1. Introduction
European ash (
Fraxinus excelsior L.) is a very valuable element of nature [
1,
2]. In forestry, the species is particularly valued for its fast growth [
3] and high quality hard wood [
4]. It is also frequently planted in urban greenery due to its high aesthetic values. European ash leaves and bark, on the other hand, are used in anti-inflammatory and anti-rheumatic herbal preparations [
5]. Alas, the
F. excelsior occurrence throughout its natural range declines, which causes extensive ecological damage to the biodiversity of forest ecosystems and significant economic loses in forestry and the wood industry [
6]. The ash tree’s decline and high mortality is due to its high vulnerability to biotic and abiotic stressors [
3], both the intensity and interactive effects of which are modified by omnipresent global climate change [
7].
The most important biotic factors posing a threat to
F. excelsior include fungi. Recently, the biggest threat to
F. excelsior in Europe has been
Hymenoscyphus fraxineus (T. Kowalski), an ascomycetous fungus [
8,
9] from Asia [
10].
H. fraxineus is a very contagious invasive species in Europe, that appeared on the continent with seedlings of Manchurian ash (
Fraxinus mandshurica Rupr.). Based on genome association studies
F. excelsior, the European ash is found to be particularly susceptible to infection by
H. fraxineus [
11]. The important characteristic of this pathogen is that it is able to infect ash seeds, seedlings, as well as older trees regardless of their biosocial position and habitat properties. The
H. fraxineus related disease is called ash dieback. The disease was observed for the first time in Poland in the early 1990s, and since then it has spread throughout Europe. The characteristic symptoms of the disease include wilting of leaves, extensive necroses on shoots and branches and gradual crown thinning and dieback. Due to the high infectious potential of
H. fraxineus and the general strategy of limiting the spread of infectious diseases adopted by State Forests (a state-owned forestry holding, administrating the vast majority of Polish forests), European ash ceased to be used in forest renewals in Poland. The disease which is ash dieback is caused by
H. fraxineus has been included in the European and Mediterranean Plant Protection Organization (EPPO) alert list containing the most important pests and pathogens threatening economically important plant species, including trees [
2,
9,
10,
12,
13,
14,
15,
16,
17,
18,
19]. Two additional fungi, that is,
Minimidochium sp. and
Thielavia basicola, belong to the most commonly occurring endophytes of European ash [
20]. Under normal conditions their colonization of ash tissues, predominantly leaves, is completely asymptomatic causing no harm to the host tree [
21]. However, when the plant is under the influence of stress caused, among other things, by toxic substances accumulated in the soil e.g., compounds of heavy metals, the plant’s immunity deteriorates significantly, as a result of which endophytes become more aggressive, which further worsens the health of the host plant. On the basis of the presented information, it is advisable to establish whether endophytes may be pathogenic. Therefore, it is desirable to conduct research that determines the pathogenic potential of fungi. The pathogenic potential of a particular fungus against a given plant species must be evaluated on a case-by-case basis. This can be accomplished relatively rapidly and easily with the use of a dual culture method involving co-culturing of fungi and
in vitro grown callus or
in vitro regenerated seedlings of a given plant. The research carried out with the use of dual cultures shows that the endophytic growth is usually retarded or completely stopped before reaching the plant co-partner whereas fungal pathogens in such a setting, e.g., those of the genus
Heterobasidion, usually entirely overgrow the plant tissues relatively fast [
22]. The observed different degree of colonization of tissue cultures by fungi is due to the fact that plant tissues cultured
in vitro release numerous secondary metabolites to the medium with various levels of antifungal activity. Under normal conditions, such a barrier is sufficient to interrupt the mycelial growth of endophytes or other saprotrophic fungi, but pathogens are able to overcome this defense and to kill the cultured tissues [
23,
24].
