Results and Discussion

Results

  1. ECM fungal Abundance across sites at different soil depths/layers

ECM fungal abundance was estimated within sites at different soil depths (Fig 4). Higher ASVs have been recorded in the top soil layers within each site as compared to the bottom layers. However, a significant difference could be seen in the wet site between the top and bottom soil layers (P=0.001) with an increase of 118 % as compared to the bottom soil layer. In addition, fungal abundance in the top soil layer was 68% higher in the wet site as compared to that of the dry site. Although not statistically significant, a 125 % increase in fungal ASVs in the topsoil layer of the dry sites has been recorded as compared to the bottom soil layer.

Fig 4: Disparities in fungal abundance across sites at different soil depths

Data represents mean The data represents the mean values of 50 replications (n=50) ± standard error. Lowercase letters indicate the statistical significance at a 95% confidence level.

2. ECM fungal richness (fungal guilds) across sites at different soil depths/layers

The ECM fungal guilds were identified from the soil samples taken from lodgepole pine stands; proportionated across different soil layers within sites. As hypothesized, a variation in fungal abundance and richness was found within sites and soil layers within. 23 fungal guilds (genera) have been identified from both sites, however, disparities in fungal richness have been observed across sites as well as between distinct soil layers (Fig 5). Interestingly, most of the fungal guilds were found in both sites with an exception of their distinct proportion within sites and soil layers. Opposingly, 5 of the ECM genera (Geopora, Hebeloma, Hygrophorus, Laccaria and Rhizopogon) were exclusively found in the dry sites. Similarly, 4 of the ECM fungal genera (Clavulina, Inocybe, Lactarius, and Sphaerosporella) were found in the wet sites only.

Fig 5: Proportion of ECM fungal guilds at different soil depths within each site

3. Soil parameters affecting ECM fungal abundance (ASVs)

Linear regression has been carried out to estimate the relationship of soil parameters with fungal abundance. The coefficient (R-square) and estimated P values indicate that ECM fungal abundance has a significant positive linear relationship with soil organic matter content (R-square=0.71; P<0.001) and Ergosterol (R-square=0.85; P<0.001) (Fig 6). In contrast, no significant effects were observed for fungal abundance as affected by soil parameters including soil pH, NH4, NO3, and PO4 content.

Fig 6: Linear regression: (A) Changes observed in ECM fungal abundance (ASVs) associated with changes in soil organic matter content (B) Changes observed in ECM fungal abundance (ASVs) associated with changes in soil ergosterol content

4. Lodgepole pine seedling growth as affected by ECM fungal application under drought and optimal growth conditions

ECM fungal inoculation has significantly improved lodgepole pine growth under both optimal growth conditions and drought stress as compared to control treatments (no inoculation) (P<0.001). However, variations have been observed between the effects of both fungal communities on seedling growth under different environmental growth conditions. ECMND (non-drought fungal communities) was more effective under optimal growth conditions as compared to ECMD (drought fungal communities) by increasing plant height, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight by 19, 23, 43, 22, and 23 % respectively (Fig 7: B, C, D, E and F). In contrast, ECMD (drought fungal communities) has significantly improved seedling growth as compared to both control treatment and ECMND under both moderate and severe drought stress conditions. Under moderate drought conditions, lodgepole pine seedlings as inoculated with ECMD have been observed with an increase in plant height, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight by 34, 44, 53, 46, and 55 % respectively as compared to control treatment (no inoculation) (Fig 7: B, C, D, E and F). Similar results were observed under severe drought conditions, however, the seedling growth is reduced with increasing levels of drought stress.

Besides, drought conditions have substantially reduced fungal colonization, however, fungal communities from drought areas were less affected as compared to that of non-drought fungal communities (Fig7 A). This perhaps indicates the adaptability of ECMD fungal communities to drought conditions.

Fig 7: Effect of ECM fungal inoculants on the growth of lodgepole pine seedlings: (A) Fungal colonization (B) Shoot fresh wt (C) Shoot dry wt (D) Root fresh wt (E) Root dry wt (F) Plant height. The data represents the mean values of 15 replications (n=15) ± standard error. Lowercase letters indicate the statistical significance at a 95% confidence level.

