrid poplar plantation in northern Poland showed symptoms of tree decline. The leaves appeared smaller, yellow-brown, and were shed prematurely. Twigs and smaller branches died without distinct cankers. Trunks decayed from the base. The phloem and xylem showed brown necrosis. Ten percent of the trees died 1–2 months after the first appearance of the symptoms. None of these symptoms were typical for known poplar diseases. The trees’ mycobiota were analysed using Illumina sequencing. A total of 69 467 and 70 218 operational taxonomic units (OTUs) were obtained from the soil and wood. Blastocladiomycota and Chytridiomycota occurred only in the soil, with very low frequencies (0.005% and 0.008%). Two taxa of Glomeromycota, with frequencies of 0.001%, occurred in the wood. In the soil and wood, the frequencies of Zygomycota were 3.631% and 0.006%, the frequencies of Ascomycota were 45.299% and 68.697%, and the frequencies of Basidiomycota were 4.119% and 2.076%. At least 400 taxa of fungi were present. The identifiable Zygomycota, Ascomycota, and Basidiomycota were represented by at least 18, 263 and 81 taxa, respectively. Many fungi were common to the soil and wood, but 160 taxa occurred only in soil and 73 occurred only in wood. The root pathogens included species of Oomycota. The vascular and parenchymal pathogens included species of Ascomycota and of Basidiomycota. The initial endophytic character of the fungi is emphasized. Soil, and possibly planting material, may be the sources of the pathogen inoculum, and climate warming is likely to be a predisposing factor. A water deficit may increase the trees’ susceptibility. The epidemiology of poplar vascular wilt reminds grapevine trunk diseases (GTD), including esca, black foot disease and Petri disease.


Introduction
Populus is a genus of deciduous trees in the family Salicaceae, native to most of the Northern Hemisphere. They are among the fastest-growing trees, and the most efficient in terms of sustainability. Poplar is significant because of: (i) its rapid production of wood (in Europe, 1 m 3 of lumber can be produced on average in 15 years, six times faster than with oak); (ii) its very versatile wood, with an excellent ratio between specific weight and mechanical features, making it suitable for furniture, plywood and the paper industry; (iii) its excellent capacity for purifying the air by capturing CO 2 and storing it in the biomass (1 ha can capture 11 t CO 2 /year); (iv) its capacity for purifying water while acting as a green filter, absorbing nitrates and sediments; (v) its potential for biofuel production using the coppicing method; (vi) the possibility for its cultivation on abandoned and degraded land, thus optimizing land use.
Poplar is an important source of wood for pulp and paper products, but mostly paper, for which worldwide production reaches 420 Mt, including 5 Mt in Poland [1]. Its wood is also suitable for use as a renewable energy source. The development of renewable sources for energy purposes has been substantially supported and promoted by a European Union Directive. Poland is obliged to obtain at least 30% of its energy from renewable sources by 2030 (Directive (EU) 2018/2001). Wood that is suitable for renewable energy includes that derived from trees grown in short-and medium-rotation plantations, often on agricultural land or non-forested areas. Plantations based on varieties of Acacia and Eucalyptus have been particularly effective in tropical countries with favourable climate and soil conditions for faster growth; Eucalyptus has produced 25 m 3 of wood per ha annually, compared with 7-8 m 3 in the temperate climate zone (1). Plantations of fast-growing trees are now also being established in the temperate zone. The most promising genus in Poland is poplar (Populus spp.), with plantations usually in short-(up to 10 years) or medium-rotation (up to 15-25 years) coppice systems [2][3][4].
Poplars are frequently attacked by microorganisms that cause discolorations, necrosis, depressions, deformations (thickening of the trunk and branches, the abnormal proliferation of the underlying phloem, the formation of the corky ridges or woody galls). Stresses predispose trees to infection by phytopathogens. Attacks on the trunk and branches of younger trees often kill the main shoot.
The In 2017, a 560 ha plantation of hybrid poplar (P. deltoides × P. nigra) in northern Poland showed symptoms of tree decline. The leaves of the diseased trees appeared smaller, turned yellow-brown, and were shed prematurely. Twigs and smaller branches died without definite cankers. The bark of the entire trunk was sunken and discolored, often loosened and split. It often fell off, exposing wet wood. The trunks decayed from the base. The phloem showed brown necrosis. Ten percent of the trees died in 1-2 months (in June) after the first appearance of the symptoms. None of the observed symptoms were typical for known poplar diseases.
The objectives of the study on the structure of the fungal communities present in the rotten wood of poplar trunks and in the soil were to: (i) determine the abundance and diversity of pathogens and other fungi; (ii) identify interactions among fungi that may contribute to the disease progress; (iii) assess associations between the disease and global warming, with consequences for host and pathogen physiology, reproduction, survival, spatial and temporal distribution, resource availability and competition.

