Results

58 Statistical analysis

For experiment 1 fresh biomass data was collected for both aboveground and belowground portions.

Data was analysed by factorial analysis of variance and significance was assigned according to Duncan test at significance level of 0,05 p-value. In order to isolate effect of different treatments on ECM and non-ECM, total biomass data of Q. ilex seedlings was analysed through relative interaction index.

For experiment 2 data was compared for total root length, total length of fine roots and number of fine roots in seedlings growing in the presence or absence of an ECM symbiont associated to different decomposed litter by factorial analysis of variance and significance assessed by Duncan test at p-values below 0.05. More-over, the increase of length and number of fine roots was analysed to test the effect of the presence of symbiont, presence and absence and period of decomposition of self-litter added and the date of sampling. Other param-eter explaining root strategical proliferation and the trajectory assumed across the date so sampling was ana-lysed by principal component analysis (PCA). All statistical analysis and plotting were performed using Sta-tistica 10 software.

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Differently, when considering the interactive effect of the treatments, significant variations are observed for both, root biomass, shoot biomass and total biomass (Table 3.2.1). In detail, increased level of growth was observed for ECM plants in unsterilized soils. Among the ECM plants, those treated with a supplement of self-litter showed the higher level of growth compared to all the other treatment. While, no significative changes are observed in Root-shoot ratio values (Figure 3.2.4).

Table 3.2.1 GLM (Generalized linear model) testing significant variations for root, shoot, total biomass and root/ shoot parameters for Quercus ilex seedlings growing in presence of Ectomycorrhizal symbiosis of P. tinctorius, litter and sterilized soil. Significant values in bold with p-value below 0,05

Treatments Root biomass Shoot Biomass Tot Biomass Shoot/Root

Biomass

F P-value F P-value F P-value F P-value

Intercept 306,5646 0,000000 275,5821 0,000000 321,0713 0,000000 709,3360 0,000000 ECM inoculum

(ECM) ^{7,7666 0,007709 3,1206 0,083943 5,2373 0,026750 1,4969 0,227373}

Sterilization (ST) 9,3355 0,003734 3,4724 0,068791 6,0607 0,017630 0,7087 0,404244 Litter (LT) 1,8658 0,178597 2,9363 0,093342 2,7795 0,102276 0,0119 0,913547 ECM x ST 16,1402 0,000216 13,1276 0,000724 15,9348 0,000234 0,0015 0,969246 ECM x LT 0,1631 0,688187 0,0502 0,823699 0,0968 0,757136 0,0313 0,860287 ST x LT 1,1878 0,281445 0,9591 0,332537 1,1677 0,285501 0,1795 0,673811 ECM x ST x LT 3,3740 0,072700 2,3372 0,133167 3,0376 0,088036 0,0850 0,771999

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Figure 3.2.4 bar plot showing root (a), shoot (b), total biomass (d) and root/ shoot (c) values for Q. ilex seedlings growing in all the combination of the three experimental conditions (presence of Ectomycorrhizal symbiosis, presence of litter and soil steriliza-tion). Lecter indicate significant difference between each experimental unit. Significance was assigned according to Duncan post-hoc test for p-values below 0,05.

The analysis of Relative interaction index (RII) reveals that the comparison among the different treat-ment operated on Q. ilex seedling, the ECM inoculation and the removal of litter promoting growth of seed-lings. Oppositely negative values are observed for the process of sterilization becoming a growth suppressor (Figure 3.2.5). When considering litter removal and sterilization as processes affecting the growth of ECM and non-ECM plants, RII reveals that for Non-ECM plants litter removal and sterilization act as growth promoting practice. Differently, in ECM plants, the effect of sterilization strongly affected in negative way the develop-ment of seedlings (Figure 3.2.6).

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Figure 3.2.5 RII (relative interaction index) explaining the effect of Ectomycorrhizal symbiotic inoculation of P. tinctorius, Litter removal and soil sterilization on growth of Q. ilex seedlings.

Figure 3.2.6 RII (relative interaction index) showing the effect of soil sterilization and litter removal on Q. ilex seedlings in presence and absence of ectomycorrhizal symbiosis with P. tinctorius

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Figure 3.2.7 detail of the ectomycorrhizal symbiosis in Q. ilex seedlings, in the upper photo is showed a portion of the root system of Q. ilex with presence of ectomycorrhizal root tips in unsterilized soil. In the lower photo is showed the root system of ectomy-corrhizal Q. ilex seedlings in sterilized soil.

63 Experiment 2

Significant changes in roots morphometry and architecture was observed comparing ECM and Non-ECM seedlings of Q. ilex. At the end of the experiment variable level of growth has been observed between ECM plants and Non-ECM plants and according to litter supplement typology.