5. Antioxidation enzyme activity as influenced by ECM fungal inoculation under optimal growth conditions and drought stress

The data seems to be very interesting. As compared to control, higher values could be seen in response variables as a treatment effect, specifically, the POL and SOD's in both lodgepole pine roots and leaves, ECMD being better specifically under drought stress conditions (moderate and severe). In contrast, minimum variability could be seen in PRO activity, particularly in lodgepole pine leaves. Interestingly, under optimal growth conditions, the PRO activity was lower in all treatments (control being the highest), but with the stress, the PRO content increased in all the treatments (although not far away from each other)

The antioxidation enzyme activity in lodgepole pine seedlings is significantly affected following the inoculation of fungal communities (P<0.001) (Fig 8). The proline content in the leaves under optimal growth conditions is, however, reduced with the inoculation of ECMND and ECMD by 13 and 9 % respectively as compared to control (no inoculation) treatment. However, under moderate drought stress conditions, both fungal treatments significantly improved proline content in the leaves. In contrast, no significant improvements in proline content could be seen for either fungal community when the drought stress level was severed (Fig 8; A, B). Besides, ECMND under severe drought stress conditions showed no significant improvements in POD (peroxidase dismutase) and SOD (superoxidase) activity in lodgepole pine leaves and roots. In contrast, ECMD has significantly improved POD and SOD activity under moderate and severe drought conditions with an exception in proline content. Under moderate drought stress, POD activity in lodgepole pine leaves and roots following the inoculation of ECMD was significantly improved by 60 and 36% respectively over the control. Similarly, SOD content in lodgepole pine leaves and roots was significantly higher in ECMD treatment as compared to the control by 69 and 57% respectively (Fig 8; C, D, E and F).

Fig 8: Effect of ECM fungal inoculants on antioxidation enzyme activity of lodgepole pine seedlings: (A) Proline content in leaves (B) Proline content in roots (C) Peroxidase dismutase activity in the leaves (D) Peroxidase dismutase activity in the roots (E) Superoxidase activity in leaves (F) Superoxidase activity in the roots. The data represents the mean values of 15 replications (n=15) ± standard error Lowercase letters indicate the statistical significance at a 95% confidence level.

Discussion

The fungal diversity has significantly varied across sites and variances have been observed between different soil layers within each site. However, in wet sites, more diversity of ECM fungal communities has been observed as compared to the dry sites. The disparities among fungal guilds and their abundance are perhaps due to the climatic conditions and optimum habitat that these sites have to offer. Given that, the fungal abundance is much higher in wet sites as compared to dry sites. In addition, the fungal communities have increased affinity for the top soil layer irrespective of the sites, which could be due to the higher content of organic matter, and perhaps, moisture content. A linear regression estimation has shown the positive relationship of fungal abundance with soil organic matter content. In contrast, on some occasions, higher fungal abundance has been observed in the bottom soil layer. Based on the data available, there is no strong evidence to justify the fungal abundance of the few genera in the bottom soil layer as none of the estimated soil parameters except soil organic matter content have an effect on fungal abundance.

The application of fungal inoculants has significantly improved lodgepole pine seedling growth and resistance under both optimal and drought-stress conditions. However, ECMD being more effective under drought stress has substantially improved seedling growth as compared to both control treatment and ECMND inoculants. Interestingly, similar improvements have been observed under optimal growth conditions as compared to control treatment with an exception of ECMND being more effective under unstressed conditions and vise versa. This could be due to fungal adaptation to either drought or non-drought conditions which let them thrive better when provided with favorable growth conditions. It is very interesting to convey that ECM fungal communities irrespective of the environmental growth conditions improve plant growth however to a different extent.

Since the experiment was conducted under controlled environmental growth conditions, the outcomes of the results are not guaranteed under natural or uncontrolled environmental growth conditions. Therefore, further research in field conditions is highly advised to attest to the outcomes of this research.