Site and Sampling
The study was carried out in the Łoża, Czarne District, Człuchów County, Pomeranian Voivodeship, northern Poland (53 • 41 29" N 17 • 04 19" E), in a 560 ha plantation of 5-6-yearold hybrid poplar (P. deltoides × P. nigra, cultivar AF2, from Italy) showing symptoms of crown decline, trunk-base decay (520 ha) and tree death (40 ha) (Figures 1 and 2). The plantation was so intensively affected that the inclusion of a control (healthy plantation) from the same area with the same conditions of climate and soil was impossible. The trees were grown at a density of 425 trees/ha (4 m × 4m spacing), and had a mean diameter of 9-10 cm at breast height. The post-agricultural soil was sandy loam, consisting of sand (60%), silt (20%) and clay (20%), with a low humus level. The former crop was rye (Secale cereale L.). The average temperature is 7.9 • C and the rainfall is 680 mm.
Five wood cores; 10 cm long and 3 cm in diam., each including bark, phloem and xylem, were sampled from the bases of the necrotic trunks of five symptomatic trees, 0 cm and 50 cm above the ground, with a Pressler borer. The core samples were surfacesterilized and ground to sawdust with a cordless SPARKY BUR2 15E drill. Additionally, five subsamples of soil were taken as cylindrical cores, 10 cm long and 5 cm in diam., from the surroundings of roots of five symptomatic trees. They were placed in sterile glass containers and refrigerated for 48 h.

DNA Extraction, Amplification and Illumina Sequencing
Five samples of sawdust were prepared from five wood cores in the SPEXTM Sam-plePrepTM Freezer/MillTM cryogenic mill. The wood's genomic DNA was extracted from each of five 30 mg heavy sawdust samples using a Plant Genomic DNA Purification Kit (Thermo Scientific, Carlsbad, California, USA). The soil's genomic DNA was extracted from each 300 mg soil subsample using a Power SoilM DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA).
The PCR reaction mixture consisted of 12.5 µL of 2 × Mix PCR (A & A Biotechnology, Gdańsk, Poland), 0.2 µM of each primer, 1.5 µL purified and diluted DNA, and 10.6 µL water. The DNA amplification was performed under the following conditions: denaturation at 94 • C for 5 min followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 30 s, elongation at 72 • C for 30 s, and a final elongation at 72 • C for 7 min. The visualization of 5-µL amplicons was performed in 1.0% agarose gel dyed with Midori Green Advance DNA (Genetics). The pooled PCR products were purified using a MinElute PCR Purification Kit (Qiagen, Hilden, Germany). The concentration of PCR products was quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and an equimolar mix of PCR products from each sample was prepared. The amplicons were sequenced using the Illumina system in the Genomic Laboratory, DNA Research Center, Rubież 46, Poznań, Poland.

Bioinformatics Analysis
A table of Operational Taxonomic Units (OTUs) was prepared by PIPITS, version 1.2.0 [8]. The read-pairs were joined with PEAR, version 0.9.6 [9], filtered with a quality threshold of q = 30 by FASTX-toolkit, version 0.0.13 (http:hannonlab.cshl.edu/fastx_toolkit/index.html, accessed on 26 April 2012) converted to the Fasta format, and merged into a single file. The prepared sequences were de-replicated, and subregions of ITS were selected with the use of ITSx, version 1.0.11 [10]. Unique sequences and those shorter than 100 bp were removed. The remaining sequences were clustered with 97% sequence identity. The resulting representative sequences for each cluster were subjected to chimera detection and removal using the UNITE UCHIME reference dataset, version 6.0 (https://unite.ut.ee/index.php (accessed on 26 April 2012)). The input sequences were then mapped onto the representative sequences, and taxonomy was assigned using RDP Classifier, version 2.10.2 [11] against the UNITE fungal ITS reference database, version 11.2 [12]. This process resulted in the creation of a table of OTUs. The sequences were identified by comparison with reference sequences from the National Center for Biotechnology Information (NCBI) database.
The abundance of fungi was defined as the average number of OTUs from five subsamples. The frequency of an individual taxon was defined as the percentage (%) of OTUs in the total number of OTUs. The similarity and relationships between the fungal communities from the soil and wood is shown by a heat map.