Apart from fungal pathogens, a number of abiotic factors may negatively affect the health condition of plants, the most important of which are drought (a factor not the subject of research in this manuscript), heavy metal pollution and high salinity of soils. An example is the research carried out on the species
Prunus sargentii (Rehder) H.Ohba (1992) and
Larix kaempferi (Lamb.) Carr., which showed that woody plants, as a result of drought show visible reduction in leaf size, diminution of photosynthetic activity of the water potential in the assimilation material, and a reduction in sap movement as a result of damage to the plant’s xylem. These changes contribute to irreversible alterations in the plant phenotype and physiology, and eventually to plant death [
25]. In addition, due to human activity, especially in many economic sectors such as agriculture or heavy industry leads to the formation of dust and gases containing heavy metals particularly harmful to plants [
26]. The accumulation and occurrence of heavy metal free ions in the forest soil is a significant factor negatively affecting the growth conditions of forest trees [
27]. Long-term observations indicate that the most dangerous elements are nickel, cadmium and lead. The interaction between these elements and plants causes the appearance of many undesirable effects. It has been proven that the excess of nickel concentrations negatively affects the plants’ overall development, resulting in chloroses and necroses of tissues [
28,
29]. In contrast, cadmium interferes with the uptake of minerals by plants [
30]. Lead adversely affects the development of a plant root systems as well as the growth and development of other plant organs [
31]. These unfavorable effects of heavy metals on living organisms and the environment have influenced the application environmental pollutants determination for use in biomonitoring.
F. excelsior, as a species susceptible to the adverse effects of heavy metals, is an excellent biomonitor of the environment. These studies on the accumulation of heavy metals in plant tissues of
F. excelsior are used to determine the actual degree of environmental pollution in regions with common ash. On this basis, the extent of damage to the plant cells caused by heavy metals can be determined [
32]. Laboratory tests conducted at cellular level of heavy metals, lead in particular, cause build-up of cell wall deposits and disruption of the organelles including thylakoid grana, which damages the metabolism of photosynthesis [
33]. Build-up of lead in the nucleolus and in mitochondria massively disrupts the structure and physiology of cells leading to their death. For this reason, plants have developed some methods to recognize heavy metals in their cells. The detection of heavy metals triggers defense mechanisms in plant tissues including production of chelator secondary metabolites. The same reaction occurs in plant callus cultures
in vitro [
34]. Through these mechanisms a certain level of heavy metal tolerance may be maintained by plant tissues. Another harmful abiotic factor discussed in this work is the salinity of the substrate. Similar to heavy metal pollution, the salinity of soils in many regions of the world has increased due to human activity and global warming. Estimates show that by 2050 the area of high salinity soils (>2000 ppm) will increase by 14% [
7,
35]. According to the Food and Agriculture Organization of the United Nations (FAO) (2010) the process of salinization and sodification of soils, which is a multi-factorial phenomenon, may occur both naturally, e.g., as a result of rising sea levels or intrusion of water from the sea, rivers or groundwater, but also be of anthropogenic origin associated, for example, with intensification of agriculture or excessive use of mineral fertilisers. It is estimated that over 1100 million ha of soils on all continents are currently affected by salinity and sodicity, of which 60% are saline soils, 26% sodic soils, and 14% sodic-saline soils. For example, Europe introduces about 1 million tonnes of salt into the environment each year, and the United States up to about 10 times more salt per year on paved surfaces, resulting in secondary salinisation. Climate change can exacerbate the salinization/sodification of soils and lead to rapid inhibition of individual plant species, as well as entire habitats. Therefore, a comprehensive understanding of how individual plants respond to salinity stress is essential to improve the salt tolerance of (crop/industrial) plants, consequently improving agricultural production, the provision of essential ecosystem services, and the achievement of the Sustainable Development Goals. Soil salinity disrupts ion homeostasis, and causes osmotic and oxidative stress (the excess of reactive oxygen species), thus affecting numerous physiological processes in the plant tissues [
36,
37]. Keeping in view the negative impact of soil salinity on plant growth and development, it is essential to identify the genetic variation and genes responsible for resistance to salinity stress in plants [
38]. Reactive oxygen species (ROS) such as singlet oxygen (
1O
2), superoxide anion (O
2-) hydroxyl radical (HO•) and hydrogen peroxide (H
2O
2) originate mostly as byproducts of oxygen metabolism, as well as in other enzymatic reactions [
36,
39]. The excess H
2O
2 or other ROS species resulting from its decay is by itself dangerous for various cell components, making efficient ROS reduction an essential process ensuring the integrity of cell structures and effective regulation of metabolic processes [
36,
37,
39,
40,
41,
42]. Activity of some antioxidant enzymes, such as catalases CAT or peroxidases POX, through regulation of H
2O
2 content influence various physiological processes [
36,
37,
39], including e.g., rhizogenesis in
Mesembryanthemum crystallinum L. [
43,
44], or organogenesis in
Acanthophyllum sordidum (Bunge ex Boiss) [
45] or
Crocus sativus L. [
46] in
in vitro cultures. The increased level of ROS generated via the transfer of excess electrons to oxygen under environmental stress conditions lead to a decline in photosynthetic efficiency, and can damage photosynthetic apparatus leading to inhibition of carbon assimilation and finally reduced plants growth.
