The origin of Epichloë endophyte - perennial ryegrass symbionts modify plant reactions to elevated concentration of Pb2+, Cd2+ and Cu2+ ions in soil

Background: The phenomenon of plant mutualistic symbiosis with microbes may have a positive effect on improvement of plant tolerance to environmental stresses. The influence of fungal endophyte of the Epichloë sp. (Clavicipitaceae) on perennial ryegrass (Lolium perenne L.) plants grown in presence of elevated concentration of heavy metal (HM) ions: Cd2+, Pb2+ and Cu2+, in soil was studied. Results: The presence of Epichloë in the host grass tissues resulted in increased accumulation of HM ions in aerial parts of plants and was dependent on host genotypes related to host plant origin. In plants with (E+) and without (E-) endophytes the hormesis effect was induced by elevated concentration of Cu2+ ions, resulting in better growth and photosynthesis, as examined by measurements of Chl a fluorescence. The obtained results indicate that based on the laboratory evaluation of the efficiency of the symbiosis, we were able to choose the best associations of perennial ryegrass with endophytes for HM phytoremediation. Conclusions: The presence of Epichloë endophytes positively affected ryegrass ability to accumulate HM ions and this accumulation was associated with the origin of Epichloë-ryegrass symbionts.


Background
Endophytes are able to colonize plant tissues and live without inducing any visible symptoms of biotic stress in plants. The Epichloë species (Clavicipitaceae, Hypocreales), are specialized fungi of cool-season grasses which can grow throughout the aerial parts of their host plants, forming systemic and predominantly asymptomatic associations, resulting in defensive mutualism (Tadych et al., 2014). In general, as a consequence of host plantmicrobe interactions, these endophytes produce a range of alkaloids and stimulate the host plant for enhanced synthesis of primary and secondary metabolites, e.g. free sugars, sugar alcohols, proline, glutamic acid, phospholipids, proteins and polysaccharides (Avila et  . Also the roots metabolism is altered in response to colonization of the aboveground parts of plants (Strehmel et al., 2016). Altogether, the mutual associations lead to changes in host plant gene expression and improve plant adaptations to environmental stresses, both: biotic (e.g. insects, herbivore animals, diseases) and abiotic (e.g. drought) (Bacon et al., 2015;Dupont et al., 2015;Rodriguez et al., 2008;Schardl et al., 2012Schardl et al., , 2013. The basic physiological aspect of the endophyte host interactions is poorly understood (Kaul et al., 2016). Dupont et al. (2015) have shown that in controlled conditions the Epichloë-ryegrass association induced more than one third of host plant genes, that was ten times more than in other plant-fungi symbiotic interactions and nearly twice more than in the case of plants infected by pathogens, as detected by microarray and RNAseq. 'Endophyte-induced' genes of the host were mostly responsible for the reprogramming of secondary metabolism at a cost to primary metabolism. Among a quarter of genes which were down regulated were genes of Calvin cycle and the tetrapyrrole pathway of chlorophyll synthesis; also the slowdown of photosynthesis was detected. In described experiment the Epichloë free seeds were germinated prior to infection under controlled conditions (Dupont et al., 2015). On the other hand Rozpądek et al. (2015) has shown that in symbiosis of Epichloë typhina-Dactylis glomerata, the photochemistry of photosystem II (PSII) and carbon assimilation were slightly improved. Earlier studies also documented weak or no influence of endophytes on photosynthesis in non-stressed hosts and defensive mutualism of the Epichloë endophyte in Achnatherum inebrians against Blumeria graminis (Clay, 1988;Bacon, 1993 modifications in plant tissues that display common characteristics with those induced by drought (Barceló and Poschenrieder, 1990). Membrane damage and altered enzyme activities lead to a wide range of secondary effects that concern practically all the physiological processes. Photosynthesis is very sensitive process due to several structural and metabolic disturbances: direct interactions of HM ions with thiol-, histidyl-and carboxylgroups of cell proteins; induction of reactive oxygen species (ROS) formation; displacement of essential cations in protein active centers (Hall, 2002;Hossain et al., 2012;Farid et al. 2013). Some ions such as Hg 2+ , Cu 2+ , Cd 2+ , Ni 2+ or Zn 2+ may substitute the central Mg 2+ ion in chlorophyll molecules, forming complexes lowering the quantum efficiency of PSII (van Asche and Clijsters, 1990;Sharma and Dietz, 2009). These circumstances affect most of parameters of chlorophyll a (Chl a) fluorescence detected by so called JIP test (Żurek et al. 2014). However it has been demonstrated that endophytes play a key role in host plant adaptation to polluted environments and that they can enhance phytoremediation by mobilizing/degrading or immobilizing contaminants in the soil, promoting plant growth, decreasing phytotoxicity and improving plants' HM ions tolerance (Soleimani et al. 2010, Li et al. 2012a). The importance of endophytic Epichloë species for ecosystems due to modulation of both below-and aboveground ecosystem processes is well recognized and accepted (Saikkonen et al., 2016).
The settlement of the polluted environment by plant-microorganisms symbionts for phytoremediation purposes has been defined as a 'low-input biotechnology for the ecosystem revitalization' (Abhilash et al., 2012).
Phytoremediation is increasingly used as a sustainable approach for soil remediation. However, methodology improvement is constantly forced due to the expected increase in phytoremediation efficacy as well as due to economic reasons. Due to complex biological interactions, currently used methods do not always give the demanded results, so further multidirectional studies are needed (Thijs et al. 2017). The research hypothesis presented in this paper indicates that the origin of Epichloë endophyte -perennial ryegrass symbionts modify plant reactions to elevated concentration of Pb 2+ , Cd 2+ and Cu 2+ ions in soil.