Statistical Analyses
The differences in the abundance of microfungi in the soil and wood were analysed with chi-squared tests (χ 2 ). The diversity between the communities of microfungi was compared with Margalef's diversity index (D Mg ), Shannon's diversity index (H), Simpson's diversity index (D), Shannon's evenness index (E) and Berger-Parker's index (d) [13].
The wood-decay Basidiomycota included the white rot fungi Armillaria mellea, Aurantiporus fissilis, Bjerkandera adusta, Chondrostereum purpureum, Hyphodontia pallidula and Peniophora, and the brown rot fungus Fomitopsis piniola. They occurred with frequencies of 0.028% in the soil and 0.62% in the wood.
The individual fungi often belonged to more than one trophic group. Margalef's index (D Mg ), Shannon's diversity index (H) and Simpson's diversity index (D) indicated greater diversity in the soil than in the wood. Shannon's evenness index (E) showed more evenness in the soil and, conversely, Berger-Parker's dominance index (d) showed more dominance of individual taxa in the wood.

Disease Characteristics
The vascular wilt of hybrid poplar appeared locally in Poland in 2017. The symptoms appeared suddenly in 5-6-year-old trees, and the disease developed very quickly, in less than 2 months. The activity of the pathogens, either already known or previously unrecognized, apparently circumvented any resistance in the host and led to the failure of the plantations. The disease was asymptomatic in its initial stage. Diagnosis at the final stage was not possible because of either: (i) the immaturity of the pathogen, or (ii) the absence of the distinctive morphological elements essential for the identification of causal fungi. Poplar diseases have a serious economic impact on wood production worldwide, and so the development of effective management strategies depends on the clear identification of the pathogens involved. The affected tissues were therefore analyzed by DNA sequencing.
The symptomatology of poplar wilt can be compared with that of some grapevine diseases, notably grapevine trunk diseases (GTD), including the esca and black foot diseases, and Petri disease [14,15]. Grapevine trunk disease symptoms include the sectorial and/or central necrosis of the trunk wood, brown streaking of the wood, cankers, and the discoloration and wilting of the foliage, which can occur suddenly [15,16]. Petri disease is a vascular disease associated with the decline and dieback of young grapevines. Typical black foot disease symptoms include stunted growth, reduced vigour, retarded or absent sprouting, sparse and chlorotic foliage with necrotic margins, wilting, dieback and death. Characteristic sunken necrotic root lesions with a reduction in root biomass and root hairs may also occur.