Among ECM seedlings, higher level of root length was observed for plants growing in undecomposed and decomposed litter (120 days) while those growing in absence of root supplement has depressed growth compared to the other. Oppositely, root growth observed in Non-ECM plants was inhibited by the presence of fresh and decomposed litter and enhanced in absence of this. Noticeably, when growing without litter, Non-ECM plants has average values of comparable to those of litter enriched Non-ECM plants. Generally, the trend described is repeated for measured variables as such as total root length, total root length of fine roots and number of fine roots (Figure 3.2.9).

Figure 3.2.8 Root systems of Q. ilex seedlings growing in presence (up) and absence (down) of ectomycorrhizal symbiosis of P.

tinctorius with three different type of litter regimes: fresh litter (0 day decomposed), aged litter (120 day decomposed) and without litter.

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Figure 3.2.9 Bar plot showing a) total root length, b) total length of fine roots and c) N° of fine root values in Q. ilex seedling growing in presence of P. tinctorius ectomycorrhizal partner with fresh litter, aged litter and with no litter. Letters indicates significant variation according to Duncan post-hoc test. Significance p-values below 0.05

The periodical measurement of root development showed that ECM plants are faster in producing roots being characterized by higher radical elongation when growing in litter enriched soil. Oppositely, Lower level of elongation is assessed in ECM plants growing without litter. Among Non-ECM plants, seedlings growing in non-enriched soil developed root metrics with similar values of those of ECM plants for the whole period. Cumulative growth trends showed that No-ECM seedlings developed best without litter starting from

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the first stages of growth until the end of the experiment. Inversely, characteristic trends are observed in Non-ECM plants growing in decomposed litter, showing root development in the initial part of the experiment higher with respect to the other sampling points, where growth are stunned or reduced compared to the begin-ning.

The negative trends of root parameters in time is observed uniquely in plants growing in presence of decomposed litter and indifferently by the presence of ECM symbiont. In detail, the decline of root elongation and proliferation is observed at the second date of sampling (1 month) in ECM-plants, rather than Non-ECM plants has a decreased ability to synthesize new roots after the first date of measurement (15 days) (Figure 3.2.10).

Figure 3.2.10 cumulative growth of Q. ilex seedlings for total root length (a), Total length of fine roots (c) and N° of fine roots (e). gain of total root length (b), Total length of fine roots (d) and N° of fine roots (f) measured each 15 days in ectomycorrhizal and non-ectomycorrhizal seedlings f Q. ilex with different litter regimes. Letters indicate significant variations assigned through Duncan post hoc test (p-value < 0,05).

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Table 3.2.3 GLM (Generalized linear model) testing significant variations in total length of fine roots, Number of fine roots and total root length of Q. ilex seedlings during a period of two months in presence of ectomycorrhizal symbiosis of P. tinctorius and conspecific litter at different stages of decomposition.

treatments Total length of fine roots N° of fine roots Total root length

F p F p F p

Intercept 252.8456 0.000000 382.8198 0.000000 432.5799 0.000000

ECM 19.0983 0.000066 22.7757 0.000017 25.5213 0.000007

Litter 0.0475 0.953665 1.7781 0.179905 0.5802 0.563646

week of growth 1.3507 0.269108 9.4895 0.000050 1.0156 0.394054

ECM*Litter 13.5504 0.000022 18.0763 0.000001 9.1856 0.000419

ECM*week of growth 1.4087 0.251707 1.2899 0.288606 0.6407 0.592594 Litter*week of growth 1.6025 0.167024 1.9464 0.092323 2.7618 0.021824 ECM*Litter*week of growth 0.5342 0.779553 1.2259 0.309768 0.9316 0.481133

Moreover, eight parameters were measured to obtain information on root architecture and strategy of proliferation in soil. Then data was reduced by means of Principal component analysis (PCA) explaining around 90 % of the total variance in the data (PCI: 68,44% and PCII 21,65%). Despite higher level of variance is explained on the first principal component, because of the effect of root growth, differentiation in the dispo-sition of variables in the second principal component appear to be the more explicative of the patterns of root proliferation and strategy. More precisely, the multivariate analysis showed that ECM plants are generally more correlated to the development of a wider root apparatus and higher ellipse area. Same strategy is assumed by litter enriched non-ECM plants that developed wider root apparatus but with lower correlation because of the scant growth. Complete inverse strategy is developed in Non-ECM plants growing with higher depth-width ratio, higher density of fine roots and higher depth (Figure 3.2.11).

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Figure 3.2.11 Principal component analysis (PCA) explaining variation in root architectures associated with ectomycorrhizal and non-ectomycorrhizal Q. ilex seedling growing with different litter regimes. In right side score plot of the variable measured to explain root system architectural changes. Left-side trajectories of associated loadings in each sampling date.