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 plant growth under conditions of elevated concentration of Pb+2, Cu+2, and Cd+2 ions in the 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). All regions except one (SWK) were characterized by relatively low concentrations of HM ions in soil: Pb2+- c.a. 9.6, Cd2+- 0.17 and Cu2+- 4.3 [mg·kg-1]. Much higher (almost twice) concentrations of HM ions have been reported by Terelak (2007) for the SWK region: Pb2+- c.a. 17.8, Cd2+- 0.37 and Cu2+- 7.6 [mg·kg-1] (Fig. 1, Suppl. Table 1).
The average endophyte incidence in perennial ryegrass plants was 79.7% (Table 1). The lowest endophyte incidence was noted in plants collected from the most northern position (POD region, ecotype 50). Low values were also noted on plants collected on MAZ (ecotypes 801 and 730) and SWK (ecotype 227). Locations characterized by a high endophyte incidence, above 90%, were noted for LUB and three ecotypes in SWK.
Analysis of biomass yields, relative chlorophyll contents and Chl a fluorescence parameters
By using the term 'ecotype' we mention a group of plants within a species that is adapted to particular environmental conditions (locality) and therefore exhibiting 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, for plants grown in the presence of HM ions dry matter yields were higher than for control plants (Fig. 2, Suppl. Fig. 2). The yield of plants grown in the presence of HM, despite the presence of endophyte in plants, were 48% higher than control at 1st cut, 342% at 2nd and 143% at 3rd cut, in average for the whole experiment total yield from HM treated plants was higher of 115% than 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 on average higher than in non-HM treated ones (Fig.3). The differences were higher for the ecotypes originated from the northern areas (ecotypes 50, 801, 131, and 685) than from the southern ones (#227, #87) (Fig. 3).
Elevated concentration of the HM in the soil was also the main source of variation of Chl a fluorescence parameters: FO, FM, FV, FV/FM, FV/FO, and (1-Vj)/Vj (Table 3). Not the ecotype nor endophyte status resulted in a significant effect of any from above mentioned Chl a fluorescence parameters. However, a significant interaction between HM presence in soil and endophyte presence in plants has been calculated for FO, FM, FV, FV/FM, FV/FO, and Area (Table 3, Figure 4). For the parameters TFM, RC/ABS, and PIABS, none of the main sources of variation nor interactions were significant, therefore they were not listed in Table 3 nor Fig. 4.
Considering interactions presented in Figure 4, perennial ryegrass plants, if grown without the addition of HM, exhibited some negative effects of endophyte presence in tissues, as reflected in lower values of FM, FV, and Area. When HM was added to the soil medium, values of the mentioned parameters increased in the presence of endophyte. However, the value of the parameter reflecting the force of light reactions of PS II (FV/FM) was significantly lower in the presence of HM in soil and endophyte in plant tissues.
Measured parameters of Chl a (FO, FM, FV) were 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) (Suppl. Fig. 3). Only one E+ ecotype, #730, was characterized by a 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 (ecotypes 45 and 273) were characterized by about twofold increase of measured parameters in the presence of HM in the soil. E+ plants, from southern locations (in order north-south ecotypes 160, 129, 227 and 87) were characterized by nearly the same changes of measured parameters in a response to HM (Suppl. Fig. 3).
Interestingly, E+ plants collected in more northern localities were characterized by a more visible decline of FV/FM and FV/F0 ratios. And, as in the case of measured parameters, E+ ecotype 730 reacted differently, by their slight increase. The ratio of FV/F0 was ≤ 4.0 in E- plants, whereas in E+ plants in 3 cases the ratio exceeded 4 (ecotypes 45, 87 and 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 have shown the 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 (F0, FV, FM, and Area), influenced such grouping.
Ecotypes with endophytes, grown without HM in soil were mostly separated on the left side of the graph, as contrary to E+ grown with the presence of HM. Negative values of factor 1, which is negatively correlated with F0, FV, FM, and Area, were ascribed to increased values of mentioned Chl a parameters. On the right side of the OX axis, along with decreasing values of Chl a parameter, points representing E+ plants grown with the addition of HM were located. This is another presentation of interaction between HM and endophyte presence.