Pathogens in Diseased Poplar Trunk
According to EN 350:2016, poplar wood is non-durable, and some studies have shown that it is highly susceptible to wood-rotting fungi [32,33].
Basidiomycetous Burgoa anomala was found in pine wood and litter [65]. Some of the fungi are, surprisingly, often common on wood in water, including sea water. This group includes Didymosphaeria futilis, Halenospora varia, Halosphaeria quadriemis, Paraphoma radicina, Trichocladium and basidiomycetous Cystobasidium [66][67][68][69][70][71][72]. Fusarium spp. were not abundant in the poplar wood, but occurred frequently in the soil. Various Fusarium spp. have been reported in Poland as causing swellings, necrosis, bark fray, reddish-purple discoloration, and ultimately the characteristic cankers in poplar [73]. Fusarium avenaceum is perhaps the most important species, first reported in the 1950s on Euramerican poplar clones in France. Since then it has spread in Europe, from central and eastern areas with a continental climate to sub-mediterranean areas, and recently to Portugal, with its oceanic climate. Neocosmospora solani (=Fusarium solani (Mart.) Sacc. (found mostly on Aigeiros and Tacamahaca poplars and intersectional hybrids) seemed to be confined to North America until it was reported in Poland [74]. Species with sporadic occurrence and of limited importance include F. lateritium Nees, observed in France and in the USA on Populus trichocarpa Torr. and A. Gray, and F. sporotrichioides Sherb., observed in eastern Europe and central Italy on Populus × euramericana. Fusarium spp., constituting a threat to young trees. Colonized trunks are susceptible to breakage, and to attacks by other bark parasites which are also active during a plantation's early years. The symptoms are not immediately visible, and mostly take the form of the disorganization of the cortical tissues in part of the trunk.
Cytospora species are cosmopolitan, facultative parasites, and appear in tree stands subjected to some form or stress, with poor agronomic management or infected by other pathogens. Infection occurs in late autumn or winter, when the host is dormant, usually behaving as a distinctly secondary parasite. The initial symptoms include brown-blackish discolorations, necrosis, depressions in the bark and underlying wood, callus production and withering. Older, sturdier tissues may develop resistance to further invasion. The disease then appears as small brown depressions bounded by distinct calluses. In the advanced stage, the bark tissues may peel away to reveal underlying stained wood [75]. Cytospora ambiens, C. chrysosperma and C. nivea (Hoffm.) Sacc., which are usually present on/in poplar wood worldwide, with their highest incidence in central and southern Italy, eastern Europe, the Near East, northern India, southern Africa (mainly in plantations) and the west-central USA (especially in Colorado), were not detected in the diseased hybrid poplars.
Paraphoma is root-associated on Populus, although P. chrysanthemicola has so far been reported only from Juniperus, Malus and herbaceous plants [97,98].The fungus can infect the leaves of certain plant species and provoke disease [99]. On poplar, it caused foliar blight [100]. The fungus can also live benignly in asymptomatic plant tissues, and has been detected or isolated from the roots of healthy plants [101].
Phaeoisaria loranthacearum has so far been reported from twigs of Loranthus europaeus in Germany [102].
Phialophora species, found very abundantly, may include P. richardsiae, a serious pathogen implicated in the Petri disease of grapevine. The significance of other Phialophora spp. potentially occurring in the diseased poplar wood should also be emphasized. They are mostly saprotrophic and common in soil and wood, in which they cause soft rot. Growth at the hyphal tip and the secretion of lignolytic enzymes (pectinase, amylase, xylanase, cellulase and mannanase) causes widened cavities in sapwood and the degradation of the wood [103,104]. They can also cause cavities in the wood and plants via an erosion-type attack [105]. The degradation of Populus tremuloides wood has been known to affect sales of commercial aspen timber. The blue staining of wood by Phialophora has also been reported [106]. The fungus is psychrotolerant (able to grow at a low temperature).
The cosmopolitan Cenococcum geophilum, one of the most frequently encountered ectomycorrhizal fungi in nature, is well recognized for its extremely wide host and habitat range [123].
Fungi of the genera Alternaria, Epicoccum, Fusarium, Cladosporium, Penicillium and Trichoderma are highly robust and ubiquitous, with an almost global distribution, occurring in the Americas, Asia, and Europe [103]. Their spores have been found in a variety of habitats, predominantly in soil of various types and in sand, often in extreme conditions. Epicoccum can grow on leaves submerged in water, even at 0 • C; hyphal growth can resume within an hour of exposure to water [104,124].
Some fungi were recorded for the first time on wood, or have been found rarely on wood. Ascomycetous Neocatenulostroma germanicum is known from pine needles, and is known to cause needle blight on Pinus mugo Turra, P. nigra Arn. ssp. pallasiania and P. sylvestris L. in Lithuania, Poland and Ukraine [44,122], but has also occurred in the soil in Poland [125]. Sydowia polyspora is so far known from the foliage of Abies spp., Pinus spp. and Pseudotsuga menziesii (Mirb.), and litter [126]. Research suggests that some of these hosts can be primary inoculum sources when located near poplar plantations [127].
Some can occur at the extreme of their host ranges. Graphium basitruncatum has been isolated from wood and soil, even in the Solomon Islands and Japan, and from a leukemic patient [140,141]. Scytalidium lignicola and Sporothrix are recognized as saprotrophic opportunists of which the lifestyle can change from plant to human or animal pathogenicity.
Oomycota with eight species of Globisporangium, two species of Phytophthora and eight species of Pythium were mostly in the soil, and were not very common. Their contribution to the development of the disease cannot be excluded. All of them are plant pathogens, which cause root rot and damping off in a multitude of species. Phytophthora plurivora Jung and Burgess, followed by P. pini Leonian, P. polonica Belbahri, E. Moralejo, Calmin and Oszako, P. lacustris Brasier, Cacciola, Nechw., Jung and Bakonyi, P. cactorum (Lebert and Cohn) Schröt, and P. gonapodyides (Petersen) Buisman. were common in three declining and three healthy poplar plantations in Serbia [142].

