Phenotyping as a tool to study the impact of seed priming and arbuscular mycorrhizal fungi on tomato response to water limitation

Abstract

This study explores the effects of natural seed priming compounds (i.e. chitosan alone and in combination with salicylic acid or melatonin) with the symbiosis of arbuscular mycorrhizal fungi (AMF) on the capability of two Italian tomato varieties (Principe Borghese and San Marzano nano) to withstand water deprivation through high-throughput plant phenotyping technology. Plant responses have been automatically evaluated by integrating physiological, morpho-biometric, and biochemical data. Under water deprivation, AMF-inoculated plants exhibited enhanced physiological performance, by reducing oxidative damage and improving stomatal function. Digital phenotyping provides a non-invasive approach to assess the effects of external factors, such as the impact of mycorrhizal fungi on plant development. RGB (visible light) imaging enables the analysis of morphological traits like plant size and growth patterns, and of colorimetric changes used as a proxy of physiological responses. Biochemical analyses revealed increased carotenoid and flavonoid content in chitosan + salicylic acid-treated plants with AMF, particularly in Principe Borghese. Genotype-dependent differences were evident in terms of fruit production, where Principe Borghese plants showed significantly more red fruits in presence of AM fungus. The results underline the potential of combined AMF and natural compound application as a sustainable strategy for improving tomato resilience to water stress, contributing to resource-efficient agricultural practices and climate change mitigation.

Keywords: arbuscular mycorrhizal symbiosis; chemical priming; high-throughput plant phenotyping; stress adaptation; target metabolite analysis; water deficit.

Figure 1.

Schematic representation of the adopted experimental set-up (A), phenotyping platform (B), and data collection and sampling times (C).

Video 1.

Detail of the daily measurement of pot weight and irrigation of tomato plants at the PHENO-Lab Platform of the ALSIA 'Metapontum Agrobios' Research Centre.

Figure 2.

Effects of seed priming treatment, either in the absence (A–D) or presence (E–H) of AM fungal inoculation, on the physiological performances of two Italian tomato genotypes (Principe Borghese, San Marzano nano) at the end of water deficit imposition. Changes in the rates of (A, B and E, F) stomatal conductance (g_s, mol H₂O m^{− 2} s⁻¹) and (C, D and G, H) leaf transpiration (E, mmol H₂O m⁻² s⁻¹) in NS and WS plants. All results are reported as mean ± SE (n = 5 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 40, residual degrees of freedom = 32). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the priming treatment (T) × stress condition (S) interaction (T × S) is significant. ns, *, **, ***: not significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. The dashed red rectangle highlights the end of the water stress imposition, whereas the grey rectangle indicates the period of recovery following plant rehydration. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress.

Figure 3.

Effects of AM fungal inoculation, independently of seed priming treatment, on the physiological performances of two Italian tomato genotypes (Principe Borghese, San Marzano nano) at the end of water deficit imposition. Changes in the rates of (A and B) stomatal conductance (g_s, mol H₂O m^{− 2} s⁻¹) and (C and D) leaf transpiration (E, mmol H₂O m⁻² s⁻¹) in NS and WS plants. All results are reported as mean ± SE (n = 20 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 80, residual degrees of freedom = 76). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the stress condition (S) x inoculation (I) interaction (S × I) is significant. ns, *, **, ***: not significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. Asterisks indicate significant differences between NMYC_WS and MYC_WS plants 6 days after rehydration (13 November 2023), according to a one-way ANOVA test. The dashed red rectangle highlights the end of the water stress imposition, whereas the grey rectangle indicates the period of recovery following plant rehydration. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress.

Figure 4.

Effects of seed priming treatment, either in the presence or absence of AM fungal inoculation, on the high-throughput phenotyping parameters (A and B) height above reference, (C and D) projected shoot area, and (E and F) solidity side view, in an Italian tomato genotype (Principe Borghese), at the end of water deficit imposition, making comparison between NS and WS plants. All are reported as mean ± SE (n = 5 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 40, residual degrees of freedom = 32). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the priming treatment (T) × stress condition (S) interaction (T × S) is significant. ns, *, **, ***: not significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. The dashed red rectangle highlights the end of the water stress imposition; whereas the grey rectangle indicates the period of recovery following plant rehydration. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress.

Figure 5.

Effects of seed priming treatment, either in the presence or absence of AM fungal inoculation, on the high-throughput phenotyping parameters (A and B) height above reference, (C and D) projected shoot area, and (E and F) solidity side view, in an Italian tomato genotype (San Marzano nano), at the end of water deficit imposition, making a comparison between NS and WS plants. All results are reported as mean ± SE (n = 5 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 40, residual degrees of freedom = 32). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the priming treatment (T) × stress condition (S) interaction (T × S) is significant. ns, *, **, ***: not significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. The dashed red rectangle highlights the end of the water stress imposition, whereas the grey rectangle indicates the period of recovery following plant rehydration. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress.

Figure 6.

Effects of AM fungal inoculation, independently of seed priming treatment, on the high-throughput phenotyping parameters: (A and B) height above reference, (C and D) projected shoot area, and (E and F) solidity side view, in two Italian genotypes (Principe Borghese, San Marzano nano), with and without AM fungal inoculation, at the end of the water deficit imposition in NS and WS plants. All results are reported as mean ± SE (n = 20 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 80, residual degrees of freedom = 76). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the stress condition (S) × inoculation (I) interaction (S × I) is significant. ns, *, **, ^{***: not} significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. The dashed red rectangle highlights the end of the water stress imposition, whereas the grey rectangle indicates the period of recovery following plant rehydration. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress.

Figure 7.

Effects of AM fungal inoculation, independently of seed priming treatment, on the yield parameters of produced ripe red fruits: (A) number of red fruits, (B) average weight of red fruits, and (C) total soluble solids, in two different Italian genotypes (Principe Borghese, San Marzano nano), either in the presence or absence of AM fungal inoculation, at the end of the water deficit treatment in NS and WS plants. All results are reported as mean ± SE (n = 20 biological replicates per condition). Statistical analysis was performed using two-way ANOVA (total observations = 80, residual degrees of freedom = 76). When present, different lower-case letters denote significant differences according to Tukey HSD test (P ≤ 0.05) and are reported only when the genotype (G) × inoculation (I) interaction (G × I) is significant. ns, *, **, ***: not significant or significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. CTRL: untreated, CHI: treated with chitosan, CH + SA: treated with chitosan + salicylic acid, CHI + MEL: treated with chitosan + melatonin, NS: not-stressed, WS: water stress, PB: Principe Borghese, SMN: San Marzano nano.