Results
It is well known that the presence of endophytes in plants can have a positive effect on improving plant tolerance to environmental stress. The research undertaken in the work focused on the impact of the presence of Epichloë endophytes in perennial ryegrass plants on their growth in conditions of elevated concentration of Pb +2 , Cu +2 and Cd +2 ions in soil.

Plant collection sites
Most of the soils on which meadows were located and from which perennial ryegrass plants were derived, were of mineral or organic type, with medium or low soil moisture content, mainly with medium or low intensity usage as pastures or for cutting (Suppl.  Table 1).

Endophyte detection
The average endophyte incidence in perennial ryegrass plants was 79.7% (Table1). The lowest endophyte incidence was noted for site at the most northern position -#50 (POD region). However, relatively low values were also noted for sites from other regions (# 801 and # 730 from MAZ and #227 from SWI). More sites of high endophyte incidence, above 90% were noted for LUB and SWI than for MAZ and POD.

Phenotyping of endophyte-infected (E+) and endophyte-free (E-) ecotypes responses to HM iones
By using term 'ecotype' we mention a group of plants within a species that is adapted to particular environmental conditions (locality) and therefore exhibits structural or physiological differences from the other members of the same species. Biomass yields were significantly affected by the ecotype and HM treatment throughout the whole experiment whereas the main effect of the endophyte was significant only for the first (after a month) and second cuts (after two months) ( Table 2). Generally, plants grown in the presence of HM ions yielded much better than control plants (Fig. 2, Suppl. Fig. 2). The yield of plants grown in the presence of HM, despite the endophyte status of plants, were 148% of control at 1 st cut, 442%, at 2 nd and 243% at 3 rd cut, in average for the whole experiment total yield from HM treated plants was 215% higher than from control plants.
Elevated concentration of the HM in the soil as well as the origin of the tested ecotypes were the main sources of variation for the relative chlorophyll content, expressed as CCI. In contrast, neither endophyte presence nor its interaction with the plant origin and HM gave a significant effect on the CCI ( Table 2). The CCI in HM treated ecotypes was in average higher than in non HM treated ones (Fig.3). The differences were higher for the ecotypes originated from the northern areas (# 50, #801, #131, #685) than from the southern ones (#227, #87) (Fig. 3).  (Table 3). Not the ecotype nor endophyte status resulted in significant effect of any from above mentioned Chl a fluorescence parameters. However, significant interaction between HM presence in soil and endophyte presence in plants has been calculated for F O , F M , F V , F V /F M , F V /F O and Area (Table 3, Figure 4). For parameters: T FM , RC/ABS and PI ABS none of main sources of variation nor interactions were significant, therefore they were not listed in Table 3 nor on Fig. 4. Figure 4, perennial ryegrass plants, if grown without addition of HM, exposed some negative effects of endophyte presence in tissues, as reflected in lower values of F M , F V and Area.