Yeasts in Diseased Poplar Trunks
Yeasts are now identified and classified almost exclusively by DNA sequence analysis, which has resulted in the discovery of many new species and taxonomic revisions.
Filamentous fungi have a key role in the decomposition of plant material because of their ability to produce a wide range of extracellular enzymes that efficiently attack the recalcitrant lignocellulose matrix. However, the presence of yeasts during the different stages of wood breakdown highlights the ecological role of these microorganisms. Yeasts have been found to produce enzymes acting on cellulose, hemicelluloses and pectin [143]. They can therefore degrade plant material. They can also be transient fungi, using products released during decomposition by other organisms. Many yeast species found in live or decaying plant parts are associated with insects that also use these habitats as feeding or breeding sites.
The general opinion is that the most abundant yeast taxa associated with decayed wood are basidiomycetous (Agaricomycotina) and xylose-assimilating species. The present data do not support this thesis. Some ascomycetous yeasts were particularly abundant in the wood, where basidiomycetous yeasts were much less frequent.
Ascomycetous Aureobasidium pullulans and Candida spp., and basidiomycetous species of Apiotrichum, Cystofilobasidium, Naganishia, Saitozyma, Solicoccozyma, Tausonia, Tremella, Trichosporon and Vishniacozyma are frequently found in decaying plant material [143]. However, variations in their abundance and diversity reflect the environment, and also correlate with the natural abundance and distribution of basidiomycetous fungi in the study areas [144]; Apiotrichum, for example, was reported as being abundant in wood decayed by Armillaria. The abundance of ascomycetous yeasts in the wood resulted from the high frequency of Nakazawaea spp., especially N. populi, which was previously found in exudates of Populus species [145].

Mycorrhiza-Forming Fungi
Mycorrhiza-forming fungi were rare, especially in the soil. Basidiomycetous species occurred, surprisingly, more often in the wood, probably as: (i) facultative biotrophic encounters that either formed mycorrhizal structures or colonized the tissues as endophytes (i.e., grew within living plant tissues, without apparent infection, but not forming true mycorrhizae or causing any disease symptoms), or (ii) saprotrophs. Transition from saprotrophy to mycorrhizal status is common in fungal development [146], and other unexpected trophic conversions within the mycobiota may be possible.

The Endophytic State/Habit/Lifestyle of Fungi
As with grapevine diseases, it is assumed that the causal fungi are endophytic, living for a time asymptomatically in the plant. Then, at some point, in association with plant stress, they modify their behaviour and become pathogenic, which leads to the expression of disease symptoms [147]. As endophytes, they would often have key positive roles in plant function and fitness [148,149]. As parasites, they are cryptic, often opportunistic pathogens, which in special conditions induce disease [150]. Their virulence may be dictated by multi-partner interactions and environmental conditions. The most favoured conditions include: (i) the presence of very vigorous plants with succulent tissues; (ii) prolonged periods of damp and wet weather; (iii) free-standing water on the leaves; (iv) injuries such as pruning and leaf wounds; (v) the presence of senescent tissues, especially older, lower leaves; (vi) frost damage; and (vii) excessive crowding. Tissues are invaded by enzyme action, and roots and stems are gradually enveloped until the vessels are eventually reached, and wilting and desiccation occur. Different lifestyles and functions may occur depending on the situation. Phoma may at first be a plant-growth-promoting fungus [151].The lifestyles of Phaeoisaria and Pyrenochaetopsis depend on secreted peptidases [121,152]. Plectosphaerella (mostly P. populi) damages poplar stems [102,152], but simultaneously induces the formation of antifungal phenolic metabolites that protect poplar against foliar pathogens [153]. Some, such as Pyrenochaeta, are weak pathogens [154], but their adaptability to different climates allows them to infect many hosts and to survive in a broad range of pH, temperature and aeration conditions and soil types. Fungi such as Ilyonectria may survive in the roots of apparently healthy (asymptomatic) poplars, where they may suppress other fungal root pathogens and help maintain tree health [27,30]. These examples show that caution is necessary in classifying fungi according to function. There is no indication that other species, uncommon on Populus or so far not detected, might be pathogenic.