Another parameter that can be used to monitor the condition of plants or plant tissues
in vitro is chlorophyll fluorescence measured as the maximum quantum efficiency of photosystem II (Fv/Fm). The method can be applied non-destructively, allowing for easy and fast assessment of the impact of various stress factors. For instance, it can be used to determine the susceptibility of tissues to various levels and types of illumination [
47].
The survival of the F. excelsior is undoubtedly endangered by the influence of many environmental factors. For this reason, the degree of threat to the F. fraxineus population in Europe from individual biotic and abiotic factors should be assessed individually. In this study, we investigate the impact of three such factors: fungi commonly occurring in tissues of F. excelsior, heavy metals and various levels of salinity. In particular, our research was to: (i) determine the pathogenic potential of three fungi commonly occurring on F. excelsior, i.e., H. fraxineus, T. basicola, and Minimidochium sp., towards F. excelsior tissues in dual cultures, (ii), determine the influence of heavy metals (nickel, cadmium and lead) on different plant tissues (iii) determine the resistance of F. excelsior tissues to various concentrations of NaCl.
The main aim of the present study was to investigate and determine the harmfulness of selected biotic and abiotic factors in in vitro culture of F. excelsior at a cellular level, excluding the negative influence of environmental factors.
The conducted laboratory tests constitute the basis for supporting and developing effective field experiments related to the protection of F. excelsior population in the forest environment.
4. Discussion
European ash is a species with a profound ecological impact, whose presence regulates the amount of light reaching the lower parts of the stand shaping the species composition and biodiversity of underbrush and groundcover communities. Its organic debris in the litter stimulates cycling of elements in forest ecosystems [
53]. However recently, the condition of
F. excelsior in Europe is weak due to a variety of biotic and abiotic environmental factors among which the most prominent is ash dieback caused by
H. fraxineus. For many European countries, the weakening of the
F. excelsior population is so pronounced that it threatens the very existence of the species. For these reasons, we conducted a series of
in vitro experiments aiming to evaluate the susceptibility of selected genotypes of
F. excelsior to various environmental stress factors. Initially, the procedure involved plant tissue cultures used to produce the test material comprising callus cultures and seedlings regenerated via indirect organogenesis. This kind of method has a great potential for use in forest sciences in a number of practical applications. As an example, the indirect somatic organogenesis was successfully used to evaluate the regenerative potential of Aleppo pine (
Pinus halepensis Mill.), as well as to select genotypes based on their susceptibility to environmental factors [
54].
A modification of standard plant tissue cultures are plant-fungus dual cultures. The method is particularly useful to evaluate the pathogenicity of various fungal species to their host without need for extensive field experiments, as it allows the disease process to be approximated in a controlled environment. Numerous studies using this technique helped to assess the course and impact of fungal diseases in trees [
55]. Therefore, we used a dual culture approach to evaluate the relative pathogenicity of two ash endophytes,
T. basicola and
Minimidochium sp. and an ash pathogen
H. fraxineus. Their pathogenicity to
F. excelsior tissues (measured as growth rate toward the plant copartner), varied significantly (
Table 1 and
Table 2).