Considering interactions presented on
When HM was added to the soil medium, values of mentioned parameters increased in the presence of endophyte. However, value of the parameter reflecting force of light reactions of PS II (F V /F M ) was significantly lower in the presence of HM in soil and endophyte in plant tissues.
Measured parameters of Chl a (F O , F M , F V ) were higher in E-, and were also only slightly influenced by HM treatment, as it has been explained by the analysis of the data ( Table 3, Suppl. Fig. 3). In leaves of E+ plants, higher values of Chl a fluorescence measured parameters were detected in the ecotypes collected from more northerly localized sites (higher latitude values), for which weaker positive reactions to HM ions were detected (Suppl. Fig. 3). Only one E+ ecotype, #730, was characterized by decrease of measured parameters. That ecotype was collected from the halfway between most north and most south locations. Two other E+ ecotypes collected south from that point (#45 and #273) were characterized by about twofold increase of measured parameters in presence of HM in the soil. E+ plants, from southern locations (in order north-south: #160, #129, #227, #87) were characterized by nearly the same changes of measured parameters in a response to HM (Suppl. Fig. 3).
On the other hand, proportions of measured parameters slightly (less than 5%) decreased in leaves of most plants grown in the presence of HM ions. Interestingly, E+ plants collected in more northern localities were characterized by more visible decline of F V /F M and F V /F 0 ratios. And, as in the case of measured parameters, E+ ecotype #730 reacted differently, by theirs slight increase. The ratio of F V /F 0 was ≤ 4.0 in E-plants, whereas in E+ plants in 3 cases the ratio exceeded 4 (# 45, #87, #873). Parameter (1-Vj)/Vj, the measure of forward electron transport, seemed to be slightly affected by HM, especially in the leaves of E+ plants.
The PCA (Principal Component Analysis) run on bases of Chl a fluorescence parameters has shown distribution of ecotypes depending on the endophyte presence mostly over the OX axis (first factor) (Fig. 5, Sup. Tab 2), which means, that mostly measured parameters, significantly correlated with the first factor (F 0 , F V , F M , and Area) influenced such grouping.

HM ions content in E+ and E-ecotypes
Analysis of variance for the data of HM ions concentration in the plant tissue revealed statistically strong influence of both, plant origin and endophyte presence in the host plant as well as their interaction. The exception was the influence of endophyte presence and Pb 2+ ions concentration in plant leaves (Table 4 and 5, Fig. 6).
The highest concentration of HM ions (sum of Pb 2+ , Cd 2+ and Cu 2+ ) was detected in the leaves of #160 E+ ecotype (102 mg·kg -1 ), whereas in the leaves of the E-plants, the concentration of HM was low (44 mg·kg -1 ). Differences in the particular ions concentration of above mentioned ecotype were as follows: almost two fold higher concentration of Pb 2+ and Cd 2+ ions and three fold of Cu 2+ in E+ plants as compared to E-. As for the Eplants, the highest concentration of Pb 2+ was detected in the ecotype #50, (43.9 mg•kg -1 ) whereas the lowest in the ecotype #227 (10.4 mg•kg -1 ). Considering E+ plants, the highest Pb 2+ concentration (40.7 mg•kg -1 ) was detected in above mentioned ecotype #160, and also high in # 685 and # 873 (33.2 and 32.7 mg•kg -1 , respectively). For all those three ecotypes Pb 2+ concentration in E+ plants was significantly higher than in Eplants.
Cadmium concentration in aerial parts of E+ ecotypes was the highest in #801 ecotype (19.8 mg kg -1 ) as well as in #45 and #685 (16.2 and 15.1 mg kg -1 , respectively) ( and Cu 2+ ions); E+ plants accumulated higher amount of HM ions from the soil than E-plants. In our experiment there were three ecotypes #160, 129 and 685 which accumulated all HM ions in higher concentration in E+ than E-. Following ecotypes: #45, 227, 273 and 873 ecotypes accumulated two different HM ions in higher concentration in E+ than E-; all above relations between HM ions concentration in E+ and E-plants in one ecotype -#730.
Four E+ ecotypes, which were the most effective in extraction of HM ions from polluted soil (#160 and 227, 129, 273) originated from SWK region.