Interactions among Fungi
Trichoderma spp. occurred at a high natural frequency in the plantation soil. They are well known for their antagonistic activity, hyperparasitism and ability to induce defensive systems in plants to other microorganisms (specifically soil microorganisms). They are used in the biological control of several pathogens. Trichoderma harzianum Rifai and T. atroviride Karst. have shown promise in controlling Botryosphaeria dieback and esca disease in vineyards and other common trunk diseases [155]. Trichoderma significantly improved grapevine root growth and decreased the incidence of fungi involved in diseases when tested in vitro or in nurseries [24,156]. Grapevine defence systems have also been induced by Oomycota. The necrosis of root systems of vine cuttings was reduced by 50% after colonization by Pythium oligandrum [157][158][159]. Other biological control agents (Aureobasidium pullulans, Cladosporium herbarum, Fusarium lateritium and Rhodotorula rubra) have been reported to be effective against grapevine trunk disease pathogens, alone or in combination with fungicides, although some were tested only in vitro or in nurseries [160]. Arbuscular mycorrhizal fungi have been shown to increase the tolerance of grapevine rootstocks to Ilyonectria spp. [161]; Glomus intraradices was the most effective [162]. Aureobasidium pullulans, P. oligandrum, Trichoderma spp. and two species of Glomeromycota, present in the poplar plantation soil, may naturally decrease the incidence of pathogens involved in disease. Mortierella elongata, also detected, has been found to manipulate poplar defenses while promoting plant growth [30].This response was particularly beneficial because it was independent of cultivars.

Soil and Planting Material as the Source of the Inoculum
The soil origin was shown to be a significant factor affecting the composition of the fungal communities and networks in Populus [149,163].
The soil was here shown to be a natural source of many vascular and parenchymal pathogens found in the affected hybrid poplars, i.e., species of ascomycetous Alternaria, Cadophora, Cladosporium, Fusarium, Ilyonectria, Nectria, Neonectria, Neopyrenochaeta Ophiostomataceae, Phoma, Pyrenochaeta, Sporothrix, Thelonectria and Verticillium, and of basidiomycetous Armillaria and Entyloma. Their presence in the soil has been associated with their occurrence on plant debris and plant roots [164]. Soil was also the main source of pathogenic Oomycota (Aphanomyces, Elongisporangium, Globisporangium, Phytophthora and Pythium), which can, generally, cause extensive and devastating root rot. The destruction of roots can lead to minor or severe wilting caused by impeded root functioning or further biotrophic infections that can become necrotrophic in response to infection pressure or environmental stress. Oomycota tend to be very generalistic and non-specific, with a wide range of susceptible host roots, including poplar [142]. The wilt results from root degradation by Oomycota and a lack of oxygen, followed by disrupted water transport. A moist habitat and low pH in forest soils favour the growth, propagation, and dispersal of Oomycota spores. At optimal temperatures (28-30 • C), some species of Globisporangium grow very fast, i.e., 2.7 cm in 24-h.
Fungi such as Collophorina, Hyalodendriella and Hyaloscypha bicolor, which occurred sporadically in the soil, whilst being biotrophic parasites, may contribute to the final wilt [165,166].
The planting material may, however, already have been infected, either systemically from infected mother poplars or by contamination during the propagation process.

