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). All regions but the last 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]. The SWK region was characterized by almost twice higher content of HM ions: Pb2+- c.a. 17.8, Cd2+- 0.37 and Cu2+- 7.6 [mg·kg-1] (Terelak, 2007) (Fig. 1, Suppl. Table 1).
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 1st cut, 442%, at 2nd and 243% at 3rd 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).
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 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 FO, FM, FV, FV/FM, FV/FO and Area (Table 3, Figure 4). For parameters: TFM, RC/ABS and PIABS none of main sources of variation nor interactions were significant, therefore they were not listed in Table 3 nor on Fig. 4.
Considering interactions presented on 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 FM, FV and Area. 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 (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 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 FV/FM and FV/F0 ratios. And, as in the case of measured parameters, E+ ecotype #730 reacted differently, by theirs slight increase. The ratio of FV/F0 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 (F0, FV, FM, 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 Pb2+ ions concentration in plant leaves (Table 4 and 5, Fig. 6).
The highest concentration of HM ions (sum of Pb2+, Cd2+ and Cu2+) 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 Pb2+ and Cd2+ ions and three fold of Cu2+ in E+ plants as compared to E-. As for the E- plants, 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 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 Pb2+ concentration in E+ plants was significantly higher than in E- plants.
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) (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. 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 form 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 was confirmed.
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 SWK region accumulated Pb2+ ions about twice more intensively than E- plants. Mean efficiency of Pb2+ uptake by Epichloë- perennial ryegrass symbionts collected from SWK region was c.a. 170%. Moreover, all Epichloë- perennial ryegrass symbionts from SWK region accumulated up to 200% Cu2+ more than E- plants. Mean efficiency of Cu2+ ions uptake by E+ ecotypes from MAZ region was 150% higher than by E- ones, from LUB region it was 120%. The highest values of Cd efficiency uptake were noted for the E+ ecotypes from MAZ region: up to 200% higher than in E- plants, with 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 our experiment there were two ecotypes: #131 and 50;
- no significant difference between E+ and E-. It was in case of # 87 (Cd2+ and Cu2+ ions), # 801 (Pb2+ and Cu2+ 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.