Discussion
There is increasing evidence that interactions of plants and microbes (including endophytes) play a critical role in metal phytoextraction and metal-mining, as they mediate different physicochemical and biological activities to facilitate ecological performances of the host plant (Muehe et al., 2015).
The results of our studies revealed considerable variation in terms of the grass-fungus association's ability to cope with elevated concentration of heavy metals ions in soil. We suggest that origin of ecotypes (i.e. place where associations grow and were shaped by natural conditions) affected their ability to accumulate heavy metal ions in aerial parts of plants. Spatial variation of mutualistic interactions between a host organism (grass plant) and infecting fungus (endophyte) by means of its intensity (endophyte frequency per locality) and production of toxic metabolite i.e. ergovaline, has been previously described ( In the current experiment we have observed the whole range of possible reactions: from E+ plants accumulating less HM than E-plants, through no effect, to increased accumulation of one, two or three HM ions from the soil by E+ plants. Detected differences resulted, probably, not only from differences in the endophyte activities but also from strong interactions between the fungus and the host plant, which arose as a result of particular conditions in an origin site. In current research, spatial aggregation of E+ plants able to uptake relatively higher amounts of the HM from the soil has been found for Pb 2+ accumulation. Perennial ryegrass ecotypes collected from the SWK region (locations below the latitude 50.84 N) demonstrated the ability for accumulation of relatively higher concentration of Pb 2+ ions in E+ plants than those from the other regions. It could be presumed that it is in line with natural concentration of Pb 2+ in the soils from this region which was concentrated in average of 17.8 mg·kg -1 of soil as compared to 9.4-10.2 mg·kg -1 of the soils from other sampling sites in our experiment (see Suppl. Table 1). Hesse et. al. (2003,2004) concluded that plant-endophyte associations are adapted to their native habitats via natural selection. As we have mentioned before, natural content of the HM, especially Pb 2+ ions, in soil was higher in the SWK region than in other regions. Probably symbionts of this origin used to accumulate more Pb 2+ than those coming from areas of low Pb 2+ concentration. The economic significance of grass-endophyte associations has been studied intensively in New Zealand in aspect of grazing ruminants (Cosgrove et al., 2010). The benefits of grass-endophyte associations can be improved by selection, and such selection can substantially alter the profile of the secondary metabolites produced by the symbiont. There are clearly established precedents for selecting endophytic fungal strains for beneficial purposes as for example disease, pests and drought resistance (Easton et al., 2001;Easton and Tapper, 2005). Many different types of endophyte-grass symbionts are used for improvement of agricultural production in New Zealand due to increased resistance to environmental stresses and consequently a better yield (Johnson et al,. 2013).

Conclusions
Tested associations (fungus + host) exposed wide variation in response to the presence of elevated concentration of lead, cadmium and cooper in soil. The presence of Epichloë sp. in perennial ryegrass tissues resulted in the increase of accumulation of cadmium and copper in aerial parts of the host plants. Phytobeneficial effect of endophytes was strongly dependent on specific host-fungal mutual associations, which in turn were the effect of the host plant's origin. To achieve the best result of phytoremediation of heavy metals, the choice of the most effective perennial ryegrass-Epichloë symbiosis should be based on their laboratory evaluation.