HM ions content in E+ and E- ecotypes
Analysis of variance (Table 4) for the data of HM ions concentration in the plant tissue revealed a 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 Pb2+ ions concentration in plant leaves (Table 5, Fig. 6).
The highest concentration of HM ions (sum of Pb2+, Cd2+ and Cu2+) was detected in the leaves of E+ variant of ecotype 160 (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 the above-mentioned ecotype were as follows: almost two-fold higher concentration of Pb2+ and Cd2+ ions and three-fold of Cu2+ in E+ plants as compared to E-. As for the E- plants of other ecotypes, the highest concentration of Pb2+ 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 Pb2+ concentration (40.7 mg∙kg-1) was detected in ecotype 160, and also high in ecotypes 685 and 873 (33.2 and 32.7 mg∙kg-1, respectively). For all those three ecotypes Pb2+ concentration in E+ plants was significantly higher than in E- plants. On the other hand, for some ecotypes, the Pb2+ ions concentration was higher in E- plants as compared to E+. The relatively low Pb2+ concentration, observed in E+ ecotypes: 730 - 10.2 [mg∙kg-1],131 - 11.0 [mg∙kg-1] and 50 - 15.7 [mg∙kg-1] were found to be significantly lower than in the corresponding E- plants. Similar relations were registered for the above-mentioned ecotypes for Cu2+ ions. Concentration of Cu2+ ions in E+ plants of ecotypes: 730, 131 and 50 was 14.6, 13.8 4.6 [mg∙kg-1], respectively. For Cd2+ no such relations were confirmed.
Cadmium concentration in aerial parts of E+ ecotypes was the highest in ecotype 801 (19.8 mg kg-1) as well as in ecotypes: 45 and 685 (16.2 and 15.1 mg kg-1, respectively) (Table 5). Similarly to relations described above for Pb2+ concentration, for all three ecotypes with relatively high Cd2+ concentration in E+ plants, the Cd2+ ions concentration was significantly higher than the concentration values found in E- plants.
High copper concentration was found in aerial parts of E+ ecotypes 160, 273 and 873 (47.9, 40.6 and 37.4 mg·kg-1, respectively). All mentioned values were significantly higher than in leaves of corresponding E- plants.
E+ plants from different regions were identified as having different efficiency in HM uptake from the soil. Four plant-endophyte symbionts out of five collected in the SWK region accumulated Pb2+ ions about twice more intensively than E- plants. The mean efficiency of Pb2+ uptake by Epichloë- perennial ryegrass symbionts collected from SWK region was 165.5%. Moreover, all Epichloë- perennial ryegrass symbionts from SWK region accumulated up to 200% Cu2+ more than E- plants. The mean efficiency of Cu2+ ions uptake by E+ ecotypes fro the MAZ region was 150% higher than by E- ones, from the LUB region it was 120%. The highest values of Cd efficiency uptake were noted for the E+ ecotypes from the MAZ region: up to 200% higher than in E- plants, with the mean for the region of about 130% (Table 5).
The effect of endophyte presence in perennial ryegrass plants resulted in different types of E+ plant reactions to elevated concentration of HM ions in the soil:
- E+ plants accumulated less HM ions from the soil than E- plants. In the experiment there were two ecotypes: 131 and 50;
- E+ and E- plants accumulated the same amounts of HM ions (no significant difference). It was in case of ecotypes: 87 (Cd2+ and Cu2+ ions), 801 (Pb2+ and Cu2+ ions);
- E+ plants accumulated a higher amount of HM ions from the soil than E- plants. In our experiment, there were three ecotypes 60, 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 the above relations between HM ions concentration in E+ and E- plants in one ecotype - 730: for Pb2+ higher concentration in E- than in E+, for Cd2+ higher concentration in E+ and Cu2+ - no difference between E+ and E-.
Four E+ ecotypes, which were the most effective in the extraction of HM ions from polluted soil (ecotypes: 160 and 227, 129, 273) were provided from the SWK region.