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. 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.
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. 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. Further, using DIVA-GIS ver. 7.1.7 software (http:///www.diva-gis.org) two bioclimatic variables: BIO3- annual mean temperature [ºC] and BIO14- annual precipitation [mm] from WorldClim database (http://www.wordclim.org/current)  were ascribed to each collection site. The average monthly climatic data, registered by the worldwide meteorological stations network in the period 1950–2000 are available at world-clim.org . Average concentration of Pb2+, Cu2+ and Cd2+ ions in soils for regions of collections sites were given on the basis of Terelak .
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, theirs 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 (52.214 N; 20.644 E). No additional treatments (fertilization, watering, chemical weed control) were applied.
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. Figure 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 (E+ [%]) 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. 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.
From each ecotype for both E + and E- forms 24 plants were planted, 3 in a 1.5 l pot containing mixture (1:2) of sterilized sand and peat substrate of the final content of: 95.1 N; 150.2 P2O5; 153.3 K2O; 55.5 MgO; 7.7 Pb2+; 0.2 Cd2+ and 2.4 Cu2+ [mg·kg− 1 of dry substrate]; pH = 6.1 and 13% of soil organic carbon (SOC). Experiment was run in unheated glasshouse, starting from late spring for 16 weeks in total, with first 7 weeks of HM treatment. HM ions were given tenfold during watering, in repetitive cycles: twice– every third day/ twice–once per week, which brought in total 20 mg of Cd2+ and 700 mg of both Pb2+ and Cu2+ ions in 1 kg− 1 of the used substrate. Finally, HM ions concentration in the substrate, as extracted by water, reached: 15.5 Cd2+; 550.9 Pb2+; 546.0 Cu2+ [mg·kg− 1]. The analysis of soil chemical properties were done by Regional Agrochemical Station in Warsaw (accredited laboratory acc. PN-EN ISO/lEC 17025:2005).
Determination of HM concentration were done as described previously (Żurek et al., 2014) by Regional Agrochemical Station in Warsaw. 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 70o C for 3 days. Three hundred [mg] of dried, ground plant material was wet-ashed using concentrated nitric acid (Merck) in a microwave system (MDS 2000, CEM, USA). For determination of total HM ions (Cd2+, Pb2+ and Cu2+) 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 (HClO4) and fluoric (HF) acids and. Amount of Cd2+, Pb2+ and Cu2+ ions were measured using inductive coupled plasma spectrometry (ICP-AES). (Spectro Analytical Instruments GmbH, Kleve, Germany).
Phenotyping of E + and E- ecotypes responses to HM iones
Biometric phenotyping of the terrestrials part of plants was done in order to determine the rate of plant growth. Three cuts of aerial parts of plants from all experimental pots were done after 1 and 2 and 4 months, at the end of the experiment followed by drying at 70o C for 3 days for determiantion of dry matter yield.
Chlorophyll Content Index (CCI) was measured with CCM200 Plus (PSI, Brno, Czech Republic), on 3 leaves per plant for total 24 plants of both forms E + and E- of each ecotype. The single result consisted of five single measurements per leaf. Chlorophyll a (Chl a) fluorescence was measured using PocketPEA portable fluorimeter (Hansatech Instruments, King’s Lynn, Norfolk, UK). Three measurements per plant (3 plants per ecotype per replication per variant) were done. Fluorescence was induced by saturating, red actinic light with energy of 3.500 µmol·m− 2·s− 1 and first 3 seconds of transient fluorescence, covering more than its exponential growing part, was registered with time intervals increasing from 10 µs within first 300 µs of the measurement up to 100 ms intervals for times longer than 0.3 sec. Measured and calculated parameters were used for interpretation of endophyte-plant interaction in the presence of HM ions. Measured parameters: FO ≈ F50µs [minimal fluorescence]; FM = FP [maximal recorded fluorescence]; tFM [time (in ms) to reach the maximal fluorescence, FM]; Area [total complementary area between the fluorescence induction curve and FM of OJIP curve]. Parameters calculated and listed by PocketPEA software: FV [maximal variable fluorescence calculated as FM – FO]; FV/FM [force of the light reactions]; RC/ABS [the amount of active reaction centers per absorption]; (1-VJ)/VJ [measure of forward electron transport]; PIABS [performance index]. Measurements were done 2 weeks after last HM ions dosing and before 2nd cut.
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. T-tests were performed at independent samples mode. Principal Component Analysis (PCA) analysis, based on correlation matrix algorithm were performed for all chlorophyll fluorescence a traits measured and calculated for all ecotypes.