Plant collection sites
Ecotypes of perennial ryegrass (Lolium perenne L.) were collected from 12 localities in Poland in a form of living plants from permanent grasslands in most cases used for cattle feeding. Different contents of HM in soil from mentioned localities has been reported by Terelak (2007). Those areas were located in Podlaskie (POD), Mazowieckie (MAZ) and Lubelskie (LUB) and Świętokrzyskie (SWK) regions located on Central European Plain, in Poland, between northern parallels 50.4˚ -53.7˚ and eastern meridians 20.4˚ -23.1˚, on elevations 70 -300 meters above the sea level (Fig. 1, Table 1). None from 12 localities were protected area, therefore no written permission were required for collection of perennial ryegrass, which is a common and not protected species. However, at each locality, field owners, managers etc., were asked for collecting permission. This is in line with our country official regulations concerning plant genetic resources collecting at in situ conditions. Each locality, apart from GPS coordinates, was described during plant collection in terms of: general habitat description, soil moisture (high, medium, low), type and intensity of usage and soil type (mineral, organic or mineral-organic), based on observations and local farmers inquiries. Average concentration of Pb 2+ , Cu 2+ and Cd 2+ ions in soils for regions of collections sites were given on the basis of Terelak (2007).

Plant collection
From five to ten plants were picked up in each locality, with the distances of 5-10 m from each other, in order to avoid clones collection. Since plants in described experiment were part of a large collection, their numbering has no ordinal values. Ecotype #50 was picked up at Podlaskie (POD) region; ecotypes numbered: 131, 685, 730, 801 and 873 at Mazowieckie (MAZ); #45 at Lubelskie (LUB) and ecotypes numbered: 87, 129, 160, 227 and 273 were collected at Świętokrzyskie (SWK) region. Collected ecotypes were replanted in a spaced nursery, with 0.5 m distances between plants in Radzików, Poland (52.21 N; 20.64 E). No additional treatments (fertilization, watering, chemical weed control) were applied.

Endophyte detection
Epichloë endophyte-perennial ryegrass symbionts were identified by rapid staining method according to Saha et al. (1988). Ten tillers from each ecotype grown in the nursery were investigated. Small epidermal strips were peeled off the adaxial surface of the leaf sheaths and placed into a drop staining solution: 0.5 % Rose bengal in 5% of ethyl alcohol to be examined under light microscope (magnitude of x100) for the presence of fungal hyphae (E+), which appeared as an intercellular, long and convoluted hyphae parallel to the leaf-sheath axis of the plant cell without forming haustorial structures (Clay and Holah, 1999; Suppl. Fig. 1). On bases of our recent discoveries and work done on perennial ryegrass endophytes (Wiewióra et al., 2015a, 2015b), fungus forming fungal hyphae inside intercellular spaces was described as belonging to genus Epichloë (Clavicipitaceae). For each location a percentage share (Ee [%]) of Epichloë-ryegrass symbionts in total number of ecotypes collected from particular site were calculated.
Twelve E+ ecotypes were selected as material for further studies. Seeds were collected from plants of those ecotypes grown in the nursery and again tested for the presence of the endophyte hyphae using Rose bengal staining method (Saha et al. 1988). Half of the seeds from each ecotype was treated with Tebuconazole (placing the seeds in a liquid suspension), a triazole fungicide to remove the endophyte from seed bulk (E-). Both: E+ and E-seeds were sown on filter paper and seedlings were transferred to 0.5 l pots filled with mixture (1:2) of sterilized sand and peat. Seedlings were grown in pots for next 4 weeks, with frequent watering and without additional fertilization.
The presence/absence of the endophyte hyphae was again confirmed on 3-4 weeks old seedlings by Rose bengal staining prior to microscopic examination of 3 tillers per each plant. For each ecotype 12 E-and 12 E+ plants were reproduced on a vegetative way: half of each set was intendent for HM treatment and half remained as a control (no HM). As a result of the final round of vegetative reproduction 24 plants per ecotype E+ and the same number per E-were used in the experiment run in fourfold repetitions per 3 plants each. Again, the endophyte status (E+/E-) was checked.