H. fraxineus and
T. basicola colonized both tissue types, callus and seedlings, in a similar manner. The growth pattern of
Minimidochium sp. was different, as its colonies preferred to grow away from the plant tissues (
Figure 2). The conducted experiment showed that selected species of endophytes have a completely different effect on callus and common ash seedlings. The results showed that the
T. basicola endophyte poses a threat to the health and development of the common ash as well as the potent pathogen
H. fraxineus. Therefore, it is desirable to carry out further experiments with the use of endophyte species, the effect of which on common ash tissue may cause plant death. These studies can be additionally supplemented by the genetic analysis of representatives
F. excelsior with the use of very precise analysis of genomic estimated breeding values (GEBVs). The GEBVs method is a very precise and accurate analysis that allows determination of the susceptibility of individuals to fungal infections. The application of the (GEBVs) method may complement the
in vitro tests of European ash tissue culture, the final result of which will be the selection of tree genotypes resistant to fungal infections [
11].
The impact of heavy metal pollution is another issue examined in this study. We compared the impact of three heavy metals on
F. excelsior callus cultures and seedlings. It turned out that the effects of cadmium and lead differed to that of nickel (
Table 4). The first two metals caused quick dieback of callus cultures, but the time necessary to kill seedlings was much longer (
Table 3,
Figure 3). For nickel, the time necessary to kill callus cultures and seedlings was very similar. The results of the research show that tissue cultures of
F. excelsior react differently to changing concentrations of heavy metals in the substrate. On the basis of the research results, it is advisable to use
F. excelsior as a biomonitor to assess the actual state of environmental pollution in urbanized areas [
32]. It is desirable to conduct laboratory research at a cellular level on the basis of which it will be possible to determine the degree of damage to the plant tissues taken from the urban environment. These studies will enable the precise determination of environmental pollution with heavy metals and allow the search for an
F. excelsior genotype resistant to industrial pollution. Taking this into account, one should focus on finding a solution that would minimize the negative impact of heavy metals on
F. excelsior. On the basis of the tests carried out, which showed the negative effect of heavy metals on the seedlings and callus of common ash, attention should be paid to the environmental factors that will limit the accumulation of heavy metals by plants. An important factor reducing the availability of heavy metal in the soil is the amount organic matter in the forest floor. This is because, many organic compounds form complexes with heavy metal ions that are not easily absorbed by plants. Numerous analyses show the significant role of deciduous trees in this process due to the large amounts of organic debris produced each year with cast leaves. In predominantly deciduous forest stands the soil condition is improved by increased pH and by accumulation of heavy metals by organic matter. Importantly, the absorption of heavy metals by trees may be significantly reduced by various management strategies and recultivation treatments. Recent analyses show that negative impact of heavy metals on various physiological processes, especially those related to photosynthesis and respiration, may be mitigated by salicylic acid (SA) treatment [
56,
57,
58]. Therefore, our studies on the susceptibility of
F. excelsior tissues to heavy metals should be expanded, including other metals (zinc, chromium, copper [
59], but also to evaluate the mitigating effect of phytohormones. This kind of result may help to develop growth regulator treatments effective in reducing the heavy metal stress in
F. excelsior and in other plants.
The last stress factor examined in our study was salinity, which was analyzed using two parameters, that is, activity of antioxidant enzymes and maximum quantum efficiency of PSII. The former analysis involved measurements of CAT and POX which was either increased or decreased, compared to the control, depending on the concentration of NaCl and on the tissue type (callus vs. seedlings). In callus cultures, NaCl concentrations of 1.0% and 1.5% inhibited the activity of both enzymes (
Figure 4 and
Figure 5) whereas, in seedlings the activity of CAT or POX was stimulated by NaCl addition in concentrations of 0.5% and 1.0% (
Figure 4 and
Figure 5). Higher concentrations of NaCl (1.5%) appear to be toxic, even for seedlings. This suggests that moderate levels of salinity stress increase the amounts of ROS being released in tissues, which in turn triggers antioxidant defense response in seedlings. The same reaction was not observed for callus cultures. Here, the addition of salt caused reduction in activity of CAT and POX enzymes suggesting the callus tissue is characterized by lower defense potential against salinity stress compared to seedlings. We showed that both seedlings and callus of ash are able to develop during slight/medium (not exceeding 0.5% or 1.0%) salt stress. Our results are in agreement with Raddi et al. [