Colonization
As in grapevine disease, poplar wilt may be a complex disease in which symptoms result from the concomitant action of several factors.
The initial stage of the disease seems to be accomplished by highly specialized vascular fungi in the plant's phloem. Their presence in the soil suggests that the infection can be soil-borne. Hyphae from established mycelia, and germ tubes developing from spores, perceive signals from root exudates. The hyphae secrete cell-wall-degrading enzymes and enter roots through wounds, at branching points, or directly through root tips. The mycelium spreads between root cortex cells to reach phloem and xylem vessels, from which the fungus travels as conidia in the sap stream, mostly upwards. The phloem and xylem become obstructed by mycelium and spores, and by plant-produced gels, gums and tyloses. Water transport to the leaves fails, and the plant wilts and dies. The fungus then invades all of the plant tissues and obtains nutrition by decomposing them. The response to the degradation of hemicellulose or lignin by the pathogen is usually the accumulation of tylose, polysaccharides and phenolic compounds (gummosis), tannins and phytoalexins. It is likely that at least a part of the external and internal symptoms are caused by phytotoxic fungal metabolites produced in decayed wood, or by the oxidation of some host-response substances. Some chemicals produced in grapevine in response to fungal infection are toxic, notably α-glucans and two naphthalenone pentaketides, scytalone and isosclerone [22]. A similar situation may be expected in poplar.
The final stage of the disease is apparently accomplished by parenchymal fungi. The spores released from reproductive structures produced in dead wood in the presence of water are dispersed by wind, potentially infecting fresh new wounds. Among the parenchymal fungi, bracket fungi (Polyporales, Basidiomycota) were, surprisingly, found only sporadically; they usually dominate communities of wood-rotting organisms. In grapevine, the phytoalexin resveratrol showed a direct antifungal effect, inhibiting the in vitro growth of two bracket species, Fomitiporia mediterranea and Stereum hirsutum. It is possible that the accumulation of certain compounds produced by poplar suppresses the colonization of wood by bracket fungi.

Effects of Climate
Up to 133 fungal species of 34 genera have so far been associated with grapevine trunk diseases worldwide [127]. The incidence of particular taxa differs between regions. All known grapevine trunk pathogens have been encountered in all grape-cultivation regions, mainly between latitudes of 30 • to 50 • , where annual mean temperatures are generally 10-20 • C [127,167]. There are conflicting reports on the effects of temperature and water stress on the incidence of grapevine trunk disease [127]. Therefore, it is not possible to assume a straightforward relationship between poplar disease and climatic conditions, particularly concerning water stress. Water stress is likely, however, to increase susceptibility. In recent years, precipitation in central Europe has often been characterized by extreme events (fog, hailstorms, thunderhails, heat waves, heavy rains, floods, winds), followed by drought. Increased humidity favours disease development. Infection by ascospores or conidia released from perithecia or pycnidia embedded in the bark or wood will be promoted by high humidity, often associated with higher temperatures; such conditions encourage the release and spread of spores, and favour spore germination [168][169][170][171]. The inoculum potential is consequently increased.
An extremely hot and dry summer (particularly August and September) occurred across Poland in 2015. The climate projections for Poland and central Europe predict further warming and the continuation of the changes already observed, including decreased precipitation and drought, especially in summer [172]. Such conditions may be expected to affect the health of poplar and other trees.

Control and Mitigation
Fungicides such as sodium arsenite or 8-hydroxyquinoline, used against esca and with the potential to control the wilt of poplar, are banned in Europe. No other highly effective treatments are available. Other chemical products and biological stimulators used in vineyards are not curative, and so only preventive methods are available in poplar plan-tations. Infections in grapevine from propagating materials can increase from 40% before cuttings are taken up to 70% after nursery processing [172]. Detection prior to planting is therefore critical to assure the longevity of newly established plantations [173]. A healthy poplar at planting is fundamental to the establishment and sustainability of a plantation. Good hygiene and wound protection are of the utmost importance. The disinfection of propagating materials with fungicides or hot water treatment (50 • C for 30 min), applied correctly to avoid plant stress and death, is advisable. Where soil constitutes the main source of the inoculum, disease management practices based on soil disinfestation and amendments, plant-based resistance to infection, and prophylactic cultural practices should be applied. Infected plant parts and infected dead wood on the soil should be removed, pruning wounds should be chemically protected, and the elimination of plant-stress factors should be taken into account.

1.
Populus hybrids may be subjected to various, thus far unidentified pathogenic agents.

2.
New diseases may be asymptomatic, at least in the initial phase.

3.
The indigenous microbiota can be involved in the development of the disease, but can also have an important role in limiting or preventing the development of pathogens.

4.
The development of new diseases is related to climate change. It can lead to the near-total disappearance of some diseases, the sudden emergence of a new pathogens, or to the fungi already present becoming pathogenic.

5.
Poplar wilt symptoms may be a consequence of various factors, the most important being climate and its effects on fungal development and the host-pathogen relationship. 6.
Fungal diseases can spread from the soil or from introduced plant material, with the latter potentially introducing them into new areas.