Pot experiment
From each ecotype for both E+ and E-forms 24 plants were planted, 3 in one 1. Pot experiment has been arranged into randomized complete block design with 4 blocks, where each ecotype was grown in 4 pots per block (3 plants per pot): two pots with E+ plants and two pots with E-plants. From those four pots, two were treated with HM solution (see below) and two were control. Pots in blocks were re-arranged during the course of the experiment due to reduce positional effect and reduce the residual or pot-to-pot variance.
Experiment was run in unheated glasshouse, starting from late spring for 16 weeks in total, with first 7 weeks of HM treatment. Seedlings were planted into pots and after three weeks of growth in unheated glasshouse first watering was applied, than watering was applied 9 times during next 36 days of vegetation. Intervals between watering usually were 4 -5 days. The whole watering brought in total 20 mg of Cd 2+ and 700 mg of both Pb 2+ and Cu 2+ ions in 1 kg -1 of the used substrate. Finally, HM ions concentration in the substrate, as extracted by water, reached: 15.5 Cd 2+ ; 550.9 Pb 2+ ; 546.0 Cu 2+ [mg·kg -1 ].

Phenotyping of E+ and E-ecotypes responses to HM ions
Biometric phenotyping of aboveground part of plants was done in order to determine the rate of plant growth. Three cuts of plants from all experimental pots were done after 1, 2 and 4 months of plant growth in pots since planting, followed by drying at 70 o C for 3 days for determination of dry matter yield. Dry biomass from each pot was collected to determine HM concentration in plants. Above measurements (CCI and Chl a) were done 2 weeks after last HM ions dosing.

Chemical analysis
Determination of HM concentration in plants and soil were done as described previously (Żurek et al., 2014) by Regional Agrochemical Station in Warsaw (accredited laboratory acc. PN-EN ISO/lEC 17025:2005). Plant material was washed with tap water and then with deionized water in an ultrasonic washer to remove all soil particles followed by drying at 70 o C for 3 days. Three hundred [mg] of dried, ground plant material was wet-washed using concentrated nitric acid (Merck) in a microwave system (MDS 2000, CEM, USA).
For determination of total HM ions (Cd 2+ , Pb 2+ and Cu 2+ ) concentration in soil, extraction of air-dried soil samples taken at the end of the experiment from each pot, ground to <0.25 mm and extracted with concentrated perchloric (HClO 4 ) and fluoric (HF) acids and. Amount of Cd 2+ , Pb 2+ and Cu 2+ ions were measured using inductive coupled plasma spectrometry (ICP-AES). (Spectro Analytical Instruments GmbH, Kleve, Germany).

Statistical Analysis
All calculations were made with STATISTICA ® 12 for Windows (StatSoft, 2014). Significance of differences were accepted with 95% of probability. Lest significant differences (LSD) were calculated according to Fisher test. Ttests were performed at independent samples mode. Two-way factorial ANOVA analysis were performed with ecotypes, presence of HM in soil and endophyte presence in plants applied as main factors. Principal Component Analysis (PCA) analysis, based on correlation matrix algorithm were performed for all chlorophyll fluorescence a traits measured and calculated for all ecotypes.

Declarations
Ethics approval and consent to participate: Not applicable.

Consent for publication: Not applicable.
Availability of data and material: The data sets supporting the results of this article are included within the article and its supplementary materials.

Competing interests:
The authors declare that they have no competing interests.
Funding: Research has been founded by the Polish Ministry of Agriculture and Rural Development, which provided the financial support. This founder had no role in the design of the study, collection, analysis or interpretation of the data or in the writing of the manuscript.
Author Contributions: B.W. and G.Ż. conceived, designed and performed the experiments, data analysis and paper writing; K.R. fluorescence studies planning and data interpretation, data analysis and paper writing; K.P. fluorescence studies and data collection. All authors have read and approved the manuscript. photosynthetic ability and dry matter production of its host Achnatherum inebrians infected by Blumeria graminis under various soil water conditions. Fungal Ecol.   Table 3 Analysis  Table 4 Analysis of variance for the effect of ecotypes, endophyte presence in the host plant and their interaction on the content of HM ions in leaves of E+ (perennial ryegrass colonized by Epichloë endophyte) and E-(endophyte free perennial ryegrass). F-values were given and significance of the effects, with probability higher than 99.9% (***).