60] who showed that
F. angustifolia (Vahl.) is able to germinate at low salinity and to tolerate temporarily moderate salinity (not exceeding 75 mM NaCl) conditions. Interestingly, the activity of CAT and POX enzymes in the control, both in callus and seedlings, varied significantly. This indicates that both enzymes play a role in organogenesis and morphogenetic differentiation of callus tissue and suggests that a certain threshold level of H
2O
2, (different for callus tissue and for seedlings) is necessary for the proper functioning of these processes. Catalase is a commonly used peroxisomal marker and plays a key role in removal of photorespiratory H
2O
2 [
61]. However, it may also reflect the activity of other peroxisomal metabolic pathways, especially those related to nitrogen metabolism, fatty acid oxidation and phytohormone biosynthesis (e.g., indolyl-3-acetic acid (IAA) and jasmonic acid (JA)) [
61,
62]. As a result of environmental changes, corner plants are under stress causing changes in their morphology, biochemistry and philosophy. Plants exposed to environmental stress such as drought produce excessive amounts of ROS in their cells. The reactive forms of telnus lead to damage to and death of plant cells and entire tissues. In order to prevent cell damage by ROS under conditions of high environmental stress, plants have evolved processes to prevent damage under stress. Plants produce substances such as sugars (fructose, sucrose), sugar alcohols (mannitol) and amino acids (proline), which act as antioxidants under stress conditions. These substances support the detoxification of ROS, support the protection of membranes and improve the stability of enzymes and proteins. Ultimately, the plant becomes resistant to abiotic factors [
63,
64]. Under environmental stress, photorespiration acts as an energy sink, preventing excessive reduction of the photosynthetic electron transport chain and photoinhibition, as well as preventing the accumulation of ROS [
60,
65]. Peroxidases in general are involved in multiple metabolic processes during the entire life cycle of plants, including the metabolism of ROS, reactive nitrogen species (RNS) and metabolism of phytohormones (e.g., auxins), as well as in the production of lignin and suberin, and in cross-linking of cell wall components [
41,
42,
66]. We can conclude that through regulation of ROS/H
2O
2 levels, CAT and POX are involved in specific signalling pathways [
42] including those involved in salt stress tolerance. In previous studies the differentiation in antioxidant enzyme activities has been shown to be linked with salt tolerance as well as morphogenic processes, organogenesis [
43], rhizogenesis [
44], totipotency potential and plant (oak) development [
67].
Maximum quantum effectiveness of PSII (Fv/Fm) was another parameter used to evaluate the level of salinity stress experienced by
F. excelsior tissues. The measurements showed that the Fv/Fm values recorded for callus cultures were always lower than those observed in seedlings, regardless of the NaCl concentration. This is, presumably, due to differences in the maturity of photosynthetic apparatus between these two types of plant material. Photosynthetic machinery in totipotent callus cells is expected to be less developed and less efficient compared to seedling leaves, where cells are organized to efficiently utilize light energy and to minimize damage caused by its excess [
68]. When examining the Fv/Fm values for a salt-treated callus, differences in the amount of damage caused by NaCl stress became visible (
Table 6). The most stable photosynthetic apparatus under these conditions was recorded for genotype G4, while the apparatus of the G3 genotype turned to be the weakest. Similar differences were not observed in seedlings, for which Fv/Fm values recorded under salinity stress did not differ statistically either among the NaCl concentrations, including the control, or the genotypes. Presumably, the proper development and maturity of the photosynthetic apparatus, on structural and molecular levels, is a much stronger factor determining the quantum effectiveness of PSII, under examined NaCl concentrations, than differences between the genotypes.
There is no doubt that the methods presented in the article as well as the results of our analyses can serve as useful basis for their application in functional research aiming to protect F. excelsior stands against negative effects of environmental factors. This is mostly due to the use of in vitro techniques for both production of larger amounts of plant material and for actual testing at cellular level and for plants in their youngest stages of development, i.e., seedlings. Such methods may potentially greatly facilitate testing of the effects of various biotic and abiotic environmental factors, as well as selection of resistant genotypes. Routine use of micropropagation techniques to produce F. excelsior seedlings, coupled with in vitro selection for increased resistance, may be a valid reason to reevaluate the current restrictions on planting of European ash in forests.